Ling-Gui-Zhu-Gan decoction inhibits cardiomyocyte pyroptosis via the NLRP3/Caspase-1 signaling pathway

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-1 beta. 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 mu g/mL) for 12 hand ATP (8 mM) for 2 h were used as modeling condition. LGZGD demonstrated a significant reduction in LDH, and IL-1 beta 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.