Chenyang Han1, Yi Yang2, Anqi Yu3, Li Guo4, Qiaobing Guan5, Heping Shen6, Qingcai Jiao7. 1. 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. Electronic address: 691513770@qq.com. 2. Department of pharmacy, The Second Affiliated Hospital of Jiaxing University, Zhejiang 314001, China. Electronic address: wasd911@126.com. 3. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, China. Electronic address: 1144900357@qq.com. 4. Department of Center Laboratory, The Second Affiliated Hospital of Jiaxing University, Zhejiang 314001, China. Electronic address: taishanlg@126.com. 5. Department of neurology, The Second Affiliated Hospital of Jiaxing University, Zhejiang 314001, China. Electronic address: guanqb@126.com. 6. Department of neurology, The Second Affiliated Hospital of Jiaxing University, Zhejiang 314001, China. Electronic address: shenhp_77@163.com. 7. State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing, 210093, China. Electronic address: jiaoqc@163.com.
Abstract
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.
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.
Authors: Tiantian Du; Jie Gao; Peilong Li; Yunshan Wang; Qiuchen Qi; Xiaoyan Liu; Juan Li; Chuanxin Wang; Lutao Du Journal: Clin Transl Med Date: 2021-08