{"title":"硫化氢通过S-硫酸化caspase-1,减轻氧化-LDL诱导的NLRP3/caspase-1/GSDMD依赖性巨噬细胞脓毒症。","authors":"Zhenli Jia, Xulin Zhang, Zhiyi Li, Hanyu Yan, Xiangqin Tian, Chenghua Luo, Ketao Ma, Ling Li, Liang Zhang","doi":"10.3892/mmr.2024.13259","DOIUrl":null,"url":null,"abstract":"<p><p>Macrophage pyroptosis mediates vascular inflammation and atherosclerosis (AS). Hydrogen sulfide (H2S) exerts a protective role in preventing inflammation and AS. However, its molecular mechanisms of regulating the pyroptosis signaling pathway and inhibiting macrophage pyroptosis remain unexplored. The present study aimed to determine whether H2S mitigates macrophage pyroptosis by downregulating the pyroptosis signaling pathway and S‑sulfhydrating caspase‑1 under the stimulation of oxidized low‑density lipoprotein (ox‑LDL), a pro‑atherosclerotic factor. Macrophages derived from THP‑1 monocytes were pre‑treated using exogenous H<sub>2</sub>S donors sodium hydrosulfide (NaHS) and D,L‑propargylglycine (PAG), a pharmacological inhibitor of endogenous H<sub>2</sub>S‑producing enzymes, alone or in combination. Subsequently, cells were stimulated with ox‑LDL or the desulfhydration reagent dithiothreitol (DTT) in the presence or absence of NaHS and/or PAG. Following treatment, the levels of H<sub>2</sub>S in THP‑1 derived macrophages were measured by a methylene blue colorimetric assay. The pyroptotic phenotype of THP‑1 cells was observed and evaluated by light microscopy, Hoechst 33342/propidium iodide fluorescent staining and lactate dehydrogenase (LDH) release assay. Caspase‑1 activity in THP‑1 cells was assayed by caspase‑1 activity assay kit. Immunofluorescence staining was used to assess the accumulation of active caspase‑1. Western blotting and ELISA were performed to determine the expression of pyroptosis‑specific markers (NLRP3, pro‑caspase‑1, caspase‑1, GSDMD and GSDMD‑N) in cells and the secretion of pyroptosis‑related cytokines [interleukin (IL)‑1β and IL‑18] in the cell‑free media, respectively. The S‑sulfhydration of pro‑caspase‑1 in cells was assessed using a biotin switch assay. ox‑LDL significantly induced macrophage pyroptosis by activating the pyroptosis signaling pathway. Inhibition of endogenous H<sub>2</sub>S synthesis by PAG augmented the pro‑pyroptotic effects of ox‑LDL. Conversely, exogenous H<sub>2</sub>S (NaHS) ameliorated ox‑LDL‑and ox‑LDL + PAG‑induced macrophage pyroptosis by suppressing the activation of the pyroptosis signaling pathway. Mechanistically, ox‑LDL and the DTT increased caspase‑1 activity and downstream events (IL‑1β and IL‑18 secretion) of the caspase‑1‑dependent pyroptosis pathway by reducing S‑sulfhydration of pro‑caspase‑1. Conversely, NaHS increased S‑sulfhydration of pro‑caspase‑1, reducing caspase‑1 activity and caspase‑1‑dependent macrophage pyroptosis. The present study demonstrated the molecular mechanism by which H2S ameliorates macrophage pyroptosis by suppressing the pyroptosis signaling pathway and S‑sulfhydration of pro‑caspase‑1, thereby suppressing the generation of active caspase-1 and activity of caspase-1.</p>","PeriodicalId":18818,"journal":{"name":"Molecular medicine reports","volume":null,"pages":null},"PeriodicalIF":3.4000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11188054/pdf/","citationCount":"0","resultStr":"{\"title\":\"Hydrogen sulfide mitigates ox‑LDL‑induced NLRP3/caspase‑1/GSDMD dependent macrophage pyroptosis by S‑sulfhydrating caspase‑1.\",\"authors\":\"Zhenli Jia, Xulin Zhang, Zhiyi Li, Hanyu Yan, Xiangqin Tian, Chenghua Luo, Ketao Ma, Ling Li, Liang Zhang\",\"doi\":\"10.3892/mmr.2024.13259\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Macrophage pyroptosis mediates vascular inflammation and atherosclerosis (AS). Hydrogen sulfide (H2S) exerts a protective role in preventing inflammation and AS. However, its molecular mechanisms of regulating the pyroptosis signaling pathway and inhibiting macrophage pyroptosis remain unexplored. The present study aimed to determine whether H2S mitigates macrophage pyroptosis by downregulating the pyroptosis signaling pathway and S‑sulfhydrating caspase‑1 under the stimulation of oxidized low‑density lipoprotein (ox‑LDL), a pro‑atherosclerotic factor. Macrophages derived from THP‑1 monocytes were pre‑treated using exogenous H<sub>2</sub>S donors sodium hydrosulfide (NaHS) and D,L‑propargylglycine (PAG), a pharmacological inhibitor of endogenous H<sub>2</sub>S‑producing enzymes, alone or in combination. Subsequently, cells were stimulated with ox‑LDL or the desulfhydration reagent dithiothreitol (DTT) in the presence or absence of NaHS and/or PAG. Following treatment, the levels of H<sub>2</sub>S in THP‑1 derived macrophages were measured by a methylene blue colorimetric assay. The pyroptotic phenotype of THP‑1 cells was observed and evaluated by light microscopy, Hoechst 33342/propidium iodide fluorescent staining and lactate dehydrogenase (LDH) release assay. Caspase‑1 activity in THP‑1 cells was assayed by caspase‑1 activity assay kit. Immunofluorescence staining was used to assess the accumulation of active caspase‑1. Western blotting and ELISA were performed to determine the expression of pyroptosis‑specific markers (NLRP3, pro‑caspase‑1, caspase‑1, GSDMD and GSDMD‑N) in cells and the secretion of pyroptosis‑related cytokines [interleukin (IL)‑1β and IL‑18] in the cell‑free media, respectively. The S‑sulfhydration of pro‑caspase‑1 in cells was assessed using a biotin switch assay. ox‑LDL significantly induced macrophage pyroptosis by activating the pyroptosis signaling pathway. Inhibition of endogenous H<sub>2</sub>S synthesis by PAG augmented the pro‑pyroptotic effects of ox‑LDL. Conversely, exogenous H<sub>2</sub>S (NaHS) ameliorated ox‑LDL‑and ox‑LDL + PAG‑induced macrophage pyroptosis by suppressing the activation of the pyroptosis signaling pathway. Mechanistically, ox‑LDL and the DTT increased caspase‑1 activity and downstream events (IL‑1β and IL‑18 secretion) of the caspase‑1‑dependent pyroptosis pathway by reducing S‑sulfhydration of pro‑caspase‑1. Conversely, NaHS increased S‑sulfhydration of pro‑caspase‑1, reducing caspase‑1 activity and caspase‑1‑dependent macrophage pyroptosis. The present study demonstrated the molecular mechanism by which H2S ameliorates macrophage pyroptosis by suppressing the pyroptosis signaling pathway and S‑sulfhydration of pro‑caspase‑1, thereby suppressing the generation of active caspase-1 and activity of caspase-1.</p>\",\"PeriodicalId\":18818,\"journal\":{\"name\":\"Molecular medicine reports\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.4000,\"publicationDate\":\"2024-08-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11188054/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular medicine reports\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.3892/mmr.2024.13259\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/6/14 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q2\",\"JCRName\":\"MEDICINE, RESEARCH & EXPERIMENTAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular medicine reports","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3892/mmr.2024.13259","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/6/14 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}
Macrophage pyroptosis mediates vascular inflammation and atherosclerosis (AS). Hydrogen sulfide (H2S) exerts a protective role in preventing inflammation and AS. However, its molecular mechanisms of regulating the pyroptosis signaling pathway and inhibiting macrophage pyroptosis remain unexplored. The present study aimed to determine whether H2S mitigates macrophage pyroptosis by downregulating the pyroptosis signaling pathway and S‑sulfhydrating caspase‑1 under the stimulation of oxidized low‑density lipoprotein (ox‑LDL), a pro‑atherosclerotic factor. Macrophages derived from THP‑1 monocytes were pre‑treated using exogenous H2S donors sodium hydrosulfide (NaHS) and D,L‑propargylglycine (PAG), a pharmacological inhibitor of endogenous H2S‑producing enzymes, alone or in combination. Subsequently, cells were stimulated with ox‑LDL or the desulfhydration reagent dithiothreitol (DTT) in the presence or absence of NaHS and/or PAG. Following treatment, the levels of H2S in THP‑1 derived macrophages were measured by a methylene blue colorimetric assay. The pyroptotic phenotype of THP‑1 cells was observed and evaluated by light microscopy, Hoechst 33342/propidium iodide fluorescent staining and lactate dehydrogenase (LDH) release assay. Caspase‑1 activity in THP‑1 cells was assayed by caspase‑1 activity assay kit. Immunofluorescence staining was used to assess the accumulation of active caspase‑1. Western blotting and ELISA were performed to determine the expression of pyroptosis‑specific markers (NLRP3, pro‑caspase‑1, caspase‑1, GSDMD and GSDMD‑N) in cells and the secretion of pyroptosis‑related cytokines [interleukin (IL)‑1β and IL‑18] in the cell‑free media, respectively. The S‑sulfhydration of pro‑caspase‑1 in cells was assessed using a biotin switch assay. ox‑LDL significantly induced macrophage pyroptosis by activating the pyroptosis signaling pathway. Inhibition of endogenous H2S synthesis by PAG augmented the pro‑pyroptotic effects of ox‑LDL. Conversely, exogenous H2S (NaHS) ameliorated ox‑LDL‑and ox‑LDL + PAG‑induced macrophage pyroptosis by suppressing the activation of the pyroptosis signaling pathway. Mechanistically, ox‑LDL and the DTT increased caspase‑1 activity and downstream events (IL‑1β and IL‑18 secretion) of the caspase‑1‑dependent pyroptosis pathway by reducing S‑sulfhydration of pro‑caspase‑1. Conversely, NaHS increased S‑sulfhydration of pro‑caspase‑1, reducing caspase‑1 activity and caspase‑1‑dependent macrophage pyroptosis. The present study demonstrated the molecular mechanism by which H2S ameliorates macrophage pyroptosis by suppressing the pyroptosis signaling pathway and S‑sulfhydration of pro‑caspase‑1, thereby suppressing the generation of active caspase-1 and activity of caspase-1.
期刊介绍:
Molecular Medicine Reports is a monthly, peer-reviewed journal available in print and online, that includes studies devoted to molecular medicine, underscoring aspects including pharmacology, pathology, genetics, neurosciences, infectious diseases, molecular cardiology and molecular surgery. In vitro and in vivo studies of experimental model systems pertaining to the mechanisms of a variety of diseases offer researchers the necessary tools and knowledge with which to aid the diagnosis and treatment of human diseases.