基于PI3K/Akt/Beclin-1信号通路探析积雪草苷对OGD/R诱导损伤H9C2心肌细胞的影响  

Effect of asiaticoside on OGD/R induced injury of H9C2 cardiomyocytes based on PI3K/Akt/Beclin-1 signaling pathway

作　　者：曹策 李玲美 韩笑[2] 王奥奥 王紫艳 李磊[2] 刘建勋 CAO Ce;LI Ling-mei;HAN Xiao;WANG Ao-ao;WANG Zi-yan;LI Lei;LIU Jian-xun(Institute of Chinese Medicine Sciences,Guangdong Pharmaceutical University,Guangzhou 510006,China;National Clinical Research Center of Traditional Chinese Medicine for Cardiovascular Diseases,China Academy of Chinese Medical Sciences,Beijing Key Laboratory of Chinese Materia Pharmacology,Institute of Basic Medical Sciences of Xiyuan Hospital,Beijing 100091,China)

机构地区：[1]广东药科大学中医药研究院,广东广州510006 [2]中国中医科学院西苑医院基础医学研究所,中药药理北京市重点实验室,国家中医心血管疾病临床医学研究中心,北京100091

出　　处：《药学学报》2023年第5期1149-1155,共7页Acta Pharmaceutica Sinica

基　　金：国家自然基金重点资助项目(82030124,82174015);中国中医科学院科技创新工程(CI2021A04609);国家中医药管理局中医药传承与创新“百千万”人才工程(岐黄工程)岐黄学者。

摘　　要：观察积雪草苷(asiaticoside,Ass)对氧糖剥夺/再复(oxygen and glucose deprivation/reperfusion,OGD/R)损伤的H9C2心肌细胞的增殖、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/Bcl-2同源结构域蛋白(Beclin-1)信号通路的干预作用,探讨其对H9C2心肌细胞的影响。选用H9C2心肌细胞作为研究对象,采用细胞增殖与活性检测试剂盒(CCK-8)法检测H9C2细胞活性。将H9C2细胞分成对照组、OGD/R组、Ass低浓度组(10μmol·L^(-1))、Ass高浓度组(80μmol·L^(-1))和Ass高浓度+氯喹组(80μmol·L^(-1)+50μmol·L^(-1))。对照组于正常条件下培养,其余各组在处理后进行氧糖剥夺4 h/再复2 h。酶联免疫吸附法检测细胞上清液中天门冬氨酸氨基转移酶(aspartate transaminase,AST)、乳酸脱氢酶(lactate dehydrogenase,LDH)、肌酸激酶(creatine kinase,CK)的含量;免疫荧光观察药物对细胞数量的影响;自噬染色检测试剂盒观察细胞自噬;分子对接技术确定Ass分子靶点;蛋白质印迹法检测PI3K、Akt、选择性自噬接头蛋白(P62)、Beclin-1的表达水平。与OGD/R组相比,Ass各组在10~80μmol·L^(-1)具有保护作用,AST、LDH和CK的含量均降低,PI3K、Akt、P62和Beclin-1蛋白表达水平降低。与Ass组相比,Ass高浓度+氯喹组的AST、LDH和CK的含量均降低,PI3K、Akt、Beclin-1和P62蛋白表达水平均降低。免疫荧光显示抑制剂组、各给药组与模型组相比均具有不同程度的保护作用。综上所述,积雪草苷能够降低OGD/R诱导的H9C2心肌细胞损伤,降低AST、LDH、CK含量和P62蛋白水平,减少细胞自噬,这可能与其抑制PI3K/Akt/Beclin-1信号通路激活密切相关。In order to investigate the effects of asiaticoside(Ass)on H9C2 cardiomyocytes,the present study examined the potential intervention of Ass on the proliferation and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/Bcl-2 homology domain protein(Beclin-1)signaling pathway in H9C2 cardiomyocytes following oxygen and glucose deprivation/reperfusion(OGD/R)injury.H9C2 cardiomyocytes were selected as the research objects,and the activity of H9C2 was detected by cell counting kit-8(CCK-8).H9C2 cells were divided into control group,OGD/R group,Ass low concentration group(10μmol·L-1),Ass high concentration group(80μmol·L-1)and Ass high concentration+chloroquine group(80μmol·L-1+50μmol·L-1).The control group was cultured under normal conditions,and the other groups were treated with oxygen and glucose deprivation for 4 h and reperfusion for 2 h.The activity and content of aspartic aminotransferase(AST),lactate dehydrogenase(LDH)and creatine kinase(CK)in the supernatant of H9C2 cardiomyocytes were detected by enzyme-linked immunosorbent assay.Autophagy staining assay kit with monodansylcadaverine(MDC)method to observe cellular autophagy;molecular docking technique to identify the molecular targets of Ass.Immunofluorescence was used to observe the effect of the drug on cell number.The expression levels of PI3K,Akt,selective autophagy adaptor protein(P62)and Beclin-1 were detected by Western blot.Compared with OGD/R group,Ass group had a protective effect from 10−80μmol·L-1,and the activities and contents of AST,LDH and CK were decreased.The protein expression levels of PI3K,Akt,P62 and Beclin-1 were decreased.Compared with the administration group,the activities and contents of AST,LDH and CK in Ass high-concentration+chloroquine group were significantly decreased,and the protein expression levels of PI3K,Akt,Beclin-1 and P62 were significantly decreased.Immunofluorescence showed that the inhibitor group and each administration group had different degrees of protective effect compared with the model group

关 键 词：积雪草苷 心肌细胞 自噬 PI3K/Akt/Beclin-1 氧糖剥夺/再复 

分 类 号：R966[医药卫生—药理学]