| Prupose : To investigate the protective effect and mechanism of hydrogen sulfide on rat hepatic fibrosis in liver ischemia-reperfusion injury.Methods:There are totally 64 SD rats to establish hepatic fibrosis rats model using bile duct ligation,We taken 32 SD rats randomly as hepatic fibrosis rat model by bile duct ligation,and the rats were divided into four groups by random number table : ① sham group(Sham group), ② hepatic ischemia-reperfusion injury group(HIRI group), ③ NaHS preconditioning group(NaHS group), ④Na HS pretreatment + PI3 K inhibitor(LY294002 group), with total hepatic ischemia and reperfusion injury. ①Sham group: Only with laparotomy to reveal liver pedicle, without blocking blood flow. ②HIRI Group: with laparotomy to reveal hepatic pedicle, and hepatic inflow occlusion using pringle maneuver to establish total hepatic ischemia model for 30 mins, and then with reperfusion for 4h. ③NaHS treatment group were injected intraperitoneally with NaHS 42 umol / kg 30 mins before HIRI, ④LY294002 NaHS group was injected with autophagy inhibitor LY294002 10umol/kg 30 mins before the tail vein before NaHS pretreatment, and the rats were sacrificed 4h after HIRI, Blood and liver tissue were taken to measure aspartate aminotransferase, alanine aminotransferase, and bilirubin using automatic biochemical analyzer;and then determine MDA levels in liver tissue using thiobarbituric acid(TBA); and SOD level of liver tissue using xanthine oxidase method.The left lateral lobe of the liver tissue was taken, then fixed with 2.5% glutaraldehyde, and dehydrated with ethanol, acetone by gradient, embedded, double stained with uranyl acetate and lead citrate, examined by transmission electron microscope, photographed,autophagy morphology and quantity was observed; The left hepatic of the lobe tissue was made into 10% tissue homogenate and autophagy specific marker protein Beclin1, LC3 B and AKt were detected with western blot in each group.Results:1, Establishment of hepatic fibrosis animal models : Total 32 SD rats were used to establish animal models with liver fibrosis in pre-experiment.8 of the SD rats were sham-operated, and 24 dealt with the the common bile duct ligation were divided into 3 groups, and then detected 5 days, 10 days,and 18 days after ligation.We detected on serum total bilirubin(TBIL), direct bilirubin(DBIL), aspartate aminotransferase(AST), alanine aminotransferase(ALT) levels, and compared these with the sham group. 5 rats were dead 18 days after the ligation with a mortality rate of 16.7%.Detection on TBIL, DBIL, AST, ALT in the ligation group were significantly higher(p <0.05)than normal rats. Masson staining of liver tissue sections with microscopic pathology revealed that most of the rats were in the Ⅲ stages of liver fibrosis in the 18 th day of ligation, indicating that liver fibrosis animal model was successful.1. Ischemia-reperfusion model: 64 SD rats were used to establish a rat model of liver cirrhosis using common bile duct ligation. 18 rats were dead after18 days of ligation,a mortality rate of 28.1%, the remaining 32 SD rats were divided into four groups randomly, with the number of sham-operated group and hepatic ischemia-reperfusion injury group being 8, and the remaining group of 8.2. Biochemical tests:4h after ischemia-reperfusion of liver fibrosis, we found that aspartate aminotransferase were significantly elevated in each group(p <0.05),comparing with sham contrast. Damage was alleviated in NaHS group than HIRI group(p < 0.05), NaHS group embraced aggravated damage compared with LY294002 group(p <0.05).3.MDA and SOD Levels: 4h fter ischemia-reperfusion of liver fibrosis, we found that SOD decreased significantly in each group(p <0.05), comparing with the sham group,MDA levels increased significantly( p <0.05)each group.Among these HIRI decreased most obviously; NaHS group had the high levels of SOD than HIRI group(p <0.05), and lower MDA levels(p <0.05); LY294002 group had lower levels of SOD than NaHS group(p <0.05), and higher MDA levels(p <0.05).4. Autophagy index detection : 1) autophagosome detection : 4h after ischemia and reperfusion of rats with liver fibrosis,we used electron microscope examination discovered that autophagy increased in each group,comparing with the sham group, NaHS group pretreated group presented autophagy reduction compared with HIRI, LY294002 pretreatment group had increased autophagy compared with NaHS group 2) The expression of Beclin1, LC3 B and AKt : 4h after ischemia and reperfusion of hepatic fibrosis in rats, Beclin1 expression levels were elevated(p <0.05) in each group compared with the sham group, NaHS preconditioning group had lower expression levels of Beclin1 compared with HIRI group(p <0.05), LY294002 intervention group had higher expression levels of Beclin1 compared with NaHS group(p <0.05); compared with the sham group,LC3 B expression levels were elevated in each group(P <0.05), NaHS group pretreated group had lower expression levels compared with HIRI LC3B(p <0.05), LY294002 intervention group had higher expression levels of LC3 B compared with NaHS pretreatment group, the difference was statistically significant(p <0.05); 4h after ischemia-reperfusion of liver fibrosis in rats, there was no significant difference(P <0.05) in Akt expression levels in HIRI group, in comparison with the sham group,NaHS pretreated group had higher expression levels of Akt compared with HIRI group(P <0.05), LY294002 intervention group had lower Akt expression levels compared with NaHS pretreatment group, the difference was statistically significant(p <0.05).Conclusion:1.Exogenous hydrogen sulfide can reduce hepatic ischemia-reperfusion injury of hepatic fibrosis in rat.2, Exogenous hydrogen sulfide can relieve liver fibrosis hepatic ischemia-reperfusion injury of hepatic fibrosis in rat by inhibiting oxidative stress.3, One of the protective mechanisms for exogenous hydrogen sulfide work on rat liver fibrosis ischemia-reperfusion injury is to inhibit autophagy via PI3 K / Akt signaling pathway. |
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