摘要
目的:探究乔松素通过调节缺氧诱导因子1α(HIF-1α)/bcl2/腺病毒E1B相互作用蛋白3(BNIP3)信号通路介导细胞自噬减轻缺氧/复氧诱导的心肌细胞损伤的机制。方法:体外培养大鼠心肌细胞H9c2,建立H/R模型后以0、25、50、100、200、300μg/mL乔松素处理24h,采用MTT法检测各处理组细胞活力,筛选出合适的药物作用浓度。将体外培养的H9c2细胞随机分为对照组、模型组、乔松素低剂量组、乔松素高剂量组、HIF-1α siRNA阴性对照组、乔松素高剂量+HIF-1α siRNA组,除对照组外其余各组建立H/R模型,然后以乔松素、HIF-1α siRNA及其阴性对照分组处理后,采用MTT法、流式细胞实验分别检测各组细胞增殖及凋亡;采用酶联免疫吸附测定法(ELISA)测量各组H9c2细胞炎性因子白细胞介素(IL)-17、IL-1β、IL-18水平;采用试剂盒测量各组H9c2细胞氧化应激因子过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、活性氧(ROS)、丙二醛(MDA)水平;采用单丹磺酰戊二胺(MDC)荧光染色法检测各组H9c2细胞自噬体的形成;采用免疫印迹法检测各组H9c2细胞自噬及HIF-1α/BNIP3通路相关蛋白表达。结果:与对照组相比,模型组细胞活力、自噬体相对含量、细胞CAT及GSH-Px水平、细胞LC3-II/LC3-I及P62、HIF-1α、BNIP3蛋白表达降低(P<0.05),凋亡率、细胞培养基上清中IL-17、IL-1β及IL-18水平、细胞ROS及MDA水平升高(P<0.05)。与模型组相比,乔松素低、高剂量组细胞活力、自噬体相对含量、细胞CAT及GSH-Px水平、细胞LC3-II/LC3-I及P62、HIF-1α、BNIP3蛋白表达均升高(P <0.05),凋亡率、细胞培养基上清中IL-17、IL-1β及IL-18水平、细胞ROS及MDA水平均降低(P<0.05);乔松素高剂量组细胞活力、自噬体相对含量、细胞CAT及GSH-Px水平、细胞LC3-II/LC3-I及P62、HIF-1α、BNIP3蛋白表达相比乔松素低剂量组进一步升高(P<0.05),凋亡率、细胞培养基上清中IL-17、IL-1β及IL-18水平、细胞ROS及MDA水平进一步降低(P<0.05);HIF-1α siRNA阴性对照组细胞各指标无显著差异(P>0.05)。与乔松素高剂量组相比,乔松素高剂量+HIF-1α siRNA组细胞活力、自噬体相对含量、细胞CAT及GSH-Px水平、细胞LC3-II/LC3-I及P62、HIF-1α、BNIP3蛋白表达降低(P<0.05),凋亡率、细胞培养基上清中IL-17、IL-1β及IL-18水平、细胞ROS及MDA水平升高(P<0.05)。结论:乔松素通过上调HIF-1α/BNIP3通路而促进自噬,进而减轻炎症与氧化应激,抑制缺氧/复氧诱导的心肌细胞凋亡,减轻细胞损伤。
Objective:To explore the mechanism of pinocembrin in reducing myocardial cell injury induced by hypoxia/reoxygenation by regulating autophagy mediated by hypoxia-inducible factor 1α(HIF-1α)/Bcl2/adenovirus E1B-interacting protein 3(BNIP3)signaling pathway.Methods:Rat cardiomyocytes H9c2 were cultured in vitro,after establishing the H/R model,the mice were treated with 0,25,50,100,200 and 300μg/mL pinocembrin for 24 hours,the cell viability of each treatment group was detected by MTT method,and the appropriate drug concentration was screened out.The H9c2 cells cultured in vitro were randomly separated into a control group,a model group,a low-dose pinocembrin group,a high-dose pinocembrin group,a HIF-1αsiRNA negative control group,and a high-dose pinocembrin+HIF-1αsiRNA group.Except for the control group,H/R models were established in the other groups,and then treated with pinocembrin,HIF-1αsiRNA and their negative controls.The proliferation and apoptosis of cells in each group were detected by MTT method and flow cytometry respectively;enzyme-linked immunosorbent assay(ELISA)was used to measure the levels of inflammatory factors interleukin(IL)-17,IL-1βand IL-18 in H9c2 cells in each group;kits were used to measure the levels of oxidative stress factors catalase(CAT),glutathione peroxidase(GSH-Px),reactive oxygen species(ROS)and malondialdehyde(MDA)in H9c2 cells in each group;monodansylpentanediamine(MDC)fluorescence staining was used to detect the formation of autophagosomes in H9c2 cells in each group;Western blotting was used to detect the expression of autophagy and HIF-1α/BNIP3 pathway-related proteins in H9c2 cells in each group.Results:Compared with the control group,the cell viability,the relative content of autophagosomes,the levels of CAT and GSH-Px,LC3-II/LC3-I,and the protein expressions of P62,HIF-1αand BNIP3 in the model group decreased(P<0.05),the apoptosis rate,the levels of IL-17,IL-1βand IL-18 in cell culture supernatant,and the levels of cellular ROS and MDA increased(P<0.05).Compared with the model group,the cell viability,the relative content of autophagosomes,the levels of CAT and GSH-Px,LC3-II/LC3-I,and the protein expressions of P62,HIF-1αand BNIP3 in the low-dose and high-dose pinocembrin groups increased(P<0.05),the apoptosis rate,the levels of IL-17,IL-1βand IL-18 in cell culture supernatant,and the levels of cellular ROS and MDA decreased(P<0.05);compared with those in the low-dose pinocembrin group,the cell viability,the relative content of autophagosomes,the levels of CAT and GSH-Px,LC3-II/LC3-I,and the protein expressions of P62,HIF-1αand BNIP3 in the high-dose pinocembrin group further increased(P<0.05),the apoptosis rate,the levels of IL-17,IL-1βand IL-18 in cell culture supernatant,and the levels of cellular ROS and MDA further decreased(P<0.05);there was no obvious difference in each index of cells in HIF-1αsiRNA negative control group(P>0.05).Compared with the high-dose pinocembrin group,the cell viability,the relative content of autophagosomes,the levels of CAT and GSH-Px,LC3-II/LC3-I,and the protein expressions of P62,HIF-1αand BNIP3 in the high-dose pinocembrin+HIF-1αsiRNA group decreased(P<0.05),the apoptosis rate,the levels of IL-17,IL-1βand IL-18 in cell culture supernatant,and the levels of cellular ROS and MDA increased(P<0.05).Conclusion:Pinocembrin promotes autophagy by up-regulating the HIF-1α/BNIP3 pathway,thereby reducing inflammation and oxidative stress,inhibiting hypoxia/reoxygenation-induced cardiomyocyte apoptosis and reducing cell injury.
作者
刘冰
方存明
马小林
LIU Bing;FANG Cunming;MA Xiaolin(Xuancheng People's Hospital,Anhui Xuancheng 242000,China)
出处
《河北医学》
CAS
2022年第11期1761-1768,共8页
Hebei Medicine
基金
安徽省宣城市科学计划项目,(编号:1817)。
作者简介
通讯作者:方存明。
