Hydrogen Sulfide Resistant L-NAME-induced Rat Liver Injury And The Mechanism Research

ABSTRACTCardiovascular disease and its injuries on the body attract more attention of clinic and research. With the improvement of living conditions, incidence and mortality rates of cardiovascular disease increases. In the crowd, hypertension and hyperlipidemia are common causes of vascular conditions. High blood pressure and hyperlipidemia can not be only as an independent risk factor for cardiovascular disease, but also an important risk factor for atherosclerosis. Atherosclerosis can aggravate hypertension and hyperlipidemia and its damage to target organ. Therefore, how to reduce cardiovascular risk is research hotspot in recent years.With the studies found that nitric oxide(NO) and carbon monoxide(CO), endogenous hydrogen sulfide(H2S) plays an important role in the cardiovascular system, and is considered as the 3rd gaseous signaling molecule in the body.Nitric oxide(NO) is considered as the endothelium-dependent relaxing factor, and was regarded as a regulation factor of internal organs and blood circulation. In addition, NO also functions in other physiological processes such as cell proliferation and nerve conduction. It is reported that inhibition of NO production can increases blood dose-dependently and eventually cause some damage. Using L-NAME to block the production of NO in rats can cause abnormality of lipid metabolism. In rat model of hypercholesterolemia, we also observed reduced plasma levels of NO and endothelium damage and therefore the incidence of atherosclerosis.Hydrogen sulfide(H2S) has been considered as a toxic gas. With continuous research, mammals can generate endogenous H2 S. H2 S products in many ways, it is currently detected via synthase cystathionine-gamma-lyase(CSE), cystathionine-beta-synthase(CBS) and the recently discovered 3-mercaptopyruvate sulfurtransferase(3-MST) and cysteine Aminotransferase(CAT). H2 S is mainly synthesized by CSE in the cardiovascular system, together with CSE and CBS in the liver. Study on the protective effects of H2 S on the cardiovascular system showed that H2 S can protect the heart ischemia-reperfusion injury, and decreases blood pressure in spontaneously hypertensive rats(SHR).Numerous studies show that liver plays a important function in stability and maintenance of the homeostasis of lipid. Liver damage often results in abnormal lipid metabolism, thereby increasing the risk of cardiovascular disease. Current research finds that H2 S protection against cirrhosis induced by CCl4 or portal vein ligation. H2 S can also protect the liver against ischemia-reperfusion injury. In rats model of L-NAME-induced hypertension, it shows not only elevated blood pressure, but also serum lipid and lipoprotein abnormalities. If we can investigate the relationship between H2 S in the liver and NO and the relationship between H2 S and cardiovascular disease risk factors, we can provide new ideas for prevention and treatment of cardiovascular disease.This research aimed at using NG-nitro-L-arginine methylester(L-NAME)-induced rat model of hypertension and liver injury, made discussion on H2 S protection against L-NAME-induced rat liver injury and mechanism of H2 S reduction of cardiovascular risk. Part I Protective effect of hydrogen sulfide in L-NAME-induced rat liverdamage and cardiovascular riskObjective: Through copies rat model of L-NAME-induced damage and mediated by exogenous H2 S donor sodium hydrosulfide(Na HS) and glibenclamide(Gli), a KATP-channel blockers, we made study on the protective effect of L-NAME-induced rat liver injury and cardiovascular risk.Methods: 36 healthy male Sprague-Dawley rats were randomly assigned into 6 groups, namely: control(con), L-NAME group, con + glibenclamide Group(con +Gli), con + sodium hydrosulfide(con +Na HS), L-NAME +Na HS and L-NAME +Na HS +Gli, 6 in each group. Sodium hydrosulfide group of rats uses sodium hydrosulfide(56 μmol/kg) daily intraperitoneal injection; Other set of group uses daily intraperitoneal injection of saline solution. L-NAME treated rats was given daily drink of L-NAME water solution. Glibenclamide in groups of rats were given prepared aqueous solution of glibenclamide for everyday drinking. Systolic pressure were measured weekly in each group. Rats were treated for 5 weeks, and serum and liver tissue was collected. TG, high density lipoprotein(HDL), low density lipoprotein(LDL), total cholesterol(CHO) and alanine aminotransferase(ALT) level was measured and liver tissues was observed under microscope, especially liver cell morphological arrangement, histologic structure stability, portal structure, lymphocytic infiltration and fat vacuoles appearance.Results: 1 evaluation of rat model: con vs. rats, the rats food intake and water intake and weight increased with the number of weeks, no significant difference was shown between groups. Rats in each group show no cases of serious illness, death. 2 the changes of blood pressure in rats: the tail artery systolic blood pressure in each group before the process showed no statistically significant differences. After the treatment, L-NAME groups showed rat tail artery systolic blood pressure increase. L-NAME blood pressure is significantly higher than the L-NAME group +Na HS Group, but L-NAME group +Na HS +Gli blood pressure showed no significant difference compared with the L-NAME group. 3 changes in liver function: serum ALT and CHO showed no statistical differences between groups. Among con group, con +Na HS group, and con +Gli group, LDL and TG content showed no statistical differences between groups. Markedly elevated LDL and TG showed in L-NAME group than in the con group. LDL and TG in L-NAME +Na HS group, L-NAME +Na HS +Gli groups was significantly lower than in the L-NAME group, but there were no statistically significant differences between this two groups. Among con group, con +Na HS group, con +Gli HDL levels, each pair showed no statistical difference. HDL in L-NAME group decreased significantly than in the con group. HDL in L-NAME +Na HS group, L-NAME +Na HS +Gli group and L-NAME group significantly increased, but there were no statistical differences between the L-NAME +Na HS group and L-NAME +Na HS +Gli group.4. the pathomorphological observation of liver: H&E stained and under microscope, lobular architecture in con group, con +Na HS group, con +Gli is clear, complete, liver cells radially arranged neatly around the central vein. In L-NAME group, liver lobules disarranged, there is hyperplasia of fibrous connective tissue, liver cells increase in size with visible cytoplasm and unequally sized circular bubble. There is ballooning degeneration of liver cells; around the central veins and visible portal inflammatory cell infiltration. In L-NAME +Na HS and L-NAME +Na HS +Gli group, histopathological changes in the liver are between con and L-NAME groups.Summary: this study confirmed that the NO blocker L-NAME causes increased blood pressure in rats, liver and serum liver function changes of relevant indices. H2 S donor Na HS can be reduced by L-NAME-induced rat high blood pressure; thus, it protects against the liver damage, reduced blood pressure effect of KATP channel antagonist glibenclamide blocked but it cannot block the effect of H2 S on the liver protection. Part II Hydrogen sulfide by regulating e NOS/AKT protection ofL-NAME-induced rat liver injuryObjective: Via copying an L-NAME-mediated rat model with applying exogenous H2 S donor Na HS and glibenclamide(Gli), we detected plasma NO and H2 S and use molecular biology techniques to study the H2 S function of adjusting the e NOS/AKT to protect L-NAME-induced liver injury in rats.Methods: 36 healthy male Sprague-Dawley rats, the animals were divided into 6 groups, namely: control(con), L-NAME group, con + glibenclamide Group(con +Gli), con + sodium hydrosulfide(con +Na HS) group, L-NAME +Na HS group, and L-NAME +Na HS +Gli group, 6 in each group. Sodium hydrosulfide group of rats uses sodium hydrosulfide(56 μmol/kg) daily intraperitoneal injection; Other set of group uses daily intraperitoneal injection of saline solution. L-NAME prepared rats was given daily drink of L-NAME water solution. Glibenclamide in groups of rats were given prepared aqueous solution of glibenclamide for everyday drinking. Rats were treated for 5 weeks, and serum and liver tissue H2 S content was measured. we also measured the content of NO in liver tissue, and e NOS, P-e NOSs1177, AKT, P-AKTs473 protein expression by Western blot method.Results: 1 changes of plasma levels of H2 S content: as for the H2 S content in serum of the group of con, con +Na HS group, and con +Gli, there was no statistically significant difference between each pair. Plasma levels of H2 S L-NAME group than in the con group decreased significantly. H2 S in L-NAME +Na HS group, L-NAME +Na HS +Gli group was significantly higher in plasma than L-NAME group, but there were no statistically significant differences between these two groups. 2 NO changes of hepatic tissue: the NO content in the liver tissue of con group, con +Na HS group, con +Gli group showed no statistically significant difference between the groups. Liver tissue NO content decreased significantly in L-NAME group than in the con group. Liver tissue NO content in L-NAME +Na HS group, L-NAME +Na HS +Gli group was significantly higher than L-NAME group, but there was no statistically significant difference between these groups. 3 Protein expression in liver: Results uses gray value compared with internal reference GADPH. In con group, con +Na HS group, con +Gli group of liver tissue, e NOS, P-e NOSs1177, AKT, P-AKTs473 protein expression were not statistically significant differences between groups. In L-NAME liver tissue, e NOS, P-e NOSs1177, AKT, P-AKTs473 protein expression were significantly lower than the con group. In L-NAME +Na HS group, L-NAME+Na HS group, L-NAME+Na HS+Gli group, P-e NOSs1177, AKT, P-AKTs473 protein expression was significantly higher than L-NAME group, but there were no statistically significant differences between the two groups.Summary: this study confirmed NO blockers L-NAME in rat plasma and NO H2 S content decreased, and downregulates expression of e NOS, P-e NOSs1177, AKT, P-AKTs473 proteins. Applying H2 S donor Na HS can upregulated NO and H2 S concentrations in L-NAME rats, upregulate NO synthesis in the NO-associated protein expression in liver. This effect cannot be blocked by glibenclamide, that proved a pathway independent of KATPchannels. It was possibly through activation of AKT-e NOS pathways. Part III Hydrogen sulfide by regulating e NOS/AKT protection ofL-NAME-induced rat liver cells injuryObjective: Through the experiments of in vitro cultured primary liver cells, we investigate whether H2 S in vitro directly regulating e NOS/AKT to shield L-NAME-induced rat liver injury via regulating phosphorylation of e NOS/AKT and increases the generation of nitric oxide in liver cells.Methods: Hepatocytes isolation and culture used Sprague-Dawley male rats by portal vein fluid perfusion to separate liver cells, and liver cell viability determined by trypan blue exclusion method. We select cultured liver cell by cell motility of more than 90%. Primary liver cells were divided into control groups(con), con+Na HS(100 μ mol/l) group, L-NAME(100 μ mol/l) group, L-NAME(100μmol/l)+Na HS(100μmol/l) group, L-NAME(100 μ mol/l)+Na HS(100 μ mol/l)+Gli(100 μ mol/l) group, L-NAME(100 μ mol/l)+Na HS(100 μ mol/l)+LY294002(20μmol/l). Drug application time is all 8 hours. Cell culture supernatant was collected to measure NO content, using specific fluorescent probe DAF-FM DA(3-Amino,4-aminomethyl-2’,7’-difluorescein, diacetate) to measure changes of intracellular NO. e NOS, P-e NOSs1177, AKT, P-AKTs473 protein expression in primary liver cells was detected by the western blot assay.Summary: This study confirmed NO blockers L-NAME in rat liver primary cell blocks NO formation, downregulated liver cells P-e NOSs1177, P-AKTs473 protein expression and e NOS /AKT phosphorylation. H2 S donor Na HS can upregulate liver primary cell NO formation and its associated protein expression. This effect cannot be blocked by glibenclamide, indicated which is independent of KATP channels. The regulation can be blocked by AKT inhibitor LY294002 indicated that H2 S upregulates NO in liver cells possibly via activation of AKT-e NOS pathway.