The Effects Of Inhibition Of The IP3R-Ca²⁺ Pathway In APAP-induced Liver Injury And Mitochondrial-Associated Endoplasmic Reticulum Membranes

Objective:To explore the effects of inhibition of the inositol 1,4,5-trisphate receptor（IP3R）-Ca²⁺pathway on acetaminophen（APAP）-induced liver injury in mice and its Mitochondrial-Associated Endoplasmic Reticulum Membranes（MAMs）.Methods:40 mice were randomly divided into control group（n=10）,model（acetaminophen）group（n=10）,IP3R inhibitor（2-aminoethoxydiphenyl borate,2-APB）group（n=10）and the Ca²⁺chelator（1,2-bis（2-aminophenoxy）ethane-N,N,N’N’-tetraacetic acid,BAPTA-AM）group（n=10）.Mice in the APAP group,2-APB group,and BAPTA-AM group were given a single intraperitoneal injection of APAP（300mg/kg）.Half an hour before APAP injection,mice in the 2-APB group were injected intraperitoneally with2-APB（20 mg/kg）and mice in the BAPTA-AM group were injected intraperitoneally with BAPTA-AM（2.5 mg/kg）.The mice in each group were executed 24 hours after intraperitoneal injection of APAP.Serum alanine aminotransferase（ALT）and aspartate aminotransferase（AST）were measured by chemical method.The pathological change of liver was observed by HE staining.The ultrastructural changes of mitochondria,endoplasmic reticulum and MAMs in liver cells were observed by transmission electron microscopy.The changes of Ca²⁺in liver tissue homogenate were measured by Calcium Assay Kit.The expression of MFN1,MFN2,IP3R1,GRP75,VDAC1 was determined by Western blot.Results:Compared with the control group,the serum ALT and AST in the model group increased（P<0.01）,Disorganized hepatocyte arrangement,hepatocyte degeneration and lobular central necrosis were observed.Breakage and disappearance of mitochondrial cristae,swelling and fracture of the endoplasmic reticulum and increase of the number of MAMs were also observed by transmission electron microscopy.The Ca²⁺level in liver tissue homogenate increased（P<0.01）.MFN1 and MFN2 protein expression decreased（P<0.01）,IP3R1、GRP75 and VDAC1 protein expression increased（P<0.01）.Compared with the model group,the serum ALT and AST in2-APB group and BAPTA-AM group decreased（P<0.01）,the liver pathological changes were significantly improved,MAMs were decreased,the content of Ca²⁺in liver homogenate decreased（P<0.01）,MFN1 and MFN2 protein expression increased（P<0.01）,and IP3R1、GRP75 and VDAC1 protein expression decreased（P<0.01）.Conclusion:Inhibition of the IP3R-Ca²⁺pathway protects against APAP-induced liver injury in mice.Its mechanism is related to reducing hepatic Ca²⁺levels,reducing the number of MAMs,and regulating the expression of MAMs-related proteins.