The Potential Utility of Aqueous Extract of Stevia Rebaudiana Bertoni in Adjunct with Metformin in Treating Diabetes Mellitus 

Introduction

Type 2 diabetes mellitus (T2DM) and metabolic syndrome have become major public health concern. There is a sharp increase in the incidence of T2DM and obesity as a result of aging, dietary habits and decreased physical activities [1]. The majority of diabetic patients consume sweeteners with low calories to decrease their calorie intake. Most artificial sweeteners, such as cyclamates and saccharine, are a source of high calorie sugars and are potential carcinogens [2]. The increased incidence of diabetes and obesity and the growing concern over the safety of some chemical sweeteners such as aspartame, cyclamate, saccharin, sucralose, etc., have stimulated the use of natural non-calorie sweeteners [3]. In addition, the treatment of T2DM is currently unsatisfactory and new agents are needed [4]. Long-term use of oral hypoglycemic agents induces desensitization to metabolic stimuli in patients with T2DM. In the clinical setting, diabetic subjects often develop secondary failure after long-term treatment with sulfonylureas, which also have numerous side effects. Currently, there is an enormous increase in the use of herbal and other alternative medicines for the treatment of diabetes all over the world.

As a traditional medicine, the plant Stevia rebaudiana Bertoni is used in the treatment of D.M among the Guarani Indians in Paraguay and Brazil [5]. Stevia and its related glycosides have been widely used all over the world as natural zero calorie/lowcalorie sweeteners. Stevia rebaudiana Bertoni is a perennial herb native to certain regions of South America (Paraguay and Brazil) [6]. In 1905, Moises S. Bertoni, an Italian scientist described stevia plant and named it “Stevia rebaudiana Bertoni” after the chemist who first refined it, Dr. Rebaudi. The Native South Americans have been using stevia extract as a sweetener and traditional medicine for several hundred years. Stevia plant proved to posses numerous therapeutic properties for hundreds of years such as antihypertensive, antiobesity and antihyperglycemic effects [7,8]. Due to the sweetness and potential therapeutic properties of its leaf; S. rebaudiana Bertoni has attracted economic and scientific interests. It is noteworthy that a multifactorial approach combining the control of blood pressure, blood glucose, and lipid lowering action is most effective in preventing diabetic complications and /or metabolic syndrome. Consequently, therapeutic agents with diversified actions, e.g, antihyperglycemic and blood pressurelowering effects are in great demand.

Metformin is an ancient herbal remedy derived from the plant Galega officinalis (French lilac), a plant traditionally employed in Europe as a drug for diabetes (DM) treatment. In 1950, Stern et al. discovered the clinical usefulness of metformin, and it was introduced into treatment of T2DM in 1957. Since then, the same preparation of metformin has remained in clinical use for over five decades [9]. Now, metformin is recommended as a first-line drug in recent treatment guidelines of the American Diabetes Association (ADA) and European Association for the Study of Diabetes (EASD) [10]. Nevertheless, the need still exists for more thorough investigation of the pharmacological activity of stevia and, particularly; its possible use and interaction with the other known marketed antidiabetic agents. Thus, the present study will be undertaken to study the antidiabetic effect of S. rebaudiana alone and in combination with other antidiabetic drugs in rats to prove a new strategy for treatment of patients with diabetes mellitus or to use it in combination with small doses of oral antidiabetic drugs in patient with overt diabetes mellitus.

Materials and Methods

Drugs and Chemicals

Streptozotocin and metformin hydrochloride (Glucophage®) powder were provided by Sigma-Aldrich Chemical Company (Germany) and pharmaceutical companies (cid company, Egypt, Assuit), respectively. Stevia leaves were purchased from.

Plant Material

Stevia (Stevia rebaudiana Bertoni) leaves were collected from Ayub Agricultural Research Institute (AARI), Faisalabad (Reference no. 606/8). Stevia leaves were washed to remove the dirt, dust and foreign material adhered to the surface. After washing, leaves of stevia were air-dried under shade at room temperature and finely powdered with the help of grinder (MJ-176-NR-3899).

Stevia Aqueous Extract Preparation

Stevioside was extracted from the dried ground leaves of stevia plant by using water extraction. The dried ground leaves of stevia were mixed with hot water (65°C) at the ratio of 1:45 (w/v) [15]. The mixture was shaken properly and kept at room temperature for 24 h. It was stirred 2–3 times a day. After 24 h, mixture was filtered through what man filter paper and the filtrate was evaporated using rotary vacuum evaporator (EYELA N-1110S 115V) at 40-45 °C.

The Evaluation of Stevioside in Stevia Leaves

Stevioside concentration was measured by Macaulay et al’s method 21. Filtered solution of stevia extract was injected to HPLC Agilent system. Stevioside (2000 mg/l with 95% purity) was used as the standard solution. According to the Area Under the Standard Peak (AUSP), stevioside concentration in the stevia plant leaves was determined Figure 1.

Experimental Animals

Fifty adults male Wistar rats weighing 150-200 g obtained from animal house of Faculty of Medicine, Assiut University were used in all experiments were housed in standard polypropylene cages (four rats per cage). Rats were allowed water and food (laboratory chow) ad libitum. The rats were kept under standard conditions of temperature (22 ± 2₀C) and relative humidity (55 ± 5%) with 12-light/12-dark cycle. Experimental design and animal handling were according to the guidelines of the Ethical Committee of the Faculty of Medicine, Assuit University, for Animal Use.

Induction of Diabetes

The diabetes was induced in the rats by a single intraperitonial injection of 230 mg/kg of nicotinamide (NA) followed by 65 mg/ kg of streptozotocin (STZ), which was freshly prepared in citrate buffer (0.1 M, pH 4.5), after an overnight fasting (Rabbani 2009). STZ-injected animals were given 20% glucose solution for 24 h to prevent initial drug-induced hypoglycemic mortality. The normal control rats received only distilled water and standard diet. Development of diabetes mellitus in the rats was confirmed by testing fasting blood glucose (FBG), after 72 h of STZ injection. The rats with FBG higher than 200 mg/dL were considered diabetic and were selected for the study.

Animal Groups and Experimental Design

Fourty male albino rats were divided into five groups of eight animals in each group each. The animal groups divided as the following:

(i) Group A: saline (Control-non diabetic)

(ii) Group B: saline (control diabetic)

(iii) Group C: diabetic rats received 250 mg/kg metformin

(iv) Group D: diabetic rats received 300 mg/kg aquatic extract of stevia

(v) Group E: diabetic rats received 300 mg/kg aquatic extract of stevia plus 250 mg/kg of metformin. The aqueous extract of stevia and metformin was dissolved in distilled water and was given orally by stomach tube in a single dose every morning for 21 days. Doses of stevia and metformin were selected depending on our preliminary evaluation and in accordance with previous studies respectively [11,12]. The blood glucose levels were measured by Accu- check glucometer on day 0, 5, 10, 15 and 21. The blood samples were collected during the period of treatment from tail vein puncture and blood glucose levels were analyzed.

Biochemical Measurements

Blood samples were collected for biochemical measurements from the orbital sinus of the overnight fasted rats before the experiment. At the end of experimental duration the overnight fasted rats were sacrificed by decapitation. Blood, kidney and liver tissues were obtained from each animal for biochemical measurements. After centrifugation of blood samples for 10 min, the serum was collected for estimation of the levels of glucose, insulin, adiponectin, TNFα, TG, cholesterol, HDL, ALT, AST, urea and creatinine. Centrifugation of kidney and liver tissues was done for estimation of MDA levels. The samples after centrifugation could be used directly or stored at -20oC until assay. Serum glucose was determined by Accu-chek Glucometer. TG, cholesterol, HDL, ALT, AST, urea and creatinine levels were determined colorimetrically by spectophotomer. Radioimmunoassay technique was adopted to determine insulin content using an Elisa kit obtained from Calbiotech., USA, by utilizing a rat insulin antiserum to determine the level of rat insulin in serum.

TNFα was determined by using an enzyme-linked immunosorbent assay (ELISA) kit obtained from (Sino Biotech Co., Ltd, by utilizing a rat TNFα antiserum to determine the level of rat TNFα in serum. The adipocytokine, adiponectin level in the rat’s serum was also determined by using an enzyme-linked immunosorbent assay (ELISA) kit obtained from Elbascience, USA. The level of MDA in kidney and liver tissues was determined colorimetrically by spectophotomer by using a commercially available kit obtained from Biodiagnostic, Egypt.

Preparation of Kidney and Liver Tissue: The liver and one kidney from each animal were kept in 10% formalin for histopathological and immunohistochemical studies. The rest of liver and other kidney were rinsed in ice-cold saline, dissected, cleaned from fat and other tissues and blotted carefully. The kidney and liver tissues were cuted into small pieces and suitable weights were homogenized in 10% w/v phosphate buffer (pH 7.4) or saline by using a motor-driven Teflon pestle. The homogenate from liver and kidney tissue were centrifuged for 10 min at 10,000 rpm and the supernatant were used for estimation of malondialdehyde (MDA) directly or stored at -20oC until assay.

Histopathological Examination: Liver and Kidney tissues were obtained from each animal after scarification at the end of experiment. Representative sections were obtained from the liver and kidney tissues. These sections were fixed in 10% buffered formalin, processed for embedding in paraffin wax by routine protocols and 5- μm thickness sections were then cut by microtome. The sections were stained with hematoxylin& eosin stain (H&E. stain). Liver sections were examined for hydropic degeneration, vascular congestion, sinusoidal dilatation, lobular inflammation, portal inflammation and necrosis. The kidney tissues were examined for renal glomerular injury, tubular degeneration and vascular congestion. The lesions scoring in both liver and kidney tissues were scored on a semi quantitative scale as follows:

a) 0= normal (no change),

b) 1= mild (1%-30%),

c) 2= moderate (31%-70%)

d) 3= severe (>70%), based on the percentages of tissues affected.

Immunohistochemical Analysis: Other sections of 3 to 5 μm thickness were cut from the previous paraffin blocks and mounted on aminopropyl triethxysilane (APSE) coated slides. Once mounted, the slides were dried to remove any water that may be trapped under the section. This was carried by leaving the slides at 60oC oven overnight. Endothelial nitric oxide synthase (eNOS) was examined immmunohistochemically by using the standardized commercially available Rabbit Polyclonal Antibody (Elabscience Biotechnology Inc, USA) according to manufacturer’s instructions. Negative control slides were done by omitting the primary antibody. A section from vascular endothelium was stained as a positive control for eNOS.

Statistical Analysis: Results were expressed as means ± S.E.M. of the mean (X ± SEM). Statistical analysis of the differences between groups was done using the one-way analysis of variance (ANOVA), followed by Bonferroni test as post hoc analysis. The level of statistical significance was taken at P < 0.05, P < 0.01 and P < 0.001. All statistics were carried out using GraphPad Prism software (GraphPad; San Diego CA, USA).

Results

Effect on Blood Glucose Levels (BGL)

The mean values of blood glucose levels in stevia treated rats were (451.7±4.4) on day0, (391.7±4.4) on day 5, (350.0±17.3) on day 10 and (276.7±8.8) on day 15, until the results became (205±14.4) on the day 21. The results showed that, there was a decrease in the mean blood glucose levels upon daily administration of stevia extract (300mg/kg) for diabetic rats in comparison to control untreated rats as shown in Table 1. The results obtained in this study were in agreement with previous observations of [11]. Its obivious from the results that the stevia extract has a prominent blood glucose lowering effect or in other words antihyperglcemic effects. The results also showed a mild decrease in the BGL in metformin treated rats, While in the combination group of stevia extract (300mg/kg) and metformin (250 mg/kg) there is prominent decrease in the mean blood glucose level in comparison to control as shown in Table 2. The decrease on the mean blood glucose level on the combination group was starting from the day 15 (237±16.5) and become more prominent on the day 21 to become (180±11.5).

Table 1: Effects of oral daily administration of Stevia extract, metformin and their combination on the blood glucose levels of diabetic rats during the period of treatment.

Table 2: Mean of score of liver injuries of the study groups.

Effect on Serum Insulin Level

The insulin levels of diabetic and normal rats are shown in Figure 1. According to the results the insulin level of diabetic control rats (5.5±1) decreased as compared to normal control rats (14.7±1.6). While administration of stevia extract 300 mg/kg/day orally to diabetic rats for three weeks showed significant increase in insulin level (42.5±4.9) (p<0.01, Figure 1) compared to control rats. Administration of metformin 250 mg/kg/day orally to diabetic rats for the same duration showed little or insignificant effect on the serum insulin level. However, the combination of (300mg/ kg/day) stevia extract and metformin (250 mg/kg/day) orally to diabetic rats for the same duration caused a significant increase in serum the insulin level (21±1.4) (p<0.01, Figure 1) compared to control rats.

Figure 1: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of insulin in diabetic rats.

Effect on Serum Adiponectin Level

Daily treatment of diabetic rats with 250 mg/kgday metformin orally for three weeks produced significant changes in the adiponectin level. Oral administration of metformin to diabetic rats significantly increased the serum adiponectin level as compared to control rats (p<0.05, Figure 2). As shown in the same figure, daily administration of 300 mg/kg/day stevia extract with 250 mg/kg/ day metformin orally to diabetic rats for the same duration produced significant changes in the serum adiponectin level in comparison to control rats (p< 0.001, Figure 2). Stevia extract signficantly increased the effect of metformin on serum adiponectin level in comparison to control rats (p< 0.001, Figure 2). The serum adiponectin was increased from metformin group value of 4.24 ± 0.13 to 5.5 ± 0.2 by concomitant treatment with stevia extract (Figure 2).

Figure 2: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum adiponectin level in diabetic rats.

Each value represents the mean ± S.E.M. of 8 observations.

* Significant difference at P< 0.05 vs. control values.

*** Significant difference at P< 0.001 vs. control values.

Effect on Serum Lipid Profile Level

Effect on Serum Cholesterol: The serum cholesterol significantly increased in diabetic rats (62±3.2) (P < 0.001, Figure 3) than normal control rats (43.5±1.4). Administration of (300 mg/ kg/day) stevia extract significantly decreased the cholesterol level (53±1) (P < 0.05, Figure 3) as compared to control rats. Treatment of diabetic rats with metformin (250 mg/kg/day) orally for three weeks significantly decreased the cholesterol level (52.5±1) (P < 0.01, Figure 3) as compared to control rats. Concurrent administration of (300 mg/kg/day) stevia extract plus metformin (250 mg/kg/day) orally to diabetic rats significantly decreased the cholesterol level (48±1.5) (P < 0.001, Figure 3) as compared to control diabetic rats for the similar duration.

Figure 3: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of cholesterol (TC) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

* Significant difference at P < 0.05vs. diabetic control values

** Significant difference at P < 0.01vs. diabetic control values

***Significant difference at P < 0.001vs. diabetic control values.

Effect on Serum Triglycerides: As shown in Figure 4, the triglycerides (TG) level significantly increased in diabetic rats (109±3.5) (P < 0.001, Figure 4) as compared to control non diabetic rats. Administration of (300 mg/kg/day) stevia extract orally for three weeks significantly decreased the (TG) level (95.9±3.6) (P < 0.05, Figure 4) in diabetic rats. Administration of (250 mg/kg/day) metformin orally for three weeks significantly decreased the (TG) level (95.7±2.5) (P < 0.05, Figure 4) in diabetic rats. concomitant administration of (300 mg/kg/day) stevia extract with metformin (250 mg/kg/day) orally in diabetic rats produced a significant decrease in the serum triglyceride as compared to untreated diabetic rats for the similar duration (90.25 ± 2.4) (p<0.001, Figure 4). The stevia extract increased the effect of metformin on the serum triglycerides.

Figure 4: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of triglycerides (TGs) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

*Signficant difference at P < 0.05 vs. control values.

***Signficant difference at P < 0.001 vs. control values.

### Signficant difference at P < 0.001 vs. control values.

Effect on Serum High Density Lipoprotein: Daily treatment of diabetic rats with 300 mg/kg/day stevia extract orally for three weeks produced a significant increase in the serum high density lipoprotein in comparison to untreated diabetic rats (p<0.001, Figure 5). Administration of (250 mg/kg/day) metformin orally to diabetic rats produced a significant increase (28.88± 1.4, p<0.05, Figure 5) in the serum high density lipoprotein in comparison to untreated diabetic rats (35.25 ± 1.6) for the same period. On the other hand, combined administration of 300 mg/kg/day stevia extract and 250 mg/kg/day metformin orally produced a significant increase in the serum high density lipoprotein in comparison to untreated diabetic rats for the same period (p<0.001, Figure 5).

Figure 5: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of high-density lipoprotein (HDL) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

* Significant difference at P< 0.05 vs. control values.

***Signficant difference at P < 0.001 vs. control values.

Effect on Liver Function (Enzymes)

Effect on Serum Aspartate Aminotransferase (AST/GOT) Activity: Administration of (250 mg/kg/day) metformin orally to diabetic rats for three weeks produced insignificant decrease in the serum AST activity as compared to untreated diabetic rats. Combined treatment of diabetic rats with (250 mg/kg/day) metformin and (300 mg/kg/day) stevia extract orally to for three weeks significantly decreased the serum AST activity as compared to untreated diabetic rats (p<0.01, Figure 6). The serum AST was decreased from metformin treated group value of (37± 4.20) to (28.10± 3) by concomitant treatment with stevia extract (Figure 6).

Figure 6: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of aspartate tranferase enzyme (AST) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

*Signficant difference at P < 0.05 vs. control values.

**Signficant difference at P < 0.01 vs. control values.

Effect on Alanine Aminotransferase (ALT/GPT) Activity: Daily treatment of diabetic rats with 300 mg/kg/day stevia extract orally for three weeks significantly decreased the activity of serum ALT in comparison to untreated diabetic rats (p<0.001, Figure 7). Similarly, administration of 250 mg/kg/day metformin orally to diabetic rats for three weeks significantly decreased the activity of serum ALT in comparison to untreated diabetic rats (p<0.05, Figure 7). Concomitant administration of (250 mg/kg/day) metformin orally with 300 mg/kg/day stevia extract orally to diabetic rats for the same duration significantly decreased the serum ALT activity as compared to untreated diabetic rats (p<0.001, Figure 7).

Figure 7: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of alanine tranferase enzyme (ALT) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

**Signficant difference at P < 0.01 vs. control values.

Effect on Kidney Function

Effect on Serum Urea: Daily treatment of diabetic rats with (250 mg/kg/day) metformin orally for three weeks produced a significant decrease in the serum urea as compared to untreated diabetic rats (p<0.01, Figure 8). Concomitant administration of 250 mg/kg/day metformin orally with 300 mg/kg/day stevia extract orally to rats produced a significant decrease in the serum urea as compared to untreated diabetic rats for the same duration (p<0.001, Figure 8).

Figure 8: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of urea in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

**Signficant difference at P < 0.01 vs. control values

***Signficant difference at P < 0.001 vs. control values

Effect on Serum Creatinine: Similarly, administration of 250 mg/kg/day metformin orally to diabetic rats for three weeks produced a significant decrease in the serum creatinine in comparison to untreated diabetic rats for the same period (p<0.05, Figure 9). Co-administration of 250 mg/kg/day metformin orally with 300 mg/kg/day stevia extract orally to diabetic rats for three weeks produced a significant decrease in the serum creatinine as compared to untreated diabetic rats (p<0.001, Figure 9).

Figure 9: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of creatinine in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

*Signficant difference at P < 0.05 vs. control values.

**Signficant difference at P < 0.01 vs. control values.

***Signficant difference at P < 0.001 vs. control values.

Effect on Serum Total Protein Concentration: Administration of 250 mg/kg/day metformin orally to diabetic rats for three weeks produced insignificant decrease in the total protein conc. in comparison to untreated diabetic rats for the same period (p> 0.05, Figure10). Combined administration of 250 mg/kg/day metformin orally with 300 mg/kg/day stevia extract orally to rats for three weeks produced a significant decrease in the total protein conc. as compared to untreated diabetic rats (p<0.01, Figure 10).

Figure10: Effect of Effect of chronic treatment with stevia extract, metformin and their combination on the serum total protein conc. in diabetic rats.

Each value represents the mean ± S.E.M. of 8 observations. (One-way ANOVA followed by Bonferroni test).

** Significant difference at P< 0.01 vs. control values.

#Significant difference at P< 0.05 vs. metformin values.

Effect on Serum Level of Tumour Necrosis Factor-α (TNF-α): Treatment of diabetic rats with 250 mg/kg/day metformin orally for three weeks produced a significant decrease in the serum tumor necrosis factor in comparison to untreated diabetic rats for the same period (p < 0.05, Figure11). Co-administration of 250 mg/kg/ day metformin with 300 mg/kg/day stevia extract orally to rats for three weeks produced a significant decrease in the serum tumor necrosis factor as compared to untreated diabetic (p<0.001, Figure 11).

Figure 11: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of tumour necrosis factor-α (TNF-α) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

* Significant difference at P< 0.05 vs. control values.

*** Significant difference at P< 0.001 vs. control values.

#Significant difference at P< 0.05 vs. metformin values.

Effect on Renal Level of Malondialdehyde (MDA): Treatment of diabetic rats with 250 mg/kg/day metformin orally for three weeks produced significant decrease in the renal MDA level in comparison to untreated diabetic rats for the same period (p<0.001, Figure 10). Combined administration of 250 mg/kg/day metformin orally with 300 mg/kg/day stevia extract orally to diabetic rats for the same duration produced a significant decrease in the renal MDA level as compared to untreated diabetic rats and metformin treated rats (p<0.001, Figure 12).

Figure 12: Effect of oral administration of 300mg/kg/day stevia extract, 250mg/kg metformin and their combination on serum level of tumour necrosis factor-α (TNF-α) in diabetic rats.

Each value represents the mean ± S.E.M. (one-way ANOVA followed by Bonferroni test).

*** Significant difference at P< 0.001 vs. control values.

### Significant difference at P< 0.05 vs. metformin values.

Histopathological Examination of The Liver Tissue: Liver sections from the normal control group were histologically unremarkable. On the contrary, sections from diabetic control rats showed significant histopathologic changes in the form of severe hydropic degeneration, sinusoidal dilatation, vascular congestion and focal areas of inflammation and necrosis. Whereas, changes indicate chronic illness as hepatic fibrosis and macrovesicular steatosis were not detected. As regard liver injuries, Improvement of the hepatic injuries was noted in stevia treated group with insignificant difference in comparison to those treated by metformin Table 3. It is of note that combination of Stevia &Metformin significantly reduced the liver injuries than those treated with Stevia alone Table 4. Also, combination of Stevia & Metformin significantly reduced the liver injuries than those treated with Metformin alone Table 4.

Table 3: Mean of score of renal injuries of the study groups.

Table 4: The difference between the mean liver and renal injuries scores in the study groups.

The Histopathological Examination of The Kidney Tissue: Sections from the kidney of the normal control group were histologically unremarkable. Alternatively, sections of diabetic control group showed features of early diabetic renal damage in the form of hydropic degeneration of the tubules, vascular congestion and mild enlargement of glomeruli with glomerular capillaries congestion. The mean of the renal injuries was summarized in Table 3. It is of note that, the mean of renal injuries were significantly higher in the diabetic rats when compared to the control group (Table 4). Lower mean of renal injuries was observed in Stevia& Metformin treated group than those treated with Metformin alone (Table 1). However, this difference was statistically insignificant (Table 4). Combination of Stevia & Metformin significantly reduced the renal injuries than those treated with Stevia alone (Table 4).

Immunohistochemical Analysis: The immunohistochemical analysis of the kidney tissue obtained from the rats after three weeks of administration of 300mg/kg/day stevia extract orally showed a decrease in eNOS protein expression (Fig.) and an increase in the protein expression of iNOS (Fig.) as compared to control animals (Fig.). Co-administration of 300 mg/kg/day stevia extract with metformin to diabetic rats increased the protein expressions of eNOS (Fig.) and iNOS (Fig.), while daily administration of 250 mg/kg/day metformin alone to diabetic rats decreased the protein expression of eNOS (Fig.) and iNOS (Fig.).

Discussion

The currently available hypoglycemic agents for management of diabetes mellitus are ineffective and have certain drawbacks. The United Kingdom Prospective Diabetes Study (UKPDS) showed that monotherapy with oral agents often fails to maintain glycemic control over time, and many patients have to switch to treatment with combinations of oral agents or insulin therapy. Therefore, there is a demand for new natural-based medicinal compounds with diversed actions. Stevia rebaudiana Bertoni is a medicinal plant with multiple potential benefits. Metformin is a widely used oral glucose-lowering drug for type 2 diabetes. The use of biguanide as a first-line drug depends on evidence showing that metformin reduces incidence of cardiovascular events as well as total mortality, in particular in mega-trials of T2DM such as the UK Prospective Diabetes Study (UKPDS) (UKPDS, 1998). In agreement with previous findings, our results demonstrate that treatment of diabetic rats with stevia extract orally at a dose (300mg/kg/day) for three weeks produced favorable effects on blood glucose levels. Similary, concurrent administration of stevia extract (300mg/kg/ day) and metformin (250mg/kg/day) orally for the same duration also reduced blood glucose levels.

The results indicated that aqueous extract from leaves of stevia rebaudiana, produced a significant long term reduction in blood glucose levels especially when combined with other hypoglycemic agents, which provide a good control of hyperglycemia in diabetic rats. This finding is collaborated with the previous researches which proved a positive effects of stevia extract on diabetic rats. This study demonstrates that stevia extract have a good efficacy in controlling diabetes not only via decreeing the blood glucose level but also via increasing the insulin level. Previous study showed that stevioside was able to regulate blood glucose levels by enhancing not only insulin secretion and sensitivity but also insulin utilization in insulin deficient rats which was due to decreased PEPCK gene expression in rat liver [13] .These results are in agreement with [14,15] who also observed that stevia aqueous extract lowered the random and fasting blood.

We observed that the effects of stevia extract on control of blood glucose are more pronounced when combined with metformin. Previous studies showed that adiponectin plays a central role in obesity, insulin resistance and type 2 diabetes. Studies have shown that adiponectin administration in humans and rodents has insulinsensitizing, anti-atherogenic, and anti-inflammatory effects, and, in certain settings, also decreases body weight [16,17]. Concerning to the effect of metformin in obesity and related diseases, metformin has found to decrease the basal metabolic index and improved inflammatory and cardiovascular-related obesity parameters especially insulin sensitivity and the adiponectin level [18]. Our results indicate that aqueous extract of stevia has a significant effect on increasing the serum level of adiponectin. Thus the combination of stevia and metformin could provide potential therapeutic targets in the treatment of obesity, insulin resistance and type 2 diabetes.

Combined administration of stevia extract and metformin Showing improvement in lipid profile, regarding serum triglycerides and cholesterol with significant reduction in their levels. Also the serum high density lipoprotein was significantly increased. Thus, the combination of stevia extract and metformin has a synergistic effect on lipid profile. Therefore, it appears that stevia extract and metformin may provide benefits in conditions associated with impaired glucose tolerance, dyslipidemia and reduced insulin sensitivity. During diabetes or insulin resistance, failure of insulinstimulated glucose uptake by fat and muscle causes high glucose concentration in blood. Since high blood glucose is susceptible to oxidation, hyperglycemia causes high ROS production and, in turn, leads to high Malondialdehyde (MDA) levels in tissues [19]. MDA is the principal and most studied product of polyunsaturated fatty acid peroxidation which plays important role in etiopathology of several chronic diseases including diabetes [20]. Elevated levels of lipid peroxidation products (MDA) were found in diabetic rats. This might be due to overproduction of peroxides and inhibition of activities of peroxidases which is a common event in diabetic rats. Reduction of renal and hepatic MDA level was noted in stevia and stevia & metformin groups.

Consistent with the results of the current study, many experimental studies indicated that the antioxidant property of stevia extract might be due to the presence of substantial amounts of flavonoids and phenolic contents [21,22]. Currently, there is renewed interest in plant-based medicines and functional foods modulating physiological effects in the prevention and cure of diabetes. On the other hand, oxidative stress along with hyperglycemia plays a major role in the pathogenesis of diabetes. Owing to the antihyperglycemic and anti-oxidative potential of stevia extract, thus the extract of stevia may be new therapeutic agent inprevention and treatment of diabetes. The serum levels of ALT and AST were decreased in diabetic rats after treatment with aqueous extract of stevia. There is a significant decrease in serum of hepatic enzymes in stevia and stevia extract & metformin groups, which was associated with a marked improvement in the liver function and structure.

Stevia extract and its polyphenol caused a significant decrease in the levels of ALT and AST serums. These results are in accordance with [24,25] who demonstrated the hepatoprotective effect of stevia extract on the liver damage of diabetic rats. It was likely that the reduced levels of ALT and AST serums by the stevia extract and its antioxidant effect are an indication of alleviation of plasma membrane damage produced by diabetes. Also, STZ kidney injury is characterized by both diagnostic indicators and histopathological analysis [27]. The levels of renal markers such as urea and creatinine were increased in the STZ treated rats [28,29]. Animal studies also reported that STZ induced nephrotoxicity which characterized by proximal tubular nephropathy, glomerular sclerosis, interstitial fibrosis, renal tubular epithelial cell necrosis, leukocyte infiltration, and apoptosis of various renal cells [30]. In agreement with these findings, our results demonstrate that STZ produced a marked elevation of the renal markers, which was associated with impairment in the kidney structure were observed in control diabetic group.

The serum urea and creatinine levels were significantly decreased in stevia and stevia extract & metformin groups. The histopathological examination of kidney tissue revealed Lower mean of renal injuries was observed in treated groups in comparison with control group. This is in accordance with similar resuts observed by previous researchers that stevia leaves extract have a significant role in alleviating kidney damage in the STZ-diabetic rats besides its hypoglycemic effect [31]. Our results support the validity of stevia leaves extract for the management of diabetes as well as diabetes-induced renal disorders. It is known that hyperglycemia is commonly associated with disturbance of lipid metabolism, leading to the increased total cholesterol and low-density lipoprotein as well as decreased high density lipoprotein levels [6,29-32]. The long-term prognosis of type 2 diabetes relies on the treatment of hyperglycemia, and on coexisting conditions, such as hypertension, dyslipidemia and obesity, as delineated in the United Kingdom Prospective Diabetes Study (UKPDS) [33]. As approximately 50% of type 2 diabetic subjects suffer from hypertension and dyslipidemia which cause a 2-3fold increased the risk of CAD [34]. Consequently, the pharmacologic intervention in type 2 diabetes should aim to lower blood glucose and also lipid concentration. Our results revealed that treatment with stevia extract significantly decreased TC and TG levels and increased HDL level in treated diabetic rats. It has been reported that most drugs which were used in the treatment of hypercholesterolemia decrease both total and HDLcholesterol levels [35,36].

However, SR aqueous extract reduced total cholesterol level and increased HDL-cholesterol level. These results are collaborated with previous results [39-42]. Studies on humans also indicated that the consumption of SR extracts increased the level of HDL and reduced the levels of cholesterol, triglycerides, and low-density lipoproteins significantly [43]. Therefore, aqueous extract from Stevia leaves could be used as natural anti-hyperlipidemic drug for the treatment of hyperlipidemia and its associated complications (Ahmad et al., 2018). Besides antihyperglycemic efficacy, metformin represents increasing evidence of a potential efficacy in improving dyslipidemia [44]. Studies have shown that metformin decreased plasma total cholesterol and TG levels and increased HDL-c level. Similar to prior observations, our current study supported that metformin significantly reduced total cholesterol and TG levels and increased HDL-cholesterol level. Thus, combination of stevia extract and metformin could provide additive benefits on subjects with dyslipidemia and overweight/obese.

It is well known that, the proinflammatory cytokine tumour necrosis factor TNF-alpha has been implicated as a causative factor in obesity-associated insulin resistance and the pathogenesis of type 2 diabetes, exerting its actions through the immune and inflammatory pathways. Wang et al., 2012 Suggested that the proinflammatory cytokine TNF-alpha was significantly downregulated together with the expressions of interleukin 6 (IL6), interleukin 1𝛽 (IL1𝛽), and interleukin 10 (IL10), among other chemotactic and pro-inflammatory cytokines. Therefore, stevioside was seen to be able to potentiate in the reduction of insulin resistance through reducing the inflammation in adipose tissues by regulating TNF𝛼. Our results indicate that stevia extract significantly reduced serum level of TNF-α. The effect of metformin on TNF𝛼 were more pronounced when it combined with stevia extract leading to a significant decrease in its level as compared to diabetic rats treated with metformin alone. These results with increasing the insulin level suggesting that the effect of stevia extract on TNF𝛼 may be the cause of increasing insulin level and decreasing the insulin resistance.

Conclusions

The present study suggests that aqueous extract from stevia leaves may decrease the blood glucose level, while serum insulin and adiponectin levels were significantly increased in diabetic rats as compared with the diabetic control rats after 3weeks study period. The aqueous extract of stevia also showed significant decrease in lipid profile, TNFα and oxidative stress marker, (MDA). Moreover, the diabetic rats treated with stevia aqueous extract exhibited values for hepatic and renal function near normal levels. These effects of stevia extract were more prominent when it is combined with metformin. It is concluded that combination of aqueous extract of stevia at a concentration 300 mg/kg plus 250 mg/kg metformin may provide an effective therapeutic anti-diabetic combination for the treatment of diabetes and its associated complications.

Acknowledgement

The present article was extracted from the thesis written by Doaa Hamdy Abd El-hamied and financially supported by Science and technology development fund (STDF) Grant No, 12667.

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Separation of Gas Acids by Layered Double Hydroxides

Opinion

Hydrocarbon synthesis processes are well known and include fixed bed, fluid bed and slurry type processes in which a synthesis gas (syngas) comprising a mixture of H₂ and CO is reacted in the presence of a suitable Fischer-Tropsch type of hydrocarbon synthesis catalyst at conditions effective to form hydrocarbons, and preferably paraffinic hydrocarbons which are solid at standard room temperature conditions of temperature and pressure. The syngas is produced by reacting a low molecular weight hydrocarbon gas with oxygen and steam via well-known processes which include partial oxidation, catalytic steam reforming and combination thereof, using a fixed or fluid catalyst bed. A preferred source of the low molecular weight hydrocarbon is natural gas in which the hydrocarbon comprises primarily methane with minor amounts of C₂ hydrocarbons, including C⁴⁺ hydrocarbons. Other natural gas components include nitrogen, carbon dioxide, water vapor and sulfur in the form of sulfur bearing compounds including H2S, mercaptans, other organic sulfides generally, carbonyl sulfide and sometimes minor amounts of carbon disulfide. Sulfur and other acid substances in the feed to a syngas generator will poison the steam reforming catalyst and result in a loss of syngas productivity.

There are several existing options available for acid substance capture however, each of these systems has its own limitations that impede the technical or economical. Selective adsorption mechanism is a promising technique considered for acid separation. A few inorganic materials such as zeolites, activated carbons, clays were found to have good adsorption capacities of gas acids. However, they are not attractive for separation from wet feeds at high temperatures due to poor hydrothermal stability. Layered double hydroxides are inorganic compounds, and in particular their layered double oxide derivatives produced on calcination have desired properties as acid adsorbents. LDHs were prepared by the co-precipitation of divalent Mg and trivalent Al ions in an alkaline solution containing NaOH and Na₂CO₃. Samples were characterized before and after calcination using various techniques such as FTIR, XRD, TGA and XPS. Different LDH samples prepared were screened to identify the samples with optimum sorption properties. Sorption was measured both volumetrically and thermogravimetrically as a function of calcination and sorption temperatures. Reversible desorption capacities were determined using vacuum and temperature cycling methods.

A thorough investigation of aspects pertaining to aging, regeneration, impact of water and sorption potentials of the remaining flue gas components including CO₂, SO₂ and H₂S was conducted during this research. The findings and discussions from this work provide following major contributions to the gas acids capture research. Significant increase in the sorption capacity of LDHs; recyclability with good sorption and desorption potential and more than 90 % regenerabilty of LDHs; mechanistic aspects of gas acid sorption identified based on heats of adsorption and temperature programmed desorption data; good hydrothermal stability and SO₂ sorption; high sorption values and consistent performance even at low partial pressures of gas. The first contribution of this work is related to the synthesis of LDHs leading to best acid sorption capacity. Using novel synthesis procedure an increase of sorption capacity was achieved. This was possible by choosing optimum reactant concentrations and conditions from the large number of LDH samples prepared and tested. In this study different Mg/Al ratios were studied and the ration 0,72 mol/ mol were found to be the optimum values leading to high sorption capacity. Further, rate of addition of metal ions and aging of the reaction mixture were also found to produce LDHs with better surface properties including higher surface area.

Calcination of LDH at appropriate temperature leading to layered double oxide formation was another important aspect that influenced sorption capacity. We have studied processes of spinel obtaining at different calcing temperature. Calcination temperature of 800^oC was found ideal for the LDHs prepared with above concentrations. Excellent regenerabilty and stability of the material after repeated vacuum and temperature cycles with good sorption and desorption potential refers to the second contribution of this work. High consistency observed in the sorption and desorption patterns after six cycles demonstrated the stability and recyclability of our material. In all the cases more than 99 % of the original sorption capacity was recovered after regeneration. Aging or storage of LDOs in the atmosphere found to produce significant structural changes leading to the reduction in the sorption capacities. An in-situ calcination method was found to be highly productive and suggested for all sorption measurements of layered double oxide to overcome these losses. High isosteric heat of sorption calculated from the sorption isotherms suggests that it is mostly a chemisorption process. Increase of sorption values with temperature up to 200^oC also proves that the sorption process involves activation energy.

TPD results also revealed that only a small amount of total sorption is a weak adsorption. Hence, it is concluded that CO2 sorption on layered double oxides is mostly a chemisorption process rather than physisorption. Finally, CO₂ sorption on layered double oxides is explained with a suitable mechanism in correlation with TPD results. Unlike with other solid sorbents such as zeolites, presence of water in the feed proved to have no adverse impact on sorption efficiency of layered double oxides. Water was found to adsorb on layered double oxide simultaneously along with CO₂ by increasing the sorption value over the maximum dry CO₂ sorption value observed at 200^oC. The water uptake of the layered double oxides which was found to be approximately equal to the difference of wet and dry CO₂ sorptions proved that water did not restrict CO₂ sorption. Consistent sorption and desorption behavior observed after six temperature cycles also revealed the hydrothermal stability of the sorbent. LDOs found to have very high sorption potential of SO₂. Even at low feed concentrations of SO₂ the sorption values were very high indicating strong affinity of SO₂ for layered double oxides. SO₂ sorption was observed to be much stronger as regeneration levels. All the other constituents of flue gas including NO2 showed no sorption property towards layered double oxide.

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Tubo-Ovarian Abscess with Bilateral Ovarian Endometriotic Cysts Caused by Edwardsiella tarda Infection 

Introduction

Edwardsiella tarda (E. tarda) is a member of the family Enterobacteriaceae, a gram negative, motile, facultatively anaerobic rod-shaped bacteria. Edwardsiella infection are rare, the most common human infection is gastroenteritis. Here we report a case of a tubo-ovarian abscess with bilateral ovarian endometriotic cysts caused by E. tarda infection without any predisposing illnesses.

Case Report

A 42-year-old Indonesian woman patient was referred to our hospital with initial diagnosis of endometrial cysts. She was admitted for further investigation with a 2 days history of lower abdominal pain, vaginal discharge with acute onset shortness breath and generalized body weakness. Medical history was negative for any chronic illnesses. She was infertile for 16 years. On her physical examination the patient was alert and oriented with a 36.50C, pulse rate of 84/minute, blood pressure of 110/80 mm Hg and respiratory rate of 26/minute. The oxygen saturation was 96% on room air. The cardiac and lungs exam was normal. Her abdomen was distended with moderate tenderness at lower quadrant area, no rebound tenderness and no palpated mass palpable. Liver and spleen were not palpable. Extremities were warm and well-perfused. Neck and the musculoskeletal examination were normal. The patient’s pelvic examination revealed negative for cervical motion tenderness or adnexal masses.

Laboratory evaluation of blood study from initial hospital showed a normal white blood cell count of 6,700/mm3 with 92% of neutrophil, hemoglobin 11.9 g/dL, hematocrit 36% and platelet count was 335,000/μL. Serum chemistry was normal. Urinalysis was blood and nitrite positive, 20-25 RBC/HPF and 15-20 WBC/ HPF. Urine pregnancy test was negative (Table 1). On admission, re-evaluation of blood study was normal and urine pregnancy test was negative. Ultrasound of abdomen and pelvis was performed, which showed free fluid in the pouch of Douglass reported as ectopic pregnancy. Patient was scheduled for emergency laparotomy with a diagnosis of ectopic pregnancy. Gentamycin 80 mg was administered as an antimicrobial prophylaxis. Operative findings revealed bilateral perforated of ovarian cysts and pelvic abscess in the pouch of Douglass. The patient underwent bilateral salpingectomy and the specimen was sent for histopathologic examination. The pus was drained and sent for bacterial culture and antibiotic susceptibility testing. Postoperatively the patient was given empirically meropenem and metronidazole.

Table 1: Initial Laboratory Report.

The histopathologic examination revealed bilateral ovarian endometriotic cysts, multiple follicular cysts of ovary and bilateral salpingo- oophoritis. The culture result revealed only a heavy growth of Edwardsiella tarda (Figure 1a & 1b). The E. tarda isolate was susceptible to all antibiotics tested (Table 2). The patient complained increasing shortness of breath then, one day after had undergone laparotomy. Her vital signs became as follows: body temperature 36.50C, blood pressure 110/90 mmHg, pulse 120 beats/min, respiratory rate 26 breaths/min, and oxygen saturation 98%. Physical examination found vesicular breath with abnormal gallop sound which was confirmed as a minimal right sided pleural effusion by chest radiography. Her ECG showed sinus tachycardia and echocardiogram suggested pulmonary embolism. The patient was treated with 1 vial intravenous injection of furosemide. The result of D-dimer and Troponin-I test were 5.0 mg/L (normal range 0.1-0.3 mg/L) and 0.05 ng/mL (normal range <0.01 ng/mL), subsequently. The antibiotic switch to Azythromycin 1x500 mg, after day-5, for 6 days later. The patient remained stable then and was discharge after for a total of 11 days hospitalized.

Table 2: Susceptibility Test.

Figure 1a: Edwarsiella tarda colony in McConkey agar, after 24 incubation at 370C.

Figure 1b: The colony were small, transparent and punctate.

Discussion

Edwardsiella tarda is an anaerobic Gram-negative bacillus associated with freshwater environments and animal. Human infections caused by E. tarda are rare but has been reported cultured in human stool, blood, urine, CSF, abscess, peritoneal fluid and wounds. In more than 80% of cases, the organism is cultured from stool specimens and is associated with gastrointestinal illness [1]. This organism, have been reported, can result in extraintestinal infections including soft- tissue infection, bacteremia, meningitis, cholecystitis, osteomyelitis, salpingitis and endocarditis. Extraintestinal infections are rare and very often associated with immune-compromised states [2]. The cases of gynecologic infection associated with E. tarda have been reported. Two women were reported with gynecologic infections that involved abscess formation and E. tarda was isolated in pure culture from a ruptured tubo-ovarian abscess [2]. A young woman with salpingitis and tubo-ovarian abscess related to colonization of the gastrointestinal tract was diagnosed after ingestion of raw fish [3].

The patient in our case report had not recently handled any wild animals or reptiles. She had no exposure to freshwater or saltwater environment, but she often ate ‘pindang’. ‘Pindang’ is a wellcooked South Sumatra cuisine made from fresh water fish such as patin fish. The patient in our case report experienced fever, abdominal pain but without diarrhea. In fact, we did not know the exact of E. tarda entry port in this patient. There was no previous report of infertility, effusion pleura and pulmonary emboli caused by E. tarda infection. Edwarsiella tarda is usually susceptible to a wide variety of antibiotics and therefore, nearly any approved drug can be used to combat the infection [4]. In our patient, the E. tarda isolate was susceptible to all antibiotics tested and patient was cured well.

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Expression of OTX1 and OTX2 in Normal and Pathologic Conditions of The Nasal Cavity 

Introduction

Homeobox genes are a family of regulatory genes coding for transcription factors, that function as marker of specific brain areas and of nuclei of the developing central nervous system [1]. OTX1 and OTX2 homeobox genes play a critical role in controlling the antero-posterior patterning during embryonic development [2] and act in specification, regionalization and terminal differentiation of the rostral part of the central nervous system [1]. The transcription factor Orthodenticle homeobox 1 (OTX1), the vertebrate homologue of the Drosophila orthodenticle (otd) gene responsible for head formation [3] is mainly involved in brain and sensory organ development [4]. Initially, OTX1 is expressed when the anterior neuroectoderm becomes prosencephalon and mesencephalon. In later embryonic development, OTX1 is expressed in the cortical ventricular zone and in the neocortex cortical plate. It is also detected in the emerging cortical plate in the most lateral zone of the telencephalon, in the ventricular area of the ganglionic eminence, and in cerebellum [1,5] In mice, OTX1 is needed for regional identity, maintenance, and patterning of forebrain, midbrain and for neuronal differentiation [6,7]. In addition, during the adulthood, OTX1 is expressed in sense organs, especially in the anterior part of the retina, where it is involved in the development of the ciliary body and in the olfactory bulb. OTX1 has additionally been found to be involved in hematopoiesis [8,2] Different studies imply a pathological role in tumor onset and/or maintenance, based on findings of its overexpression in medulloblastomas [9,10], in aggressive non-Hodgkin lymphomas [4], breast carcinomas [11,12] and colorectal cancers [13].

Orthodenticle homeobox 2 (OTX2) is comparably involved in rostral head development, playing a critical role in forebrain and eye development [14,15]. This gene is expressed in the diencephalon, in the mesencephalon and in the epithelium of the choroid plexus [1]. At later embryonic stages, OTX2 is expressed in different regions of the brain, including the hippocampal angle, the pineal gland and also the cerebellum, and it is also located in the inner eye, retina and olfactory system [3,15,16], in both vomero-nasal organ and the major olfactory epithelium [17]. OTX2 mutations lead to severe ocular defects (anophthalmia, microphthalmia, optic nerve or optic chiasm hypoplasia and retinal dystrophies) even associated with brain malformations such as the otocephaly-dysgnathia complex [18] or pituitary abnormalities [19]. In cancer, OTX2 has been found to be highly expressed in medulloblastomas [10,15,20]. It was demonstrated by array comparative genomic hybridization (aCGH) that the olfactory neuroblastoma carry altered karyotype with high levels of aneuploidy [21]. Moreover, it was proved that OTX2 and Crx genes are expressed in retinoblastoma tumors as markers of differentiation in these tumors [22,23].

Despite the involvement of both OTX genes in the development of the olfactory system very few studies showed a localization of OTX1 and/or OTX2 expression in human nasal mucosa, paranasal sinuses and nasopharynx neither in physiological nor in tumoral conditions. Sinonasal carcinomas are infrequent neoplasms, that count less than 3% of tumors arising from head and neck area. Diagnosis and treatment are difficult because of their low incidence, histological diversity, non-specific symptoms, location and staging [24]. The disease occurs in approximately 1/100000 inhabitants per year and the mean age of appearance is between 60 and 70 years. Despite progresses in surgical techniques and radiotherapy, the management of the disease is difficult and complex, leading to high morbidity and mortality.

In recent years, with the advancement of molecular diagnostic methods, the attention has been focused on developing individualized target therapies for treating these different types of cancer. The most frequent alterations associated to head and neck tumors are mutations in TP53, EGFR, HER2, KRAS, BRAF and WNT. TP53 is often mutated (18-77%) in the initial stage of head and neck tumors, highly in sinonasal adenocarcinomas (SNAs) and sinonasal squamous cell carcinomas (SNSCCs), and it has been associated with increased chemoresistance both in SNSCCs and intestinal type SNA (ITAC) [25,24]. EGFR and HER2 play a key role in the pathogenesis of SNSCCs and the overexpression of these genes is associated with poor prognosis and greater relapse rates. Moreover, EGFR has been showed to be upregulated in ITAC, in 33% of the cases [26]. The activation status of KRAS and BRAF is important because it determines resistance to therapies against EGFR; fortunately, mutations in KRAS or BRAF in SNC are minimal, so we can suppose that these genes play a limited role in the oncogenesis of head and neck tumors [24]. Some authors detected the expression of WNT in patients affected by ITAC [27].

It has been demonstrated that OTX1 and OTX2 genes are highly expressed in sinonasal mucosa, both in the ciliated pseudostratified respiratory-type epithelium and in the submucosal glandular cells [28]. Moreover, OTX2 has been shown to selectively drive the expression of the TAp63 isoform and to have no effect on the transcription of ΔNp63. The activation of TAp63 can directly induce the Notch patway [29]. The high expression of the ΔNp63 isoform has been correlated with a poor prognosis in HNSCC type of cancer [30]. This action is generally known to be counteracted by TAp63, acting as a dominant negative repressor of ΔNp63. Nasal polyps share biological pathways with neoplastic forms; in fact, various studies showed that patients with nasal polyps have a higher risk to relapse after endonasal surgery [31], but a lower risk of metastatization. The expression of OTX2 in nasal polyps, leading to TAp63 activation, could explain their low grade of transformation and metastatization.

Pirrone C et al. showed an upregulation of OTX mRNA levels in different epithelial and neuroectodermal neoplasms, including inverted papilloma (IP), pleomorphic adenoma (PA), poorly differentiated neuroendocrine carcinoma (PDNEC), adenoid cystic carcinoma (ACC), squamous cell carcinoma (SqCC), olfactory neuroblastoma (ON) and in non-intestinal type adenocarcinoma (NITAC). In particular, OTX1 seems to be more expressed in SqCC and NITAC; by contrast, OTX2 was more frequently upregulated in neuroendocrine neoplasms and ON. Finally, both OTX1 and OTX2 have been demonstrated to be co-expressed in ACC and PA [32]. Thus, OTX genes may be involved in both maintenance and patterning of sinonasal mucosa and in tumor differentiation and development.

Conclusion

The increasing knowledge about the molecular pathways that underlies their carcinogenesis may help to identify prognostic and chemoradiotherapy response predictive marker, to optimize existing treatments. Taken together the upregulation of OTX1 and/ or OTX2 in neoplastic tissue, compared to normal mucosa, suggest that the activation of OTX factors is involved in the pathogenesis of different types of sinonasal carcinomas and potentially represent therapeutic targets, in parallel with the most common molecular biomarker.

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The Role of a Specific Microbiome in Ovarian Cancer Development 

Introduction

The term microbiome was first used at the turn of the 20^th and 21^st centuries by the Noble prize winner Joshua Ladeberg and should be understood not only as a description of a simple set of organisms occurring in a given environment, but above all as a set of their genomes interacting with each other and the place where are located [1]. An attempt to detect the dependence between the presence and functioning of a particular microbiome inhabiting the human body on the oncogenesis process is currently one of the main research directions combining such sciences as microbiology, genetics and clinical oncology. Research into the definition of this relationship has in recent years led to the emergence of the term oncobiome, pointing to the direct role and relationship of specific organisms inhabiting the human body to the process of carcinogenesis [2]. It is believed that microorganisms can modulate or influence the formation of up to 20% of tumors [3]. The example of Helicobacter pylori bacteria, whose eradication from the stomach mucosa allows to significantly reduce the risk of cancer development or vaccination against oncogenic HPV viruses in the prevention of cervical cancer, shows how prospective and future research can assess individual oncobiome living in the human body [4]. Physiologically, the upper part of the female genital organs is a microbiologically sterile environment. Ovaries and the surrounding fallopian tubes may, however, be the site of numerous infections that take place after both acute and chronic infections. The infection may occur, among others through direct spread in the case of infections of nearby organs, eg during appendicitis or salpingitis. Most infections, however, are ascending and are associated with primary infection of the lower genital tract. Oncobiome research is mainly about colorectal cancer, although in recent years there has been scientific work combining specific microbes and specific microbiota with gynecological tumors. In this article, we conducted a short review of the literature on the microbiome and its connection with ovarian tumors [5-7].

Ovarian Cancer and Pelvic Inflammatory Disease

Numerous infectious agents that cause pelvic inflammatory disease, both acute and chronic, make the sterile environment of the upper part of the female reproductive system a place exposed to specific, pathogenic flora, both bacterial and viral. For many years, there have been reports of an increased risk of ovarian cancer in the population of women who underwent pelvic inflammatory disease [8,9]. A meta-analysis summarizing 13 case-control studies showed an increased risk of borderline malignancy in women who underwent pelvic inflammatory disease (PID). This risk increased with the increase in the number of PID episodes and was twice as large if the patient underwent PID, at least twice during life [10]. A similar relationship regarding the association of PID episodes with an increased risk of ovarian cancer was noted in a meta-analysis of 6 cohort studies and 7 case-control studies. Serum ovarian cancer occurred statistically significantly more frequently in patients who had an episode of PID in their lifetime [11]. These studies assessed only the general and broadly understood pelvic inflammatory disease on the process of oncogenesis in ovarian cancer, and thus the impact of acute and chronic infection of the upper reproductive tract that can be caused by numerous microorganisms. They did not assess the presence of a specific flora, but only indicated the process of chronic inflammation that would initiate the process of tumor formation.

Ovarian Cancer and Viruses

Most studies on a specific microbiome and its effect on oncogenesis in ovarian cancer relate to human papillomavirus- HPV virus. In a meta-analysis conducted in 2013 involving 24 studies on the presence of genetic material of different subtypes of the HPV family virus, a high frequency of viral DNA was found in tumor tissue, thus drawing conclusions about the possible impact of HPV infection on the tumor initiation process [12]. In the studies evaluating genetic material of the virus, the presence of subtypes with high oncogenic potential was found - HPV-16, HPV-18, HPV- 45 [13,14]. However, the role of HPV infection in ovarian cancer oncogenesis remains unclear and arouses much controversy among researchers mainly due to data based on few patient groups and data from Asian populations, where the prevalence of HPV infection is definitely higher than in the European population [12]. In a pioneering ovarian cancer oncobiome study published in 2017 researches identified the genetic material of a unique microbiome coexisting with malignant serous tumors using microarrays. In the case of viruses in the analyzed material, the highest hybridization signal was obtained for the Retroviridae Virus signatures, followed by Hepadnaviridae, Papillomaviridae and Flaviviridae. Data published in the cited publication indicate a significant disturbance of the virome present in cancer tissues. Firstly, they indicate completely different families of viruses in the analyzed cancer samples in relation to control samples. Numerous viral signatures, among others Anelloviridae, Astroviridae, Birnaviridae, Bornaviridae, Caliciviridae, Hepadnaviridae were detected at a significant level only in cancer samples. Similarly, in the case of HPV virus, specific molecular signatures of subtypes with high oncogenic potential, i.e. HPV-16 and HPV-18 were detected only in tumor samples and were not found in control group samples [15].

Ovarian Cancer and Bacteria

The main pathogenic factors associated with ovarian cancer and their potential influence on the oncogenesis process are bacteria-Chlamydia Trachomatis and Mycoplasma Genitalium [16]. They are sexually transmitted bacteria that can cause persistent infections in the upper reproductive tract. Both in the case of Ch. trachomatis and M. genitalium, the suggested mechanism for promoting carcinogenesis is the inhibition of apoptosis in infected cells. Ch. trachomatis can also cause changes in the genome of host cells through the production of reactive oxygen species and the inhibition of dsDNA repair mechanisms [17,18]. The presence of Chlamydia Trachomatis genetic material in tissues of invasive ovarian cancer may suggest the effect of infection on the oncogenesis process. In a case-control study conducted in India, the presence of Ch. trachomati DNA was confirmed in 85% of invasive ovarian cancer samples and the presence of genetic material of this bacterium in control samples including healthy tissue of the female gonad [19]. Similar results were observed in a study published in 2018 [20].

However, the research results are ambiguous. In one of the studies, no genetic material of this microorganism was found in any of the invasive ovarian cancer samples [21]. Similar ambiguous conclusions concern the search for Mycoplasma genitalium DNA. In a study of 27 ovarian cancer samples, this pathogen was demonstrated in 16 of them, which is approx. 59% [22]. In two other studies, however, no correlation was found between M. genitalium infection and this tumor [21,23]. New light on bacterial oncobiome associated with ovarian cancer is being shed by the study already published in 2017 by a group of scientists led by Dr. Sagarika Banerjee. As in the case of viruses, also in the case of bacteria, the results of the research indicated clearly different molecular signatures in tumor tissues as compared to control tissues. The presented study allowed to determine the unique bacterial microbiome present only in cancer samples. The dominant types of bacteria were: Proteobacteria (52%) and Firmicutes (22%). Significantly more bacterial signatures were found in tumor tissues as compared to control samples [15].

Ovarian Cancer and Fungi

The only study that managed to identify the specific fungal microbiota that may be associated with ovarian cancer is the study of the Dr Banerjee group already mentioned. The study detected 18 fungal signatures that were present only in cancerous samples. They were, among others signatures corresponding to Pneumocystis, Acremonium, Cladophialophora, Malassezia and Microsporidia Pleistophora. In contrast, the highest signal in hybridization was obtained for the 18S Cladosporium rRNA [15]. Unfortunately, there are no other studies in which the effect and presence of specific fungal flora in the process of carcinogenesis in ovarian cancer will be assessed.

Summary

Human microbiome are still discovered in the context of physiological processes taking place in the human body. A look at this world that is not fully understood in the context of pathology of diseases and even their initiation may result in the development of numerous new therapeutic points that could be used not only to treat but also to prevent disease. New research looking for specific and specific microorganisms related to the oncogenesis process indicating numerous quantitative and qualitative disturbances of the microbiome in cancer tissues shows how important influence can be exerted by infectious agents on this process. Already today, some researchers point to the possibility of vaccination against HPV not only in the context of prevention of cervical cancer, but also of ovarian cancer [14]. The detection and linking of specific organisms with the initiation of cancer processes or their promotion would be of particular importance in the case of cancers such as ovarian cancer, which has not been screened so far and whose pathogenesis has remained so far enigmatic.

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Disposition of Pathogenic Flora in the Development of Nonspecific Ulcerative Colitis 

 

Introduction

Ulcerative colitis refers to the number of diffuse chronic recidivating diseases of the colon, which mainly affected its mucous membrane [1-5]. UC meets throughout the world. In year 3 to 15 of diagnosed new observations on 100 000 of the population, and the incidence of reaches 50-80. Men and women have equally often. The first peak of the detect ability of age from 20 years old do 40, the second 60-70 years (2.7). At present, there is no clear information about the etiology and pathogenesis of this disease. It is expected that the development of wheatgrass is the breakdown of immunological tolerance to intestinal antigens. The result is a loss of immunological control of inflammation in the wall of the small and large intestines (2.6). It is believed that this pathological process normally resists low doses of endogenous Gljukokortikosteroidov (GKS), which in a cage are associated with the

This complex enters the nucleus of the cell and is in contact with DNK elements in the region are glukokortikoida specific genes. The result is a suppression gene encoding the transcription of inflammatory proteins, especially signaling molecules cascade MAPK (mitogen-activated protein kinas). Parallel synthesis inhibitor-IkBa amplifies a key transcription factor NFkB, allowing specific RNK transport education is sup-pressed (m-RNK) and shortened the period of their half-life. Since m-RNK is responsible for regula-tion of synthesis and release of inflammatory cytokines TNF-a, IFN-y, IL-23, IL-17of others in-volved in the inflammatory response, in appointing GCS level their decline [6-8]. This also reduces education arachidonic acid and its subsequent metabolism with the formation of leukotrienes and prostaglandins [9]. When UC holds a mixture of inflammatory reaction involving the t-helper cells as the 1^st and 2^nd types (6.8). The most often considered to be a hereditary predisposition to the development of autoimmune inflammation in the mucosa of the colon in response to sowing surface microorganisms and viruses, as well as contact the impact food. This opinion is based on the frequent combination of UC with other autoimmune processes (3.8). The discovery of same in colon mucosa of IgG-antibody to epithelial cells and p-ANCA only strengthened the position of the supporters of this hypothesis. Completed studies to determine the ratio of t-lymphocytes in the mucosa of the colon indicate violation agent’s interactions activated CD4 and CD8 lymphocytes. Normal epithelial cells stimulate the predominantly CD8-T cells. While at UC they activate CD4 lymphocytes exclusively and is accompanied by IL-2 secretion of lymphocytes and stimulation of macrophages in the complement system. Identified and family history of UC, with first-line relatives fall sick more often than the average population, as well as the risk factors they have clearly seen the use of oral contraceptives, as well as features nutrition and psychosocial problems (3.5). Literature data suggest an important role of normal intestinal microflora in the adaptation reaction of human organism to the age changing his life (3.8). In view of these data, the reason becomes clear when you change syn-drome pathological development of microflora of the colon. At a young age caused for one reason or another, intestinal symbiosis is accompanied by diarrhea, coupled with the brodilnym process. Every fifth patient in this age group are marked with allergic dermatitis, occurring against the backdrop of autonomic expressed violations.

Dysbacteriosis of the same link with a postponed intestinal infection and long treatment with antibiotics. Performed bacteriological researches in this group of patients indicate expressed growth suppression of Escherichia coli with a simultaneous settlement of the lu-men of the colon of conditionally pathogenic microflora (enterobacteria, citrobakterii, Klebsiella, Pro-teus, fungi kind Candida, gemolizirujushhie strains of Escherichia, etc.). Individuals have the same maturity (40-59 years), in which there is persistent constipation alternating with diarrhea, bacterial painting Calla proportion of functionally defective (lactosenegative and enzymatically attenuated) strains of e. coli, which occurs against the backdrop of moderate decline bifidumbakterij growth. The same persons over the age of 60 years, suffering persistent constipation, the feces noted a sharp de-cline in the obligate microflora (Lactobacillus bifidum and Lactobacillus) while increasing the level of conditionally pathogenic microflora [2]. Infections often cause the development and exacerbation of wheatgrass, because one way or another damaged mucosa easily kontaminiruetsja pathogenic microflora (3.8). UC have detected symptoms of a enteric 20% of patients. They include Nodular Erythema, gangrenoznaya Pyoderma, inflammatory eye diseases, arthritis, ankylosing spondylitis, respiratory dysfunction, Myositis, Vasculitis, Glomerulonephritis, and other pathological processes outside the walls of the colon guts (3.5). If the role is conditionally pathogenic microflora in the development of the UC clearly understood, here’s its settlement mechanism of the colon remains unclear. The definition of these ways and was the purpose of this study.

Material and Methods

Watched 38 patients with UC had expressed enteric (kostnoarticulate) symptoms manifesta-tions of the disease, which has resulted in their hospitalization in the casualty department. Only in the course of the survey, they had identified the true cause of the painful condition, i.e. the UC. All of these patients, whose age was from 42 to 68 years, suffered from distorting the artrozami joints of the lower limbs and ankylosing spondylitis. Men was 16 (42.2%). Upon admission to the hospital all patients stressed doctors’ osteoarticular pathologies and umalchivali about the problems associated with the Act of defecation. Only through the 2-3 days after hospitalization, they began to bring complaints of liquid stool mixed with blood and mucus in the stool. Frequency defekacij reaches 5-6 times per day. In patients suspected of having perpetuated was food toksikonfekcija and diagnostic measures have been made. When you run rectoromanoscopy, attention was drawn to the existence of patients with redness, maceration, and cracks in the perianal region. Endoscopic study found the typical symptoms of chronic (or relapsing, or continuously-relapsing) wheatgrass. Bacteriological study of tissue taken from the surface of the detritus Armenian revealed microbial Association of Klebsiella, Proteus, gemolizirujushhih esherihi and etc. After verification of diagnosis was assembled a de-tailed life in this direction. It was found that all patients with adolescence suffered from constipation.

The Chair was only a day, and sometimes through the 2-3 days. With age the constipation has become persistent nature and without enemas achieved defecation them failed. Patients often resorted to staging of salt, soap, oil and other enemas, which appeared in the left iliac region pain and tenesmus after defecation. In the last year before the hospitalization in the stake appeared mucus and blood veins. For medical assistance. These changes in the Act of urinating associated with frequent taking enemas. Osteoarticular diseases they have began to develop after 30 years. After ascertaining the true causes of painful condition, patients were placed in a specialized unit. During the specific treatment, with the use of corticosteroids, 2 of them (5.2%) microperforation occurred altered sigmoid colon walls. They were transferred to the Department of surgery with primary acute peritonitis phenomena. Both patients was the primary submersible kolokoloanastomoz.

Result

These 2 patients of postoperative complications were observed. After the stitches have been medication they have continued in the therapeutic Department. The remaining 36 patients of such surgical complications were observed. Treatment took place in the therapeutic Department and had a positive effect is difficult. Continued to harass pain in the joints and spine.

Discussion

Development of a pathological process in these patients can be associated with a dislocation of the virulent Microbe flora of anus the Canal in the lumen of the colon that occurred during staging enemas. Resort to this method of release of the rectum from the faeces of patients forced to constant constipation, and they had them with adolescence. The aggravation of the same they have UC on the 2-3 day after the hospital casualty department could explain the sharp changes the nature of power and of psycho-emotional loads. Duration of intensive integrated inpatient treatment ranged from 18 to 22 days, and then was an outpatient. Spring and autumn came.

Conclusion

Thus, as a factor that contributes to the development of UC may include deployment of microbial flora of the channel anus in the lumen of the colon that occurs when setting enemas. Clearly an allergic and autoimmune nature of its origin. Disease is tenacious in nature and it is difficult to treat and, for this reason, the focus should be paid to prevention. Behind the seeming enema bezobidnostju hide terrible consequences. For this reason the issue of defecation everyone should pay close attention to and follow its daily accomplishment, through a balanced diet and active lifestyle.

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