Atractylenolide III Attenuates Apoptosis in H9c2 Cells by Inhibiting Endoplasmic Reticulum Stress through the GRP78/PERK/CHOP Signaling Pathway.
Atractylenolide III Attenuates Apoptosis in H9c2 Cells by Inhibiting Endoplasmic Reticulum Stress through the GRP78/PERK/CHOP Signaling Pathway.
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DOI：
10.1155/2022/1149231
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发表时间：
2022
影响因子：
--
通讯作者：
Huang, Jin-Ling
中科院分区：
文献类型：
--
作者：
Zuo, Meng-Yu;Tang, Tong-Juan;Wang, Xiang;Gu, Jin-Fan;Wang, Liang;Chen, Jian;Yao, Juan;Li, Xiang-Yang;Zhou, Peng;Huang, Jin-Ling
本研究旨在探讨白术内酯III(ATL-III)对内质网应激(ERS)损伤、衣霉素(TM)诱导的H9c2心肌细胞凋亡及GRP78/PERK/CHOP信号通路的影响。用分子对接的方法预测ATL-Ⅲ与ERS中的关键蛋白GRP78、PERK、IREα和ATF6的结合亲和力。然后，通过体外实验对分子对接结果进行验证。用TM建立H9c2细胞ERS损伤模型。用四甲基偶氮唑盐比色法检测细胞存活率，Hoechst/PI双染法和流式细胞仪检测细胞凋亡率。免疫印迹法检测GRP78、PERK、eIF2、α、ATF4、CHOP、BAX、Bcl2、Caspase3的蛋白表达水平。逆转录-定量聚合酶链式反应检测GRP78、CHOP、PERK、eIF2、α、ATF4mRNA水平。此外，利用GRP78抑制剂(4-苯基丁酸，4-PBA)和PERK抑制剂(GSK2656157)进一步研究了其作用机理。结果表明，ATL-III与GRP78有较好的结合亲和力，其中与PERK的结合亲和力最好。与模型组比较，ATL-III能提高H9c2细胞的存活率，降低凋亡率，下调Bax和Caspase-3的表达，上调Bcl2的表达。此外，ATL-III还下调GRP78、CHOP、PERK、eIF2、α和ATF4的蛋白和基因表达水平，与4-PBA的抑制作用一致。ATL-Ⅲ还降低了PERK、eIF2α、ATF4、CHOP、BAX和Caspase-3的表达水平，而上调了Bcl2的表达，这与GSK2656157一致。综上所述，ATL-III可通过调节GRP78/PERK/CHOP信号通路，抑制TM诱导的ERS损伤和H9c2心肌细胞的凋亡，具有心肌保护作用。
The objective of this study was to determine the effect of atractylenolide III (ATL-III) on endoplasmic reticulum stress (ERS) injury, H9c2 cardiomyocyte apoptosis induced by tunicamycin (TM), and the GRP78/PERK/CHOP signaling pathway. Molecular docking was applied to predict the binding affinity of ATL-III to the key proteins GRP78, PERK, IREα, and ATF6 in ERS. Then, in vitro experiments were used to verify the molecular docking results. ERS injury model of H9c2 cells was established by TM. Cell viability was detected by MTT assay, and apoptosis was detected by Hoechst/PI double staining and flow cytometry. Protein expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, and Caspase-3 were detected by Western blot. And mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4 were detected by RT-qPCR. Moreover, the mechanism was further studied by using GRP78 inhibitor (4-phenylbutyric acid, 4-PBA), and PERK inhibitor (GSK2656157). The results showed that ATL-III had a good binding affinity with GRP78, and the best binding affinity was with PERK. ATL-III increased the viability of H9c2 cells, decreased the apoptosis rate, downregulated Bax and Caspase-3, and increased Bcl-2 compared with the model group. Moreover, ATL-III downregulated the protein and mRNA levels of GRP78, CHOP, PERK, eIF2α, and ATF4, consistent with the inhibition of 4-PBA. ATL-III also decreased the expression levels of PERK, eIF2α, ATF4, CHOP, Bax, and Caspase-3, while increasing the expression of Bcl-2, which is consistent with GSK2656157. Taken together, ATL-III could inhibit TM-induced ERS injury and H9c2 cardiomyocyte apoptosis by regulating the GRP78/PERK/CHOP signaling pathway and has myocardial protection.
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