促红细胞生成素抑制血管紧张素Ⅱ诱导的大鼠心脏成纤维细胞中转化生长因子β1和胶原的表达

目的 探讨促红细胞生成素(EPO)对血管紧张素Ⅱ(AngⅡ)诱导的心脏成纤维细胞(CF)中转化生长因子(TGF)-β1蛋白表达和胶原生成的影响,以及磷脂酰肌醇-3-激酶(PD-K)/Akt信号途径和一氧化氮合酶(NOS)在其中的作用.方法 应用胰酶和胶原酶双酶法分离培养新生大鼠CF细胞,应用EPO、Ang Ⅱ、PI3-K抑制剂LY294002、NOS抑制剂L-NAME等不同因素干预.ELISA法检测CF中Ⅰ型和Ⅲ型胶原的浓度.化学酶法检测CF培养液中的NO浓度以及NOS总的活性及其亚型的活性.Western blot检测Akt、p-Akt、内皮型一氧化氮合酶(eNOS)、iNOS和TGF-β1蛋白的表达.结果 EPO剂量依赖性的抑制Ang Ⅱ诱导的CF培养液中Ⅰ型和Ⅲ型胶原表达以及提高NO的浓度.10 U/ml的EPO对Ⅰ型和Ⅲ型胶原浓度的抑制分别达到了28%和46%,同时NO浓度则提高了154%.EPO也显著抑制了Ang Ⅱ促CF中TGF-β1蛋白的表达,同时Akt的磷酸化水平显著提高,并促进eNOS蛋白的表达.应用LY294002使eNOS蛋白表达水平明显下降,培养液中的NO浓度也随之下降.L-NAME不能降低eNOS蛋白表达,但抑制了NO的生成.EPO抑制Ang Ⅱ诱导的CF中TGF-β1蛋白的表达以及Ⅰ型和Ⅲ型胶原合成作用均能被二者阻断.结论 EPO可抑制Ang Ⅱ诱导的新生大鼠CF中TGF-β1的表达以及Ⅰ型和Ⅲ型胶原表达,可能是通过激活PI3-K/Akt信号途径促使CF中eNOS表达,从而促进NO的表达来实现.

Erythropoietin suppresses the expressions of TGF-β1 and collagen in rat cardiac fibroblasts induced by angiotensin Ⅱ

Objective Recent studies have shown cardiac protection effects of erythropoietin (EPO).The present experiment was designed to investigate the effects of EPO on TGF-β1, nitric oxide synthase ( NOS), collagen contents induced by angiotensin Ⅱ ( Ang Ⅱ ) in rat cardiac fibroblasts (CFs) and explore the roles of PI3-K/Akt signaling pathway on related effects.Methods Neonatal rat CFs was isolated by collgenase and trypsinase digestion methods.PBS, EPO, Ang Ⅱ in the absence or presence of LY294002, an inhibitor of PI3-K, or L-NAME, an inhibitor of NOS, were added to CFs and cultured for 24 hours.The concentration of collagen Ⅰ and collagen Ⅲ in culture medium were quantitated by ELISA.The levels of nitric oxide (NO) and the activities of NOS as well as NOS isofonns were measured by chemical enzymic method.Western blot was applied to detecting the protein expressions of Akt, p-Akt, eNOS, iNOS, and TGF-β1.Results The concentrations of collagen Ⅰ and collagen Ⅲ in CFs culture medium were significantly increased while the level of NO was significantly decreased by Ang Ⅱ and these changes were significantly suppressed by EPO in a dose dependent manner.The effects of EPO on eNOS and NO could be blocked by LY294002.L-NAME could block EPO's effect on NO but not on the eNOS expression.The suppression effects on expressions of TGF-β1 and collagen by Ang Ⅱ in CFs were blocked by both LY294002 and L-NAME.conclusion EPO suppresses the upregulated expressions of TGF-β1 and increased production of collagen induced by Ang Ⅱ through activating the PI3-K/Akt signaling pathway in neonatal rat CFs.