Connexin43敲降对血红素诱导的星形胶质细胞凋亡和铁死亡的影响

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摘要目的：探讨星形胶质细胞（astrocyte,ASC）的缝隙连接蛋白Connexin43（Cx43）在调控细胞凋亡和铁死亡中的作用及机制。方法: 采用氧化血红素（hemin）处理ASC建立模拟脑出血后细胞凋亡和铁死亡的细胞模型，通过干扰慢病毒敲降ASC中Cx43表达，观察细胞对hemin诱导的凋亡和铁死亡的敏感性。通过基因集富集分析（gene set enrichment analysis，GSEA）预测Cx43参与调控细胞凋亡和铁死亡的可能机制。通过对Hippo信号通路的关键效应分子Yes相关联蛋白（Yes associated protein,YAP）进行免疫荧光染色以及使用抑制剂干预，对生信分析预测的信号通路进行验证。结果: 与对照组相比，hemin处理引起细胞凋亡和铁死亡比例显著增加（P<0.05）；相比单纯hemin处理组和阴性对照组（Cx43NC），Cx43敲降组（Cx43KD）细胞凋亡比例显著降低（P<0.05），铁死亡比例显著升高（P<0.05）。生信分析结果显示，Cx43与Hippo信号通路、铁离子结合基因集、脂质过氧化通路、缺氧应激等通路显著相关，进一步转录因子调控网络分析发现转录增强相关结构域（transcriptional enhanced associate domain，TEAD）转录因子家族中TEAD3可能参与调控Cx43相关性下游信号转导通路。免疫荧光染色结果显示，Cx43KD组YAP出现核转位，采用抑制剂阻断YAP和TEAD结合后，Cx43敲降引起的细胞凋亡抵抗和铁死亡增敏得到部分逆转。结论: Cx43敲降通过抑制Hippo通路，促进YAP核转位，与TEAD3结合形成转录复合体，调控ASC对hemin诱导的细胞凋亡抵抗和铁死亡增敏。

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Abstract：［Abstract］Objective： To explore the role and mechanism of Connexin43 (Cx43), a gap junctional protein of astrocytes (ASC), in regulating apoptosis and ferroptosis. Methods： ASC was treated with 25 μmol/L hemin to establish a cell model that mimics apoptosis and ferroptosis after intracerebral hemorrhage. Lentivirus was used to knock down the expression of Cx43 in ASC, and the sensitivity of cells to hemininduced apoptosis and ferroptosis was evaluated. Through gene set enrichment analysis (GSEA), the possible mechanism of Cx43 involved in the regulation of apoptosis and ferroptosis was predicted. The signaling pathway predicted by the bioinformatics analysis was verified by immunofluorescence staining of Yes associated protein (YAP) and intervention with the YAP inhibitor Verteporfin (Vp). Results： Compared with the control group, hemin treatment caused a significant increase in the proportion of apoptosis and ferroptosis (P<0.05). The Cx43 knockdown group (Cx43KD) had a significantly lower percentage of apoptosis (P<0.05), and higher percentage of ferroptosis (P<0.05). Bioinformatics analysis showed that Cx43 was significantly related to Hippo signaling pathway, iron binding gene set, lipid peroxidation pathway, hypoxic stress pathways. Further transcription factor regulatory network analysis found that TEAD3 in the TEAD transcription factor family may be involved. Immunofluorescence staining showed that YAP was translocated to the nuclei in the Cx43KD group. Blocking the combination of YAP and TEAD with Vp, the apoptosis resistance and ferroptosis sensitization induced by Cx43 knockdown were partially reversed. Conclusion： Cx43 knockdown exerts ASC with resistance to hemin-induced apoptosis and the sensitivity to hemin-induced ferroptosis by inhibiting the Hippo pathway.

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收稿日期: 2022-05-20

基金资助:

通讯作者: 卢小东（通讯作者），教授，硕士生导师，E-mail： xdglu@ujs.edu.cn；杨勇（通讯作者），博士，主治医师，E-mail： yangyong@gdph.org.cn

作者简介: 国家自然科学基金青年基金资助项目（81901250）；广东省自然科学基金资助项目（2019A1515010104）；广东省人民医院高水平医院建设登峰计划——青苗项目（DFJH201924）

引用本文:

陆聪，桂如林，吕广钊，等.. Connexin43敲降对血红素诱导的星形胶质细胞凋亡和铁死亡的影响[J]. 江苏大学学报:医学版, 2023, 33(01): 29-36. LU Cong1, GUI Rulin1, LYU Guangzhao2, ZHANG Zhijian1, LU Xiaodong1, YANG Yong2. Knocking down Connexin43 in astrocytes increases the resistance of

hemin-induced apoptosis and the sensitivity of ferroptosis

. Journal of Jiangsu University(Medicine Edition), 2023, 33(01): 29-36.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2023/V33/I01/29

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