二苯乙烯苷对TGF-β1诱导新生大鼠心肌成纤维细胞增殖和胶原合成的影响及其机制
Effects and mechanisms of stilbene glucoside on the proliferation and collagen synthesis of cardiac fibroblasts stimulated by TGF-β1
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摘要: 探讨二苯乙烯苷(2，3，5，4′-四羟基二苯乙烯-2-O-β-D葡糖苷，TSG)对转化生长因子β1(TGF-β1)诱导的新生大鼠心肌成纤维细胞(CFB)增殖和胶原合成的影响，并研究其相关机制。用消化法培养新生SD大鼠CFB，建立TGF-β1诱导新生大鼠CFB纤维化模型。采用5-溴脱氧尿嘧啶核苷(BrdU)掺入法检测CFB增殖情况；乳酸脱氢酶(LDH)法检测药物对细胞毒性作用；羟脯氨酸测定检测CFB胶原含量；流式细胞仪测定细胞周期；Western blot法检测α-平滑肌肌动蛋白(α-SMA)、增殖细胞核抗原(PCNA)、Ⅰ型胶原(COLⅠ)、Ⅲ型胶原(COL Ⅲ)、p-Smad2、Smad2、p-Smad3、Smad3、p-ERK、ERK、p-P38、P38、p-JNK、JNK的蛋白表达。结果表明，终浓度为0.1~100 μmol/L的TSG无明显细胞毒性作用；在一定浓度范围内，TSG可明显抑制TGF-β1诱导的CFB增殖和胶原合成，抑制细胞由G0/G1期向S期的转变，抑制TGF-β1诱导的CFB核内PCNA的蛋白表达，降低α-SMA、COLⅠ和COL Ⅲ的过度表达，并抑制TGF-β1诱导的Smad3、ERK和P38的磷酸化。因此推测一定浓度的TSG能抑制TGF-β1诱导的CFB增殖和胶原合成，其机制可能与干预Smad3、ERK和P38信号通路有关。Abstract: The aim of this study was to investigate the effect of stilbene glucoside(2, 3, 4′, 5-tetrahydroxystilbene-2-O-β-D glucoside, TSG)on the cell proliferation and collagen synthesis in cultured neonatal rat cardiac fibroblasts(CFBs)stimulated by transforming growth factor-β1(TGF-β1)and to explore the underlying mechanisms. CFB were cultured from neonatal rats of 1-3 days and treated with TGF-β1. CFB proliferation was analyzed by 5-bromo-2-deoxyuridine(BrdU)incorporation assay. The synthesis of collagen was examined by measurement of the hydroxyproline concentration. Flow cytometry used to examined cell cycle of CFBs treated with TSG. The expressions of α-smooth muscle actin, proliferating cell nuclear antigen(PCNA), collagen Ⅰ, collagen Ⅲ, p-Smad2, Smad2, p-Smad3, Smad3, p-ERK, ERK, p-P38, P38, p-JNK, JNK were measured by Western blot. The results of the present study showed that TSG(0. 1-100 μmol/L)to exerted no significant cytotoxicity on the cells. Within a concentration coverage, TSG inhibited CFB proliferation and collagen synthesis with inhibition of cell cycle transition from G0 /G1 phase to S phase. Furthermore, TSG decreased the protein expressions of PCNA, α-smooth muscle actin, collagen Ⅰ, Collagen Ⅲ, p-Smad3, p-ERK and p-P38 stimulated by TGF-β1. Taken together, TSG significantly inhibited CFB proliferation and collagen synthesis stimulated by TGF-β1, though, at least in part, by suppression of Smad3, ERK and P38 activation.
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