OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

中文翻译：

---

马芬尼抑制细胞焦亡和炎症因子释放的机制研究[J].

目的本研究旨在研究马芬尼 (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。方法 脂多糖 (LPS) 和尼日利亚菌素用于诱导小鼠骨髓源性巨噬细胞 (iBMDM) 和小鼠小胶质细胞 (BV2) 的细胞焦亡。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）检测炎症因子的表达。培养基。MAF 用生物素标记，随后进行下拉测定以检测其与 GSDMD 的结合。GSDMD-Asp275 位点进一步突变以验证 MAF 与 GSDMD 的结合。最后，MAF 对炎症因子释放和小胶质细胞活化的影响在 APP/PS12 动物模型中得到证实。结果 MAF 可抑制 iBMDM 和小胶质细胞 BV2 的细胞焦亡，减少炎症因子的释放。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，减少炎症因子的释放。MAF可降低APP/PS1小鼠脑脊液和外周血炎症因子水平，抑制小胶质细胞活化。结论 MAF调控炎症反应的机制与抑制细胞焦亡有关。MAF 可以通过直接结合 GSDMD 来抑制 GSDMD 裂解。