Treatment With Hydrogen Sulfide Prevents Liver Injury Of NAFLD Induced By Diet In Rats

Objective：Non-alcoholic fatty liver disease （NAFLD） is the most major chronic liver dysfunctionin the world and its prevalence in the China is increasing with epidemics of obesity,diabetes, and metabolic syndrome. NAFLD is defined as the spectrum of benign fatty liver（steatosis） to necro-inflammation and fibrosis or non-alcoholic steatohepatitis （NASH） thatcan lead to cirrhosis and hepatocellular cancer, in the absence of excessive alcoholingestion.In the transition from benign steatosis to NASH,“two hit theory” is assumed to berequired, with the first hit being lipid accumulation in hepatocytes increasing the sensitivityof the liver to the second hit such as oxidative stress and pro-inflammatory cytokines,which are expected to be the promising targets in the treatment of this disease.Hydrogen sulfide （H₂S） was best known as a foul smelling and toxic gas before beingrecognized as an important mediator in several biological systems including neurological,cardiovascular, and gastrointestinal systems. H₂S is endogenously produced in mammaliantissues by cystathionine γ-lyase （CSE） and cystathionine β-synthase （CBS）.Given that H₂S displays anti-oxidative, anti-inflammatory and cytoprotective activities,an anti-fibrotic effect against pulmonary fibrosis, and a protective effect against CCl4-induced injury in liver, we hypothesized that H₂S might attenuate the methionine-choline-deficient diet （MCD） or high-fat diet （HFD） induced NASH in rats.Methods：Part I:1. A MCD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） MCD-fed rats. Measurement of H₂Slevels in plasma and livers of rats was performed by using ELIT Ion Analyzer. The mRNAexpression and protein expression of CBS and CSE were determined by RT-PCR method and Western blotting analysis, respectively.2. A HFD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） HFD-fed rats. Measurement of H₂Slevels in plasma and livers of rats was performed by using ELIT Ion Analyzer. The mRNAexpression and protein expression of CBS and CSE were determined by RT-PCR methodand Western blotting analysis, respectively.Part II:1. A MCD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） MCD-diet-fed rats;（3） MCD-diet-fedrats treated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, adonor of H₂S）. Assessments of steatohepatitis by HE staining, apoptosis by TUNEL, ALTand AST activities were performed to investigate the effect of H₂S on MCD-inducedNASH.2. A HFD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） HFD-diet-fed rats;（3） HFD-diet-fed ratstreated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, a donor ofH₂S）. Assessments of steatohepatitis by HE staining, apoptosis by TUNEL, ALT and ASTactivities and insulin resistant were performed to investigate the effect of H₂S onHFD-induced NASH.Part III:1. A MCD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） MCD-diet-fed rats;（3） MCD-diet-fedrats treated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, adonor of H₂S）. Oxidative stress, inflammation, and expression profiles of fatty acidmetabolism genes in livers were measured to investigate the mechanism of the H₂Sprotective effect on HFD-induced NASH.2.A HFD rat model was prepared. Rats were divided into three experimental groupsand fed for8weeks as follows:（1） control rats;（2） HFD-diet-fed rats;（3） HFD-diet-fed ratstreated with NaHS （intraperitoneal injection of0.1ml/kg/d of0.28mol/l NaHS, a donor ofH₂S）. Oxidative stress, inflammation, and expression profiles of fatty acid metabolism genes in livers were measured to investigate the mechanism of the H₂S protective effect onHFD-induced NASH.Results：Part I:1. After3weeks, MCD led to reduced plasma H₂S level and hepatic H₂S content inrats. MCD suppressed CBS and CSE mRNA and protein expression in livers of rats.2. After6weeks, HFD led to reduced plasma H₂S level and hepatic H₂S content inrats. HFD suppressed CBS and CSE mRNA and protein expression in livers of rats.Part II:1. Effect of treatment with exogenous H₂S in MCD-fed rats[1] Treatment with exogenous H₂S in MCD-fed rats increased plasma H₂S levels andhepatic H₂S content, further decreased hepatic CSE expression, but had no significant effecton hepatic CBS expression.[2] Serum levels of glucose, total cholesterol, and triglycerides were lower inMCD-fed rats than that in control rats. Treatment of MCD-fed rats with H₂S had nosignificant effect on serum glucose, total cholesterol, and triglycerides.[3] Hepatic content of cholesterol and triglycerides were higher in MCD-fed rats thanthat in control rats. Treatment with H₂S in MCD-fed rats resulted in a significant reductionof hepatic cholesterol and triglycerides.[4] HE staining revealed macrovesicular steatosis and inflammation in the livers ofMCD-fed rats, which was attenuated by treatment with H₂S. The increased TUNEL-positive cells revealed increased apoptosis in the livers of MCD-fed rats, which wasalleviated by treatment with H₂S. MCD led to increased activities of ALT and AST in serum,which was restored partly by treatment with H₂S.2. Effect of treatment with exogenous H₂S in HFD-fed rats[1] Treatment with exogenous H₂S in HFD-fed rats increased plasma H₂S levels andhepatic H₂S content, further decreased hepatic CSE expression, but had no significant effecton hepatic CBS expression.[2] Serum levels of glucose, total cholesterol, and triglycerides were higher inHFD-fed rats than that in control rats. Treatment of HFD-fed rats with H₂S reduced serum glucose, but had no significant effect on total cholesterol, and triglycerides in serum.[3] Hepatic content of cholesterol and triglycerides were higher in HFD-fed rats thanthat in control rats. Treatment with H₂S in HFD-fed rats resulted in a significant reductionof hepatic cholesterol and triglycerides.[4] HE staining revealed macrovesicular steatosis and inflammation in the livers ofHFD-fed rats, which was attenuated by treatment with H₂S. The increased TUNEL-positivecells revealed increased apoptosis in the livers of HFD-fed rats, which was alleviated bytreatment with H₂S. HFD led to increased activities of ALT and AST in serum, which wasrestored partly by treatment with H₂S.Part III:1. Effect of treatment with exogenous H₂S in MCD-fed rats[1] MCD led to increased MDA formation in livers, which were restored by treatmentwith H₂S. Hepatic mRNA and protein expression of CYP2E1and HO-1were higher inMCD-fed rats than that in control rats. Treatment with H₂S reduced CYP2E1expression,but further increased HO-1expression in livers of MCD-fed rats.[2] MCD induced hepatic NFκB activation, which was suppressed by treatment withH₂S. MCD led to upregulation of hepatic mRNA and protein expression of TNF-α and IL-6,which was restored by treatment with H₂S.[3] Hepatic mRNA levels of PPARα, SREBP-1c, FAS, and L-FABP weredownregulated; hepatic mRNA levels of CD36, TLR-2, and TLR-4were upregulated inMCD rats. Treatment of MCD-fed rats with H₂S increased hepatic mRNA levels of PPARαand L-FABP, and reduced hepatic mRNA levels of CD36, SREBP-1c, FAS, TLR-2, andTLR-4.2. Effect of treatment with exogenous H₂S in HFD-fed rats[1] HFD led to increased MDA formation in livers, which were restored by treatmentwith H₂S. Hepatic mRNA and protein expression of CYP2E1and HO-1were higher inHFD-fed rats than that in control rats. Treatment with H₂S reduced CYP2E1expression, butfurther increased HO-1expression in livers of HFD-fed rats.[2] HFD induced hepatic NFκB activation, which was suppressed by treatment withH₂S. HFD led to upregulation of hepatic mRNA and protein expression of TNF-α and IL-6,which was restored by treatment with H₂S. [3] Hepatic mRNA levels of PPARα, SREBP-1c, FAS, and L-FABP weredownregulated; hepatic mRNA levels of CD36, TLR-2, and TLR-4were upregulated inHFD rats. Treatment of HFD-fed rats with H₂S increased hepatic mRNA levels of PPARαand L-FABP, and reduced hepatic mRNA levels of CD36, SREBP-1c, FAS, TLR-2, andTLR-4.Conclusion：Endogenous H₂S formation was found suppressed in development of NASH inducedby MCD and HFD in rats.Treatment with H₂S could attenuate MCD and HFD-induced NASH in rats.Treatment with H₂S could abate MCD and HFD-induced oxidative stress in livers.Treatment with H₂S could inhibit MCD and HFD-induced inflammation in livers.Treatment with H₂S could attenuate HFD-induced insulin resistant in rats.Treatment with H₂S could alleviate MCD and HFD-induced lipid accumulation andhave a beneficial modulation on expression profiles of fatty acid metabolism genes inlivers.