Influences Of HRS On Hepatocyte Autophagy During Laparoscopic Hepatic IRI Combined With Partial Hepatectomy In Miniature Pigs

As a key and frequently occurring surgical complication,hepatic ischemia-reperfusion injury?IRI?has been a unavoidable pathophysiological phenomena in liver hepatectomy and hepatic transplantation.So preventing hepatic IRI is a challenge in clinical treatment.H₂ is widely used in the study of IRI complications due to its selective anti-oxidation,anti-apoptosis and anti-inflammatory effects.However,the biological effects of H₂ in IRI still need to be further studied.Therefore this study dissolved hydrogen saturation in physiological saline by a hydrogen generator to make hydrogen-rich saline?HRS?to study the influences of HRS on hepatocyte autophagy during laparoscopic hepatic IRI combined partial hepatectomy and the protective effect of HRS against laparoscopic hepatic IRI combined partial hepatectomy.This study established an associated injury laparoscopic surgery model in miniature pigs.Eighteen miniature pigs were randomly divided into three groups:Sham operation group?group sham?,model group?group IRI?,and HRS intervention group?group HRS?.The same anesthesia,pneumoperitoneum and the same surgical access were used in all groups.The Sham group was anesthetized and pneumoperitoneum established only,and the laparoscopic flip the liver leaf without any other manipulation.IRI group and HRS group performed the right hepatic ischemia60min and left hemihepatectomy after the conventional anesthesia,the establishment of pneumoperitoneum and surgical access,HRS group received hepatic portal vein cannulation after ischemia,and HRS?10 mL/kg?was administered intravenously 10 min before reperfusion.Intravenous infusion of HRS was performed on the 1st,2nd and 3rd postoperative day?10 mL/kg?.Every group was monitored the respiratory,anesthesia,heart rate,blood pressure,body temperature and other physiological indicators during the surgery to ensure the safety of surgery.Samples of hepatic tissue and serum were collected at preoperative,at the time of reperfusion?0h?,3 h of reperfusion?3h?,1 d and 3 d post-reperfusion?1d and 3d?for examination.The liver function?AST,ALT,ALP?at various time points was detected by biochemical methods.The oxidative stress markers?MDA,GSH,CAT,SOD?in liver tissues were assessed by kit method.The expression of autophagy related genes?Beclin1,P62,ATG5,ATG12 and mTOR?in liver tissues were detected by Real-time PCR.Western blot was used to detect the expression of autophagy related protein?Beclin1,P62 and LC3B?in liver tissue.The expression of LC3B protein in liver tissue was detected by immunohistochemistry.The changes of hepatocyte sub-microstructure were observed by transmission electronmicroscopy?TEM?.The results showed that the three groups of laparoscopic surgery were successfully completed without serious complications.Compared with Sham group,in IRI group,the indexes of AST,ALT and ALP in serum were increased at 0h,3h and 1d and the difference was extremely significantly?P<0.01?;TME observation showed that mitochondrial swelling and endoplasmic reticulum expansion occurred in 3h;the oxidative stress index showed that the content of SOD,CAT and GSH in liver tissue decreased and the content of MDA increased at 0h,3h and 1d and the difference was extremely significantly?P<0.01?;the mRNA expression of autophagy-related genes showed that Beclin 1,ATG5 and ATG12 genes mRNA expression were increased,while mTOR and P62 genes mRNA expression were decreased at 0h,3h and 1d and the difference was extremely significantly?P<0.01?;Western Blot results showed that the protein expression of Beclin1 and LC3B were increased and the protein expression of P62 in liver tissue was decreased at 0h and 3h and the difference was extremely significantly?P<0.01?;the positive expression of LC3B protein immunohistochemistry in liver tissue at 3h in IRI group was higher than that of in Sham group and the difference was extremely significantly?P<0.01?.After HRS intervention,compared with IRI group,the liver function indexes in HRS group were significantly lower at 1d and the difference was extremely significantly?P<0.01?;and the submicroscopic structural damage of hepatocytes at 3h under electron microscope was lighter;the levels of SOD,CAT and GSH in liver tissue were increased and the level of MDA was decreased at3h and 1d and the difference was significantly or extremely significantly?P<0.05 or P<0.01?;the mRNA expression of Beclin 1,ATG5 and ATG12 were decreased and the mRNA expression of mTOR and P62 was increased,the protein expression of LC3B and Beclin 1 were decreased and the protein expression of P62 was increased at 3h and the difference was significantly or extremely significantly?P<0.05 or P<0.01?.The above experimental results show that:1.The intervention of HRS can reduce liver function injury and oxidative stress during laparoscopic hepatic IRI combined with partial hepatectomy injury,and it has the protective effects on the liver.2.The intervention of HRS can reduce the increase of hepatocyte autophagy caused by laparoscopic hepatic IRI combined with partial hepatectomy injury.