神经节苷脂预处理对布比卡因诱导N2a细胞焦亡的影响及其机制

Effect of ganglioside pretreatment on bupivacaine-induced pyroptosis of N2a cells and its mechanism

目的观察神经节苷脂(GM-1)预处理对布比卡因诱导N2a细胞焦亡的影响,并探讨其机制。方法取对数生长期N2a细胞随机分为3组:布比卡因组(B组)加入终浓度为900μmol/L的布比卡因培养24 h,GM-1预处理组(G+B组)加入5μmol/L GM-1预处理24 h后再加入900μmol/L的布比卡因继续培养24 h,对照组(C组)不加入GM-1或布比卡因。取各组细胞,分别用倒置相差显微镜、扫描电镜观察各组细胞形态;采用CCK-8法测算细胞存活率,以细胞存活率表示细胞活力;采用TUNEL染色试验测算TUNEL染色阳性细胞率,以TUNEL染色阳性细胞率表示细胞凋亡率;采用乳酸脱氢酶(LDH)释放实验测算各组细胞中LDH含量;采用天冬氨酰特异性半胱氨酰蛋白酶1(Caspase-1)活性实验测算各组细胞中Caspase-1活性;采用Western blotting法检测各组细胞焦亡相关蛋白细胞炎症小体活化蛋白质-gasdermin D(GSDMD)、NOD样受体蛋白3(NLRP3)和Caspase-1蛋白表达量。结果C组细胞胞体形态多样且饱满、形态规则,细胞膜完整且光滑;B组细胞皱缩变圆,细胞突触折断、消失,产生凋亡小体样的细胞突起囊泡;G+B组细胞损伤形态明显减轻,未见细胞膜的完整性丧失及微孔形成。与C组相比,B组细胞活力下降,细胞凋亡率升高,LDH含量升高,Caspase-1活性升高,GSDMD、NLRP3、Caspase-1蛋白相对表达量均升高(P均<0.05);与B组相比,G+B组细胞活力升高,细胞凋亡率下降,LDH含量下降,Caspase-1活性下降,GSDMD、NLRP3、Caspase-1蛋白相对表达量均降低(P均<0.05)。结论布比卡因可通过提高N2a细胞膜通透性,导致细胞活力下降、LDH释放、Caspase-1活性和细胞凋亡率升高,促进细胞焦亡,其机制可能与GSDMD,NLRP3,Caspase-1蛋白相对表达量升高有关;GM-1预处理可抑制布比卡因诱导的N2a细胞焦亡过程,其机制可能与降低GSDMD、NLRP3、Cas⁃pase-1蛋白相对表达量有关。

Objective To investigate the effect and molecular mechanism of ganglioside(GM-1)pretreatment on bu⁃pivacaine-induced pyroptosis of N2a cells.Methods N2a cells in the logarithmic phase were randomly divided into three groups:the bupivacaine group(B group,N2a cells were cultured with a final concentration of 900μmol/L bupiva⁃caine for 24 h),GM-1 pretreatment group(G+B group,after pretreatment with 5μmol/L GM-1 for 24 h,N2a cells were cultured with 900μmol/L bupivacaine for 24 h),and control group(C group,N2a cells were treated without GM-1 or bu⁃pivacaine).The cell morphology of each group was observed by the microscope and scanning electron microscope.Cell via⁃bility was measured by CCK-8.TUNEL staining test was used to measure the TUNEL staining positive cell rate,and the rate of TUNEL stained positive cells indicated the rate of apoptosis.The lactate dehydrogenase(LDH)content was mea⁃sured by LDH release experiment;the Caspase-1 activity experiment was used to calculate the Caspase-1 activity.Western blotting was used to detect the protein expression levels of inflammasome activating protein-gasdermin D(GSDMD),Nodlike receptor pyrin containing 3(NLRP3)and Caspase-1.Results The cell bodies of the C group were diverse and full with regular morphology,and the cell membrane was intact and smooth;cells of the B group shrunk and became round,and cell synapses were broken and disappeared,and apoptotic body-like cell protruding vesicles were produced;the cellu⁃lar damage in morphology was significantly reduced,and there was no loss of cell membrane integrity and micropore forma⁃tion in the G+B group.Compared with the C group,the viability of B group decreased,apoptosis rate and LDH content in⁃creased and the relative expression of GSDMD,NLRP3,and Caspase-1 protein significantly increased(all P<0.05).Compared with B group,the cell viability of G+B group increased,apoptosis rate,LDH content and Caspase-1 activity de⁃creased,and the relative expression levels of GSDMD,NLRP3,and Caspase-1 protein decreased(all P<0.05).Conclu⁃sions Bupivacaine can increase N2a cell membrane permeability,decrease cell viability and LDH release,increase Cas⁃pase-1 activity and apoptosis rate,and promote pyroptosis;the mechanism may be related to the increase in the relative protein expression of GSDMD,NLRP3,and Caspase-1.GM-1 pretreatment can inhibit the process of bupivacaine-in⁃duced pyroptosis of N2a cells,and the mechanism may be related to the decrease of the relative protein expression of GSD⁃MD,NLRP3,and Caspase-1.