布比卡因通过组织蛋白酶B激活NLRP3炎性体诱导N2a细胞毒性

Bupivacaine contributes to neurotoxicity in N2a cells by activating NLRP3 inflammasome via Cathepsin B

目的:探讨组织蛋白酶B及NLRP3炎性体在布比卡因诱导N2a细胞神经毒性中的作用及分子机制。方法:将N2a细胞随机分为4组进行干预,空白对照组(C组):完全培养基培养,无任何药物干预;单纯抑制剂组(CA组):10μmol/L的CA-074-me预处理1 h后不加任何药物处理;CTSB抑制剂预处理+布比卡因组(CA+B组):10μmol/L的CA-074-me预处理1 h后,应用0.9 mmol/L的布比卡因培养12 h;布比卡因组(B组):0.9 mmol/L的布比卡因培养12 h。采用CCK-8法测定细胞活力;LDH释放实验检测上清液LDH活性;免疫荧光法检测NLRP3蛋白水平;western blotting法检测CTSB以及NLRP3炎性体活化相关因子NLRP3、cleaved-caspase-1和N-GSDMD蛋白表达。结果:与C组比较,CA组细胞活力、上清液LDH活性以及CTSB、NLRP3、cleaved-caspase-1、N-GSDMD蛋白表达比较,差异均无统计学意义(均P>0.05);B组细胞存活率明显降低,LDH释放增多,CTSB蛋白含量增加,炎性体活化相关蛋白NLRP3、cleaved-caspase-1和N-GSDMD表达水平明显升高(均P<0.05)。与B组比较,CA+B组细胞存活率增加,LDH释放减少,CTSB蛋白含量降低,炎性体活化相关蛋白NLRP3、cleaved-caspase-1和NGSDMD表达水平明显降低(均P<0.05)。结论:布比卡因可能通过CTSB激活NLRP3炎性体活化诱导N2a细胞损伤。

Objective:To investigate the role and molecular mechanism of Cathepsin B(CTSB)and NLRP3 inflammasomein bupivacaine-induced neurotoxicity in N2a cells.Methods:N2a cells were cultured in vitro and randomly divided into four groups.The control group(C group)was grown in complete medium without any drug intervention;Simple inhibitor group(CA group):after 10μmol/L CA-074-me pretreatment for 1 h,N2a cells were cultured in complete medium;CTSB inhibitor pretreatment+bupivacaine group(CA+B group):10μmol/L CA-074-me pretreated for 1 h,N2a cells were cultured with 0.9mmol/L bupivacaine for 12 h;Bupivacaine group(B group):N2a cells were cultured with 0.9mmol/L bupivacaine for 24 h.CCK-8 method was applied to assess the cell viability.LDH release experiment was conducted to measure the LDH activity in supernatant.Immunofluorescence method was used to detect the level of NLRP3 expression,and western blotting was employed to detect the expressions of CTSB and inflammasome-dependent factors NLRP3,cleaved-caspase-1 and N-GSDMD.Results:There were no statistic differences in cell viability,LDH release and the expressions of CTSB,NLRP3,cleaved-caspase-1 and N-GSDMD protein between C and CAgroups(P>0.05).Compared with the C group,the cell viability was decreased significantly,while the LDH releaseand the expression levelsof CTSB,NLRP3,cleaved-caspase-1 and N-GSDMD were increasedin B group(P<0.05).Compared with B group,the cell viability improved,the release of LDH decreased,and the expression of CTSB,NLRP3,cleaved-caspase-1 and N-GSDMD decreased significantly in CA+B group(P<0.05).Conclusion:Bupivacaine-induced neurotoxicity may be caused by the activation of NLRP3 inflammasome via CTSB in N2a cells.