摘要
该文探讨人参皂苷Rg_(1)通过微小RNA155(miR-155)/神经源性基因Notch同源蛋白1(neurogenic gene Notch homologous protein 1,Notch1)/发状分裂相关增强子1(hairy and enhancer of split 1,Hes1)通路调控细胞自噬减轻HL-1心肌细胞缺氧复氧(hypoxia/reoxygenation,H/R)损伤的具体机制。构建H/R损伤HL-1细胞模型,使用人参皂苷Rg_(1)和(或)Notch1抑制剂(DAPT)、miR-155 mimics处理细胞。cell counting kit(CCK)-8检测H/R损伤HL-1细胞的相对活力;乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒检测细胞培养基上清液中LDH的含量;透射电镜观察细胞自噬小体;MDC法检测细胞自噬水平;荧光定量聚合酶链式反应检测miR-155、Notch1、Hes1、微管相关蛋白1轻链3(microtubule-associated protein1 light chain 3,LC3)的mRNA水平;蛋白免疫印迹法检测Notch1、Hes1、LC3Ⅰ、LC3Ⅱ的蛋白表达水平。结果显示,H/R损伤后HL-1细胞活性降低、LDH漏出量增加,细胞内自噬小体增多,LC3的mRNA水平和LC3Ⅱ/LC3Ⅰ比值升高;人参皂苷Rg_(1)能提高细胞活性,减少LDH漏出量,减少细胞内自噬小体数量,降低细胞内LC3的mRNA水平,降低LC3Ⅱ/LC3Ⅰ比值,通过抑制细胞自噬发挥心肌细胞保护作用,Notch1抑制剂或miR-155过表达则抑制人参皂苷Rg_(1)的作用,促进细胞自噬,加重HL-1细胞H/R损伤;人参皂苷Rg_(1)抑制H/R损伤造成的Notch1、Hes1 mRNA水平和蛋白表达降低、miR-155 mRNA水平升高,Notch1抑制剂或miR-155过表达则呈现相反作用。综上所述,人参皂苷Rg_(1)通过miR-155/Notch1/Hes1通路调控自噬减轻HL-1心肌细胞H/R损伤。
This article explored the specific mechanism by which ginsenoside Rg_(1) regulates cellular autophagy to attenuate hypoxia/reoxygenation(H/R)injury in HL-1 cardiomyocytes through the microRNA155(miR-155)/neurogenic gene Notch homologous protein 1(Notch1)/hairy and enhancer of split 1(Hes1)pathway.An HL-1 cell model with H/R injury was constructed,and ginsenoside Rg_(1) and/or Notch1 inhibitor DAPT and miR-155 mimics were used to treat cells.Cell counting kit(CCK)-8 was used to detect the relative viability of HL-1 cells with H/R injury.The lactate dehydrogenase(LDH)content in cell culture medium supernatant was detected by using an LDH assay kit,and autophagosome in cells was observed by transmission electron microscopy. The level of autophagy in cells was detected through the mono-dansyl-cadaverine (MDC)detection method. Fluorescence quantitative polymerase chain reaction was used to detect the mRNA levels of miR-155,Notch1, Hes1, and microtubule-associated protein1 light chain 3 (LC3), and Western blotting was used to detect the proteinexpression levels of Notch1, Hes1, LC3Ⅰ, and LC3Ⅱ. The results show that after H/R injury, the activity of HL -1 cellsdecreases, and LDH leakage increases. Besides, the number of intracellular autophagosomes increases, and the mRNA levelof LC3 and the LC3Ⅱ/LC3Ⅰ ratio are elevated. In addition, ginsenoside Rg 1 can increase cell activity, decrease LDH leakageand the number of intracellular autophagosomes, and reduce the mRNA level of LC3 and the LC3Ⅱ/LC3Ⅰ ratio. Therefore, itplays a cardioprotective role by inhibiting autophagy, and Notch1 inhibitor or miR-155 overexpression can inhibit the effectof ginsenoside Rg_(1), promote autophagy, and aggravate H/R injury in HL-1 cells. Ginsenoside Rg_(1) can inhibit the reduction ofNotch1 and Hes1 mRNA levels and protein expressions and the increase in miR-155 mRNA levels caused by H/R injury,while Notch1 inhibitors or miR-155 overexpression show the opposite effect. In summary, ginsenoside Rg_(1) can regulateautophagy through the miR-155/Notch1/Hes1 pathway to alleviate H/R injury in HL-1 cardiomyocytes.
作者
张会雨
辛高杰
陈原原
曹策
崔晓珊
高佳明
郭浩
付建华
ZHANG Hui-yu;XIN Gao-jie;CHEN Yuan-yuan;CAO Ce;CUI Xiao-shan;GAO Jia-ming;GUO Hao;FU Jianhua(School of Traditional Chinese Medicine,Binzhou Medical College,Yantai 264003,China;Institute of Basic Medicine,Xiyuan Hospital,China Academy of Traditional Chinese Medicine,Beijing 100091,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第23期6450-6458,共9页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82074060,82174219)
中国中医科学院科技创新工程项目(CI2021A00912,CI2021A04602)
国家中医药管理局高水平中医药重点学科建设项目(zyyzdxk-2023231)。
