State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing, 210093, China; Department of pharmacy, The Second Affiliated Hospital of Jiaxing University, Zhejiang 314001, China.
Department of pharmacy, The Second Affiliated Hospital of Jiaxing University, Zhejiang 314001, China.
Eur J Pharm Sci. 2020 Apr 30;147:105303. doi: 10.1016/j.ejps.2020.105303. Epub 2020 Mar 12.
The present study was designed to investigate the roles and mechanism of mafenide (MAF) in targeted inhibition of Gasdermin D (GSDMD) cleavage and in suppressing pyroptosis.
Lipopolysaccharide (LPS) and Nigericin were used to induce pyroptosis in mouse bone marrow-derived macrophages (iBMDM) and mouse microglia (BV2). Lactate dehydrogenase (LDH) release rate and Propidium Iodide (PI) uptake rate were used to detect cytotoxicity, Western blot was used to detect the protein expression, and Enzyme-linked immunosorbent assay (ELISA) was utilized to detect the expression of inflammatory factors from culture medium. MAF was labeled with biotin and subsequently subjected to Pull-down assay to detect its binding to GSDMD. GSDMD-Asp275 site was further mutated to validate the binding of MAF to GSDMD. Finally, the effects of MAF on inflammatory factor release and microglial activation were confirmed in the APP/PS12 animal model.
MAF could inhibit pyroptosis in iBMDM and microglia BV2, and decrease the release of inflammatory factors. MAF could inhibit GSDMD cleavage by directly binding to the GSDMD-Asp275 site, while the expression of p30-GSDMD was simultaneously down-regulated and the release of inflammatory factors was decreased. MAF could reduce the levels of inflammatory factors in cerebrospinal fluid and peripheral blood of APP/PS1 mice, and suppress the activation of microglia.
The mechanism underlying the regulation of MAF on inflammatory response was correlated with the inhibition of pyroptosis. MAF could inhibit GSDMD cleavage by directly binding to GSDMD.
本研究旨在探讨磺胺嘧啶（MAF）在靶向抑制 Gasdermin D（GSDMD）裂解和抑制细胞焦亡中的作用和机制。
采用脂多糖（LPS）和 Nigericin 诱导小鼠骨髓来源巨噬细胞（iBMDM）和小鼠小胶质细胞（BV2）发生细胞焦亡。采用乳酸脱氢酶（LDH）释放率和碘化丙啶（PI）摄取率检测细胞毒性，Western blot 检测蛋白表达，酶联免疫吸附试验（ELISA）检测培养基中炎性因子的表达。用生物素标记 MAF，随后进行 Pull-down 实验检测其与 GSDMD 的结合。进一步突变 GSDMD-Asp275 位点以验证 MAF 与 GSDMD 的结合。最后，在 APP/PS1 动物模型中验证 MAF 对炎性因子释放和小胶质细胞激活的影响。
MAF 可抑制 iBMDM 和 microglia BV2 中的细胞焦亡，减少炎性因子的释放。MAF 可通过直接结合 GSDMD-Asp275 位点抑制 GSDMD 裂解，同时下调 p30-GSDMD 的表达并减少炎性因子的释放。MAF 可降低 APP/PS1 小鼠脑脊液和外周血中炎性因子的水平，并抑制小胶质细胞的激活。
MAF 调节炎症反应的机制与抑制细胞焦亡有关。MAF 可通过直接结合 GSDMD 抑制 GSDMD 裂解。
