Huaier attenuates the adverse effects of pyroptosis by regulating the methylation of rat mesangial cells: an in vitro study

Abstract

Background Pyroptosis is a highly programmed inflammatory cell death process that represents an innate immune response. In this study, the occurrence of pyroptosis in rat mesangial cells (RMCs) and the effect of Huaier (Trametes robiniophia Murr) on this process were investigated. Methods RMCs were incubated with OX7 antibodies (0.5 μg/ml, 2.5 μg/ml, 10 μg/ml), normal rat serum (NRS) and Huaier (1 mg/ml, 5 mg/ml, 10 mg/ml). RMC morphology was observed under a light microscope and by immunofluorescence. Lactate dehydrogenase (LDH) release was assessed using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit. Western blot assays were performed, and then the RMCs were incubated with the methylase DNMT3B and the demethylase 5-aza-2′-deoxycytidine. Results Morphological, LDH, immunofluorescence and western blot analyses showed that RMCs were lysed when stimulated with OX7 antibodies and NRS. RMC lysis released inflammatory cytokines (interleukin-18, interleukin-1β, monocyte chemoattractant protein-1 and intracellular adhesion molecule-1), and Huaier protected RMCs by controlling lysis and the levels of inflammatory cytokines. Lysis was mediated by pyroptosis due to the positive expression of GSDME. The methylase DNMT3B reduced the expression of GSDME induced by OX7 together with NRS. Furthermore, Huaier significantly suppressed the expression of GSDME, which was increased by 5-aza-2’-deoxycytidine. Conclusions Pyroptosis might occur in RMCs, and Huaier can protect RMCs by upregulating the methylation of a group of molecules.