脂联素抑制β1-肾上腺素受体自身抗体诱导的大鼠H9c2心肌细胞自噬流下降  

Adiponectin inhibits decrease in autophagy of rat H9c2 cardiomyocytes induced by β1-adrenergic receptor autoantibodies

作　　者：孙聪 郭帅 宁娜 侯晓鸿 王昌图 原媛 王晓晖 王丽 SUN Cong;GUO Shuai;NING Na;HOU Xiao-hong;Wang Chang-tu;YUAN Yuan;WANG Xiao-hui;WANG Li(Department of Pathology,School of Basic Medical Sciences,Shanxi Medical University,Tai⁃yuan 030001,China;Laboratory of Morphology,School of Basic Medical Sciences,Shanxi Medical University,Tai⁃yuan 030001,China)

机构地区：[1]山西医科大学基础医学院病理学教研室,山西太原030001 [2]山西医科大学基础医学院形态学实验室,山西太原030001

出　　处：《中国病理生理杂志》2020年第5期796-802,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31871177);山西省高等学校科技创新项目(No.201802059)。

摘　　要：目的:观察脂联素抑制β1-肾上腺素受体(β1-AR)自身抗体(β1-AA)诱导的大鼠心肌细胞自噬流下降的作用及机制。方法:SD大鼠随机分为免疫组和对照(control)组,每组6只;H9c2大鼠心肌细胞分为control组、β1-AA组、β1-AA+脂联素组、脂联素组、β1-AA+脂联素+Compound C组和β1-AA+Compound C组。合成β1-AR细胞外第2环(β1-AR-ECII)抗原肽段,主动免疫SD大鼠并采用亲和层析法纯化大鼠血清中β1-AA;CCK-8法检测H9c2心肌细胞活力;real-time PCR检测心肌细胞LC3B和beclin-1的m RNA水平;Western blot检测心肌细胞LC3-II、P62、AMP活化蛋白激酶(AMPK)和磷酸化AMPK(p-AMPK)的蛋白水平。结果:与control组相比,10μg/L脂联素预处理可显著抑制β1-AA诱导的心肌细胞活力下降(P<0.05);脂联素预处理可显著逆转β1-AA诱导的心肌细胞LC3B和beclin-1 m RNA水平下降、LC3-II蛋白表达水平下降及P62蛋白的累积(P<0.05);脂联素可逆转β1-AA诱导的心肌细胞AMPK磷酸化水平下降(P<0.05)。加入AMPK抑制剂Compound C预处理,脂联素则不能逆转β1-AA诱导的心肌细胞LC3B和beclin-1 m RNA及LC3-II蛋白水平降低(P<0.05),但仍可减少P62蛋白累积(P<0.05)。结论:脂联素抑制β1-AA诱导的H9c2大鼠心肌细胞自噬流降低,其机制部分依赖AMPK途径。AIM:To investigate whether adiponectin inhibits the decrease in autophagy of rat H9c2 cardiomyocytes induced byβ1-adrenergic receptor(β1-AR)autoantibodies(β1-AA),and to explore its mechanism.METHODS:SD rats were actively immunized withβ1-AR extracellular second loop(β1-AR-ECII)antigen peptide.Affinity chromatography was used to purifyβ1-AA in serum of the SD rats.The viability of H9c2 cells was measured by CCK-8 assay.The m RNA levels of LC3B and beclin-1 in the H9c2 cells were detected by real-time PCR.The protein levels of LC3-II,P62,AMP-activated protein kinase(AMPK)and phosphorylated AMPK(p-AMPK)were determined by Western blot.RESULTS:Pretreatment with adiponectin at 10μg/L for 1 h reversed the decreased viability of H9c2 cells induced byβ1-AA.Compared with control group,β1-AA decreased the m RNA expression of LC3B and beclin-1,decreased the protein level of LC3-II,and increased the expression of P62 protein in the H9c2 cells,suggesting thatβ1-AA decreased the autophagic flux in cardiomyocytes.Adiponectin obviously reversedβ1-AA-induced decline in autophagic flux,and upregulated the phosphorylation level of AMPK decreased byβ1-AA.Treatment with AMPK inhibitor Compound C for 30min,we observed that the m RNA expression of LC3B and beclin-1 and the protein level of LC3-II in the H9c2 cells decreased byβ1-AA were not effectively reversed by adiponectin,but the increase in P62 protein expression was still effectively reversed,indicating that adiponectin increased autophagosome production dependent on the AMPK pathway,but increased autophagosome clearance independent on the AMPK pathway.CONCLUSION:Adiponectin inhibits the decreased autophagy of H9c2 cardiomyocytes induced byβ1-AA.

关 键 词：脂联素 β1-肾上腺素受体自身抗体 自噬 AMP活化蛋白激酶 心肌细胞 

分 类 号：R363.2[医药卫生—病理学] R329.25[医药卫生—基础医学]