通心络含药血清对高葡萄糖环境下大鼠心肌成纤维细胞增殖的影响

To study the effect of Tongxinluo drug-containing serum on the proliferation of rat cardiac fibroblasts under high glucose

关键词:  大鼠心肌成纤维细胞  甘露醇  含药血清  TGF-β1/p38MAPK/CREB信号通路

Key words:  rat cardiac fibroblasts  mannitol  drug-containing serum  TGF-β1/p38MAPK/CREB signalling pathway

摘要:

[目的] 基于转化生长因子-β1（TGF-β1）-p38丝裂原活化蛋白激酶（p38MAPK）-环磷腺苷效应元件结合蛋白（CREB）信号通路探讨通心络含药血清对大鼠高葡萄糖环境下心肌成纤维细胞增殖的影响。[方法] 原代分离大鼠心肌成纤维细胞，运用免疫荧光染色鉴定细胞。采用甘露醇与葡萄糖作用于细胞不同时间节点，通过细胞增殖及毒性检测（CCK-8）方法检测细胞活力筛选最佳造模时间，造模成功后使用不同浓度的通心络含药血清及阳性药物——缬沙坦含药血清处理细胞。CCK-8方法检测细胞增殖，流式细胞仪检测细胞凋亡，免疫荧光染色检测TGF-β1蛋白表达水平，酶联免疫吸附（ELISA）法检测大鼠心肌组织中半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）、B淋巴细胞瘤-2（Bcl-2）和TGF-β1表达水平，蛋白免疫印迹（Western blot）法检测TGF-β1、磷酸化p38MAPK（p-p38MAPK）、磷酸化CREB（p-CREB）蛋白表达水平。[结果] 免疫荧光检测波形蛋白（Vimentin）的表达可以成功鉴定大鼠心肌成纤维细胞，阳性率达到90%以上。54.5 mmol/L甘露醇与25 mmol/L葡萄糖作用于大鼠心肌成纤维细胞的最佳造模时间为48 h。结果表明，模型组细胞增殖力、凋亡率较对照组升高，Caspase-3、TGF-β1、Bcl-2、p-p38MAPK及p-CREB的蛋白表达增加；不同浓度含药血清组及阳性药物含药血清组凋亡率较模型组降低，Caspase-3、Bcl-2、TGF-β1、p-p38MAPK及p-CREB的蛋白表达降低，差异具有统计学意义（P<0.05）。[结论] 采用甘露醇与葡萄糖可以导致大鼠心肌成纤维细胞损伤，通过不同剂量的通心络含药血清及阳性对照药物——缬沙坦含药血清处理均可以对大鼠心肌成纤维细胞发挥保护作用，其中中剂量通心络含药血清对高葡萄糖环境下大鼠心肌成纤维细胞的修复效果最佳，并可能通过激活TGF-β1/p38MAPK/CREB信号通路发挥保护作用。

Abstract:

[Objective] To investigate the effect of tongxinluo（TXL） containing serum on the proliferation of rat cardiac fibroblasts under high glucose，based on the transforming growth factor β1-p38 mitogen-activated protein kinase-cyclophosphate-adenosine receptor binding protein（TGF-β1/p38MAPK/CREB） signaling pathway. [Methods] Primary rat cardiac fibroblasts were isolated and immunofluorescence staining was used to identify the cells. After successful modelling，the cells were treated with different concentrations of tongxinluo containing serum and positive drug valsartan containing serum，cell proliferation was detected by CCK-8，apoptosis was detected by flow assay，TGF-β1 protein expression was detected by immunofluorescence staining，and TGF-β1 protein expression was detected by enzyme-linked immunosorbent assay（ELISA）. Enzyme-linked immunosorbent assay（ELISA） for cysteine aspartate protease 3（Caspase-3），B lymphocyte 2（Bcl-2） and transforming growth factor β1（TGF-β1） expression levels in rat myocardial tissue and protein blotting assay（Western blot） for TGF-β1，p-p38MAPK，p-CREB protein expression levels. [Results] Immunofluorescence detection of vimentin expression could successfully identify rat cardiac fibroblasts with a positive rate of more than 90%. The 54.5 mmol/L mannitol and 25 mmol/L glucose were applied to rat cardiac fibroblasts for an optimal modelling time of 48 h. The results showed that cell proliferation and apoptosis rates in the model group were significantly increased compared with those in the control group，and the protein expression of Caspase-3，TGF-β1，Bcl-2，p-p38MAPK and p-CREB were significantly increased compared with those in the model group，Bcl-2，p-p38MAPK and p-CREB. The apoptosis rate of different concentrations of drug-containing serum group and positive drug-containing serum group was significantly lower than that of the model group，and the protein expression of caspase-3，Bcl-2，TGF-β1，p-p38MAPK and p-CREB was significantly lower，with statistically significant differences（P<0.05）. [Conclusion] The use of mannitol and glucose can lead to myocardial fibroblast damage in rats，and treatment with different doses of Tongxinluo containing serum and positive control valsartan containing serum can play a protective role in rat myocardial fibroblasts，in which the dose of Tongxinluo containing serum has the best effect on the repair of rat myocardial fibroblasts with high glucose，and may play a protective role in myocardial fibroblasts through activation of the TGF-β1-p38 MAPK-CREB signalling pathway. CREB signalling pathway.