摘要
目的探究缺氧是否通过肌醇依赖酶1α(inositol requires enzymes1α,IRE1α)介导的内质网应激(endoplasmic reticulum stress,ERS)激活应激活化蛋白激酶(JNK)通路参与调控小鼠肺动脉平滑肌细胞的增殖和凋亡。方法体外培养小鼠肺动脉平滑肌细胞系(MPASMCs),构建缺氧MPASMCs模型,分别检测缺氧诱导因子1α(HIF-1α)、ERS关键基因葡萄糖调节蛋白78(GRP78)以及JNK通路基因的表达;用siRNA转染敲低IRE1α表达,用JNK通路特异性抑制剂SP600125抑制JNK通路,XBP-1s质粒转染增加XBP-1s表达,CCK8实验以及增殖细胞核抗原(PCNA)检测观察细胞增殖水平,流式细胞分型以及凋亡相关基因B淋巴细胞瘤-2基因(B-cell lymphoma-2,BCL-2)、Bcl-2相关X蛋白(BAX)检测观察细胞凋亡水平。结果缺氧培养后的MPASMCs中HIF-1α表达明显上调;在缺氧后GRP78、磷酸化IRE1α(P-IRE1α)以及磷酸化JNK(P-JNK)表达量均上升,提示IRE1α介导的ERS以及JNK通路被激活,Si-IRE1a抑制IRE1α表达后,P-JNK表达减低,说明JNK通路被抑制;缺氧组PCNA蛋白水平上调,结合CCK8实验提示细胞增殖水平上调,缺氧组促凋亡蛋白BAX表达下调,凋亡抑制基因BCL-2蛋白水平下调,结合流式细胞分型结果提示凋亡水平减低,SiIRE1a抑制IRE1α表达后,上述变化被延缓;用SP600125处理细胞后,也可部分延缓缺氧所引起的促增殖抗凋亡改变;常氧状态下过表达XBP-1s激活了JNK通路,同时出现了类似缺氧的改变。结论缺氧激活IRE1α介导的内质网应激,进而通过JNK通路促进小鼠肺动脉平滑肌细胞增殖并抑制其凋亡。
Objective To investigate whether hypoxia induces endoplasmic reticulum stress(ERS)through inositol-dependent enzyme 1α(IRE1α)and activates JNK pathway to participate in the proliferation and apoptosis of pulmonary artery smooth muscle cells in mice.Methods Mouse pulmonary artery smooth muscle cell line(MPASMCs)was cultured in vitro to establish the hypoxic MPASMCs model.The expression of hypoxia-inducible factor-1α(HIF-1α),glucose-regulated protein 78(GRP78)and JNK pathway genes were detected.The expression of IRE1αwas knocked down by siRNA transfection,JNK pathway specifi c inhibitor 1,9-pyrazoxanolone and pyrazoxanolone(SP600125)was used to inhibit JNK pathway,and XBP-1s plasmid was transfected to increase the expression of XBP-1s.CCK8 assay and proliferating cell nuclear antigen(PCNA)protein detection were used to observe the cell proliferation.Cell apoptosis was detected by fl ow cytometry and the expression of B-cell lymphoma-2(BCL-2)and BCL-2 associated X protein(BAX)protein.Results HIF-1αexpression was signifi cantly up-regulated in MPASMCs cultured under hypoxia.The expression of GRP78,phosphorylated IRE1α(P-IRE1α)and phosphorylated JNK(P-JNK)increased after hypoxia,indicating that ERS and JNK pathways mediated by IRE1αwere activated.When IRE1αexpression was inhibited by si-IRE1a,the expression of P-JNK decreased,indicating that JNK pathway was inhibited.The expression of PCNA protein was up-regulated in the hypoxia group,and CCK8 assay indicated that the cell proliferation was up-regulated.The expression of BAX and BCL-2 protein were down-regulated in the hypoxia group,and the level of apoptosis was down-regulated.The above changes were delayed after SiIRE1a inhibited the expression of IRE1α.Treatment with SP600125 could also partially delay the pro-proliferation and anti-apoptosis changes induced by hypoxia.Overexpression of XBP-1s under normoxia activated the JNK pathway,accompanied by hypoxia-like changes.Conclusions Hypoxia activates IRE1α-mediated endoplasmic reticulum stress,which promo
作者
史薪炜
钱骏
袁超
史阳阳
王玮
孙凯
Shi XinWei;Qian Jun;Yuan Chao;Shi YangYang;Wang Wei;Sun Kai(Department of Emergency Medicine,The First Affiliated Hospital of Nanjing Medical University,Nanjing 210000,China;Department of Emergency,Nanjing Drum Tower Hospital,Nanjing University Medical School,Nanjing 210000,China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2023年第8期1083-1089,共7页
Chinese Journal of Emergency Medicine
