瑞芬太尼诱导的SH-SY5Y细胞中PKCε与MOR相互作用的研究

Research on the interaction between PKCεand MOR induced by remifentanil in SH-SY5Y cells

目的利用人神经母瘤细胞(SH-SY5Y),探讨瑞芬太尼对蛋白激酶Cε(PKCε)和μ阿片受体(MOR)之间相互作用的影响以及PKCε对瑞芬太尼诱导MOR内化的调节作用。方法设置对照组和瑞芬太尼处理组(R2 h组),R2 h组采用10μmol/L瑞芬太尼处理SH-SY5Y细胞2 h。采用细胞免疫荧光观察MOR蛋白分布变化,免疫印迹检测MOR蛋白膜浆组分表达变化;细胞免疫荧光检测PKCε与MOR共定位情况,免疫共沉淀检测瑞芬太尼作用不同时间后PKCε与MOR相互作用变化;构建PKCε、MOR及PKCε截短蛋白的原核表达载体并诱导表达和纯化蛋白,采用pull-down实验检测两者直接相互作用情况。分别通过PKCε特异性激动剂和抑制剂预处理,观察激活或抑制PKCε后对瑞芬太尼诱导MOR内化的影响。结果免疫荧光结果显示,与对照组比较,R2 h组MOR显著内化[(0.72±0.07)vs.(12.57±1.10)],差异有统计学意义(t=8.90,P<0.0001)。免疫印迹结果显示,胞膜MOR蛋白显著减少,胞浆组分MOR蛋白增加。细胞免疫荧光共染显示,SH-SY5Y细胞中的PKCε与MOR存在共定位,而免疫共沉淀结果表明瑞芬太尼可增强PKCε和MOR的相互作用。GST pull-down结果显示,PKCε与MOR有直接相互作用,且主要结合区域是PKCε的催化区域。与R2 h组比较,PKCε特异性抑制剂预孵育30 min显著减少瑞芬太尼诱导的MOR内化[(1.00±0.05)vs.(0.76±0.03)],而激动剂处理则促进MOR的内化[(1.00±0.05)vs.(1.21±0.06)],差异有统计学意义(F=22.80,P<0.05)。蛋白免疫印迹结果进一步显示,εv1-2抑制MOR膜蛋白的下调(F=31.54,P<0.05)和浆蛋白的增加(F=28.25,P<0.05),而DCPLA则作用相反。结论PKCε通过与MOR直接相互作用调节瑞芬太尼诱导的SH-SY5Y细胞中的MOR内化,为瑞芬太尼诱导MOR内化机制提供新的科学依据,对寻求防治阿片类药物副作用靶点具有潜在的临床意义。

Objective To investigate the effect of remifentanil on the interaction between protein kinase Cε(PKCε)andμopioid receptor(MOR)and the regulatory effect of PKCεon MOR internalization induced by remifentanil by using human neuroblastoma cells(SH-SY5Y).Methods SH-SY5Y cells were treated with 10μmol/L remifentanil for 2 hours.The distribution of MOR protein was observed by immunofluorescence;the expression of MOR protein membrane was detected by Western blotting;the co-localization of PKCεand MOR was detected by cellular immunofluorescence,and the interaction between PKCεand MOR was detected by co-immunoprecipitation assay.The prokaryotic expression vectors of PKCεand MOR and the truncated expression of PKCεwere constructed,and the two proteins were induced and purified.The direct interaction between them was detected by pull-down assay.The effects of activation or inhibition of PKCεon MOR internalization induced by remifentanil were observed by pretreatment with specific agonists and inhibitors of PKCε,respectively.Results The immunofluorescence results showed that MOR was significantly internalized in SH-SY5Y cells treated with 10μmol/L remifentanil for 2 hours[(0.72±0.07)vs.(12.57±1.10)(t=8.90,P<0.0001)].Western blotting showed that the membrane MOR protein decreased significantly after remifentanil treatment for 2 hours,while the cytoplasm MOR protein increased.In addition,cellular immunofluorescence co-staining showed that PKCεwas co-located with MOR in SH-SY5Y cells,and remifentanil enhanced the interaction between PKCεand MOR.GST pull-down results shows that there was a direct interaction between PKCεand MOR,and the main binding region was the catalytic region of PKCε.Compared with R2h group,preincubation with PKCεspecific inhibitor for 30 min significantly reduced MOR internalization induced by remifentanil[(1.00±0.05)vs.(0.76±0.03)],while agonist treatment promoted MOR internalization[(1.00±0.05)vs.(1.21±0.06)],the differences were statistically significant(F=22.80,P<0.05).Western blotting further showed thatεv1-2 inhibited the down-regulation of MOR membrane protein(F=31.54,P<0.05)and the increase of plasma protein(F=28.25,P<0.05),while DCPLA had the opposite effect.Conclusion PKCεcould regulate MOR internalization induced by remifentanil in SH-SY5Y cells by interacting with MOR directly.