三七总皂苷通过调节miR-16/TXNIP通路改善心肌细胞氧化应激损伤的机制研究

The Mechanism of Panax Notoginseng Saponins on Protecting Cardiomyocytes from Oxidative Stress by Regulating MiR-16/TXNIP Pathway

目的探究三七总皂苷(PNS)通过调节miR-16/硫氧还蛋白互作蛋白(TXNIP)通路改善心肌细胞氧化应激损伤的作用机制。方法体外培养H9c2细胞,分为空白组(常规培养)、模型组(H_(2)O_(2)处理2h)、PNS组(H_(2)O_(2)处理2 h+PNS作用)、PNS+miR-NC组(转染anti-miR-NC+H_(2)O_(2)处理2h+PNS作用)、PNS+miR-16 inhibitor组(转染miR-16 inhibitor+H_(2)O_(2)处理2 h+PNS作用)。MTT法、PI法和TUNEL法检测细胞增殖、坏死和凋亡情况。酶联免疫试剂盒检测乳酸脱氢酶(LDH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平。荧光法检测细胞内活性氧簇(ROS)水平。Western blot法检测TXNIP蛋白表达。结果与模型组细胞相比,PNS组细胞增殖活性(F=28.610,P<0.001)和miR-16相对表达量增加(F=29.048,P<0.001),凋亡率和坏死率(F=84.628,67.087,P<0.05)、DCFH-DA荧光强度(F=204.478,P<0.05)和TXNIP蛋白表达量(F=17.823,P<0.05)降低;细胞培养上清中LDH、MDA含量降低(F=55.387、92.506,P<0.05),同时SOD、GSH-Px水平升高(F=71.713、39.641,P<0.05)。但是与PNS组比较,PNS+miR-16 inhibitor组细胞增殖活性降低,凋亡率和坏死率、DCFH-DA荧光强度和TXNIP蛋白表达量增加;细胞培养上清中LDH、MDA含量增加,同时SOD、GSH-Px水平降低。经荧光素酶报告基因确认,TXNIP mRNA是miR-16下游靶基因。结论PNS能够改善心肌细胞的氧化应激损伤,其机制与上调miR-16表达进而靶向抑制下游TXNIP蛋白活性有关。

Objective To investigate the mechanism of Panax Notoginseng Saponins(PNS)protecting cardiomyocytes from oxidative stress by regulating miR-16/thioredoxin-interacting protein(TXNIP)pathway.Methods H9c2 cells were cultured in vitro and divided into the blank group(conventional culture),model group(treated with H_(2)O_(2) for 2 h),PNS group(treated with H_(2)O_(2) for 2h+PNS),PNS+miR-NC group(transfected with anti-miR-NC+H_(2)O_(2) for 2h+PNS),and PNS+mir-16 inhibitor group(transfected with miR-16 inhibitor+H_(2)O_(2) for 2h+PNS).MTT method,PI method and TUNEL method were applied to measure rates of cell proliferation,necrosis and apoptosis.The levels of lactate dehydrogenase(LDH),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH PX)were detected by ELISA kit.The level of reactive oxygen species(ROS)was detected by fluorescence method.The expression level of TXNIP protein was detected by western blot.Results Compared with the model group,the proliferation activity(F=28.610,P<0.001)and the relative expression of miR-16(F=29.048,P<0.001)in the PNS group were increased,while the apoptosis rate and necrosis rate(F=84.628,67.087,P<0.05),DCFH-DA fluorescence intensity(F=204.478,P<0.05)and TXNIP protein expression(F=17.823,P<0.05)were decreased in the PNS group,and the contents of LDH and MDA in the supernatant were decreased(F=55.387,92.506,P<0.05).The levels of GSH PX and GSH-Px increased(F=71.713,39.641,P<0.05).However,compared with the PNS group,the proliferation activity of the PNS+miR-16 inhibitor group decreased,while the apoptosis rate and necrosis rate,DCFH-DA fluorescence intensity and TXNIP protein expression increased.LDH and MDA contents in the cell culture supernatant increased,while SOD and GSH-Px levels decreased.It was confirmed by luciferase reporter gene assay that TXNIP mRNA was the downstream targeted gene of miR-16.Conclusion Panax Notoginseng Saponins can improve the oxidative stress injury of cardiomyocytes,and its mechanism is related to the up-regulation of miR-16 expression and the targeted inhibition of downstream TXNIP protein activity.