瑞马唑仑调节NLRP3/caspase-1/GSDMD信号通路对LPS诱导的神经元焦亡的影响  

Effect of remimazolam on LPS-induced neurons necrosis by regulating the NLRP3/caspase-1/GSDMD signaling pathway

作　　者：耿长振 王利[1] 叶兰 GENG Changzhen;WANG Li;YE Lan(Department of Anesthesiology,Shanghai Public Health Clinical Center,Shanghai 201500,China)

机构地区：[1]上海市公共卫生临床中心麻醉科,上海201500

出　　处：《检验医学与临床》2024年第16期2436-2441,共6页Laboratory Medicine and Clinic

摘　　要：目的 探讨瑞马唑仑(REM)调节Nod样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白水解酶-1(Caspase-1)/消皮素D(GSDMD)信号通路对脂多糖(LPS)诱导的神经元焦亡的影响。方法 将对数期小鼠海马神经元HT22细胞随机分为正常对照组(Ctrl组)、LPS诱导组(LPS组,10 mg/L LPS)、低浓度REM组(REM-低组,160μmol/L REM)、高浓度REM组(REM-高组,320μmol/L REM)和REM-高+NLRP3激活剂尼日利亚菌素组(REM-高+尼日利亚菌素组,320μmol/L REM+10μmol/L尼日利亚菌素)。除Ctrl组外,其余各组HT22细胞均使用LPS进行诱导。各组加药处理后,采用细胞计数试剂盒8测定HT22细胞的吸光度(A值)。采用Hoechst33342/碘化吡啶(PI)染色检测HT22细胞焦亡率。采用酶联免疫吸附试验检测HT22细胞上清液中白细胞介素(IL)-1β、IL-6和肿瘤坏死因子-α(TNF-α)水平。采用实时荧光定量聚合酶链反应检测HT22细胞中NLRP3信使RNA(mRNA)、Caspase-1 mRNA、GSDMD mRNA表达水平。采用蛋白质印迹法检测HT22细胞中NLRP3、Caspase-1、GSDMD和凋亡相关斑点样蛋白(ASC)表达水平。结果 LPS组HT22细胞48、72 h的A_(450)值显著低于Ctrl组(P<0.05),而细胞焦亡率、上清液中IL-1β、IL-6、TNF-α水平,以及NLRP3、Caspase-1、GSDMD mRNA和蛋白表达水平、ASC蛋白表达水平均显著高于Ctrl组(P<0.05)。REM-低组和REM-高组HT22细胞48、72 h的A_(450)值显著高于LPS组,且REM-高组高于REM-低组,差异均有统计学意义(P<0.05),而REM-低组和REM-高组HT22细胞焦亡率、上清液中IL-1β、IL-6、TNF-α水平,以及NLRP3、Caspase-1、GSDMD mRNA和蛋白表达水平、ASC蛋白表达水平显著低于LPS组,且REM-高组低于REM-低组,差异均有统计学意义(P<0.05)。REM-高+尼日利亚菌素组HT22细胞48、72 h的A_(450)值显著低于REM-高组(P<0.05),而细胞焦亡率、上清液中IL-1β、IL-6、TNF-α水平,以及NLRP3、Caspase-1、GSDMD mRNA和蛋白表达水平、ASC蛋白表达水平均显著高于REM-高组(P<0.05)。结论 Objective To investigate the effect of remimazolam(REM)on lipopolysaccharide(LPS)induced neurons pyroptosis by regulating the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(Caspase-1)/gasdermin D(GSDMD)signaling pathway.Methods Logarithmic phase mouse hippocampal neurons HT22 cells were randomly separated into a normal control group(Ctrl group),an LPS induction group(LPS group,10 mg/L LPS),a low concentration REM group(REM low group,160μmol/L REM),a high concentration REM group(REM high group,320μmol/L REM)and a REM high+NLRP3 activator Nigerian antibiotic group(REM high+Nigerian antibiotic group,320μmol/L REM+10μmol/L Nigerian antibiotic).Except for the Ctrl group,HT22 cells in all other groups were induced by LPS.After each group was treated with medication,the CCK-8 method was applied to determine the absorbancy(A value)of HT22 cells.Hoechst33342/pyridine iodide(PI)staining was applied to detect the level of pyroptosis rate in HT22 cells.Enzyme linked immunosorbent assay(ELISA)was applied to detect the levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)in the supernatant of HT22 cells.Real-time quantitative polymerasechain reaction was applied to detect the expression levels of NLRP3 message RNA(mRNA),Caspase-1 mRNA and GSDMD mRNA in HT22 cells.Western blot was applied to detect the expression of NLRP3,Caspase-1,GSDMD and apoptosis-associated speck-like protein containing a CARD(ASC)in HT22 cells.Results The A 450 values at 48 h and 72 h in HT22 cells in the LPS group were obviously lower than those in the Ctrl group(P<0.05),while the cell apoptosis rate,IL-1β,IL-6,and TNF-αlevels in the supernatant,NLRP3,Caspase-1,GSDMD mRNA and protein expression levels,and ASC protein expression level were obviously higher than those in the Ctrl group(P<0.05).The A 450 values at 48 h and 72 h in HT22 cells in the REM low group and REM high group were obviously higher than those in the LPS group,which in the REM high group were obviousl

关 键 词：瑞马唑仑 Nod样受体蛋白3/半胱氨酸天冬氨酸蛋白水解酶-1/消皮素D信号通路 脂多糖 神经元 焦亡 

分 类 号：R965[医药卫生—药理学] R446.8[医药卫生—药学]