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-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.