The activation of NOD-like receptor protein 3 (NLRP3) inflammasome-dependent pyroptosis has been shown to play a vital role in the pathology of manganese (Mn)-induced neurotoxicity. Sodium para-aminosalicylic acid (PAS-Na) has a positive effect on the treatment of manganism. However, the mechanism is still unclear. We hypothesized that PAS-Na might act through NLRP3. The microglial cell line BV2 and male Sprague-Dawley rats were used to investigate the impacts of PAS-Na on Mn-induced NLRP3 inflammasome-dependent pyroptosis. The related protein of the NF-κB pathway and NLRP3-inflammasome-dependent pyroptosis was detected by western blot. The reactive oxygen species and mitochondrial membrane potential were detected by immunofluorescence staining and flow cytometry. The activation of microglia and the gasdermin D (GSDMD) were detected by immunofluorescence staining. Our results showed that Mn treatment induced oxidative stress and activated the NF-κB pathway by increasing the phosphorylation of p65 and IkB-α in BV2 cells and in the basal ganglia of rats. PAS-Na could alleviate Mn-induced oxidative stress damage by inhibiting ROS generation, increasing mitochondrial membrane potential and ATP levels, thereby reducing the phosphorylation of p65 and IkB-α. Besides, Mn treatment could activate the NLRP3 pathway and promote the secretion of IL-18 and IL-1β, mediating pyroptosis in BV2 cells and in the basal ganglia and hippocampus of rats. But an inhibitor of NF-κb (JSH-23) treatment could significantly reduce LDH release, the expression of NLRP3 and Cleaved CASP1 protein and IL-1β and IL-18 mRNA level in BV2 cells. Interestingly, the effect of PAS-Na treatment in Mn-treated BV2 cells is similar to those of JSH-23. Besides, immunofluorescence results showed that PAS-Na reduced the increase number of activated microglia, which stained positively for GSDMD. PAS-Na antagonized Mn-induced NLRP3 inflammasome dependent pyroptosis through inhibiting NF-κB pathway activation and oxidative stress.

中文翻译：

---

对氨基水杨酸钠通过抑制 NF-κB 通路激活和氧化应激来抑制锰诱导的 NLRP3 炎症小体依赖性细胞焦亡

NOD 样受体蛋白 3 (NLRP3) 炎症小体依赖性细胞焦亡的激活已被证明在锰 (Mn) 诱导的神经毒性病理学中起着至关重要的作用。对氨基水杨酸钠 (PAS-Na) 对治疗锰中毒有积极作用。然而，其机制仍不清楚。我们假设 PAS-Na 可能通过 NLRP3 起作用。使用小胶质细胞系 BV2 和雄性 Sprague-Dawley 大鼠研究 PAS-Na 对 Mn 诱导的 NLRP3 炎症小体依赖性细胞焦亡的影响。Western blot检测NF-κB通路相关蛋白和NLRP3-炎症小体依赖性细胞焦亡。通过免疫荧光染色和流式细胞术检测活性氧和线粒体膜电位。通过免疫荧光染色检测小胶质细胞和gasdermin D（GSDMD）的活化。我们的结果表明，锰处理通过增加 BV2 细胞和大鼠基底神经节中 p65 和 IkB-α 的磷酸化来诱导氧化应激并激活 NF-κB 通路。PAS-Na 可以通过抑制 ROS 的产生、增加线粒体膜电位和 ATP 水平来减轻 Mn 诱导的氧化应激损伤，从而降低 p65 和 IkB-α 的磷酸化。此外，Mn 处理可激活 NLRP3 通路并促进 IL-18 和 IL-1β 的分泌，介导 BV2 细胞以及大鼠基底节和海马的细胞焦亡。但是NF-κb抑制剂（JSH-23）治疗可以显着减少LDH的释放，BV2细胞中NLRP3和Cleaved CASP1蛋白的表达以及IL-1β和IL-18 mRNA水平。有趣的是，PAS-Na 处理在 Mn 处理的 BV2 细胞中的效果与 JSH-23 的效果相似。此外，免疫荧光结果表明，PAS-Na 减少了激活的小胶质细胞数量的增加，这些小胶质细胞对 GSDMD 染色呈阳性。PAS-Na 通过抑制 NF-κB 通路激活和氧化应激来拮抗 Mn 诱导的 NLRP3 炎症小体依赖性细胞焦亡。