原花青素下调miR-181a抑制颗粒细胞氧化损伤的作用机制

Grape seed procyanidins protect granulosa cells from oxidative stress-induced injury by down-regulating miR-181a expression

旨在研究葡萄籽原花青素(grape seed procyanidins, GSP)抑制颗粒细胞氧化损伤的作用及其可能机制。腹腔注射3-硝基丙酸(3-nitropropionic acid, 3-NP)诱导小鼠卵泡氧化损伤,并补充GSP进行干预处理;体外分离培养小鼠卵巢颗粒细胞,通过抑制或过表达miR-181a,研究原花青素B2(grape seed procyanidin B2,GSPB2)通过下调miR-181a抑制颗粒细胞氧化损伤的作用。采用定量PCR方法检测miR-181a表达水平,应用四甲基偶氮唑盐(methylthiazolyl tetrazolium, MTT)法检测细胞活力,利用免疫组化法检测活化天冬氨酸蛋白水解酶-3(cleaved caspase-3),应用酶标仪法检测半胱氨酸天冬氨酸蛋白酶-3(caspase-3)活性,采用双荧光素酶报告基因检测miR-181a和转化生长因子β受体Ⅰ(TGFBR1)的靶向调控关系,使用原位末端标记法(TUNEL)染色检测颗粒细胞凋亡情况,应用Western blot法检测TGFBR1蛋白水平。结果显示:在动物试验中,与对照组相比,3-NP组颗粒细胞活力显著降低(P<0.05);同时caspase-3活性、cleaved caspase-3蛋白及miR-181a表达明显升高(P<0.05);而GSP干预处理可扭转上述各指标的改变(P<0.05)。双荧光素酶报告试验结果显示,TGFBR1为miR-181a的靶基因;进一步分析发现,与对照组相比,miR-181a模拟物转染后TGFBR1蛋白表达和细胞活力均显著降低(P<0.05),而miR-181a抑制剂转染后TGFBR1蛋白表达和细胞活力均显著升高(P<0.05)。在细胞试验中,与对照组比较,H2O2组细胞中miR-181a表达升高,TGFBR1表达降低(P<0.05),细胞活力下降,caspase-3活性和细胞凋亡率均显著升高;而GSPB2预处理可扭转上述各指标的改变(P<0.05)。综上,GSP可抑制颗粒细胞的氧化损伤,其作用可能是通过抑制miR-181a表达而调控TGFBR1表达实现。

In order to explore the protective effect of grape seed procyanidins(GSP)on oxidative damage of follicular granulosa cells and the possible protective molecular mechanism of GSP,a follicular oxidative stress model on mice was established by intraperitoneal injection of 3-nitropropionic acid(3-NP),and GSP and GSPB2 were given to the rodents by feeding and culture medium to prevent oxidative stress-in-duced granulosa cell injury and to determine their effects on the expression of miR-181a.The relative expression of miR-181a was detected by qPCR.MTT assay were used to assess the proliferation of granulosa cells.The cleaved caspase-3 immunohistochemical staining and caspase-3 activity in granulosa cell were performed.The dual luciferase reporter system was used to test whether TGFBR1 was the target gene of miR-181a.The terminal transferase-mediated DNA nick-end labeling(TUNEL)assay was used to detect granulosa cell apoptosis.The protein levels of cleaved caspase-3 and TGFBR1 were determined by Western blot.The results were as follows:In the in vivo experiment,compared with the control group,the granulosa cell viability was significantly decreased(P<0.05),the caspase-3 activity and the expres-sion levels of cleaved caspase-3 protein and miR-181a were increased(P<0.05)in the granulosa cells from the 3-NP group.Further-more,TGFBR1 was confirmed as a direct target of miR-181a by bioinformatics analysis and luciferase assays.Compared with the NC group,the expression levels of TGFBR1 mRNA and the proteins were significantly reduced in the miR-181a mimics group(P<0.05).Compared with the H_(2)O_(2) group,the granulosa cell viability was significantly reduced,and the number of TUNEL positive granulosa cells was signifi-cantly increased in the miR-181a inhibitor group with H_(2)O_(2) treatment(P<0.05).In the in vitro experiment,compared with control group,the miR-181a expression level,the caspase-3 activity and the apoptosis rate were significantly increased,and the TGFBR1 expression level and the granulosa cell viability were significantly decreased in the granulosa cells from the H2O2 group.(P<0.05)Further research found that GSPB2 significantly alleviated the above conditions induced by H2O2(P<0.05).The above results indicated that GSP inhibited the oxi-dative damage of granulosa cells,and its effect might be achieved by inhibiting the expression of miR-181a and by regulating the expression of TGFBR1.