参竹心康汤调控Wnt/β-catenin信号通路减轻阿霉素诱导的心肌细胞损伤机制

Mechanism of Shenzhu Xinkang Decoction alleviating adriamycin-induced myocardial cell injury by regulating Wnt/β-catenin signal pathway

关键词:  参竹心康汤  Wnt/β-catenin信号通路  阿霉素  凋亡  氧化应激  心肌细胞

Key words:  Shenzhu Xinkang Decoction  Wnt/β-catenin signal pathway  adriamycin  apoptosis  oxidative stress  cardiac muscle cells

摘要:

[目的] 探究参竹心康汤（SZXK）是否通过调控Wnt/β-catenin信号通路减轻阿霉素（Dox）诱导的心肌细胞损伤。[方法] 体外培养大鼠H9c2心肌细胞，将其分为对照组（Control组）、Dox组（1 μmol/L Dox刺激H9c2细胞24 h）、SZXK低剂量组（SZXK-L组，20 μmol/L）、SZXK中剂量组（SZXK-M组，40 μmol/L）、SZXK高剂量组（SZXK-H组，80 μmol/L）、SZXK高+中剂量组（SZXK-H+M组，MSAB，Wnt/β-catenin通路抑制剂）。通过噻唑蓝（MTT）比色法检测SZXK对H9c2细胞的毒性，采用细胞计数试剂盒8（CCK-8）检测细胞活力，流式细胞术检测细胞凋亡，蛋白免疫印迹法（Western blot）检测B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）、Wnt/β-catenin信号通路蛋白的表达水平。试剂盒检测肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和活性氧（ROS）、超氧化物歧化酶（SOD）、丙二醛（MDA）水平。[结果] 与Control组相比，模型组的细胞活力、Bcl-2及SOD水平显著下降，细胞凋亡率、Bax水平、ROS、MDA、IL-6、TNF-α、IL-1β水平升高（P<0.05），提示心肌细胞损伤模型建立成功。而相比模型组，SZXK呈剂量依赖性提升心肌细胞活力，抑制细胞凋亡，缓解氧化应激损伤及炎症反应。此外，与Control组相比，Dox组的Wnt3a和β-catenin核蛋白水平显著降低，β-catenin质蛋白水平升高（P<0.05）；而SZXK处理后，心肌细胞Wnt3a和β-catenin核蛋白水平显著升高，β-catenin质蛋白水平降低，且变化成剂量依赖性（P<0.05）。而抑制Wnt/β-catenin通路部分逆转SZXK对Dox诱导的心肌细胞损伤的改善作用。[结论] SZXK可能通过激活Wnt/β-catenin通路发挥对Dox诱导心肌细胞损伤的保护作用。

Abstract:

[Objective] To investigate whether Shenzhu Xinkang Decoction（SZXK） can alleviate adriamycin（Dox） -induced myocardial cell injury by regulating Wnt/β-catenin signal pathway. [Methods] Rat H9c2 cardiomyocytes were cultured in vitro and divided into the following groups：control group，Dox group（H9c2 cells were stimulated with 1 μmol/L Dox for 24 h），SZXK-L group（low dose，20 μmol/L），SZXK-M group（medium dose，40 μmol/L），SZXK-H group（high dose，80 μmol/L），SZXK-H+M group（MSAB，Wnt/β-catenin pathway inhibitor）. The cytotoxicity of SZXK on H9c2 cells was detected by methyl thiazol tetrazolium（MTT） assay，cell viability was detected by cell counting kit-8（CCK-8），and cell apoptosis was detected by flow cytometry. Western blot was used to detect the expression levels of B-cell lymphoma-2（Bcl-2），Bcl-2 associated X protein（Bax） and Wnt/β-catenin signal pathway proteins. The levels of tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β），interleukin-6（IL-6），reactive oxygen species（ROS），superoxide dismutase（SOD） and malondialdehyde（MDA） were detected by kits. [Results] Compared with the control group，the cell viability，Bcl-2 and SOD levels in the model group were significantly decreased，while the apoptosis rate，Bax level，ROS，MDA，IL-6，TNF-α and IL-1β levels were increased（P<0.05），indicating that the model of myocardial cell injury was successfully established. Compared with the model group，SZXK dose-dependently increased cardiomyocyte viability，inhibited cell apoptosis，and alleviated oxidative stress injury and inflammatory response. In addition，compared with the Control group，the levels of Wnt3a and β-catenin nuclear protein in the Dox group were significantly decreased，and the level of β-catenin protein was increased（P<0.05）. After SZXK treatment，the Wnt3a and β-catenin nuclear protein levels were significantly increased，and the β-catenin protein level was decreased in a dose-dependent manner（P<0.05）. Inhibition of Wnt/β-catenin pathway partially reversed the protective effect of SZXK on DOX-induced myocardial cell injury. [Conclusion] SZXK may exert a protective effect on DOX-induced cardiomyocyte injury by activating the Wnt/β-catenin signal pathway.