槲皮素通过介导JNK信号通路抑制线粒体凋亡途径的神经保护作用

Quercetin plays a neuroprotective role in inhibiting mitochondrial apoptosis by mediating JNK signaling pathway

目的研究槲皮素(quercetin,Que)通过调节JNK信号通路抑制Aβ25-35引起PC12细胞线粒体损伤的作用机制。方法培养PC12细胞,使用Aβ25-35诱导其构建阿尔茨海默病(Alzheimer′s disease,AD)受损模型,Que、17β-雌二醇(17β-E2)和金雀异黄素(genistein,Gen)进行干预,并设定SP600125(JNK特异性抑制剂)预处理组。Fluo-4 AM染料荧光检测PC12细胞钙离子;Rhodamine123染色液检测PC12细胞线粒体膜电位;Caspase-3活性检测Caspase-3酶活性;免疫荧光检测p-JNK蛋白表达;Western blot法检测ERα、ERβ、p-JNK、JNK、Bcl-W、Bim和Cytochrome C蛋白表达。并使用雌激素受体α/β拮抗剂MPP/PHTPP和SP600125进行干预,探讨Que发挥神经保护介导的分子机制。结果与Aβ25-35组相比,Aβ25-35+Que组能提高线粒体膜电位(P<0.01);降低钙离子浓度(P<0.01)。免疫荧光、Caspase-3检测和Western blot结果显示,与Aβ25-35组相比,Que可以逆转Aβ25-35诱导的ERα表达降低(P<0.01),当加入MPP抑制Que与雌激素受体α结合后,Caspase-3表达增加(P<0.05);Que抑制p-JNK表达(P<0.01);降低促凋亡蛋白Bim的表达(P<0.01),减少Cyt C的释放(P<0.01),促进抗凋亡蛋白Bcl-W的表达(P<0.01),当加入SP600125后,Que对Bcl-W、Bim和Cyt C的作用和SP600125对Aβ25-35诱导PC12细胞线粒体凋亡途径的保护作用机制相同(P<0.01)。结论Que可通过介导JNK信号通路抑制线粒体凋亡,进而发挥神经保护作用。

Aim To study the mechanism of quercetin(Que)inhibiting mitochondrial damage induced by Aβ25-35 in PC12 cells by regulating JNK signaling pathway.Methods PC12 cells were cultured and induced by Aβ25-35 to construct AD damaged model.Que,17β-estradiol(17β-E2)and genistein(Gen)were used to intervene,and SP600125(JNK specific inhibitor)was pretreated.Fluo-4 AM dye was used to detect calcium ions in PC12 cells.The mitochondrial membrane potential of PC12 cells was detected by Rhodamine123 staining solution.The activity of Caspase-3 enzyme was detected.p-JNK protein expression was detected by immunofluorescence.The protein expressions of ERα,ERβ,p-JNK,JNK,Bcl-W,Bim and cytochrome C were detected by Western blot.Estrogen receptorα/βantagonists MPP/PHTPP and SP600125 were used to intervene and explore the molecular mechanism of Que′s neuroprotective mediation.Results Compared with the Aβ25-35 group,the Aβ25-35+Que group could increase mitochondrial membrane potential(P<0.01).Calcium concentration decreased(P<0.01).Immunofluorescence,Caspase-3 detection and Western blot results showed that Que could reverse the decreased expression of ERαinduced by Aβ25-35 compared with the Aβ25-35 group(P<0.01).When MPP was added to inhibit the binding of Que to estrogen receptorα,the expression of Caspase-3 increased(P<0.05);Que inhibited P-JNK expression(P<0.01).The expression of pro-apoptotic protein Bim was reduced(P<0.01),while the release of Cyt C was raised(P<0.01),and the expression of anti-apoptotic protein Bcl-W was promoted(P<0.01).The effect of Que on Bcl-W,Bim and Cyt C was the same as that of SP600125 on the mitochondrial apoptosis pathway induced by Aβ25-35 in PC12 cells(P<0.01).Conclusion Que can inhibit mitochondrial apoptosis by mediating JNK signaling pathway,thereby playinga neuroprotective role.