Ministry of Education Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Shihezi University, Shihezi, Xinjiang 832003, P.R. China.
Department of Blood Transfusion, Shenzhen Children's Hospital, Shenzhen, Guangdong 518034, P.R. China.
Mol Med Rep. 2024 Aug;30(2). doi: 10.3892/mmr.2024.13259. Epub 2024 Jun 14.
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 HS donors sodium hydrosulfide (NaHS) and D,L‑propargylglycine (PAG), a pharmacological inhibitor of endogenous HS‑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 HS 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 HS synthesis by PAG augmented the pro‑pyroptotic effects of ox‑LDL. Conversely, exogenous HS (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.
巨噬细胞焦亡介导血管炎症和动脉粥样硬化（AS）。硫化氢（H2S）在预防炎症和 AS 方面发挥着保护作用。然而，其调节细胞焦亡信号通路并抑制巨噬细胞焦亡的分子机制仍未被探索。本研究旨在确定 H2S 是否通过下调细胞焦亡信号通路和氧化型低密度脂蛋白（ox-LDL）刺激下的胱冬肽酶-1 的 S-硫氢化作用来减轻巨噬细胞焦亡，ox-LDL 是一种促动脉粥样硬化因子。用外源性 HS 供体硫氢化钠（NaHS）和内源性 HS 产生酶的药理学抑制剂 D,L-炔丙基甘氨酸（PAG）单独或联合预处理源自 THP-1 单核细胞的巨噬细胞。随后，用 ox-LDL 或脱硫剂二硫苏糖醇（DTT）在存在或不存在 NaHS 和/或 PAG 的情况下刺激细胞。处理后，通过亚甲蓝比色法测量 THP-1 衍生巨噬细胞中的 HS 水平。通过光镜、Hoechst 33342/碘化丙啶荧光染色和乳酸脱氢酶（LDH）释放测定观察和评估 THP-1 细胞的焦亡表型。通过胱冬肽酶-1 活性测定试剂盒测定 THP-1 细胞中的胱冬肽酶-1 活性。免疫荧光染色用于评估活性胱冬肽酶-1 的积累。通过 Western blot 和 ELISA 分别测定细胞中焦亡特异性标志物（NLRP3、前胱冬肽酶-1、胱冬肽酶-1、GSDMD 和 GSDMD-N）的表达和细胞无细胞培养基中焦亡相关细胞因子[白细胞介素（IL）-1β和 IL-18]的分泌。使用生物素交换测定法评估细胞中前胱冬肽酶-1 的 S-硫氢化作用。ox-LDL 通过激活细胞焦亡信号通路显著诱导巨噬细胞焦亡。PAG 抑制内源性 HS 合成增强了 ox-LDL 的促焦亡作用。相反，外源性 HS（NaHS）通过抑制细胞焦亡信号通路的激活减轻 ox-LDL 和 ox-LDL+PAG 诱导的巨噬细胞焦亡。在机制上，ox-LDL 和 DTT 通过减少前胱冬肽酶-1 的 S-硫氢化作用，增加胱冬肽酶-1 依赖性细胞焦亡途径的胱冬肽酶-1 活性和下游事件（IL-1β 和 IL-18 分泌）。相反，NaHS 增加了前胱冬肽酶-1 的 S-硫氢化作用，降低了胱冬肽酶-1 活性和胱冬肽酶-1 依赖性巨噬细胞焦亡。本研究表明，H2S 通过抑制细胞焦亡信号通路和前胱冬肽酶-1 的 S-硫氢化作用来减轻巨噬细胞焦亡的分子机制，从而抑制活性胱冬肽酶-1 的产生和胱冬肽酶-1 的活性。
