| Objective:To establish a HIRI model in rats and observe the changes of oxidative stress,Hcy-related metabolic enzymes in HIRI,and the effect of Ganlong capsules on HIRI pretreatment,to lay the foundation for subsequent research.Methods:1.Randomly divide 60 SD rats into Sham group,HI group and HIR group,among which HIR group was set with 4 reperfusion time periods,including HIR3 h group,HIR6 h group,HIR12 h and HIR24 h group,10 rats in each group.Establish the rat HIRI model,observe the survival and state of the rat and the pathology with HE staining of liver tissue,and determine the establishment of the rat HIRI model.2.Plasma AST,ALT activity and HA,LN content in liver tissues were detected in the rat HIRI model,and real-time fluorescence quantitative PCR was used to determine the expression levels of OPN andα-SMA mRNA in liver tissues to observe the liver injury in rats.3.To detect the activity of XOD,GSH-Px,CAT,NOS and SOD in the liver tissue of rat HIRI model liver tissue,and the content of GSH,NO,MDA to verify that oxidative stress response is involved in the occurrence of HIRI.4.The content of Hcy in liver tissue of rat HIRI model was detected.Real-time fluorescence quantitative PCR was used to determine the expression levels of MS,CβS,MTHFR,BHMT and ER-αmRNA in rat liver tissues,and to evaluate the role of Hcy,metabolism and ER-αin HIRI.5.Forty SPF SD rats were randomly divided into 5 groups,namely normal control group,model group,Ganlong Capsule（GL）Low,Medium and High dose groups(60,120,240 mg·kg-1).The number of animals in each group was 8.Rats in each group were intragastrically administered with a corresponding dose of drug or saline at a dosage of1 m L·100 g-1,once per day for 14 days.After the last administration,each group performed corresponding operations,and observed the pathological changes of HE staining in liver tissue,detected the activity of AST and ALT in plasma,measured the effect of Long Capsule on rat liver ischemic preconditioning.Results:1.The blood supply to the left and middle lobes of the rat liver was blocked by half-hepatic blood flow blocking method,and about 70%of the rat liver tissue was ischemia for 90 min.Reperfusion of blood flow was established to establish the HIRI model to successfully induce liver Damage,and the modeling success rate reached83.3%.Compared with the Sham group and the HI group,the differences of liver organ coefficients in the HIR group were not statistically significant（P>0.05）.2.The results of HE staining showed that the structure of the hepatic tissue confluence area and the central vein could be observed normally and intact under the microscope in the Sham group.The hepatocytes in the group were arranged irregularly,unevenly distributed,with a small amount of degeneration and necrosis,the liver sinus was squeezed and deformed and there was obvious congestion,and there was less inflammatory cell infiltration;the hepatocytes in the HIR3 h group and HIR6 h group were not aligned Non-uniform,with a small amount of degeneration and necrosis,hepatic sinusoidal deformation and a large number of erythrocyte retention,with a small amount of inflammatory cell infiltration;liver cells in the HIR12 h group and HIR24 h group are unevenly arranged and uniformly distributed,with different sizes and varying degrees Degeneration,edema and necrosis,liver sinus squeeze deformation or even disappear and red blood cell stasis.The liver tissue injury in the HIR group was the most severe in the HIR24 h group,with hepatocellular edema and focal necrosis,and a large amount of inflammatory cell infiltration.3.Compared with the Sham group and the HI group,after a certain period of hepatic ischemia and reperfusion in the rat,the XOD activity,NO,and MDA content in the liver tissue of the HIR group increased to varying degrees,and the increase in the HIR24 h group was most obvious.It was statistically significant（P<0.05,P<0.01）,while GSH content and GSH-Px,NOS,CAT,and SOD activity decreased to varying degrees,with the most obvious decline in the HIR6 h group and HIR12 h group,and the difference was statistically significant（P<0.05,P<0.01）.Compared with the Sham group,the SOD and GSH-Px activity in the liver tissue of the HI group decreased,the difference was significant and statistically significant（P<0.01）,while the changes in the GSH,NO,MDA content and XOD,CAT,NOS activity were not statistically different Academic significance（P>0.05）.4.Compared with the Sham group,the plasma AST and ALT activities of the HI group and the HIR group were significantly increased to varying degrees,and the differences were statistically significant（P<0.01）,especially in the HIR6 h group and the HIR12 h group The increase in vitality is most obvious;compared with the HI group,the AST vitality of the rat plasma in the HIR3 h,HIR6 h and HIR12 h groups and the ALT vitality of the HIR group plasma increased to varying degrees,the difference was statistically different（P<0.05）.Compared with Sham group and HI group,the contents of HA and LN in liver tissues of rats in HIR12 h group and HIR24 h group were significantly increased,the difference was significant and statistically significant（P<0.01）.5.RT-q PCR results showed that compared with Sham group and HI group,the expression of OPN mRNA in liver tissues of HIR6 h group,HIR12 h group and HIR24h group were significantly increased in different degrees.The expression levels of SMA mRNA also increased significantly in different degrees.Among them,the expression levels of OPN andα-SMA mRNA increased significantly in the HIR24 h group.The expression levels ofα-SMA and OPN mRNA in liver tissue of HI group were also increased compared with Sham group,but the difference was not statistically significant（P>0.05）.6.Compared with the Sham group and the HI group,the Hcy content in the liver tissues of the HIR12 h group and the HIR24 h group were significantly increased,the difference was significant and statistically significant（P<0.01）,of which the HIR24 h group had the highest content.The content of Hcy in liver tissue of HI group was higher than that of Sham group,but the difference was not statistically significant（P>0.05）.Compared with the Sham group,the MS mRNA expression in the liver tissue of the HI group and the HIR group decreased to varying degrees,and the CβS mRNA expression increased;the HTHF6 h group,HIR12 h group,and HIR24 h group rat liver tissue MTHFR mRNA expression The expression level and the expression level of BHMT mRNA in the liver tissue of the HIR group increased to varying degrees,and the differences were statistically significant（P<0.05）.The expression of MS mRNA decreased most obviously at 12 h of reperfusion,the expression of CβS and MTHFR mRNA increased most significantly at 24 h of reperfusion,and the expression of BHMT mRNA increased most obviously at 6 h of reperfusion.Compared with the HI group,the mRNA expression of MS mRNA in the liver tissues of the HIR3 h group,HIR6 h group,and HIR12 h group all decreased,while the MS mRNA expression in the HIR24 h group increased,the difference was significant and statistically significant（P<0.01）;The expression of CβS mRNA in liver tissue of HIR24 h group increased significantly,the difference was significant and statistically significant（P<0.01）;The expression of MTHFR and BHMT mRNA in liver tissue of HIR group increased,but the difference was not statistically significant Significance（P>0.05）.7.The study found that,compared with the model group,the liver tissue pathological morphology of rats in different concentrations of Ganlong Capsules were improved to varying degrees;the plasma AST and ALT activity of the GL middle-dose group was significantly reduced（P<0.01）;SOD activity in liver tissue of rats in GL low,medium and high dose groups increased differently（P<0.05）,MDA content decreased（P<0.05）,liver tissue activity and Hcy content in GL medium and high dose groups I want to reduce it significantly（P<0.01）;the HA and LN content in the liver tissue of the drug-administration group is only statistically significant in the GL Medium-dose group（P<0.05）.Conclusion:1.Rat hepatic ischemia-reperfusion can cause liver injure,and reperfusion aggravates liver injure.2.It has been verified that oxidative stress is involved in the occurrence of HIRI.Among them,preliminary contact between Hcy and HIRI found that Hcy may produce a large amount of reactive oxygen species to damage liver tissue through self-oxidation.3.In the rat HIRI model with a reperfusion time of 24 hours,the oxidative stress injury becomes more severe with the reperfusion time.4.HIRI leads to abnormal expression of MS gene,which leads to disorder of Hcy methylation metabolism,resulting in accumulation of Hcy in the body.5.A certain dose of Ganlong capsule pretreatment of HIRI in rats can reduce the production of ROS and the self-oxidation of Hcy,as well as improve the antioxidant effect and reduce the damage of liver cells. |
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