Effect Of HRS On ERS In Laparoscopic Hepatic IRI With Partial Hepatectomy In Minipigs

Hepatic ischemia-reperfusion injury(IRI)is a clinically urgent problem to be solved.Recent studies have shown that endoplasmic reticulum stress(ERS)plays an important role in hepatic ischemia-reperfusion.Intervention on the ERS response pathway may provide new protection for liver IRI.Hydrogen-rich saline(HRS)has anti-oxidation,anti-inflammatory and anti-apoptotic effects,and has extensive protective effects on various organs of IRI.Recent studies have found that HRS can reduce liver IRI by inhibiting ERS.The mechanism is not yet clear,and HRS has not been reported for the study of ERS in hepatic ischemia-reperfusion minihepatic model established by laparoscopic technique.In this study,laparoscopic surgery was used to establish a miniature liver model of hepatic ischemia-reperfusion combined with hepatectomy.HRS intervention was applyed in the modle.By tracking the change of hepatic histology and ERS related indicators after intervention,the effects of HRS on ERS after hepatic ischemia-reperfusion combined with partial hepatectomy were discussed.In this experiment,18 healthy minipigs were randomly divided into three groups: sham operation group(Sham group),model group(IRI group)and HRS intervention group(HRS group)(n=6).In the Sham group,only the hepatic lobe was turned and the pneumoperitoneum was maintained for three hours.The IRI group was treated with laparoscopic surgery to establish the right hepatic ischemia for 60 min with the left hemihepatectomy model.The HRS group was established with the surgical model and the portal vein catheter was placed 10 minutes before reperfusion.HRS(10 mL/kg)was injected at 10 min before reperfusion,6 h after reperfusion,1 d,2 d,and 3 d after surgery.Each group received preoperative reperfusion,immediate reperfusion,immediate postoperative,immediate postoperative,postoperative 1 d,and postoperative 3 d.Liver tissue was obtained by laparoscopic surgery.HE staining and electron microscopy were performed.Real-time fluorescence quantitative PCR,immunohistochemistry,and Western blot and other methods were used to detect changes in ERS parameters in the liver.Specific examination indicators included: endoplasmic reticulum protein core protein(GRP78),unfolded protein response(UPR)signal molecules(PERK,IRE1,ATF6,XBP1 s,p-eIF2α),ERS apoptosis-related proteins(ATF4,CHOP,p-JNK).The results showed that:(1)Changes in liver pathological lesions: HE staining showed that different degrees of hepatic tissue injury were observed at various time points in the IRI group compared to the Sham group: Destruction of hepatocyte cords,swelling of cells,vacuolar degeneration,presence of necrotic lesions and inflammatory cell infiltration.Compared with the IRI group,the hepatocytes in the HRS group were basically cord-like,and the hepatocyte necrosis and inflammatory infiltration in the HRS group were significantly reduced.The results of electron microscopy at 1 day after surgery showed that the microstructure of hepatocytes in Sham group was normal,and the mitochondria and endoplasmic reticulum of hepatocytes in IRI group were swollen and the structure was destroyed and glycogen was reduced.The mitochondrial structure of hepatocytes in HRS group was normal and the endoplasmic reticulum was slightly swollen.GRP78 is a core protein of the endoplasmic reticulum.To study the effect of HRS on ERS,we examined changes in GRP78 expression levels in liver tissue: The expression level of GRP78 mRNA in IRI group was significantly higher than that in Sham group at immediately after reperfusion,immediately after operation,1 day after surgery,and 3 days after operation(P<0.01),And it was significantly higher than that of HRS group(P<0.01)at immediately after reperfusion,immediately after reperfusion,1 day after surgery,and 3 days after operation.Besides of immediately after reperfusion,the expression level of GRP78 protein in IRI group was significantly higher than that in Sham group at all time points(P<0.01).The expression level of GRP78 protein in IRI group was significantly lower than IRI group at immediately after reperfusion,1 day after surgery,and 3 days after surgery(P<0.01).To further explore the mechanism of HRS on ERS,we examined the mRNA and protein expression of UPR signaling molecules: The expression levels of PERK,IRE1,and ATF6 mRNA in IRI group were significantly higher than those in Sham group and HRS group at immediately after reperfusion,immediately after surgery,and 1 day after surgery(P<0.05 or P<0.01);PERK,IRE1,ATF6 protein expression levels in IRI group was significantly higher than Sham group at immediately after reperfusion,immediately after surgery,and 1 day after surgery,It significantly higher than HRS group at 1 day after surgery(P<0.05 or P<0.01).The end result of ERS will lead to the production of endoplasmic reticulum apoptosis.For this reason,we tested the ERS apoptosis-related proteins: IRI group was significantly higher than that of Sham group at immediately after surgery,1 day after surgery,and 3 days after operation.It was significantly higher than that of HRS group immediately at immediately after surgery and 1 day after operation(P<0.05 or P<0.01).In summary:(1)HRS significantly improved hepatic pathological tissue damage after hepatic ischemia-reperfusion and partial hepatectomy,and improved the ultrastructure of hepatocytes.It shows that HRS has a protective effect on liver injury.(2)HRS can inhibit the upregulation of endoplasmic reticulum core protein caused by liver injury,inhibit UPR three signal pathways,and then inhibit the expression of endoplasmic reticulum apoptotic protein.It shows that RHS can inhibit ERS in liver injury.