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

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 REM可能通过下调NLRP3/Caspase-1/GSDMD信号通路减轻LPS诱导的神经元焦亡。

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 obviously higher than those in the REM low group,and the differences were statistically significant(P<0.05).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 in the REM low group and REM high group were obviously lower than those in the LPS group,which in the REM high group were obviously lower than those in the REM low group,and the differences were statistically significant(P<0.05).The A_(450) values at 48 h and 72 h in HT22 cells in the REM high+Nigerian antibiotic group were obviously lower than those in the REM high 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 in the REM high+Nigerian antibiotic group were obviously higher than those in the REM high group(P<0.05).Conclusion REM may alleviate LPS induced neuronal pyroptosis by downregulating the NLRP3/Caspase-1/GSDMD signaling pathway.