加参方对TGF-β1诱导的大鼠心肌成纤维细胞的细胞增殖、胶原分泌及分化的实验研究

Effects of Jia-Shen Prescription on TGF-β1 Induced Cell Proliferation, Collagen Secretion and Differentiation of Cardiac Fibroblasts in Rats

目的:研究加参方对转化生长因子β1(Transforming Growth Factor-β1,TGF-β1)诱导的大鼠心肌成纤维细胞的细胞增殖、胶原分泌及分化的影响,同时对其机制进行初步探讨。方法:出生1-3天的SD大鼠,无菌条件下取出心脏,胰酶消化心肌组织,分离心肌成纤维细胞(Cardiac Fibroblasts,CFs)。采用10 ng·m L-1浓度的TGF-β1诱导CFs,0.25 mg·m L-1浓度的加参方药液作用于CFs 24 h。采用CCK-8计数法检测加参方对CFs细胞增殖的影响;免疫荧光法检测加参方对CFsα-平滑肌肌动蛋白(α-Smooth Muscle Actin,α-SMA)及Ⅰ型胶原的影响;羟脯氨酸测定法检测加参方对CFs胶原分泌的影响;实时荧光定量PCR(Real Time-PCR,RT-PCR)检测加参方对CFs Smad2、Smad3 m RNA表达的影响;蛋白印迹法(Western blot)检测加参方对CFs磷酸化Smad2、磷酸化Smad3(Phosphorylated-Smad2/3,P-Smad2/3)蛋白表达的影响。结果:与模型组相比,加参方可明显抑制CFs的细胞增殖(P<0.05),降低α-SMA及Ⅰ型胶原的表达量(P<0.05),减少CFs的胶原分泌(P<0.05)。同时,加参方组Smad2、Smad3 m RNA较模型组明显下调(P<0.05),P-Smad2、P-Smad3蛋白表达量也明显下降(P<0.05)。结论:加参方对TGF-β1诱导的大鼠心肌成纤维细胞的细胞增殖、胶原分泌及向肌成纤维细胞的分化具有抑制作用,这可能与加参方抑制TGF-β1/Smads信号通路的激活有关。

This study was aimed to determine the effects of Jia-Shen Prescription（JSP） on transforming growth factor-β1（TGF-β1） induced cell proliferation, collagen secretion and differentiation of cardiac fibroblasts（CFs） in rats. At the same time, its possible mechanism was also explored. The heart of 1-3 days SD rat was removed under aseptic conditions. The trypsin was used to digest cardiac tissues. And then, CFs were obtained. CRs was induced with 10 ng·m L-1 TGF-β1. The 0.25 mg·m L-1 JSP was used with CFs for 24 hours. CCK-8 assay was used to determine the effects of JSP on cell proliferation of CFs. Immunofluorescence was used to detect the effects of JSP on the expression levels of α-smooth muscle actin（α-SMA） and type I collagen. Hydroxyproline assay was used to detect the effects of JSP on CFs collagen secretion. Real time-PCR（RT-PCR） was used to detect the effects of JSP on m RNA expression of Smad2 and Smad3 in CFs. Western blot was used to detect the effects of JSP on the protein expression of phosphorylated-Smad2/3（p-Smad2/3）. The results showed that compared to the model group, JSP significantly inhibited cell proliferation（P0.05） and collagen secretion（P0.05）, as well as reduced the expression levels of α-SMA and type I collagen of CFs（P0.05）. Meanwhile, the m RNA expressions of Smad2 and Smad3 in the JSP group were significantly downregulated compared to the model group（P0.05）. The protein expressions of p-Smad2 and p-Smad3 were also obviously decreased（P0.05）. It was concluded that JSP inhibited cell proliferation, collagen secretion and differentiation of CFs in rats induced by TGF-β1, which may be related to the inhibition of TGF-β1/Smads signaling way.