摘要
目的:观察丹参酮ⅡA促进氧糖剥夺/再复(OGD/R)损伤后H9C2心肌细胞再生作用机制。方法:H9C2细胞分为空白对照组、模型对照组(OGD/R)和丹参酮ⅡA 10、20、40μmol/L组;CCK-8法筛选丹参酮ⅡA对OGD/R损伤H9C2心肌细胞的保护浓度;试剂盒检测丹参酮ⅡA对乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)、肌酸激酶(CK)的影响;免疫荧光观察H9C2细胞EdU的增殖情况;划痕试验检测H9C2细胞的伤口愈合能力;转录组高通量测序探索丹参酮ⅡA对OGD/R损伤后H9C2心肌细胞再生机制;分子对接检测丹参酮ⅡA和差异蛋白的结合情况;Real-time PCR法验证差异基因Ccnd1/Plk1/Ccne2的mRNA的相对表达。结果:与OGD/R组比较,丹参酮ⅡA 10、20、40μmol/L组可以减轻OGD/R对H9C2心肌细胞带来的损伤,降低LDH、CK、CK-MB活力(P<0.01),促进心肌细胞再生和伤口愈合(P<0.05或P<0.01)。转录组测序筛选出差异基因623个;GO和KEGG富集分析结果显示,丹参酮ⅡA回调差异基因主要影响蛋白质折叠(protein folding)、核糖核蛋白复合体(ribonucleoprotein complex)、未折叠蛋白质结合(unfolded protein binding)等生物进程和内质网中的蛋白质加工(protein processing in endoplasmic reticulum)、FoxO信号通路(foxO signaling pathway)、细胞周期(cell cycle)等信号通路;分子对接结果显示,丹参酮ⅡA与核心差异蛋白的结合能均<6 kcal/mol;核心差异基因Ccnd1/Plk1/Ccne2 mRNA的相对表达与转录组测序结果一致。结论:丹参酮ⅡA可以介导CCND1/PLK1/CCNE2调节细胞周期,促进OGD/R损伤后H9C2心肌细胞再生。
Objective:To observe the mechanism by which TanshinoneⅡA(TanⅡA)promotes the regeneration of H9C2 cardiomyocytes after oxygen glucose deprivation/reoxygenation(OGD/R)injury.Methods:The experiment was divided into a normal control(NC)group,an OGD/R group,and TanⅡA groups at concentrations of 10,20,and 40μmol/L.The CCK-8 assay was used to screen the protective concentrations of TanⅡA on OGD/R-injured H9C2 cardiomyocytes.Kits were used to detect the effects of TanⅡA on lactate dehydrogenase(LDH),creatine kinase MB(CK-MB),and creatine kinase(CK).Immunofluorescence was used to observe EdU proliferation in H9C2 cells.The wound-healing assay was used to assess the wound-healing ability of H9C2 cells.Transcriptome high-throughput sequencing was performed to explore the regenerative mechanisms of TanⅡA on H9C2 cardiomyocytes after OGD/R injury.Molecular docking was employed to analyze the binding between TanⅡA and differentially expressed proteins.Real-time polymerase chain reaction(real-time PCR)was used to validate the relative mRNA expression of differentially expressed genes Ccnd1/Plk1/Ccne2.Results:Compared with the OGD/R group,TanⅡA groups alleviated the damage caused by OGD/R to H9C2 cardiomyocytes,reduced LDH,CK,and CK-MB levels(P<0.01),and promoted cardiomyocyte regeneration and wound healing(P<0.01,P<0.05).Transcriptome sequencing screened 623 differentially expressed genes.GO and KEGG enrichment analyses reveal that the regulation of differentially expressed genes by TanⅡA mainly affected biological processes such as protein folding,ribonucleoprotein complex,and unfolded protein binding,as well as pathways including protein processing in the endoplasmic reticulum,the FoxO signaling pathway,and the cell cycle.Molecular docking results show that the binding energies between TanⅡA and core differentially expressed proteins were all less than 6 kcal/mol.The relative mRNA expressions of Ccnd1/Plk1/Ccne2 were consistent with the transcriptome sequencing results.Conclusion:TanⅡA can mediate CCND1/PLK1/CCNE2 to regulate the cell cycle,thereby promoting the regeneration of H9C2 cardiomyocytes after OGD/R injury.
作者
曹策
辛高杰
刘子馨
张会雨
陈原原
郭帆
彭涵
李磊
付建华
刘建勋
CAO Ce;XIN Gaojie;LIU Zixin;ZHANG Huiyu;CHEN Yuanyuan;GUO Fan;PENG Han;LI Lei;FU Jianhua;LIU Jianxun(National Clinical Research Center of Traditional Chinese Medicine for Cardiovascular Diseases,Beijing Key Laboratory of Chinese Materia Pharmacology,Institute of Basic Medical Sciences of Xiyuan Hospital of China Academy of Chinese Medical Sciences,,Beijing 100091)
出处
《中药药理与临床》
北大核心
2025年第10期52-57,共6页
Pharmacology and Clinics of Chinese Materia Medica
基金
国家自然基金重点资助项目(编号:82030124、82174015)
中国中医科学院西苑医院能力提升项目(编号:XYZX0303-03)
中国中医科学院科技创新工程(编号:CI2021A04609)。
