酸性鞘磷脂酶对非酒精性脂肪肝病的作用及PPARα-PGC-1α通路的影响  

Effect of acid sphingomyelinase on nonalcoholic fatty liver disease via PPARα-PGC-1αpathway

作　　者：方伟进 樊志强 龚慧 刘瑞娇 宋立莹[1,2] 王春江[1,2] 刘世坤[1,2] FANG Wei-jin;FAN Zhi-qiang;GONG Hui;LIU Rui-jiao;SONG Li-ying;WANG Chun-jiang;LIU Shi-kun(Dept of Pharmacy,The Third Xiangya Hospital,Central South University,Changsha 410013,China;Center of Clinical Pharmacology,The Third Xiangya Hospital,Central South University,Changsha 410013,China)

机构地区：[1]中南大学湘雅三医院药学部,湖南长沙410013 [2]中南大学湘雅三医院临床药理中心,湖南长沙410013

出　　处：《中国药理学通报》2023年第1期57-65,共9页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金(No81900344);湖南省卫生计生课题(No20190926)。

摘　　要：目的观察酸性鞘磷脂酶(acid sphingomyelinase,ASMase)在高脂诱导小鼠非酒精性脂肪肝病中的作用及对PPARα-PGC-1α通路的调控作用。方法构建基于C57BL/6背景的ASMase敲除小鼠,与野生型小鼠杂交后,获得封闭群杂合子(ASMase^(+/-))用于制备NAFLD模型。实验分4组,野生型正常对照组(WT+Chow)、野生型高脂组(WT+HFD)、ASMase杂合子正常对照组(ASMase^(+/-)+Chow)、ASMase杂合子高脂组(ASMase^(+/-)+HFD)。对照组给予基础饲料饲养12周,高脂组给予高脂饲料饲养12周。实验结束后,生化法检测血清和肝脏脂质水平及肝功能变化;油红染色、HE染色、Masson染色和天狼星红染色分别观察肝脏中脂肪堆积和形态学变化;采用Amplex TM Red Sphingomyelinase试剂盒检测ASMase活性;Western blot检测ASMase、PPARα及其辅激活因子PGC-1α、下游基因CPT1的蛋白表达。结果WT+HFD组小鼠出现高胆固醇血症和肝功能异常,肝脏甘油三酯(TG)水平较WT+Chow组小鼠明显升高(P<0.05或P<0.01),肝细胞出现脂肪变性、空泡样变和肝纤维化,同时肝脏ASMase蛋白表达和活性升高(P<0.01或P<0.001),但CPT1、PPARα和PGC-1α蛋白表达较WT+Chow组无统计学意义;ASMase^(+/-)+HFD组小鼠肝脏TG含量较WT+HFD组明显降低,空泡样变和肝纤维化程度改善,并上调PPARα及其靶基因PGC-1α和CPT1的蛋白表达。结论ASMase促进肝脂肪变性和肝纤维化,可能与其抑制PPARα-PGC-1α通路有关。Aim To investigate the effects of acid sphingomyelinase(ASMase)on high-fat induced nonalcoholic fatty liver disease in mice and its regulation of PPARα-PGC-1αpathway.Methods ASMase knockout mice based on C57BL/6 background were constructed.Closed group heterozygotes were obtained through hybridized with wild-type mice(ASMase^(+/-)),together with the littermate WT mice were prepared for NAFLD model in this study.The experiment was divided into four groups:WT+Chow:the WT mice were fed with normal diet for 12 weeks;WT+HFD:the WT mice were fed with high-fat diet for 12 weeks;ASMase^(+/-)+Chow:the ASMase^(+/-)mice were fed with normal diet for 12 weeks;ASMase^(+/-)+HFD:the ASMase^(+/-)mice were fed with high fat diet for 12 weeks.Biochemical method was used to detect serum TC,TG and liver TC,TG contents and liver function such as ALT and AST.Oil red staining,HE staining,Masson staining and Sirius red staining were performed to detect liver lipid accumulation,hepatocyte morphology and liver fibrosis.Amplex TM red sphingomyelinase kit was applied to detect ASMase activity.Western blot was performed to detect protein expressions of ASMase,PPARα,PGC-1αand CPT1.Results WT+HFD group displayed hypercholesterolemia and liver dysfunction.Levels of liver triglyceride(TG)were significantly higher than those in WT+Chow group(P<0.05 or P<0.01).Meanwhile,the hepatocytes showed marked steatosis,balloon-like changes,and fibrosis.Protein expression and activity of ASMase in liver increased significantly(P<0.01 or P<0.001),whereas CPT1,PPARαand PGC-1αexpressions were not statistically significant compared with matched control group.Heterozygously ASMase-deficient mice reduced the elevated liver TG induced by HFD,as well as improving balloon-like changes and liver fibrosis.Furthermore,the expressions of PPARα,PGC-1αand CPT1 were up-regulated in ASMase^(+/-)+HFD mice compared with WT+Chow group.Conclusions ASMase promotes hepatic steatosis and fibrosis,which may be related to its inhibition of PPARα-PGC-1αpathway.

关 键 词：非酒精性脂肪肝 酸性鞘磷脂酶 过氧化物酶体 肝纤维化 脂肪变性 信号通路 

分 类 号：R-332[医药卫生] R322.47R344.53R575R575.2