Apelin-17通过激活β1/2-arrestin-ERK1/2信号通路降低MPP+诱导内质网应激引起SH-SY5Y细胞的凋亡

Apelin-17 Attenuates ER Stress-associated Apoptosis of MPP-treated SHSY5Y Cells by Activatingβ-arrestin-ERK1/2 Signaling

内质网应激(endoplasmic reticulum stress,ERS)与帕金森病(Parkinson’s disease,PD)发展进程密切相关。Apelin是已被证实具有明显神经保护作用的内源性多肽,但其保护机制尚不清楚。该研究探讨Apelin-17降低1-甲基-4-苯基吡啶(MPP+)对SH-SY5Y细胞凋亡的分子机制。实时无标记动态细胞分析(real time cell analysis,RTCA)实验数据表明,0.1μmol/L Apelin-17预处理2 h,MPP+诱导的SH-SY5Y细胞(Apelin-17预处理组)指数明显高于MPP+处理组(P<0.05),PD98059与β1/2-arrestin siRNA实验组的细胞指数显著低于Apelin-17预处理组(P<0.05)。流式细胞仪结果显示,Apelin-17预处理组细胞凋亡率低于MPP+处理组(P<0.05),而PD98059与β1/2-arrestin siRNA实验组的细胞凋亡率高于Apelin-17预处理组(P<0.05)。CCK-8试剂盒检测结果显示,Apelin-17预处理组的细胞活力相对于MPP+处理组明显升高(P<0.05),与Apelin-17预处理组比较,PD98059与β1/2-arrestin siRNA实验组的细胞活力显著降低(P<0.05)。LDH检测数据表明,与MPP+处理组比较,Apelin-17预处理组的乳酸脱氢酶释放量显著降低(P<0.05),而PD98059与β1/2-arrestin siRNA实验组乳酸脱氢酶释放量显著高于Apelin-17预处理组(P<0.05)。Western印迹结果证明,与MPP+处理比较,Apelin-17预处理组显著降低了内质网应激反应中PERK独立介导的信号通路中p-eIF2α、ATF4、CHOP的表达水平(P<0.05),与凋亡密切相关的切割胱天蛋白酶12(cleaved-caspase-12)蛋白表达也显著下调,而抑制细胞凋亡相关的Bcl-2蛋白表达水平显著升高(P<0.05)。同时,与促进细胞增殖密切相关蛋白Phospho-pERK1/2的表达水平显著升高(P<0.05)。ERK1/2信号通路抑制剂PD98059加入Apelin-17预处理后会反转上述实验现象,同时加入β1/2-arrestin siRNA后,Phospho-pERK1/2与Bcl-2蛋白表达水平相较于Apelin-17预处理组显著降低(P<0.05),而CHOP的表达水平升高(P<0.05)。综上所述,本文的研究结果表明,Apelin-17通过β1/2-arrestin信号通路上调Phospho-pERK1/2与Bcl-2蛋白质表达水平,同时抑制内质网应激中p-eIF2α、ATF4和CHOP蛋白质表达,进而降低MPP+对SH-SY5Y细胞的毒性。

Endoplasmic reticulum stress(ERS)is closely related to the development of Parkinson’s disease(PD).Apelin,an endogenous peptide,has a remarkable neuroprotective effect.However,its protective mechanism is still unclear.In the present study,we investigate the molecular mechanism of Apelin-17 reducing the apoptosis of SH-SY5Y cells induced by 1-methyl-4-phenylpyridine(MPP+).Real time cell analysis(RTCA)data showed that the CI values of SH-SY5Y cells pretreated with 100 nmol/L Apelin-17 for 2 h and then co-cultured with MPP+for a further 24 h(Apelin-17 pretreatment group)was significantly higher than that of the group only treated with MPP+(P<0.05),and the CI values of PD98059 andβ1/2-arrestin siRNA group were marked decreased compared with Apelin-17 pretreatment group(P<0.05).The results of flow cytometry demonstrated that the apoptosis rate of Apelin-17 pretreatment group was markedly reduced compared with only MPP+-treated group,however,PD98059 andβ1/2-arrestin siRNA attenuated the protective effect of Apelin-17(P<0.05).The results of CCK-8 assay indicated that the viability of Apelin-17 pretreatment group was increased compared with only MPP+-treated group,the protective effect of Apelin-17 was abolished by treatment of the cells with PD98059 orβ1/2-arrestin siRNAs(P<0.05).The data of LDH assay demonstrated that the level of LDH in the culture medium of SH-SY5Y cells which were incubated with Apelin-17 prior to MPP+treatment was significantly lower than that in the culture medium of cells treated with MPP+only,however,the level of LDH in the culture medium of Apelin-17 pretreatment group was increased by treatment with PD98059 orβ1/2-arrestin siRNA(P<0.05).Western blot results predicated that Apelin-17 significantly decreased the expression levels of ERS-related PERK-dependent p-eIF2α,ATF4,CHOP and GRP78,compared with only MPP+treated group,downregulated the expression levels of the apoptosis protein cleaved-caspase-12,and upregulated the expression levels of Bcl-2 and Phospho-pERK1/2(P<0.05).The level of pERK1/2 and Bcl-2 was lower in the MPP+-treated group exposed to PD98059 and Apelin-17 than that exposed to Apelin-17 only.In addition,the Apelin-17-induced upregulation of Bcl-2 and downregulation of CHOP were abolished by treatment withβ1-arrestin orβ2-arrestin siRNA.Taken together,these data indicate that the rescue effects of Apelin-17 on MPP+-mediated apoptosis and levels of pERK1/2,Bcl-2,peIF2α,ATF4,and CHOP are mediated byβ1/2-arrestin signaling.