The Effect Of HRS On Hepatocyte Apoptosis Induced By Laparoscopic Hepatic IRI Combined With Partial Hepatectomy In Miniature Pigs

Laparoscopy has the advantages of less trauma,less pain and quick recovery after operation.The safety and feasibility of laparoscopic surgery has been verified in the field of liver surgery.However,hepatic ischemia reperfusion injury also occurs in laparoscopic hepatectomy.How to reduce hepatic ischemia reperfusion injury is an urgent problem to be solved in the field of liver surgery.Most of the protective studies of hepatic ischemia reperfusion injury are confined to the experimental animal models such as rabbits and rats.The current model cannot completely simulate the operating environment of laparoscopic surgery.Establishing a model of liver injury under laparoscopy is the premise for further research.Hydrogen is a natural antioxidant,which can selectively scavenge reactive oxygen species and protect tissues and organs.Therefore,in this study,the model of hepatic ischemia reperfusion combined with partial hepatectomy was established by laparoscopy and explore the effect of hydrogen rich saline(HRS)on hepatocyte apoptosis after operation.In this study,18 miniature pigs were randomly divided into sham operation group(Sham group),model group(IRI group),and HRS intervention group(HRS group).In the sham group,only the pneumoperitoneum was established and the hepatic lobes were flipped.Animals in the IRI and HRS groups were subjected to left hepatectomy after right hepatic ischemia for 60 min.HRS(10 ml/kg)was injected into the portal vein 10 min before reperfusion and at 1 d,2 d,and 3 d post-operation.Liver tissue samples and blood samples were collected before surgery,immediately after surgery,1 d,3 d,and 7 d after operation.The change of liver function indexs detected with a kit.Bax,Bcl-2,Fas,Fasl mRNA transcriptional level and protein expression were measured by real time fluorescence quantitative PCR and Western Blot,and the expression levels of Caspase-3,8 and 9 in liver tissue were detected by Caspase activity detection kit.The TUNEL method was used to detect the hepatocyte apoptosis index in liver tissue,and the ultrastructure of hepatocytes was observed by transmission electron microscope.The study successfully complete all groups of operations and recovered well after surgery.(1)The results of liver function test showed that: The levels of ALT,AST in IRI group and HRS group were significantly higher than those in Sham group immediately after operation,1 d and 3 d after operation(P<0.05 or P<0.01),and TBIL level was significantly increased at 1 d and 3 d after operation(P<0.01);The levels of ALT and TBIL in HRS group were significantly lower than those in IRI group at 1 d and 3 d after operation(P<0.05 or P<0.01),and AST level was significantly decreased immediately after operation,1 d and 3 d after operation(P<0.05 or P<0.01).(2)Results of hepatocyte apoptosis index: Compared with Sham group,the apoptosis index of IRI group and HRS group were significantly increased immediately after surgery,1 d and 3 d after operation(P<0.05 or P<0.01);Upon HRS treatment,the apoptosis index was significantly decreased at 1 d and 3 d after operation when compared with IRI group(P<0.05).(3)Ultrastructural changes of hepatocytes: Compared with Sham group,the ultrastructure of liver tissue in IRI group and HRS group were obviously changed.The degree of damage in HRS group was less than that in IRI group.(4)Results of mRNA transcription level and protein expression of apoptosis-related factors: Compared with Sham group,Bax mRNA transcription level and protein expression in IRI group and HRS group increased significantly(P<0.05 or P<0.01),Bcl-2 mRNA transcription level and protein expression was significantly decreased immediately after surgery and 1 d after operation(P<0.05 or P<0.01).The ratio of Bax/Bcl-2 mRNA and protein in IRI group and HRS group was significantly higher than that in Sham group immediately after surgery,1 d and 3 d after operation(P<0.05 or P<0.01);Upon HRS treatment,Bax expression was significantly decreased(P<0.05 or P<0.01)and Bcl-2 was significantly increased(P<0.05 or P<0.01)immediately after surgery and 1 d after operation when compared with IRI group.The ratio of Bax/Bcl-2 in HRS group was significantly lower than that in IRI group immediately after surgery and 1 d after operation(P<0.01).Fas mRNA transcription level and protein expression in IRI group and HRS group were significantly higher than those in Sham group immediately after surgery and 1 d after operation(P<0.05 or P<0.01).Fasl mRNA transcription level and protein expression in IRI group and HRS group were significantly increased at 1 d and 3 d after operation when compared with Sham group(P<0.05 or P<0.01).Upon HRS treatment,Fas,Fasl mRNA transcription level and protein expression were significantly decreased at 1 d and 3 d after operation when compared with IRI group(P<0.05 or P<0.01).(5)Caspase activity test results: Compared with Sham group,Caspase-3,8 and 9 activities in IRI group were significantly increased at 1 d and 3 d postoperatively(P<0.01),and Caspase-3 and 9 activities in HRS group were significantly increased at 1 d postoperatively(P<0.01);Compared with IRI group,Caspase-3 and 8 activities in HRS group was significantly decreased at 1 d and 3 d postoperatively(P<0.05 or P<0.01),and Caspase-9 activity was significantly reduced immediately after surgery and 1 d postoperatively(P<0.05 or P<0.01).In summary,the following conclusions were drawn:(1)HRS can promote the recovery of liver function after operation,and it has protective effect on laparoscopic hepatic ischemia reperfusion combined with partial hepatectomy in miniature pigs.(2)HRS can reduce the number of hepatocyte apoptosis induced by laparoscopic hepatic ischemia reperfusion combined with partial hepatectomy in miniature pigs,which plays a protective role in inhibiting hepatocyte apoptosis.(3)HRS can inhibit the hepatocyte apoptosis induced by laparoscopic hepatic ischemia reperfusion combined with partial hepatectomy in miniature pigs through down-regulating Bax,Fas,Fasl mRNA and protein expression,up-regulating Bcl-2 mRNA and protein expression,and reducing Caspase-3,Caspase-8 and Caspase-9 activities.