Zuo Meng-Yu, Tang Tong-Juan, Zhou Peng, Wang Xiang, Ding Rui, Gu Jin-Fan, Chen Jian, Wang Liang, Yao Juan, Li Xiang-Yang, Huang Jin-Ling
School of Chinese Medicine, Anhui University of Chinese Medicine Hefei 230012, China.
Institute of Integrated Chinese and Western Medicine, Anhui Academy of Traditional Chinese Medicine Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2022 Aug;47(16):4436-4445. doi: 10.19540/j.cnki.cjcmm.20220304.401.
This study aims to investigate the effect of atractylenolide Ⅲ(ATL-Ⅲ) on hydrogen peroxide(H_2O_2)-induced endoplasmic reticulum stress and apoptosis of H9 c2 cells via the ROS/GRP78/caspase-12 signaling pathway.The binding activity of ATL-Ⅲ to GRP78 was determined by molecular docking.The result showed that ATL-Ⅲ had a good binding activity to GRP78, and the binding activity of ATL-Ⅲ was stronger than that of its specific inhibitor.The endoplasmic reticulum stress model of H9 c2 was established by H_2O_2(100 μmol·L(-1)) treatment.Five groups were designed: blank control group, model group, and ATL-Ⅲ(15, 30, and 60 μmol·L(-1)) groups.Apoptosis was detected by Hoechst/PI double staining and flow cytometry.The levels of superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) were measured by colorimetry.The levels of reactive oxygen species(ROS) and calcium(Ca(2+)) in cytoplasm were determined by the fluorescence probe DCFH-DA and the calcium fluorescence probe Flou-4, respectively.The protein levels of GRP78, caspase-12, and caspase-3 were determined by Western blot, and the mRNA levels of GRP78 and caspase-12 by RT-qPCR.N-acetyl-L-cysteine(NAC) and 4-phenylbutyric acid(4-PBA) were respectively used to inhibit ROS and GRP78, and then the mechanism of ATL-Ⅲ in protecting the cells from endoplasmic reticulum stress induced by H_2O_2 were deduced.ATL-Ⅲ(15, 30, and 60 μmol·L(-1)) decreased the apoptosis rate and ROS, MDA, and LDH levels(P<0.01), increased the SOD activity(P<0.01), and down-regulated the protein levels of GRP78, caspase-12, and caspase-3 and the mRNA levels of GRP78 and caspase-12(P<0.05).The addition of NAC decreased the apoptosis rate and ROS, MDA, GRP78, caspase-12, and caspase-3 levels(P<0.01), while it elevated the SOD level(P<0.01).The addition of 4-PBA also decreased the apoptosis rate and the levels of GRP78, caspase-12, caspase-3, and Ca~(2+)(P<0.01).The effect of inhibitors were consistent with that of ATL-Ⅲ.In conclusion, ATL-Ⅲ can protect H9 c2 cardiomyocytes by regulating ROS/GRP78/caspase-12 signaling pathway to inhibit H_2O_2-induced endoplasmic reticulum stress and apoptosis.
本研究旨在探讨白术内酯Ⅲ（ATL-Ⅲ）通过ROS/GRP78/caspase-12信号通路对过氧化氢（H₂O₂）诱导的H9 c2细胞内质网应激和凋亡的影响。通过分子对接确定ATL-Ⅲ与GRP78的结合活性。结果表明，ATL-Ⅲ与GRP78具有良好的结合活性，且其结合活性强于其特异性抑制剂。用H₂O₂（100 μmol·L⁻¹）处理建立H9 c2内质网应激模型。设计五组：空白对照组、模型组、ATL-Ⅲ（15、30和60 μmol·L⁻¹）组。采用Hoechst/PI双染和流式细胞术检测细胞凋亡。用比色法测定超氧化物歧化酶（SOD）、丙二醛（MDA）和乳酸脱氢酶（LDH）水平。分别用荧光探针DCFH-DA和钙荧光探针Flou-4测定细胞质中活性氧（ROS）和钙（Ca²⁺）水平。用Western blot检测GRP78、caspase-12和caspase-3的蛋白水平，用RT-qPCR检测GRP78和caspase-12的mRNA水平。分别用N-乙酰-L-半胱氨酸（NAC）和4-苯基丁酸（4-PBA）抑制ROS和GRP78，进而推断ATL-Ⅲ保护细胞免受H₂O₂诱导的内质网应激的机制。ATL-Ⅲ（15、30和60 μmol·L⁻¹）降低了细胞凋亡率以及ROS、MDA和LDH水平（P＜0.01），提高了SOD活性（P＜0.01），下调了GRP78、caspase-12和caspase-3的蛋白水平以及GRP78和caspase-12的mRNA水平（P＜0.05）。加入NAC降低了细胞凋亡率以及ROS、MDA、GRP78、caspase-12和caspase-3水平（P＜0.01），同时提高了SOD水平（P＜0.01）。加入4-PBA也降低了细胞凋亡率以及GRP78、caspase-12、caspase-3和Ca²⁺水平（P＜0.01）。抑制剂的作用与ATL-Ⅲ一致。综上所述，ATL-Ⅲ可通过调节ROS/GRP78/caspase-12信号通路保护H9 c2心肌细胞，抑制H₂O₂诱导的内质网应激和凋亡。
