程序性坏死抑制剂Nec-1对铅致BV2细胞损伤的影响

Effect of programmed necrosis inhibitor Nec-1 on lead-induced BV2 cell injury

[背景]程序性坏死与神经退行性疾病的发生和发展关系密切,但铅是否引起神经细胞程序性坏死尚未见报道。[目的]探讨程序性坏死在铅诱导神经小胶质细胞(BV2细胞)损伤中的作用及其抑制剂对铅诱导BV2细胞损伤的影响。[方法]取生长良好的对数生长期BV2细胞,经0、1、5、10、25、50、100、200μmol·L^(-1)醋酸铅分别处理12、24、36、48 h后,采用噻唑蓝法测定细胞存活率。BV2细胞经0、25、50、100μmol·L^(-1)醋酸铅处理24 h后,分别采用酶联免疫吸附试验法、蛋白质免疫印迹法和流式细胞术测定细胞内肿瘤坏死因子-α(TNF-α)、受体相互作用蛋白激酶3(RIPK3)、受体相互作用蛋白激酶1(RIPK1)和混合谱系激酶结构域样蛋白(MLKL)的表达情况;并经RIPK1抑制剂Nec-1预处理1 h,检测Nec-1对铅诱导BV2细胞损伤的影响。[结果]BV2细胞的存活率随着铅染毒浓度的增加(r_(12 h)=−0.995,r_(24 h)=−0.984,r_(36 h)=−0.983,r_(48 h)=–0.981,均P<0.01)而下降,在5μmol·L^(-1)醋酸铅染毒下,细胞存活率也随着染毒时间的延长(r=–0.994,P<0.01)而下降。与对照组比较,同一染毒时间,当铅染毒浓度达到10μmol·L^(-1)时即可引起BV2细胞存活率下降(P<0.01)。与对照组比较,25、50、100μmol·L^(-1)铅处理组细胞程序性坏死相关蛋白RIPK1、MLKL蛋白表达升高(P<0.05或0.01),并伴有炎症细胞因子TNF-α含量升高,以100μmol·L^(-1)醋酸铅组最高(P<0.01);50、100μmol·L^(-1)醋酸铅处理组p-RIPK1、p-MLKL及RIPK3表达水平均升高(P<0.01)。此外,与相应染铅组比较,Nec-1预处理使染铅BV2细胞活性升高,坏死及晚凋率降低(P<0.05或0.01)。[结论]铅可致BV2细胞活性降低,坏死率升高,并伴有RIPK3和RIPK1、MLKL蛋白及其磷酸化的表达上调,而RIPK1抑制剂Nec-1对铅引起的BV2细胞损伤具有一定的干预作用,提示程序性坏死可能在铅神经毒性中发挥着重要作用。

[Background]Programmed necrosis is closely related to the occurrence and development of neurodegenerative diseases,but whether lead causes programmed cell necrosis has not been reported.[Objective]This experiment is designed to probe into the function of programmed necrosis and the effect of its inhibitor on lead-induced microglia(BV2 cell)injury.[Methods]The BV2 cells at logarithmic growth phase were treated with 0,1,5,10,25,50,100,and 200μmol·L^(-1) lead acetate for 12,24,36,and 48 h,respectively,and methylthiazolyldiphenyltetrazolium bromide(MTT)was used to determine cell viability.After treatment with 0,25,50,and 100μmol·L^(-1) lead acetate for 24 h,enzyme-linked immunosorbent assay,Western blotting,and flow cytometry were used to determine the expressions of tumor necrosis factor-α(TNF-α),receptor-interacting protein kinase 3(RIPK3),receptor-interacting protein kinase 1(RIPK1),and mixed lineage kinase domain-like protein(MLKL)in the cells,and the effect of RIPK1 inhibitor Nec-1 pretreatment on lead-induced BV2 cell injury.[Results]The BV2 cell viability decreased with higher lead concentration(r_(12 h)=−0.995,r_(24 h)=−0.984,r_(36 h)=−0.983,r_(48 h)=−0.981,all P<0.01)and time extension(only for 5μmol·L^(-1) lead acetate,r=−0.994,P<0.01).Compared with the control group,the BV2 cell viability decreased at the same exposure time when the concentration of lead was above 10μmol·L^(-1)(P<0.01).Compared with the control group,the expressions of RIPK1 and MLKL were increased in the 25,50,and 100μmol·L^(-1) lead groups(P<0.05 or 0.01),accompanied by an increase in the contents of inflammatory cytokine TNF-α,especially in the 100μmol·L^(-1) lead group,the increment was the highest(P<0.01).The expression levels of p-RIPK1 and p-MLKL in BV2 cells were both increased when the concentration of lead acetate was above 50μmol·L^(-1)(P<0.01).In addition,pretreatment with Nec-1 increased the cell viability rate and decreased the necrosis and late apoptosis rate of BV2 cells exposed to lead compared with corresponding lead exposure groups(P<0.05).[Conclusions]Lead can reduce BV2 cell viability,increase necrosis rate,and up-regulate the expressions of RIPK1,RIPK3,amd MLKL,and the phosphorylation levels of RIPK1 and MLKL.The RIPK1 inhibitor Nec-1 has an intervention effect on lead-induced damage in BV2 cells,indicating that programmed necrosis may play a role in lead neurotoxicity.