The Effects Of The Interaction Of COX-2 And Endoplasmic Reticulum Stress On Hepatic Ischemia-reperfusion Injury In Mice

Objective:The hepatic ischemia-reperfusion injury(HIRI)mouse model was established to investigate the effects of cyclooxygenase-2(COX-2)and endoplasmic reticulum stress(ERS)on HIRI and their interaction in HIRI.Methods:C57BL/6J male mice were divided into four groups: sham-operated group(Sham group),hepatic ischemia-reperfusion group(HIR group),hepatic ischemia-reperfusion+NS398(COX-2 inhibitor)pretreatment group(HIR+NS398 group),and hepatic ischemia-reperfusion+Salubrinal(endoplasmic reticulum stress inhibitor)pretreatment group(HIR+Sal group).The HIRI mouse model was established by clamping the hepatic artery and portal vein in the liver’s left lobe and middle lobe,blocking about 70% of the liver blood,restoring perfusion after 90 min of ischemia,and closing the abdominal cavity.Sal(1 mg/kg)was given intraperitoneally one hour before ischemia in the HIR+Sal group,and NS398(30 mg/kg)was given intraperitoneally one hour before ischemia in the HIR+NS398 group.In the Sham and HIR groups,1% DMSO(10 ml/kg)was administered intraperitoneally one hour before ischemia,and the mice were taken after12 hour of reperfusion.The levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)in the serum and the superoxide dismutase(SOD)activity and malondialdehyde(MDA)in the liver tissues were measured by colorimetric method;The levels of GRP78,CHOP,and PTGS2 mRNA in liver tissues were measured by RT-qPCR;The stories of GRP78,CHOP,p-eIF2α,and COX-2 proteins expression in liver tissues were detected by Western Blot;The differential terms of GRP78,CHOP and COX-2proteins in liver tissues were detected by immunofluorescence;The morphological changes of liver tissues were observed by H&E staining.The apoptosis of liver tissues was observed by TUNEL staining.Results:1.Liver function: compared with the Sham group,the serum ALT and AST levels of mice in the HIR group were significantly increased(P<0.05).Compared with the HIR group,mice’s serum ALT and AST levels significantly decreased after pretreatment with Sal or NS398(P<0.05).2.Liver histopathology: H&E staining showed that the hepatic lobules of the Sham group were intact,the central vein and the portal area were usual,and the liver cells were intact and neatly arranged in a cord-like pattern.In the HIR group,there was an absence of a central vein,mass death of liver cells,congestion in hepatic sinuses,and infiltration of many inflammatory cells.Compared with the HIR group,the degree of hepatic sinusoidal stasis,inflammatory cell infiltration was reduced and the number of hepatic cell death was decreased after pretreatment with Sal or NS398.The Suzuki score was used to evaluate the liver tissue damage,and the results suggested that the score of the HIR group was significantly higher than the Sham group(P<0.05).Compared with the HIR group,the scores decreased after pretreatment with Sal or NS398(P<0.05).3.TUNEL staining: Compared with the Sham group,many apoptotic cells were observed in the HIR group(P<0.05).The number of apoptotic cells in Sal or NS398 pretreated groups was significantly lower than in the HIR group(P<0.05).4.Oxidative stress level: Compared with the Sham group,SOD activity in liver tissues of mice in the HIR group was decreased(P<0.05),and MDA content was increased(P<0.05).Compared with the HIR group,SOD activity was increased(P<0.05),and MDA content was decreased(P<0.05)after pretreatment with Sal or NS398.5.Expression of endoplasmic reticulum stress indicators: RT-qPCR results showed that the mRNA levels of GRP78 and CHOP in the HIR group were significantly higher than in the Sham group(P<0.05).After pretreatment with Sal or NS398,the above genes showed a downward trend compared with the HIR group(P<0.05).Western Blot results showed that compared with the Sham group,the protein expressions of p-eIF2α、GRP78,and CHOP were increased in the HIR group compared to the Sham group(P<0.05).Compared with the HIR group,the protein expression level of p-eIF2α was increased after pretreatment with Sal(P<0.05),while the protein expressions of GRP78 and CHOP were decreased(P<0.05).After pretreatment with NS398,the protein expressions of GRP78 and CHOP were decreased compared with the HIR group(P<0.05).The immunofluorescence results in paraffin sections of liver tissues indicated that compared with the Sham group,the fluorescence intensities of GRP78 and CHOP proteins were increased in the HIR group(P<0.05).After pretreatment with Sal or NS398,the fluorescence intensities of the above proteins were decreased compared with the HIR group(P<0.05).6.COX-2 expression: RT-qPCR results showed that compared with the Sham group,the mRNA level of PTGS2 in the HIR group was significantly increased(P<0.05).After pretreatment with Sal or NS398,the mRNA level of PTGS2 was significantly decreased compared with the HIR group(P<0.05).Western Blot results showed that the expression of COX-2 protein was increased in the HIR group compared with the Sham group(P<0.05).after pretreatment with Sal or NS398,the protein expression of COX-2 was decreased compared with the HIR group(P<0.05).Immunofluorescence results in paraffin sections of liver tissues suggested that the fluorescence intensity of COX-2protein in the HIR group was higher than that in the Sham group(P<0.05).After pretreatment with Sal or NS398,the fluorescence intensity of COX-2 protein was decreased compared with the HIR group(P<0.05).Conclusion:1.Inhibition of COX-2 attenuates oxidative stress and apoptosis to protect against hepatic ischemia-reperfusion injury in mice.2.Inhibition of ERS attenuates oxidative stress and apoptosis to protect against hepatic ischemia-reperfusion injury in mice.3.COX-2 interacts with ERS to mediate liver injury during hepatic ischemiareperfusion in mice.