二脒那秦激活ACE2对非酒精性脂肪肝病大鼠肝线粒体影响研究

The Research on the Effects of ACE2 Activated by Diminazene Aceturate on Mitochondria in the Liver of Rats with Non-alcoholic Fatty Liver Disease

旨在通过建立高脂饮食诱导的非酒精性脂肪肝病(non-alcoholic fatty liver disease,NAFLD)模型,研究二脒那秦(DIZE)激活血管紧张素转换酶2(ACE2)对线粒体结构和功能的影响。试验选取26只雄性SD大鼠,随机选取9只大鼠饲喂普通饲料作为对照组(CON组),其余大鼠饲喂高脂饲料进行造模,4周后,随机各取1只进行处理,经病理组织学检查确认成功建立NAFLD模型。造模成功的大鼠随机分为模型组(MOD组)和二脒那秦组(DIZE组)(每组8只),MOD组继续饲喂高脂饲料同时灌胃3 mL·d^(-1)生理盐水,DIZE组饲喂高脂饲料并灌胃15 mg·(kg·d)^(-1)DIZE,CON组继续饲喂普通饲料并灌胃等量生理盐水。试验至第10周,采集大鼠血清和肝组织样品进行试验:1)检测血清中TG、TC含量;2)ELISA法测定肝组织中AngⅡ和Ang1-7含量;3)Western blot法检测ACE2,线粒体融合蛋白(Mfn1、OPA1),分裂蛋白(Fis1、Drp1)及解偶联蛋白1(UCP1)的蛋白表达水平;4)RT-qPCR法检测线粒体生物发生和线粒体呼吸链复合物相关基因的mRNA表达水平。结果表明:1)高脂饲料饲喂4周后大鼠HE染色肝脏出现明显的脂肪沉积,成功建立NAFLD模型;2)与CON组相比,MOD组大鼠血清中的TG、TC含量显著升高(P<0.05),DIZE组则较MOD组显著降低(P<0.05);3)与CON组相比,MOD组ACE2蛋白表达水平极显著降低(P<0.01),AngⅡ/Ang1-7比值显著升高(P<0.05),DIZE组则较MOD组ACE2蛋白表达水平极显著升高,AngⅡ/Ang1-7比值极显著降低(P<0.01);4)与CON组相比,MOD组线粒体融合、分裂及UCP1蛋白表达水平极显著或显著降低(P<0.01或P<0.05),DIZE组则较MOD组极显著升高(P<0.01);5)与CON组相比,MOD组线粒体生物发生和线粒体呼吸链复合物Ⅰ和Ⅲ基因mRNA表达水平极显著下调(P<0.01),DIZE组则较MOD组极显著或显著上调(P<0.01或P<0.05)。综上表明,DIZE能够激活ACE2继而缓解由高脂饮食引起的对线粒体生物发生、融合、分裂以及呼吸链相关指标的抑制作用,从而达到治疗NAFLD的效果。本研究为DIZE治疗NAFLD提供新思路,DIZE作为老药新用具有潜在的研究价值。

The article aims to study the effects of diminazene aceturate(DIZE)activation of Angiotensin-converting enzyme 2(ACE2)on mitochondrial structure and function by establishing a high-fat diet-induced non-alcoholic fatty liver disease(NAFLD)model.Twenty-six male SD rats were selected,nine rats were fed with basal feed as a contral group,the remaining rats were fed with high-fat feed for modeling.After 4 weeks,the NAFLD model was successfully established by the confirmation of pathohistological examination.Then,the rats with successful modeling were randomly divided into Model group and DIZE group(eight rats per group),Model group rats were continued to be fed hight-fat feed and treated with 3 mL saline by intragastric administration,DIZE group rats were treated with 15 mg·(kg·d)^(-1)DIZE as gavage.The contral group continued to be fed with common feed,and the same amount of normal saline was gavage administered.In the tenth week,serum and liver samples were collected from rats for the following tests:1)The kit was used to detect TG and TC content in serum;2)The content of AngII and Ang1-7 in liver tissues were determined by ELISA;3)Western blotting method was used to test the protein expression of ACE2,mitochondrial fusion protein(Mfn1,OPA1),fission protein(Fis1,Drp1)and uncoupling protein 1(UCP1);4)RT-qPCR to test mRNA expression of mitochondrial biogenesis and mitochondrial respiratory chain complex-related genes.Results:1)The fat deposition was found in HE staining after 4 weeks of high-fat feeding,obviously,which indicates that the NAFLD modeling of rats was successfully established;2)Compared with the CON group,the serum levels of TG and TC in the MOD group rats were significantly higher(P<0.05),while the DIZE group was significantly lower(P<0.05)compared with the MOD group;3)Compared with CON group,ACE2 protein expression level in MOD group was extremely significant decreased(P<0.01),and the ratio of AngⅡ/Ang1-7 was significantly increased(P<0.05),however,compared with model group,the results were opposite in DIZE group(P<0.01);4)The expression levels of fusion,fission and UCP1 protein were significantly or extremely significant reduced in model group compared to the control group(P<0.01 or P<0.05),DIZE could upregulate the expression levels of fusion,fission and UCP1 protein extremely(P<0.01);5)Compared with the CON group,the expression levels of mRNA in mitochondrial biogenesis and mitochondrial respiratory chain complex I and III genes were extremely significant downregulated in the MOD group(P<0.01),nevertheless,compared with model group,DIZE group shows the opposite results(P<0.01 or P<0.05).These results indicated that DIZE can activate ACE2 and then alleviate the inhibition of mitochondrial biogenesis,fusion,fission and respiratory chain associated with high-fat diet to treat NAFLD.The results demonstrated that DIZE could activate ACE2,thereby alleviating the inhibitory effects on mitochondrial biogenesis,fusion,fission,and respiratory chain-related parameters induced by a high-fat diet,ultimately achieving therapeutic effects for NAFLD.This study elucidates a new idea of DIZE for the treatment of non-alcoholic fatty liver disease,which manifests DIZE has potential research value as a new use of an old drug.