抑制IP3R-Ca^(2+)途径对对乙酰氨基酚所致肝损伤及其线粒体内质网结构偶联的影响  

The effects of inhibition of the IP3R-Ca^(2+) pathway in APAP-induced liver injury and mitochondrial-associated endoplasmic reticulum membranes

作　　者：徐王婷 宋育林[1] Xu Wangting;Song Yulin(Dept of Gastroenterology,The First Affiliated Hospital of Anhui Medical University,The Key Laboratory of Digestive Disease of Anhui Province,Hefei 230022)

机构地区：[1]安徽医科大学第一附属医院消化科,安徽省消化系统疾病重点实验室,合肥230022

出　　处：《安徽医科大学学报》2023年第7期1077-1081,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽高校自然科学研究项目(编号:KJ2016A337);2016—2018年安徽高校科研平台创新团队建设项目-药物性肝损伤团队(编号:皖教秘科[2015]49号)。

摘　　要：目的探讨抑制肌醇-1,4,5-三磷酸受体(IP3R)-Ca^(2+)途径对对乙酰氨基酚(APAP)所致肝损伤及其线粒体内质网结构偶联(MAMs)的影响。方法40只SPF级雄性C57BL/6小鼠随机分为正常对照组、模型组、IP3R抑制剂(2-APB)组、钙离子螯合剂(BAPTA-AM)组,每组10只。模型组、2-APB组、BAPTA-AM组单次腹腔注射APAP(300 mg/kg)。注射APAP前30 min,2-APB组腹腔注射2-APB(20 mg/kg),BAPTA-AM组腹腔注射BAPTA-AM(2.5 mg/kg)。腹腔注射APAP 24 h后处死各组小鼠。测定血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平;HE染色观察肝脏病理变化;透射电镜观察肝细胞中线粒体、内质网结构及MAMs变化;测定肝组织匀浆中的Ca^(2+)水平;Western blot测定肝组织中线粒体融合蛋白1(MFN1)、线粒体融合蛋白2(MFN2)、1,4,5-三磷酸肌醇受体1(IP3R1)、葡萄糖调节蛋白75(GRP75)蛋白表达水平。结果与正常对照组相比,模型组小鼠血清ALT及AST水平有所升高(P<0.01),肝脏组织病理学可见肝细胞排列紊乱,并出现肝细胞变性、小叶中心性坏死;透射电镜示线粒体嵴断裂、消失,内质网肿胀断裂,MAMs数量增加;肝组织匀浆中Ca^(2+)含量增加(P<0.01);MFN1、MFN2蛋白表达降低(P<0.01),IP3R1及GRP75蛋白表达增加(P<0.01)。与模型组相比,2-APB组和BAPTA-AM组小鼠血清ALT、AST水平下降(P<0.01),肝脏病理变化明显改善,MAMs减少,肝组织匀浆中Ca^(2+)含量减少(P<0.01),MFN1、MFN2的蛋白表达水平上调(P<0.01),IP3R1及GRP75蛋白表达降低(P<0.01)。结论抑制IP3R-Ca^(2+)途径可以保护APAP所致的肝损伤,其机制与降低肝脏Ca^(2+)水平、减少MAMs数量、调节MAMs相关蛋白的表达有关。Objective To explore the effects of inhibition of the inositol 1,4,5-trisphate receptor(IP3R)-Ca^(2+)pathway on acetaminophen(APAP)-induced liver injury in mice and its mitochondrial-associated endoplasmic reticulum membranes(MAMs).Methods 40 SPF-rated male C57BL/6 mice were randomly divided into control group(n=10),model group(n=10),IP3R inhibitor(2-aminoethoxydiphenyl borate,2-APB)group(n=10)and the Ca^(2+)chelator[1,2-bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid,BAPTA-AM]group(n=10).Mice in the model group,2-APB group,and BAPTA-AM group were given a single intraperitoneal injection of APAP(300 mg/kg).Half an hour before APAP injection,mice in the 2-APB group were injected intraperitoneally with 2-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 Ca^(2+)in liver tissue homogenate were measured by calcium assay kit.The protein expression levels of mitofusin 1(MFN1),mitofusin 2(MFN2),inositol 1,4,5-trisphate receptor(IP3R1),glucose regulated protein 75(GRP75)were detected by Western blot.Results Compared with the control group,the serum ALT and AST in the model group increased(P0.01).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 Ca^(2+)level in liver tissue homogenate increased(P<0.01).MFN1 and MFN2 protein expression decreased(P<0.01),IP3R1 and GRP75 protein expression increased(P<0.01).Compared with the

关 键 词：对乙酰氨基酚 肝损伤 2-氨基乙酯二苯基硼酸 1 2-双(2-氨基苯氧基)-乙烷-N N N′N′-四乙酸 肌醇-1 4 5-三磷酸受体 钙离子 线粒体内质网结构偶联 

分 类 号：R575[医药卫生—消化系统] R99[医药卫生—内科学]