PNU介导Seipin调控BV2细胞降解Aβ寡聚体的机制研究

The Aβdegradation mechanism research based on PNU mediated Seipin expression effect on BV2 cell

目的探索在β-淀粉样蛋白(Aβ)寡聚体处理的BV2小胶质细胞模型中α7烟碱型乙酰胆碱受体的激动剂PNU282987(PNU)对Seipin蛋白表达及其介导自噬的影响。方法采用CCK8法检测不同浓度Aβ处理的BV2细胞的存活率筛选Aβ浓度;设置正常组(未做任何处理)和Aβ组(Aβ孵育24 h),采用Western blot法检测细胞经Aβ处理后Seipin蛋白的表达水平;设置Aβ组(Aβ孵育24 h)、Aβ+PNU组(先加入PNU处理18 h、再加入Aβ孵育24 h),采用Western blot法检测Aβ的表达水平;设置正常组(未做任何处理)、PNU组(PNU处理18 h),采用Western blot检测Seipin、自噬标志物微管相关蛋白1轻链3(LC3)和Beclin-1的表达;将合成的RNAi慢病毒感染BV2细胞,设置阴性对照组(Negative control,NC)、靶向Seipin小干扰RNA(Small interfering RNA;si-Seipin)组,观察Seipin被干扰情况;设置NC+Aβ组(加入NC和Aβ共同孵育)、NC+Aβ+PNU组(加入NC、Aβ和PNU共同孵育)及si-Seipin+Aβ+PNU组(加入si-Seipin、Aβ和PNU共同孵育),采用Western blot检测Aβ和LC3的表达。结果CCK8结果显示随着Aβ浓度的增加,BV2细胞存活率逐渐降低,浓度为10μmol/L时、细胞存活率为70%,故选择该Aβ浓度为造模浓度;Western blot结果显示,与正常组相比,Aβ处理的BV2细胞Seipin蛋白表达水平下降(P<0.05);与Aβ组相比,Aβ+PNU组BV2细胞中Aβ蛋白表达水平明显减少(P<0.05);与正常组相比,PNU组BV2细胞中Seipin、LC3-Ⅱ和Beclin-1表达水平升高(P<0.05);与NC组相比,si-Seipin组BV2细胞中Seipin蛋白水平下降(P<0.05);与NC+Aβ组相比较,NC+Aβ+PNU组BV2细胞中Aβ表达水平明显降低、LC3-Ⅱ表达水平明显升高(P<0.01);与NC+Aβ+PNU组比较,si-Seipin+Aβ+PNU组BV2细胞中Aβ表达水平明显升高、LC3-Ⅱ表达水平明显降低(P<0.01)。结论Aβ处理BV2细胞后Seipin降低,PNU能够抑制Aβ的表达,其机制可能与PNU上调Seipin增强BV2细胞的自噬、降解Aβ有关。

Objective To investigate the effect of PNU282987(PNU),of BV2 microglia cell modelα7nAChR treated in Aβoligomer,on the Seipin protein expression and mediated autophagy.Methods Using different concentrations of Aβto treat BV2,and CCK8 was used to screen the optimal concentration for modeling.The expression of Seipin was detected in normal group(no treatment)and Aβgroup(treated for 24 h with Aβ).The expression of Aβwas detected in Aβgroup(treated for 24 h with Aβ)and Aβ+PNU group(treated for 24 h with Aβafter PNU treatment for 18 h).The expression of Seipin,LC3,and Beclin-1 were observed in normal group(no treatment)and PNU group(treated for 18 h with PNU).Infecting BV2 with synthesized RNAi lentivirus,establishing the negative control group(NC),small interfering RNA group(si-Seipin)to observe interference on Seipin.The expression of Aβand LC3 were detected by Western blot on NC+Aβgroup(incubated with both NC and Aβ),NC+Aβ+PNU group(incubated with NC,Aβand PNU)and si-Seipin+Aβ+PNU group(incubated with si-Seipin,Aβ,and PNU).Results CCK8 results showed that with the increase of Aβconcentration,BV2 viability was gradually decreased.At the concentration of 10μmol/L,the cell viablity rate was 70%,which was selected as the model concentration.The results of protein analysis showed that the level of Seipin protein in BV2 decreased after Aβtreatment compared with the control group(P<0.05).Compared with Aβgroup,Aβprotein expression was decreased significantly in Aβ+PNU group(P<0.05).Compared with the normal group,the expression levels of protein(Seipin,LC3-Ⅱand Beclin-1)were increased in PNU treated group(P<0.05).Compared with negative control group,the levels of Seipin was decreased in si-Seipin group(P<0.05).Compared with NC+Aβgroup,Aβin BV2 was downregulated and LC3-Ⅱexpression level was upregulated in NC+Aβ+PNU group(P<0.01).Compared with NC+Aβ+PNU group,Aβexpression was upregulated significantly and LC3-Ⅱwas downregulated obviously in si-Seipin+Aβ+PNU group(P<0.01).Conclusion The expression of Seipin protein is downregulated in Aβtreated BV2,while PNU treatment can inhibit the expression of Aβ.This mechanism may be associated with the upregulation of Seipin mediated by PNU,thereby enhancing the degradation of Aβoligomer by autophagy.