Etanercept protects rat cardiomyocytes against hypertrophy by regulating inflammatory cytokines secretion and cell apoptosis

We aimed to investigate the effect of etanercept, a tumor necrosis factor-α (TNF-α) inhibitor, on rat cardiomyocyte hypertrophy and its underlying mechanism. Primary neonatal rat cardiomyocytes were isolated from Sprague-Dawley rats. The model of rat cardiomyocyte hypertrophy was induced by endothelin, and then treated with different concentrations of etanercept (1, 10, and 50 μM). After treatment, cell counts, viability and cell apoptosis were evaluated. The mRNA levels of myocardial hypertrophy marker genes, including atrial natriuretic factor (ANF), matrix metalloproteinase (MMP)-9 and MMP-13, were detected by qRT-PCR, and the expressions of apoptosis-related proteins (Bcl-2 and Bax) were measured by western blotting. The protein levels of transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) were determined using enzyme linked immunosorbent assay (ELISA) kits. In the present study, TNF-α level in cardiomyocytes with hypertrophy was significantly enhanced (P<0.05). Compared to the model group, cell number and viability were significantly increased and ratio of apoptotic cells was reduced by etanercept (P<0.05, P<0.01, or P<0.001). In addition, etanercept remarkably reduced the mRNA levels of ANF, MMP-9 and MMP-13, inhibited the expression of Bax, and increased the expression of Bcl-2 compared to the model group (P<0.05). ELISA results further showed that etanercept lowered the levels of IL-1β, IL-6, LIF and CT-1 but not TGF-β1 compared to the model group (P<0.05). Etanercept may protect rat cardiomyocytes from hypertrophy by inhibiting inflammatory cytokines secretion and cell apoptosis.

本研究旨在探讨肿瘤坏死因子-α（TNF-α）抑制剂依那西普（etanercept）对大鼠心肌细胞肥大的影响及其潜在作用机制。本研究从斯普拉格-道利（Sprague-Dawley）大鼠体内分离得到原代新生大鼠心肌细胞，采用内皮素诱导建立大鼠心肌细胞肥大模型，随后以1、10、50 μM三种不同浓度的依那西普进行干预处理。干预结束后，对细胞计数、细胞活力及细胞凋亡水平进行检测评估；通过qRT-PCR检测心肌肥大标志物基因（包括心房利钠因子（atrial natriuretic factor, ANF）、基质金属蛋白酶（matrix metalloproteinase, MMP）-9及MMP-13）的mRNA表达水平，通过Western印迹法检测凋亡相关蛋白Bcl-2与Bax的表达量；采用酶联免疫吸附测定（enzyme linked immunosorbent assay, ELISA）试剂盒检测转化生长因子-β1（TGF-β1）、白细胞介素（IL）-1β、IL-6、白血病抑制因子（LIF）及心肌营养素-1（CT-1）的蛋白水平。本研究结果显示，肥大状态的心肌细胞内TNF-α水平显著升高（P<0.05）。与模型组相比，依那西普处理可显著提升细胞数量与细胞活力，并降低凋亡细胞占比（P<0.05、P<0.01或P<0.001）；此外，相较于模型组，依那西普可显著下调ANF、MMP-9及MMP-13的mRNA表达水平，抑制Bax蛋白表达并上调Bcl-2蛋白表达（P<0.05）。ELISA检测结果进一步证实，与模型组相比，依那西普可降低IL-1β、IL-6、LIF及CT-1的蛋白水平，但对TGF-β1无显著影响（P<0.05）。综上，依那西普可能通过抑制炎症因子分泌与细胞凋亡，发挥对大鼠心肌细胞肥大的保护作用。