苓桂术甘汤通过NLRP3/半胱天冬酶-1信号通路抑制心肌细胞焦亡。
Ling-Gui-Zhu-Gan decoction inhibits cardiomyocyte pyroptosis via the NLRP3/Caspase-1 signaling pathway.
作者信息
Zhao Xiao-Ni, Ding Hui-Min, Ma Yao-Yao, Wang Liang, Zhou Peng
机构信息
Department of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, Anhui 230012, China.
Department of Integrated Traditional Chinese and Western Medicine, Anhui University of Chinese Medicine, Hefei, Anhui 230012, China; Research Institute of Integrated Traditional Chinese and Western Medicine, Anhui Academy of Chinese Medicine, Hefei, Anhui 230012, China.
出版信息
Tissue Cell. 2024 Dec;91:102588. doi: 10.1016/j.tice.2024.102588. Epub 2024 Oct 20.
OBJECTIVE
The objective of this study was to investigate the protective mechanism of Ling-Gui-Zhu-Gan decoction (LGZGD) against LPS-ATP-induced pyroptosis in H9c2 cells.
METHODS
LPS and ATP were used to induce pyroptosis in the H9c2 cell, and the cells were divided into the control, model and LGZGD groups. LDH level was detected using a colorimetric assay. ELISA was used to detect the expressions of IL-1β. Flow cytometry was utilized to observe apoptosis, while Hoechst/PI staining was used to detect pyroptosis. Immunofluorescence was employed to observe the expression levels of NLRP3 in cardiomyocytes, and RT-PCR was used to detect NLRP3, Caspase-1, GSDMD, and ASC mRNA expression. The cells were separated into seven groups: control, model, LGZGD, MCC950, LGZGD+MCC950, Nigericin and LGZGD+Nigericin. The mRNA and protein expressions were determined by RT-PCR and Western blot.
RESULTS
LPS (10 μg/mL) for 12 h and ATP (8 mM) for 2 h were used as modeling condition. LGZGD demonstrated a significant reduction in LDH, and IL-1β levels (P<0.05, P<0.01). LGZGD dramatically reduced apoptosis rate, inhibited pyroptosis, decreased the fluorescence expressions of NLRP3, and reduced the mRNA expressions of NLRP3, ASC, Caspase-1, and GSDMD (P<0.01). Further mechanism studies showed that NLRP3, ASC, Caspase-1, and GSDMD decreased significantly when combined with NLRP3 inhibitor MCC950. Furthermore, LGZGD was able to effectively reverse the upregulation of protein and gene expression of Nigericin group (P<0.01).
CONCLUSION
LGZGD inhibits LPS-ATP-induced pyroptosis in H9c2 cell via the NLRP3/Caspase-1 signaling pathway.
目的
本研究旨在探讨苓桂术甘汤（LGZGD）对脂多糖-三磷酸腺苷（LPS-ATP）诱导的H9c2细胞焦亡的保护机制。
方法
采用LPS和ATP诱导H9c2细胞发生焦亡，并将细胞分为对照组、模型组和LGZGD组。采用比色法检测乳酸脱氢酶（LDH）水平。采用酶联免疫吸附测定（ELISA）法检测白细胞介素-1β（IL-1β）的表达。利用流式细胞术观察细胞凋亡，同时采用Hoechst/碘化丙啶（PI）染色检测焦亡。采用免疫荧光法观察心肌细胞中NLR家族含pyrin结构域蛋白3（NLRP3）的表达水平，并采用逆转录-聚合酶链反应（RT-PCR）检测NLRP3、半胱天冬酶-1（Caspase-1）、Gasdermin D（GSDMD）和凋亡相关斑点样蛋白（ASC）的信使核糖核酸（mRNA）表达。将细胞分为七组：对照组、模型组、LGZGD组、MCC950组、LGZGD+MCC950组、尼日利亚菌素组和LGZGD+尼日利亚菌素组。通过RT-PCR和蛋白质免疫印迹法检测mRNA和蛋白质表达。
结果
采用10μg/mL的LPS作用12小时和8mM的ATP作用2小时作为建模条件。LGZGD组的LDH和IL-1β水平显著降低（P<0.05，P<0.01）。LGZGD组显著降低了凋亡率，抑制了焦亡，降低了NLRP3的荧光表达，并降低了NLRP3、ASC、Caspase-1和GSDMD的mRNA表达（P<0.01）。进一步的机制研究表明，与NLRP3抑制剂MCC950联合使用时，NLRP3、ASC、Caspase-1和GSDMD显著降低。此外，LGZGD能够有效逆转尼日利亚菌素组蛋白质和基因表达的上调（P<0.01）。
结论
LGZGD通过NLRP3/Caspase-1信号通路抑制LPS-ATP诱导的H9c2细胞焦亡。
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