Friday, October 23, 2026

Efficient Strategies for the Reuse of Surgical and FFP2/ KN95 Face Masks during the COVID-19 Pandemic: Home-Made and Ecological Choices for the Community

Introduction

The health emergency caused by SARS-CoV-2 has required intervention measures for respiratory protection in healthcare workers and generally in the community, to prevent the spread of the viral infection. SARS-CoV-2 is a positive-sense, single-stranded RNA virus that is highly contagious [1]. The virus uses specific proteins (S-Proteins) to attach certain kinds of receptors in the epithelial cells of the respiratory tract [1]. The S protein mediates the viral entry into the host cells by binding to the host Angiotensinconverting enzyme 2 (ACE2) receptor which is highly concentrated in the airway epithelial cells. Droplets and aerosol particles produced during breathing may contain the viral load in variable quantities [2]. The SARS- CoV-2 could stay viable for 3h in aerosols highlighting the crucial role of the use of the face masks to avoid the infection [3]. Some studies demonstrated that the SARS CoV-2 could survive for 72h on plastic, from about 4 to 48h on metallic surfaces, and for 24h on cardboards [3]. Moreover, the SARS- CoV-2 is not similar to the influenza or other common Coronavirus as the spread of the viral infection is not blocked by warm weather, and according to the latest research, the virus outbreaks along restricted latitudes, temperature, and humidity [4].

Some epidemiological studies showed that for human beings the most common risk factors includes age, male sex, obesity, smoking, and co-morbid chronic conditions such as hypertension, type 2 diabetes mellitus, and others [5-7]. As usual during initial phases of pandemics it turns to be crucial to develop a contingency plan to compensate shortage of consumables due to the ramp up of the contagious [8,9]. As a matter of fact, some strategies to decontaminate and reuse the masks several times are needed. These methods should be performed in total safety with the aim to reduce the costs associated with the purchase of the protective devices. Moreover, the regeneration of the masks reduces the polluting impact on the environment [10]. The decontamination procedure should be done adequately in order to preserve the filtration proprieties [11]. A surgical mask is designed to protect the wearer from splash or spray while the filtration capacity is variable depending on the materials and the number of layers. The surgical mask does not fit around the face at the superior level compared to other type of masks such as the FFP2 face masks. Indeed, differently from the surgical masks, the FFP2-face masks have filter capacity ≥ 94% of airborne particles (≤ 5 μm in diameter) whereas the KN95- face masks have filter capacity ≥ 95% of airborne particles (Table 1). Both masks fit well around the face of the wearers [12]. The FFP2/KN95 face masks consist of a number of variable layers of fabric (multilayer masks) and they have generally four or six layers. The filtration grades for KN95-face masks (made in China) are quite similar to the FFP2 respirators (European Union).

Table 1: Filtration grade of the Chinese KN95 and European FFP2 masks.

A standard surgical face mask has 0.3-10 μm pore size while Coronavirus has a diameter from 0.06 to 0.14 μm and it has got a spherical shape [13]. The surgical masks should not be used by the healthcare workers who are in contact with COVID-19-positive patients. In this case, the healthcare workers ought to wear the FFP2/ KN95 face masks. Most of the filtering materials used for the FFP2 face masks are synthetic, such as non-woven fabrics (TNT) based on materials as polypropylene (PP), polyethylene terephthalate (PET), and polyamide (PA). The outer layer is usually made of TNT spunbond (S) which is treated with hydrophobic treatment while the intermediate layer is often made of TNT meltblown (M) which performs the main filtering function of the mask. The inner layer, which is in contact with the face and protects the skin from the filtering layer, is made of spunbond [14]. The meltblown layer is composed of polypropylene microfibers with variable diameters (from 1 to 10 μm) which create a three-dimensional network Figure 1. Our aim is to suggest strategies for the decontamination of disposable respiratory devices for the community or the hospital settings. In particular, we focused on methods for the regeneration of surgical and FFP2/KN95 face masks (without respiratory filters) used in companies as well as at home. Furthermore, a large chapter should be reserved to numerous counterfeit certification masks present on the world market during pandemic period, considering that these masks lead to the increasing of new cases [15].

Figure 1:

a. Comparison between the different layers and materials used to make the surgical masks and

b. The FFP2/KN95 face masks.

Materials and Methods

Samples

We focused on the regeneration of two types of disposable masks: surgical masks (Jiangsu Province Jianerkang Medical Dressing Co., LTD China) and FFP2/KN95 face masks (KN95 Yiwubiweikang labor protection products Co., Ltd-China and FFP2 BLS 502 PPE-R/02.075 version 1 COVID-19, Italy). We tested a total of 24 masks: 12 surgical masks and 12 FFP2/KN95 face masks.

The Regeneration Methods for both Surgical and KN95/FFP2 Face Masks

Thermostatically Controlled Water Bath

We tested 2 surgical masks and 2 KN95/FFP2 face masks which were worn by the operator for about 8 hours. The masks were immersed in hot water thermostatically controlled bath/5 Liters (VWR International S.r.l, Italy) at 80°C (constant temperature). The temperature was monitored by the display on the thermostat bath and a mercury thermometer inserted in the tank of the water bath. The masks were soaked in the water bath at different times (15 minutes, 30 minutes, and 120 minutes), and immediately after, they were shaken to remove of water and air-dried.

Washing Machine

We tested 4 surgical and 4 KN95/FFP2 face masks, worn by the operator for about 8 hours and we washed them using a common laundry detergent in a quick washing cycle machine (LG Electronics S.p.a, Italy) at 60°C for 30 minutes.

Steam Iron

We analyzed 4 surgical and 4 FFP2/KN95 face masks worn by the operator for about 8 hours which were regenerated using a domestic steam iron machine; in particular, we used Vaporella Forever 615 P:4 Bar 80 G/min (Polti SPA, Italy). The masks were not placed in direct contact with the surface of the steam iron but they were placed under a double layer of cotton obtained by folding a cloth (Figure 2). The iron was placed on a 2.5 cm high perforated metal support (0705/04 centrifuge plate adapter (Eppendorf-S.r.l, Italy). The support was arranged above the double layer cotton in order to allow the steam to radiate correctly into the area containing the mask. Two digital and one mercury thermometers (probes) were used to detect the temperature reached in the different points of the masks. The first probe (P1) was positioned inside the masks (area of nose and mouth) while the second probe (P2) was positioned between the outer layer of the masks and the cotton double layer. Finally, the third probe (P3) was placed in the area surrounding the steam iron (Figure 2). The steam flux was sprayed over the area where the masks were placed for 35 minutes, and the temperatures detected are shown in the Table 2.

Table 2: The temperatures detected by three probes.

Figure 2: Materials used for the steam iron regenerative process.

Autoclave

We tested 4 masks and 4 FFP2/KN95 face masks worn by the operator for about 8 hours and we regenerated all of them using an autoclave in our laboratory. The masks were wrapped in a single layer of aluminum foil (used for food) and placed inside the autoclave (Vapormatic 770-Asal S.r.l, Italy) for a full cycle at 121°C 1 ATM for 35 minutes.

Tests to Assess the Effectiveness of Regeneration Procedures

Evaluation of the Fibers by Light Microscope: We examined 24 masks (12 surgical and 12 FFP2/KN95 face masks). For this purpose, we carried out an optical microscope survey for each regeneration method (thermostatically controlled water bath, washing machine, steam iron, and autoclave) to verify any possible potential damage to the fibers caused by these procedures. The surgical masks are usually made up of three layers (internal, intermediate, and external) and we analyzed all the layers by the excision of a tissue fragment of about 0.5 cm2 using a scalpel. Each fragment was placed on a microscope slide, covered with a slide, and observed at 10x magnification (Figure 3a). The same procedure was repeated for the four layers of the FFP2/KN95 face masks (Figure 3b).

Evaluation of the Waterproof Property: We tested 24 masks (12 surgical and 12 FFP2/KN95 face masks). The water repellent properties of the masks represent a crucial point that is essential to prevent and blocking microorganisms and viruses that can be contained in the droplets released during conversation and breathing. Primo vacuum filter system (250 mL-0.22 μm PES Hydrophilic 1/pkg, Euroclone, Italy) was used to test the maintenance of the waterproof properties. A vacuum filter system was a common method used previously for the sterile filtration of laboratory reagents, buffers, solutions and media. Each type of mask was put inside the Vacuum Filtration Tops with the outer surface facing upwards (Figures 4a-4e). We obtained 500 milliliters of coloured solution by mixing 250 mL of crystal violet and 250 mL of distillate water. We poured the colored solution inside the outer surface of the masks (Figures 4b-4f). Finally, the vacuum aspirator was activated and we observed the inner surface of the masks to determine their waterproofness (Figure 4c). Finally, both surgical and FFP2/KN95 face masks were subjected to a flow of running water to determine their wettability (Figures 4d, 4g & 4h).

Qualitative Fit Testing: This test was conducted to evaluate the ability of 4 surgical and 4 FFP2/KN95 face masks to filter test agents aerosolized in the air (Figure 5). The substances used as testing agents had a pungent (wine vinegar), bitter (coffee), and sweet (vanillin) taste-odor. We aimed to verify the absence of a specific flavor in the mouth or nose. The qualitative fit test had the purpose of evaluating the ability of the masks to maintain an adequate fit before and after regeneration [16]. In addition, this experiment was done following the OHSA (Occupational Safety and Health Administration) guidelines which indicate a series of physical exercises to emulate the work activity while wearing the mask, and to reproduce the stresses associated with the movement of the workers. While standing, the subject was asked to perform a series of 1-minute exercises: normal breathing, deep breathing, turning head side to side, moving head up and down, reading from a prepared text, jogging in place, and normal breathing [14,17,18].

Figure 3:

a) Comparison between the layers of an unused surgical mask and a steam regenerated mask.

b) Comparison between the layers of an unused FFP2/KN95 face mask and a steam regenerated mask.

Figure 4: Waterproof test procedure. The first line represents the procedure used for the FFP2/KN95 face masks while the second line represents the procedure used for the surgical masks.

Figure 5: Qualitative fit testing. The test agents were areodispersed in the air by a nebuliser.

Evaluation of the Efficacy to Inactivate Microbiological Agents: The microbiological tests were been carried out using eSwabs 480C (Copan Diagnostic Inc., California, USA). We tested four regenerate surgical and four regenerated FFP2/KN95 face masks. The procedure was performed under a sterile hood in order to avoid any potential cross contamination of whatever environmental nature. We previously moistened the inner surface of each mask with a sterile physiological solution (Sodium Chloride 0.9% B. Braun Melsungen AG, Germany). Then, the flocked swabs were rubbed along the entire internal surface of the surgical and FFP2/KN95 face masks. Immediately after sampling, we placed the flocked swabs inside the vials containing liquid amies preservation medium. We performed the microbiological investigations searching for the followings strains: Gram negative bacteriae with resistance to carbapenems (CRO), Staphylococcus Aureus resistant to methicillin (MRSA), Pseudomonas Aeruginosa (PAE), and Medical Yeasts (MY). It should be stated, therefore, that to pass the swab (pharyngeal type) inside the regenerated masks and conducing tests could not totally guarantee that the regeneration is absolutely good, due to the fact that the above-mentioned colonies could be not present on the inner surface of those used masks prior to the regeneration.

Results

Evaluation of the Fibers by Light Microscope

The regenerated masks (surgical and FFP2/KN95 face masks) analyzed under the light microscope showed the original weave as well as the morphology by observing all the layers (Figures 3a & 3b), therefore in the future we expect do extend experimentations with quantitative analysis (e.g., pore measures, filament diameters).

Evaluation of the Waterproof Property

Both surgical and FFP2/KN95 face masks are suitable for directly blocking the droplets during breathing. We demonstrated that the regenerated masks contained useful properties. Indeed, the water repellency remained unchanged in the regenerated masks with respect to the unused masks, even if we should outline that «water repellency», alias hydrophobicity, is measured with the contact angles and it is not synonymous with being fully «waterproof» when the dye passes through the vacuum filter (Figures 4d, 4g & 4h).

Qualitative Fit Testing

We demonstrated that the surgical masks due to their conformation were not able to isolate the operator from the environment odours because they do not perfectly adhere to the face before and after the regeneration process. On the other hand, we observed that the unused FFP2/KN95 face masks as well as the regenerated ones were able to partially block the smells. Thus, our qualitative fit test showed that the FFP2/KN95 face masks remained comparable to the unused masks.

Evaluation of the Efficacy to Inactivate Microbiological Agents

The microbiological evaluation tests came back negative to the CRO, MRSA, PAE & MY (Table 3).

Table 3: Microbiological tests of the regenerated surgical masks (1,2,3,4) and FFP2/KN95 face masks (5,6,7,8).

Discussion

Despite the discovery of vaccines, our research focuses on two main issues: the wastefulness of economic resources and the ecological environment dangers connected to the waste masks. For these reasons, we decided to report four easy and cost-effective approaches to test both surgical and FFP2/KN-95 face masks in order to reuse them several times. As a matter of fact, in the global market there are several types of masks, and the choice of the mask depends on the use made by the operator. We focused on both surgical and FFP2/KN95 face masks because these two kinds of masks were easily to find on the market during the pandemic. Our experimental tests showed that our methods used to regenerate surgical and FFP2/KN95 face masks, did not damage the fibers of the masks suggesting that the regeneration methods are suitable. The decontamination procedures used to regenerate surgical and FFP2/KN95 face masks are scientifically validated by the current literature [19-21]. Abraham et al. reported the proper sterilization of the physical objects that have been contaminated by SARS-CoV-2 [22]. In agree with the World Health Organization (WHO) guidelines, the authors comply with a 4log reduction of the viral load (SARSCoV- 2) with a treatment at 56°C for 15-minutes exposures (www. who.int/csr/sars/survival_2003_05_04/en/).

Starting from this premise, we decided to exploit four regeneration methods based on heating treatments as follows: thermostatically controlled water bath, washing machine, steam iron, and autoclave. We chose to increase the exposure time of all the heating treatment of the masks as well as the temperature, in order to ensure the elimination of the SARS CoV-2 from the treated masks. Wang et al. have already done a procedure to decontaminate the masks using a tank of hot water bath. Instead, our thermostatically controlled water bath was based on three hot water soaking cycles (15 minutes, 30 minutes, and 120 minutes). Compared to the investigations made by Rowan who showed a method to regenerate face cloths by using a washing machine, we applied the same method to four surgical and four FFP2/KN95 face masks using a softening detergent that improved the adhesion to the face. We would like to point out that using a domestic washing machine cycle at 60°C, the power of the decontamination is not only represented by the high temperature but also by the addition of a detergent which can inactivates viruses (https://www.cdc.gov/ coronavirus/2019-ncov/community/disinfecting-building-facility. html, Abraham JP). As far as we know, our steam iron method used for the regeneration of the masks is innovative and it is easily accessible by a large part of the community. Indeed, the metal support used in our test in order to avoid a direct contact between the iron and the surface of the mask, can be easily emulated using a common kitchen tool such as a rectangular perforated steel hot pad.

As well, our autoclave regeneration method which was applied to both surgical and FFP2/KN95 face masks was supported by Seresirikachorn et al. but with different results. The authors used the same procedure to decontaminate the N95 FFRs masks. After a full cycle in an autoclave at 121°C, the outer layers of N95 FFRs masks were deformed. On the contrary, we tested four surgical and four FFP2/KN95 facial masks for a full cycle at 121°C/ at 1 ATM for 35 minutes, and we did not observe any morphological change in the fibers of all the masks using the light microscope. Moreover, we decided to check the absence of microbiological agents in the regenerated masks in order to assess the safety of our methods of decontamination.

We analysed the more common microorganisms such as Gram negative/Gram positive bacteriae and Medical Yeasts which can infect the nasopharynx tract. For each regeneration mask, we carried out a microbiological test which demonstrated that all the masks were free of microbiological loads. Notably, we performed the four regeneration processes on each mask twice. Thus, each regenerated mask was worn for about 8 hours and then they were regenerated for the second time. These latest masks did not show any physical deformations, nether alteration in the elastic bands nor the metal strips. As far as we know, there is no data regarding the re-use of a mask after processes of regeneration by steam iron procedure as shown above. The critical point of all the four regenerative methods concerns the removal of the make-up residues. The washing machine and the thermostatically controlled water bath were not able to remove the stains of cosmetics from the masks. Not even, the high temperatures reached with the regeneration methods such as the autoclave (121°C) or the steam iron (82°C - 84°C), were able to eliminate the spots. On the other hand, we treated both stained surgical and FFP2/KN95 face masks with acetone and a degreaser to remove the makeup residues but with poor results.

In conclusion, we highlighted that all the regeneration methods with the exception of the autoclave, can be emulated at home, hospital, or research institutes. This makes it possible to greatly reduce the costs associated with the purchase of new masks and the amount of the biological waste released into the environment. In addition, our regenerative methods offer the community to sanitize surgical and FFP2/KN95 face masks at least twice, thus extending the half-life of the masks. Finally, we have introduced the steam iron as a new regenerated procedure among the common regeneration methods.

We are currently suggesting introducing our regeneration methods such as the autoclave and the washing machine as standard decontamination procedures of the surgical and FFP2/ KN95 face masks for healthcare workers in our public hospital in Milan (Italy).

BJSTRAngelaRoy

Thursday, October 15, 2026

Liver Fatty Acids Profile of Rats Fed Ketogenic Diet

Introduction

The ketogenic diet (KD) ‒ characterized by a low carbohydrate content and high-fat levels ‒induces a metabolic shift to the use of ketones as a source of energy for cells. It is also defined as a carbohydrate-restricted diet with a 4:1 ratio of fats to proteins and carbohydrates [1,2]. Several clinical studies have shown that KD a reduction of midlife mortality and a prolongation of health due to its ability to control body weight and its beneficial effects in the prevention of type 2 diabetes, cancer, and neurological diseases [3- 6]. Recently, in a rat model of irritable bowel syndrome (IBS), KD was able to restore intestinal cell damage through the upregulation of endocannabinoids receptors (CB1-R and CB2-R) [7] improving intestinal permeability. Besides, the KD administration positively affected gastrointestinal mucosa in IBS rats and elicited molecular compensatory mechanisms able to reduce inflammation and oxidative stress in the gut, restoring mitochondrial function and baseline autophagy [7,8].

Literature data demonstrate that KD exerts beneficial effects on subjects with obesity [9,10]. Significant weight loss was associated with reduced visceral fat mass in overweight and obese patients treated with KD [11]. Moreover, consumption of a low-carbohydrate KD is involved in a control blood pressure values and the serum levels of total cholesterol, low-density lipoprotein (LDL), and triglycerides [12]. Besides, literature suggested KD as an effective way to treat non-alcoholic fatty liver disease (NAFLD) [13]. During the improvement of human NAFLD induced by a short-term KD (six days), liver redox status significantly changed. It appears that KD, at least as a short-term treatment, can significantly reduce liver fat content and insulin resistance [13]. However, in rodents, different studies have reported that KD can promote the progression of liver steatosis [14,15]. The greater palatability of high-fat diets justifies the greater food consumption by these animals, consequently increasing body weight [16]. High-fat diets seem to favor the fat accumulation in hepatocytes associated with increased lipid peroxidation metabolites in hepatic tissue [3,16]. Notably, the pro-oxidative environment caused by enhanced levels of dietary lipids, promotes the development and progression of steatosis in the liver [17]. The liver has a prominent role in carbohydrate and lipid metabolisms so that excessive amounts of free fatty acids in hepatocytes cause fatty liver, insulin resistance, and lipotoxicity [18-20].

Fatty acids constitute essential components of membranes that influence their physiological functions [21] and their tissue composition is the result of food intake, lifestyle, and environmental factors [22,23]. Based on this evidence and in line with the existing literature background, the purpose of the present study was to investigate whether a high-fat KD with beneficial effects at intestinal levels may affect liver function in Wistar rats. Through a lipidomic approach, the study was aimed to understand the relationship between KD and fatty acid profile of liver tissue and investigate the clinical implications that occur.

Materials and Methods

Animals and Treatment

Twenty-five Wistar rats (5 weeks of age) were used in this study. All the animals received care in compliance with the “Guide for the Care and Use of Laboratory Animals” and the experimental procedures were approved by the Italian Ministry of Health (approval date: 28 November 2018, n. 901/2018-PR). The animals, at weaning, were divided at random into two groups and treated for ten weeks. Particularly, 12 animals were fed a standard diet (control group, CTR group), and 13 were fed a low-carbohydrate, high-fat KD (KD group). Diets (4RF21 standard diet and ketogenic diet) were purchased by Mucedola Srl, Settimo Milanese, Italy, and their composition is shown in Table 1. No sign of suffering, stress, or pain was observed in the animals during the experiment. At sacrifice, by anesthetic overdose, the liver from each animal was collected and stored at -80°C until assayed.

Table 1: Main constituents of Standard Diet and Ketogenic Diet.

Fatty Acids Analysis

Liver tissue fatty acids analysis was carried out using Fatty Acid Extraction Kit Low Standard, according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, U.S.A.). Briefly, 0.15 g of the liver tissue was homogenized in 3 ml of extraction solvent. After homogenization, 5 ml of aqueous buffer were added, and the sample was filtered by a syringe. The eluted fraction containing the lipid extract was trans-esterified by adding boron trifluoride-methanol solution and hexane and incubated at 95° C for 1 hour. After adding hexane, the samples were vortexed and centrifuged at 500 x g for 5 minutes and the fatty acid methyl esters contained in the top hexane layer were evaporated. The dried pellet was reconstituted with 100 μl of hexane and injected into a gas chromatography equipment (Thermo Fisher Scientific, Milan, Italy). The quantification of fatty acids was carried out as previously described [24].

Western Blotting Analysis

Fifty μg of liver total proteins were subjected to 4-12% SDSPAGE polyacrylamide gel electrophoresis and transferred onto the PVDF membrane (Bio-Rad Laboratories, Milan, Italy). Subsequently, the membrane was incubated overnight at 4°C with the following primary antibodies: NAD-dependent deacetylase sirtuin-1 (SIRT1) (Bio-Rad Laboratories, Milan, Italy), peroxisome proliferatoractivated receptor-γ (PPAR-γ) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), tumor necrosis factor (TNF-α) (Immunological Science), human neutrophil cytochrome b-light chain (p22/phox) (Abcam, Cambridge, UK), β-Actin (Cell Signaling Technology, Beverly, MA, U.S.A.). Then, the membrane was washed with TBS-T, and the secondary antibody conjugated with horseradish peroxidaseconjugated rabbit or mouse (Bio-Rad Laboratories) was used, where appropriate. The signal of each protein was visualized using the ChemiDoc™ system (Bio-Rad, Milan, Italy) and the Image Lab software. All data were normalized against β-actin expression.

Determination of Enzymatic Antioxidant Activity and Lipid Peroxidation

The levels of [Cu-Zn] Superoxide dismutase (SOD 1) and the Mitochondrial Superoxide dismutase (SOD 2) in the rat liver tissue were measured with Rat SOD 1 and Rat SOD 2 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (Fine Test, Wuhan, China), respectively. Lipid peroxidation was measured in the rat liver by determining malondialdehyde (MDA), using the Rat MDA Assay Kit ELISA (My Bio Source, San Diego, CA, U.S.A.) by following the manufacturer’s instructions.

Statistical Analysis

Sample size calculation has been performed having a fixed power of 80% and a first type error of α of 0.025 (α ½ = 0.025); then, a minimum number of 6 animals for each experimental group was needed. The statistical differences among the two rat groups were evaluated by the student t-test and they were considered significant with a p-value <0.05.

Results

The Effects of the Ketogenic Diet on Body Weight and Liver Tissue Fatty Acids Profile

Figure 1: Total levels of omega-3- and omega-6- PUFAs, and the omega-6/omega-3- PUFAs ratio in rat liver tissue of the CTR and KD group. All data are expressed as mean ± standard deviation (SD) of three different experiments. p-Value indicates significant differences among the groups (p < 0.05, Student t-test); *** p< 0.001.

Table 2: Characteristics of experimental rat groups.

Note: All data are expressed as mean ± standard deviation (SD); p-Value indicates significant differences among the groups (p < 0.05, Student t-test); ns: not significant.

Table 3: Mean percentage of fatty acid composition in liver tissue of the rats treated with the standard diet (CTR group) and KD diet (KD group).

Note: All data are expressed as mean ± standard deviation (SD); p-Value indicates significant differences among the groups (p < 0.05, Student t-test); ns: not significant.

Table 2 shows the characteristics of two experimental rat groups. Compared to the CTR group, a significant increase in body weight was observed in animals after KD treatment (352.7±83.84 vs 454.0±132.2, p=0.033). Interestingly, the administration of KD affected the tissue fatty acids profile in the liver (Table 3). After 10 weeks of KD treatment, the levels of single saturated fatty acids (SFAs), such as palmitic and stearic acid, and single monounsaturated fatty acids (MUFAs) were significantly decreased compared to CTR group. A significant decrease was also observed for the content of arachidonic acid (AA), an omega-6 polyunsaturated fatty acid. In contrast, linoleic acid (LA), an essential omega-6 fatty acid, was significantly increased in liver tissue from KD-fed rats, probably due to the higher oils content in the KD. After KD treatment, all main omega-3 polyunsaturated fatty acids, except for eicosapentaenoic acid (EPA), significantly increased in the liver tissue. Figure 1 summarizes the differences in the levels of total omega-6-, omega-3- Polyunsaturated fatty acids (PUFAs), and the omega-6/omega-3- PUFAs ratio detected between the two groups of rats. In particular, the significant increase in total omega-6- PUFAs justified the increase of omega-6/omega-3- PUFAs ratio observed in the KD group. Saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs). Data are expressed as mean ± standard deviation (SD) of three different experiments; p-Value indicates significant differences among the groups (p < 0.05, Student t-test); *** p<0,0001, ns: not significant.

The Effects of the Ketogenic Diet on Liver Protein Levels

The effects of KD administration on the rat liver levels of SIRT1, TNF-α, PPAR-γ, NFκB, and p22/phox are shown in Figure 2. The group receiving the KD treatment presented higher levels of TNF-α, PPAR-γ, NFκB, and p22/phox protein than the control group. In contrast, a statistically significant reduction in SIRT1 protein levels was observed in rats fed KD. The significant increase of the PPAR-γ and p22/phox protein expression demonstrated that KD caused an inflammatory environment in the liver, which was also justified by a significant reduction in SIRT1 protein.

The Effects of the Ketogenic Diet on Enzymatic Antioxidant Activity and Lipid Peroxidation

The analysis of enzymatic antioxidant activity based on the determination of SOD 1 and SOD 2 demonstrated that the rats fed KD showed higher value than the controls (Figure 3). No difference was observed in the hepatic MDA levels between the two experimental rat groups (Figure 4), suggesting that the higher degree of inflammation in the liver tissue occurred without significant changes in the oxidative status.

Figure 2: Western blotting analysis of SIRT1 (a), TNF-α (b), PPAR-γ (c), NFκB (d) and p22/phox (e) protein expression, normalized with β-Actin protein, in CTR and KD group. Panel f shows representative blots evaluated in two experimental groups. All data are expressed as mean ± standard deviation (SD) of three different experiments. p-Value indicates significant differences among the groups (p < 0.05, Student t-test); ** p<0.001 and *** p< 0.0001.

Figure 3: SOD 1 (panel a) and SOD 2 (panel b) levels in the CTR and KD groups. All data are expressed as mean ± standard deviation (SD) of three different experiments. ** p<0.001 and *** p< 0.0001.

Figure 4: MDA levels in CTR and KD groups. All data are expressed as mean ± standard deviation (SD) of three different experiments.

Discussion

The effects of KD on liver metabolism and function can be mixed. The present study shows that KD, on the one hand, promotes the increase of tissue inflammation in the liver, on the other, it activates defense mechanisms to protect the liver environment. Significant changes in liver tissue fatty acids profile have been observed after KD intake in rats. Most of these changes can be considered as environmental factors that lead to improving or counteracting tissue inflammation. The anti-inflammatory effects of KD are confirmed by the decrease of the AA content in liver tissue. While the increase of total omega-6-PUFAs detected in rats fed KD is probably due exclusively to the levels of LA derived by the diet. Several studies showed that the high-LA diet could cause no significant increase in AA [25,26]; the LA derived from the diet could be less exposed to enzymatic turnover and, therefore, AA production starting from its precursor is reduced. SIRT1 is known to regulate several downstream pathways eliciting antiinflammatory effects [27,28]. Higher SIRT1 protein levels have been observed during calorie restriction in tissues, as the brain, muscles, liver, and kidneys [29-31]. SIRT1 seems to regulate both liver and adipose tissue inflammation, and its protective function in high-fatinduced liver pathology has been demonstrated in SIRT1+/- mice [32]. Different studies have reported that the expression of SIRT1 is downregulated in most metabolic disorders, including obesity [29,33], demonstrating that the reduction of SIRT1 expression is often linked to an upregulation of pro-inflammatory cytokines, as TNF-α.

Here, KD treatment exerted a reduction in liver SIRT1 protein levels and a concomitant induction of TNF-α, NFκB, and p22/phox protein expression. In a normal condition, NFκB has been observed in the cytoplasm of hepatocytes in a latent form but, when exposed to pro-inflammatory stimuli, it participates in the activation of tissue inflammation [31]. In addition, the upregulation of NFκB was reported to be closely connected with the overexpression of the p22/phox protein [32]. Experimental studies have also shown that all these proteins are linked together, actively intervening in inflammatory processes [34-36]. The significant induction of PPAR-γ, detected in liver tissue of rats fed KD agreed with other studies showing increased PPAR-γ expression following excessive fat intake [37,38]. PPAR-γ is a transcription factor that controls hepatic fatty acids metabolism and, therefore, it results in enhanced hepatic steatosis and abnormal conditions of liver function [39].

Simultaneously, the diet-induced pro-inflammatory state in the rat liver was somewhat offset by increased antioxidant activity, increased total PUFA levels, and a significant reduction in SFA and MUFA. In the present study, the beneficial effect of KD is confirmed by the significant increase in SOD 1 and SOD 2 (Figure 3), in rats fed KD compared to the control. SODs are present in all aerobic living cells since O2 is a common product of oxygen metabolic reactions. SOD 1 is the main intracellular form of SOD and accounts for 80% of the total SOD protein. Previously, some studies declared SOD 1 to be predominantly cytosolic [40]. Nevertheless, later studies have found it in the whole cell, including in the mitochondrial intermembrane space and nucleus [41]. On the other hand, SOD 2 is located exclusively in the mitochondrial matrix (MM) [41]. Together, SODs provide to remove the damaging O2- free radicals from cells and tissues.

In addition to their conventional function as O2- detoxification enzymes, emerging evidence suggests that SOD 1 and SOD 2 are actively implicated in the modulation of several cellular processes [42,43]. One well-recognized mechanism is related to H2O2˗the dismutating product of O2- by SOD˗, can act as a second messenger in growth and metabolic processes regulation [44]. Thus, by increasing SODs, KD can induce antioxidant enzyme activity in liver tissue, probably in response to pro-inflammatory dietary stimuli. The tissue fatty acids profile observed in the present study after KD treatment agrees with experimental evidence underlying the role of SFAs and MUFAs in regulating cell membrane fluidity [45]. A low content of SFAs and MUFAs seems to improve the fluidity of the cell membrane and allows to counteract a possible cell damage, rapidly [45].

Moreover, the therapeutic potential of PUFAs, mainly omega-3 PUFAs, on metabolic liver diseases is well known [46]. EPA and Docosahexaenoic acid (DHA) also seem to exert protective effects against bile acids-induced cell death in human hepatocytes [47,48]. In addition, diet rich in omega-3 PUFAs are recommended for its benefits in reducing platelet adherence [49], lowing cholesterol, and reducing pro-inflammation and pro-oxidation mediators [50]. Several investigations described that the first line of defense of the cell against environmental stresses is the membrane and its content in SFAs, MUFAs, and PUFAs is important for the correct physiological functions [23]. The current study shows the possibility that the KD, through the lipidome, could improve the diet-induced inflammatory state of liver tissue. Herein, this finding implicates a significant and novel role for the tissue lipids profile in regulating complex metabolic pathways in the liver.

Conclusion

The positive effects of KD in rat liver tissue support the role of diet in modulating tissue fatty acids composition and in regulating molecular pathways related to counteracting inflammation. The clinical relevance of the study consists in suggesting a controlled use of KD in the subjects with metabolic disorders, likely for a short period, to avoid a diet-induced inflammatory effect in the liver.

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