L-ergothioneine and Metformin Alleviates Liver Injury in Experimental Type-2 Diabetic Rats via Reduction of Oxidative Stress, Inammation, and Hypertriglyceridemia

Diabetic-induced liver toxicity is a serious complication that cause signicant metabolic dysfunction. L-ergothioneine (L-egt) is a bioactive nutraceutical obtained from mushrooms and certain food products, with reported cytoprotective, antioxidant and anti-inammatory properties and potential to improve ecacy of existing therapy. Thus, this study evaluates the effects of L-egt, and/or metformin, on diabetes-induced liver injury. Diabetes was induced in male Sprague-Dawley rats using 10% fructose for two weeks, followed by a single low dose streptozotocin (STZ, 40 mg/kg i.p) injection. After induction of diabetes, animals were treated either with de-ionized water (DW), L-egt (35 mg/kg bwt), metformin (500 mg/kg bwt), or a combination of L-egt and metformin orally for seven weeks. Body weight and glucose were monitored during the experiment. At the completion of experiment, blood samples were collected, and liver tissue was excised for biochemical analysis, enzyme-link immunosorbent assay (ELISA) of various liver function biomarkers, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of genes associated with inammation, oxidative stress, and lipid metabolism, as well as histopathological evaluation. Diabetic animals showed liver hypertrophy, increased liver injury, hepatic triglycerides, oxidative stress, and hepatic inammation. However, L-egt, and/or metformin, improved glycemic control, reduced liver injury, triglycerides, oxidative damage, inammatory injury, and normalize liver histology by upregulating Nrf2/Sirt1, downregulate NF-kB/TGF-B1, and reduce SREBP1c/FAS expression. In conclusion, these results showed that combination of L-egt and metformin improve therapeutic ecacy than either treatment alone. Thus, L-egt can be used as an adjuvant to mitigate diabetes-induced liver complication. SIRT1-PGC-1α/Nrf2 pathway together with selective inhibition of aldose reductase makes compound hr5F a potential agent the diabetic complications.


Introduction
Type-2 diabetes (T2D), a chronic metabolic disorder of global prevalence, is drastically increasing in developing and industrialized countries due to excessive caloric intake, sedentary lifestyle, poor diagnosis, and disease management. The etiology and progression of T2D are multifaceted and frequently associated with obesity, hypertriglyceridemia, insulin resistance (IR), and compromised insulin L-ergothioneine (L-egt), an adaptive antioxidant obtained from mushroom and some meat products (e.g., kidney and liver), has been shown to exert antioxidant and anti-in ammatory activities as well as exhibit adaptive cytoprotective function by accumulating at the site of injury to protect against tissue damage . Also, the hepatoprotective effect of biguanides, e.g., Metformin, may result from its potency to regulate glycemic index by reducing hepatic gluconeogenesis and increased glucose uptake (Rena et al., 2017). However, metformin is associated with side effects, and patients still present with liver complications despite glycemic control, suggesting that metformin alone does not confer overall effective treatment. Therefore, this study evaluated the role of L-egt, with or without metformin, on liver injury in a rat model of type-2 diabetes.

Drugs and chemicals
Pure L-egt was obtained from Tetrahedron limited, Paris, France. QPCR iTAQ SYBR Green and cDNA synthesis kits were purchased from Lasec (Cape Town, South Africa). Primers were synthesized by Inqaba Biotec (Pretoria, South Africa). Metformin was obtained from a local pharmacy (Pharmed, South Africa). All other chemicals, reagents, and equipment were procured from standard commercial suppliers and high analytical grades.

Experimental animals and ethical approval
Thirty-six (36) male Sprague-Dawley rats (175 ± 20 g) were obtained from the Biomedical Research Unit, Westville Campus, University of KwaZulu-Natal (UKZN), South-Africa and were housed in a room with standard laboratory conditions (12 hours light-dark cycles; temperature 23 ± 1°C, 40-60% humidity). The animals were allowed access to rat feed and water ad libitum for an acclimatization period of one week before the experiment. All animal and experimental procedures were approved by the Animal Research Ethics Committee (AREC) of the University of KwaZulu-Natal, Durban, South Africa (Ethic number: AREC/006/019D).

Experimental design
After acclimatization, the animals were randomly divided into two major groups: the non-diabetic (n = 10) and the diabetic (n = 20) groups. All animals in the diabetic group were treated with fructose and streptozotocin (STZ) to induce type-2 diabetes using the established model described by (Wilson and Islam, 2012). Brie y, the animals were supplied 10% fructose in drinking water ad-libitum for two weeks to induce IR and later injected (i.p.) 40mg/kg bwt STZ freshly prepared in 0.1M citrate buffer. The animals in the non-diabetic group were injected with the same volume of 0.1M citrate buffer. Animals with nonfasting blood glucose levels of > 16.7mmol/L after one-week post-STZ injection were con rmed diabetic (Srinivasan et al., 2005) and included in the study. After successful diabetes induction, the non-diabetic animals were subdivided into two groups, while the diabetic animals were subdivided into four groups as follows. Groups 1 and 3 were administered de-ionized water (1ml/100g), groups 2 and 4 were administered L-egt (35mg/kg bwt), group 5 was administered metformin (500mg/kg bwt), while group 6 was administered a combination of L-egt and metformin. The dosage of L-egt used in this study was based on previous in-vivo studies using this nutraceutical (Tang et al., 2018, Williamson et al., 2020). All treatments were done daily by oral gavage and lasted for seven weeks.

Blood and tissue collection
After the seven-week treatment period, all animals were sacri ced by decapitation and blood was immediately collected into a serum vacutainer EDTA bottle and allowed to stand for 30 mins. The blood was then centrifuged at 3000rpm for 10mins at 4 0 C to obtain serum. The serum samples obtained were stored in the bio-freezer (Snijers Scienti c, Holland) at -80 0 C until used for biochemical analysis.
Afterward, incisions were made along the linea alba of the anterior abdominal wall to excise the liver. This organ weighed, rinsed with cold normal saline, and snap-frozen in liquid nitrogen before been stored in the bio-freezer at -80 0 C until used for analysis. Liver tissue was xed in 10% neutral-buffered formalin for histological assessment.

Analysis of Bodyweight and liver index
Body weight was monitored using a sensitive electronic weighing scale (Metler, Greifensee, Switzerland). The liver index (use to assess liver hypertrophy) was calculated as the ratio of harvested liver weight to the body weight and expressed in percentage i.e.

Liver index = Liver weight × 100
Body weight

Preparation of liver homogenates
The liver was thawed and homogenized in 10% phosphate buffer (0.1M, pH7.4, 1:9 w/v). The homogenates were centrifuged a 600g for 10min to remove cell debris. The supernatant was subsequently centrifuged at 10,000g for 20mins to obtain the cytosolic fraction, which was used immediately for biochemical analyses.

Biochemical analysis
The serum concentration of liver enzymes (AST, ALT, and ALP) and triglyceride were analyzed at an accredited pathology laboratory (Global Clinic and Viral laboratories, Amazimtoti, South Africa). The concentration of triglyceride was also measured in the liver homogenate using an automatic biochemical analyzer. Blood glucose was measured at the end of the experiment using a glucometer (Accu-Chek Performa, USA). Serum insulin levels were measured by ELISA kits (Mercodia kit), and used to evaluate homeostasis model assessment of insulin resistance (HOMA-IR), which was calculated as previously described by (Matthews et al., 1985) using the following formula: HOMA-IR = fasting serum insulin (mU/L) X fasting blood glucose (mg/dl).
Rt-PCR Analysis of SREBP1c, FAS, Nrf2, Sirt1, NF-kB and TGF-β1 mRNA expression The relative mRNA expression of SREBP1c, FAS, Nrf2, Sirt1, NF-kB, and TGF-β1 was quanti ed in the liver homogenates and analyzed using a light cycler. Total RNA was isolated in the liver using TRIzol reagent (40mg of tissue/mL, Trizol reagent). The isolated RNA quantity was determined by measuring absorbance at 260/280nm using nanodrop ND-1000 spectrophotometer (Thermo scienti c, Johannesburg, South Africa). The total RNA was converted into cDNA using iScript cDNA synthesis kit, Life Science research (Biorad, South Africa) following manufacturers' instruction. The complete reaction mixture was incubated on SimpliAmp™ thermal cycler, Applied Biosystems (Thermo Fischer Scienti c), using the following reaction condition; priming 5mins at 25 0 C, reverse transcription 20mins at 46 0 C, and RT inactivation 1min at 95 0 C. Real-time polymerase chain reaction (RT-PCR) was done using iTaq Universal SYBR Green supermix (Biorad, CA, USA) as uorescent dye on a light cycler 96 RT-PCR system (Roche, Mannheim, Germany). RT-qPCR was performed in a 10µL reaction volume containing 5µL SYBR Green Master Mix, 1µL of each primer, 1µL of nuclease-free water, and 2µL of cDNA template. The primer sequences used are provided in Table-1. The purity and speci city of ampli ed PCR products were veri ed by melting curves generated at the end of each PCR. Relative mRNA expression was calculated using the 2 −ΔΔct method (Livak and Schmittgen, 2001) and normalized in relation to the endogenous control expression, GAPDH. The primers sets were homology searched using an NCBI BLAST search to ensure that they were speci c.
Histopathological analysis of Liver Liver specimens were embedded in para n wax after dehydration in a graded series of ethanol and cleared in xylene. Serial sections were done using a rotary microtome; liver slices of 5-µm thick were xed on a slide and stained with hematoxylin &eosin (H&E). The stained sections were visualized and captured using a nanozoomer S360 digital slide scanner (Hamamatsu Photonics, Japan) and nanozoomer digital pathology version 2.8 software for analysis by a pathologist.

Statistical analysis
Data were reported as mean ± SEM. GraphPad Prism Software version 7 (San Diego, CA) was used for statistical analysis. The differences between means were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test to determine the difference between groups. Statistical signi cance between groups was considered at P < 0.05.

Results
Effect body weight, liver hypertrophy, blood glucose, HOMA-IR, and serum TG in T2DM-rats  The effects of L-egt treatment with or without metformin for seven weeks on biomarkers of liver injury (ALP, AST, and ALT) and liver triglyceride in type-2 diabetic rats were presented in  Effect of L-egt with or without metformin on liver enzymes, and triglycerides in T2DM-rats. *** p < 0.

Effect on lipid peroxidation and antioxidant enzymes
As presented in Fig. 1a- Effects on liver in ammatory biomarkers: TNF-, MCP-1, and TGF-β1.
Morphological evaluation of the liver sections after seven weeks of treatment showed that L-egt, with or without metformin, alleviates hepatic injury in T2DM animals, as shown in Fig. 4a-f. The photomicrograph of liver sections in NC and NE groups showed normal liver histoarchitecture with normal morphology of the central vein and hepatic sinusoids ( Fig. 4a and b). The liver section in the DC animal (Fig. 4c) showed liver injury characterized by disrupted hepatic sinusoids, congested central vein with mild hepatocyte degeneration compared to the NC rats. Administration of L-egt (DE) or metformin (DM), reduced sinusoid disruption and congestion of the central vein ( Fig. 4d and 4e resp). In contrast, the liver section in DEM animals shows similar histoarchitecture with NC animals (Fig. 5f).

Discussion
This study aimed to examine the bene ts of L-ergothioneine, with or without metformin, on liver injury in a type-2 diabetic rat model. Both hyperglycemia and IR increase the production of reactive oxygen species (ROS) that alter the structure and functions of vital organs (including the liver) with the resultant pathogenesis of diabetic complications. In this regard, the use of natural compounds with signi cant bioactive potential have attracted greater interest due to their reduced side effect, increased accessibility and e cacy against the molecular and cellular triggers involved in diabetic complications (Choudhury et al., 2017, Gothai et al., 2016).
A signi cant reduction in body weight has been reported in poorly managed diabetes (Magalhães et al., 2019). A similar observation was reported in this study where the DC rats show substantial weight reduction compared to NC. This reduction could be correlated with metabolic derangements associated with poor glucose utilization, thereby resulting in excess catabolism of adipose tissue, breakdown of structural proteins and reduced protein synthesis in all tissues, thereby causing muscle wasting. In this study, synergistic administration of L-egt and metformin to diabetic rats improves body weight. This suggests that this treatment regimen could halt some metabolic derangements associated with muscle wasting and loss of adipose tissues, to reduce weight loss during diabetes.
In this study, liver hypertrophy was seen in the DC rats compared to the NC rats, and this was in accordance with other studies where increased liver weight was reported in diabetic rats ( may result from the ability of L-egt to enhance membrane integrity in the hepatocytes to reduce leakage of liver enzymes into circulation. Notably, there was no signi cant change in the serum level of liver enzymes in the NE rats when compared with NC rats suggesting that L-egt does not alter liver functions in the normal rats and provides further credence to its safety evaluation.
In this study, hypertriglyceridemia observed in DC rats indicates a substantial alteration in fatty acid metabolism, which is another risk factor for diabetic complications. This result is in accordance with similar studies that reported a signi cant association between increased serum triglycerides (TG) and type-2 diabetes ( . In the present study, the reduced MDA in the L-egt treated groups suggests that L-egt may inhibit oxidative degradation of the lipid bilayers in the cell membrane to enhance cellular integrity and the viability of membrane proteins that helps in cellular communication. The increased antioxidant enzymes (SOD, CAT, and GSH) work synergistically to reduce the deleterious effect of free radicals in the tissues. An increased SOD level in the liver homogenates facilitates the detoxi cation of free radicals by enhancing the conversion of superoxide into hydrogen peroxide, while CAT helps to neutralize the free radicals by degrading hydrogen peroxide into water and oxygen molecule.
Increased GSH reduces hydrogen peroxides to water and other lipid peroxides to alcohol, usually in the mitochondrial and cytosol (Ighodaro and Akinloye, 2018). In this study, administration of L-egt, with or without metformin, increased the production and e cacy of these antioxidant enzymes in diabetic rats, thereby protects against oxidative damage, enhance cell integrity, and complement the e cacy of antioxidant enzymes against ROS. The improved antioxidant defense system observed in the L-egt treated group may result from the upregulation of Sirt1 and Nrf2 genes, which are the major transcription factors in the antioxidant signaling pathways. Nrf2 is a master regulator of cellular antioxidant response that stimulates the production of phase II cytoprotective antioxidant genes (e.g., heme-oxygenase-1 and NAD(P)H oxidase-1) and ROS-detoxifying enzyme (e.g., GSH, SOD, CAT GPx) to mediate redox balance

Conclusion
In conclusion, this study showed that L-egt can alleviate oxidative damage by upregulating Sirt1/Nrf2 expression and its downstream antioxidant molecules, downregulate NF-kB and TGF-β1 expression to reduce hepatic in ammation and brosis as well as reduce SREBP1c and FAS expression to lower hypertriglyceridemia and attenuate hepatocyte fatty acid accumulation to protect liver function during diabetes. Thus, supplementation of L-egt could be used as an adjuvant regimen with metformin therapy in the early stage of diabetes to prevent the development or alter the progression of liver complications associated with diabetes. However, further studies to evaluate the status of L-egt transporters and protein  DM=diabetic plus metformin and DEM= diabetic plus L-egt plus metformin (supplemental data).