西红花酸对心肌成纤维细胞增殖和胶原合成的影响

目的: 探讨西红花酸(crocetin)对血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)诱导的心肌成纤维细胞(cardiac fibroblasts, CFB)增殖和胶原合成的影响及其作用机制。方法: 体外培养新生SD大鼠CFB,建立AngⅡ诱导新生大鼠CFB纤维化模型;MTT法检测CFB的增殖;羟脯氨酸测定检测CFB胶原含量;流式细胞分析仪测定细胞周期;实时荧光定量PCR(Q-PCR)检测Ⅰ型、Ⅲ型胶原蛋白、间质胶原酶(MMP-2)、组织金属蛋白酶抑制因子-1(TIMP-1)、转化生长因子β₁(TGF-β₁)的基因表达;蛋白免疫印迹(Western Blot)检测细胞P27^kip1 (P27)蛋白的表达。结果: (1)在一定浓度范围里,crocetin能抑制AngⅡ引起的CFB增殖和胶原合成,并呈剂量依赖;(2)CFB G₀/G₁期百分率随crocetin浓度增加而增加,S期、G₂/M期百分率和增殖指数随crocetin浓度增加而减少,与AngⅡ组相比差异有统计学意义(P<0.05 或P<0.01);(3)Crocetin能明显降低Ⅰ型、Ⅲ型胶原、TIMP1、TGF-β₁的表达,提高MMP-2的表达;(4)Crocetin能增加P27蛋白表达。结论: Crocetin通过抑制AngⅡ诱导的CFB增殖和胶原合成起到抗心肌纤维化作用,其机制可能与细胞因子分泌和对基质金属蛋白酶的调节有关。

Effects of crocetin on the proliferation and collagen synthesis of cardiac fibroblasts

AIM: To investigate the effects of crocetin on proliferation and collagen synthesis in cultured neonatal rat cardiac fibroblasts (CFB) stimulated by angiotensinⅡ(AngⅡ), and to explore the action mechanism of crocetin. METHODS: We used cultured cardiac fibroblasts from neonatal 1-3 days rat and treating with AngⅡ to induce fibrosis model. The effects of crocetin on proliferation of CFB were observed by MTT assay, synthesis of collagen was observed by the hydroxyproline concentration measured. Cell cyclin distribution was determined with flow cytometre. CollagenⅠ, collagen Ⅲ, matrix metalloproteinase (MMP-2), the tissue inhibitor metalloproteinase (TIMP-1) and TGF-β₁ mRNA expression were examined by Q-PCR. The expression of P27 was tested by Western blotting. RESULTS: Within a concentration coverage, crocetin inhibited CFB proliferation and collagen synthesis induced by AngⅡ in a dose-dependent manner. The percentage of cells in G₀/G₁ phase in crocetin groups increased with concentration, the percentage of cells in S and G₂/M phases and the proliferation index in crocetin groups decreased with concentration, which compared with AngⅡ group difference was statistically significant (P<0.05, P<0.01, respectively). Crocetin decreased the levels of CollagenⅠmRNA, Collagen Ⅲ mRNA, TIMP1 mRNA and TGF-β₁ mRNA expression, but increased MMP-2 mRNA expression. ④Crocetin decreased the protein expression of P27 stimulated by AngⅡ. CONCLUSION: Crocetin inhibited CFB proliferation and collagen synthesis stimulated by AngⅡ, which related to the cytokines excreting and the regulation of extracellular matrix.