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

作　　者：尹健 赵馨 贾彤[3] Yin Jian;Zhao Xin;Jia Tong(Department of Anesthesiology,the First Hospital of Zhangjiakou,Hebei Province 075000;不详)

机构地区：[1]河北省张家口市第一医院麻醉科,075000 [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

出　　处：《卒中与神经疾病》2024年第2期192-200,共9页Stroke and Nervous Diseases

基　　金：张家口市2018年市级科技计划项目(编号为1821152H)。

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0~200μg/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,1μg/mL LPS)、利多卡因低水平组(Lido-L组,2μg/mL Lido+1μg/mL LPS)、利多卡因中水平组(Lido-M组,20μg/mL Lido+1μg/mL LPS)、利多卡因高水平组(Lido-H组,200μg/mL Lido+1μg/mL LPS)和利多卡因高水平+STING激活剂DMXAA组(Lido-H+DMXAA组,200μg/mL Lido+1μg/mL LPS+100μg/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(Cyclooxyge-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(Cyclic guanosine monophosphate synthase,cGAS)、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0~200μg/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NLRP3蛋白表达水平显著增高,IL-10水平和Arg-1阳Objective To investigate the effects of lidocaine on lipopolysaccharide(LPS)-induced inflammatory injury in microglia and the underlying mechanism through modulation of the cyclic guanosine-adenylate 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 200μg/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 control group,LPS induction group(LPS group,1μg/mL LPS),low-concentration group(Lido-L group,2μg/mL Lido+1μg/mL LPS),medium-concentration group(Lido-M group,20μg/mL Lido+1μg/mL LPS),high-concentration group(Lido-H group,200μg/mL Lido+1μg/mL LPS),and high-concentration Lido+STING activator(Lido-H+DMXAA group,200μg/mL Lido+1μg/mL LPS+100μg/mL DMXAA).The Griess method was used to detect NO levels.Enzyme-linked immunosorbent assays(ELISAs)were used to detect monocyte chemotactic protein-1(MCP-1),prostaglandin E2(PGE2),cyclooxygenase-2(COX-2),tumor necrosis factor-alpha(TNF-α),interleukin(IL)-10,and IL-1βlevels.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 0μg/mL to 200μg/mL.For subsequent experiments,Lidocaine concentrations of 2,20,and 200μg/mL were selected.Compared to those in the control group,the LPS group exhibited significant increases in apoptosis 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 c

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 

分 类 号：R741[医药卫生—神经病学与精神病学]