摘要
目的探讨甲基莲心碱(Nef)在β淀粉样蛋白(Aβ1-42)损伤大鼠肾上腺髓质嗜铬细胞瘤细胞PC12的作用及其分子机制。方法将PC12细胞分为对照组、模型组(4μg/mL Aβ1-42培养24 h)和干预组(低、中、高剂量甲基莲心碱)。MTT法检测PC12细胞存活;流式细胞计量术检测细胞凋亡;Western blot检测水通道蛋白4(AQP4)、Bcl-2和Bax表达量;qPCR检测细胞中miR-29a-3p和AQP4 mRNA表达。生物学信息预测和双荧光素酶基因报告分析miR-29a-3p和AQP4的靶向关系。在模型组转染miR-29a-3p、si-AQP4,或转染anti-miR-29a-3p并进行高剂量Nef干预,考察其对Aβ1-42损伤PC12细胞存活和凋亡的影响。结果与模型组相比,甲基莲心碱组细胞存活率和Bcl-2、miR-29a-3p表达明显升高,细胞凋亡率和Bax、AQP4 mRNA和蛋白表达明显降低(P<0.05)。AQP4是miR-29a-3p的靶基因。miR-29a-3p过表达和抑制AQP4表达均显著提高PC12细胞存活率和Bcl-2表达量(P<0.05),降低细胞凋亡率和Bax蛋白水平(P<0.05)。抑制miR-29a-3p表达逆转甲基莲心碱对Aβ1-42损伤PC12细胞存活、Bcl-2蛋白水平的促进作用,以及逆转甲基莲心碱对细胞凋亡率、Bax蛋白表达的抑制作用。结论甲基莲心碱通过miR-29a-3p/AQP4抑制Aβ1-42所致PC12细胞损伤,促进细胞存活,并抑制细胞凋亡。
Objective To explore the role and molecular mechanism of Neferine(Nef) on PC12 cells injury induced by Aβ1-42. Methods PC12 cells were divided into control group, model group(4 μg/mL Aβ1-42 cultured for 24 h) and low, medium and high-dose neferine intervention group. The PC12 cell viability was determined by MTT method and cell apoptosis was detected by flow cytometry. Protein expression of aquaporin4(AQP4), Bcl-2 and Bax was analyzed by Western Blot, and miR-29 a-3 p, AQP4 mRNA were detected by qPCR. Biological information prediction and dual luciferase gene report analyzed the targeting relationship between miR-29 a-3 p and AQP4. miR-29 a-3 p, si-AQP4 were transfected into PC12 cells induced by Aβ1-42, or anti-miR-29 a-3 p was transfected into cells and performed high-dose Nef intervention to investigate the effects on cell survival and apoptosisin PC12 cells injury induced by Aβ1-42. Results Compared with the model group, the cell viability rate, Bcl-2 and miR-29 a-3 p expression of neferine group were significantly increased, and the cell apoptosis rate, Bax, AQP4 mRNA and protein of neferine group expression were significantly decreased(P<0.05). AQP4 was the target gene of miR-29 a-3 p. Both over-expression of miR-29 a-3 p and inhibition of AQP4 expression significantly increased the viability rate of PC12 cells injury induced by Aβ1-42 and the expression level of Bcl-2, and decreased the apoptosis rate and Bax protein level. Inhibitiion of expression of miR-29 a-3 p reversed the promotion effect of Neferine on the survival of PC12 cell injury induced by Aβ1-42 and the level of Bcl-2 protein, as well as the inhibition effect ofNeferine on the apoptosis rate and Bax protein expression. Conclusions Neferine inhibits PC12 cell injury induced by Aβ1-42, promotes cell survival and inhibits apoptosis by miR-29 a-3 p/AQP4.
作者
曾利敏
鲁召辉
周丽平
朱超霞
ZENG Li-min;LU Zhao-hui;ZHOU Li-ping;ZHU Chao-xia(Zhengzhou Seventh People s Hospital,Zhengzhou 450000,China)
出处
《基础医学与临床》
CSCD
2020年第1期83-91,共9页
Basic and Clinical Medicine
基金
河南省自然科学基金162300410188
