温阳通脉方通过NRF2/HO-1通路对缺氧/复氧损伤H9c2心肌细胞氧化应激的保护作用

Protective Effect of Wenyang Tongmai Formula (温阳通脉方) against Oxidative Stress in H9c2 Cardiomyocytes via NRF2/HO-1 Pathway

目的:基于核因子NF-E2相关因子2(NRF2)/血红素加氧酶-1(HO-1)信号通路探讨温阳通脉方含药血清保护H9c2心肌细胞缺氧/复氧(H/R)后氧化应激损伤机制。方法:制备大鼠的空白血清和温阳通脉方的含药血清,以H9c2心肌细胞为研究对象,用氯化钴(CoCl_(2))及新鲜培养基制备缺氧/复氧(H/R)模型。将细胞分为:空白对照组、模型对照组、13 g/kg温阳通脉方含药血清5%、10%、20%组。用CCK-8法检测细胞活性;比色法检测细胞超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)的活力或含量;DCFH-DA探针法检测细胞活性氧(ROS)的水平;Western blot法检测低氧诱导因子(HIF-1α)、核因子E2相关因子2(NRF2)、血红素加氧酶1(HO-1)的蛋白表达;Hoechst33342荧光染色法检测细胞凋亡率和形态学变化。结果:与空白对照组比较,模型对照组细胞活性明显降低,MDA、LDH含量、ROS水平明显增高,SOD活力明显降低(P<0.05);HIF-1α、NRF2、HO-1蛋白表达明显上调;核碎裂细胞增加,凋亡率明显增加(P<0.05);与模型对照组比较,温阳通脉方含药血清5%、10%、20%组均能明显改善缺氧/复氧损伤H9c2细胞活性,减少MDA、LDH含量(P<0.05),增加SOD活力(P<0.05),减少ROS含量,下调HIF-1α蛋白表达(P<0.05);不规则形态、核碎裂细胞和凋亡率减少(P<0.05);温阳通脉方20%含药血清组ROS水平、NRF2、HO-1蛋白表达明显上调(P<0.05)。结论:温阳通脉方能减轻缺氧/复氧诱导的H9c2细胞氧化应激损伤,分子机制可能与调节NRF2/HO-1通路有关。

Objective:To investigate the mechanism of Wenyang Tongmai Formula(温阳通脉方)(WTF)against oxidative stress in H9 c2 cells after hypoxia/reoxygenation(H/R)injury based on the nuclear factor NF-E2-related factor 2(NRF2)/heme oxygenase-1(HO-1)signaling pathway.Methods:Blank serum and WTF-containing serum were prepared and then CoCl_(2) and fresh medium were used to induce H/R injury in H9 c2 cells.Cells were classified into blank control group(CON group),model group(H/R group),and 13 g/kg WTF-containing serum groups(5%,10%,20%).Cell Counting Kit-8(CCK-8)assay was used to detect cell viability.The content of malondialdehyde(MDA),superoxide dismutase(SOD),and lactate dehydrogenase(LDH)was measured by colorimetry.The level of reactive oxygen species(ROS)was detected by DCFH-DA probe.The protein expression of hypoxia-inducible factor-1α(HIF-1α),NRF2,and HO-1 was determined by Western blotting.The apoptosis rate and morphological changes were detected by Hoechst 33342 staining.Results:Compared with CON group,H/R group displayed low cell viability,high content of MDA and LDH,high ROS level(P<0.05),low SOD activity(P<0.05),high protein expression of HIF-1α,NRF2,and HO-1,many cells with fragmentation of nuclei,and high apoptosis rate(P<0.05).Compared with H/R group,WTF-containing serum(5%,10%,20%)improved the viability of H9 c2 cells with H/R injury,decreased the content of MDA and LDH(P<0.05),enhanced the SOD activity(P<0.05),and reduced ROS content,HIF-1αprotein expression(P<0.05),cells with irregular morphology and nuclear fragmentation,and apoptosis rate of cells(P<0.05).The 20%WTF-containing serum up-regulated the ROS level and the protein expression of NRF2 and HO-1(P<0.05).Conclusion:WTF can alleviate the oxidative stress of H9 c2 cells with H/R injury by regulating the NRF2/HO-1 pathway.