利多卡因调节cGAS-STING信号通路对LPS诱导的小胶质细胞炎性损伤的影响
Impacts of lidocaine on LPS-induced inflammatory injury of microglia by regulating cGAS-STING signaling pathway
尹健 1赵馨 2贾彤3
作者信息
- 1. 075000 河北省张家口市第一医院麻醉科
- 2. 张家口市第五医院麻醉科
- 3. 河北北方学院附属第一医院麻醉科
- 折叠
摘要
目的 探讨利多卡因(Lidocaine,Lido)通过调节环鸟苷酸-腺苷酸合成酶(Cyclic guanosine monophosphate synthase,cGAS)-干扰素基因刺激因子(Stimulator of interferon genes,STING)信号通路对脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞炎性损伤的影响及其作用机制.方法 0～200 μg/mL的利多卡因处理LPS诱导的BV2小胶质细胞24 h,四甲基偶氮唑蓝(Methylthiazolyl-tetrazolium,MTT)测定细胞活性,选择最佳药物水平;将BV2细胞分为对照组(Control组)、LPS诱导组(LPS组,1 μg/mL LPS)、利多卡因低水平组(Lido-L 组,2 μg/mL Lido+1 μg/mLLPS)、利多卡因中水平组(Lido-M 组,20 μg/mL Lido+1μg/mL LPS)、利多卡因高水平组(Lido-H组,200 μg/mL Lido+1 μg/mL LPS)和利多卡因高水平+STING激活剂 DMXAA 组(Lido-H+DMXAA 组,200 μg/mL Lido+1 μg/mL LPS+100 μg/mL DMXAA);Griess法检测NO水平;酶联免疫吸附法((Enzyme-linked immunosorbent assay,ELISA)检测单核细胞趋化蛋白-1(Monocyte ehemoattractant protein-1,MCP-1)、前列腺素 E2(Prostaglandin E2,PGE2)、环氧化酶 2(Cyclooxy-ge-nase 2,COX-2)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)、白细胞介素(Interleukin,IL)-10 和 IL-1β水平;Hoechst染色检测细胞凋亡;免疫荧光检测钙离子结合调节因子(Ionized calcium-binding adapter molecule 1,Iba-1)和精氨酸酶-1(Arginase-1,Arg-1)表达水平;Western blot检测环鸟苷酸-腺苷酸合成酶(Cy-clic guanosine monophosphate synthase,cG AS)、干扰素基因刺激因子(Stimulator of interferon genes,STING)、核苷酸结合寡聚化结构域样受体 3(Nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)蛋白水平.结果 0～200 μg/mL的利多卡因能增加LPS诱导的BV2细胞活力,选择2、20和200μg/mL的利多卡因进行后续实验.与Control组比较,LPS组BV2细胞出现大量细胞发生凋亡现象,TNF-α,IL-1β、一氧化氮(Nitric oxide,NO)、PGE2,MCP-1 和 COX-2 水平、Iba-1 阳性表达率及 Cgas,STING 和 NL-RP3蛋白表达水平显著增高,IL-10水平和Arg-1阳性表达率显著降低(P＜0.05);与LPS组比较,Lido-L组、Lido-M 组和 Lido-H 组 BV2 细胞凋亡逐渐减少,TNF-α,IL-1 β,NO,PGE2,MCP-1 和 COX-2 水平、Iba-1 阳性表达率及cGAS,STING和NLRP3蛋白表达水平显著降低,IL-10水平和Arg-1阳性表达率显著增高,且呈水平依赖性(P＜0.05);与Lido-H组比较,Lido-H+DMXAA组BV2细胞凋亡增加,TNF-α,IL-1β,NO,PGE2,MCP-1和COX-2水平、Iba-1阳性表达率及cGAS,STING和NLRP3蛋白表达水平显著增高,IL-10水平和Arg-1阳性表达率显著降低(P＜0.05).结论 利多卡因对LPS诱导的小胶质细胞炎性损伤的保护作用机制可能与抑制cGAS-STING信号通路的激活有关.
Abstract
Objective To investigate the effects of lidocaine on lipopolysaccharide(LPS)-induced in-flammatory injury in microglia and the underlying mechanism through modulation of the cyclic guanosine-ade-nylate synthase-interferon gene-stimulating factor(cGAS-STING)signaling pathway.Methods In the present study,LPS-induced BV2 microglia were subjected to different concentrations of lidocaine(ranging from 0 to 200 µg/mL)for 24 hours.To assess cell activity and identify the most effective drug concentration,a methyl thiazolyl tetrazolium(MTT)assay was performed.The BV2 cells were divided into different groups:the con-trol group,LPS induction group(LPS group,1 μg/mL LPS),low-concentration group(Lido-L group,2 μg/mL Lido+1 μg/mL LPS),medium-concentration group(Lido-M group,20 μg/mL Lido+1 μg/mL LPS),high-concentration group(Lido-H group,200 μg/mL Lido+1 μg/mL LPS),and high-concentration Lido+STING activator(Lido-H+DMXAA group,200 μg/mL Lido+1 μg/mL LPS+100 μg/mL DMXAA).The Griess method was used to detect NO levels.Enzyme-linked immunosorbent assays(ELISAs)were used to de-tect monocyte chemotactic protein-1(MCP-1),prostaglandin E2(PGE2),cyclooxygenase-2(COX-2),tumor necrosis factor-alpha(TNF-α),interleukin(IL)-10,and IL-1β levels.Hoechst staining was used to detect cell apoptosis.The expression levels of ionized calcium-binding adapter molecule 1(Iba-1)and arginase-1(Arg-1)were determined using immunofluorescence.Western blot analysis was performed to measure the protein levels of cGAS,STING,and nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3).Results The activity of LPS-treated BV2 cells increased in the presence of Lidocaine at concentrations ranging from 0 μg/mL to 200 μg/mL.For subsequent experiments,Lidocaine concentrations of 2,20,and 200 μg/mL were selected.Compared to those in the control group,the LPS group exhibited significant increases in apop-tosis in BV2 cells;elevated levels of TNF-α,IL-1β,nitric oxide(NO),PGE2,MCP-1,and COX-2;increased expression of Iba-1;and increased expression of the cGAS,STING,and NLRP3 proteins,while the level of IL-10 and the percentage of cells positive for Arg-1 were noticeably lower(P＜0.05).Compared to those in the LPS group,the apoptosis of BV2 cells in the Lido-L,Lido-M,and Lido-H groups gradually decreased,as did the levels of TNF-α,IL-1β,NO,PGE2,MCP-1,and COX-2;the expression of Iba-1;and the expression of the cGAS,STING,and NLRP3 proteins.Conversely,the level of IL-10 and the percentage of cells positive for Arg-1 increased in a concentration-dependent manner(P＜0.05).Compared to those in the Lido-H group,the Lido-H+DMXAA group exhibited greater apoptosis in BV2 cells;elevated levels of TNF-α,IL-1β,NO,PGE2,MCP-1,and COX-2;increased expression of Iba-1;and increased expression of the cGAS,STING,and NLRP3 proteins,while the level of IL-10 and the percentage of cells positive for Arg-1 were noticeably lower(P＜0.05).Conclusion The mechanism by which lidocaine protects against LPS-induced inflammatory injury in microglia may be related to the inhibition of cGAS-STING signaling pathway activation.
关键词
利多卡因/小胶质细胞/脂多糖/环鸟苷酸-腺苷酸合成酶-干扰素基因刺激因子信号通路/炎性损伤Key words
Lidocaine/Microglia/Lipopolysaccharide/Cyclic guanosine monophosphate synthase-stimulator of interferon genes signaling pathway/Inflammatory injury引用本文复制引用
基金项目
张家口市市级科技计划(2018)(1821152H)
出版年
2024
NETL
NSTL
万方数据