目的研究越橘中原花青素对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导新生大鼠心肌成纤维细胞(cardiac fibroblast,CFb)增殖的影响及其作用机制。方法用AngⅡ诱导新生大鼠CFb增殖,采用MTT法检测细胞增殖,ELISA法测定Ⅰ型胶原、Ⅲ型胶原及转化生长因子β1(TGF-β1)的量,硝酸还原酶法、分光光度法检测NO、一氧化氮合酶(i NOS)活性,流式细胞仪检测细胞周期,免疫细胞化学法测定p27蛋白的量。结果原花青素(25、50、100 mg/L)可抑制AngⅡ诱导的CFb增殖、胶原合成增加及TGF-β1蛋白量增多,并明显提高CFb NO的量、i NOS的活性,同模型组比较差异有显著性(P<0.05、0.01),且呈现剂量依赖性。流式细胞仪检测表明G0/G1期细胞比例随原花青素质量浓度增加而增加,S期细胞比例随原花青素质量浓度增加而减少,与模型组相比差异有显著性(P<0.05、0.01),免疫细胞化学染色结果也表明原花青素可增加p27的蛋白表达,与模型组相比具有统计学意义(P<0.01)。结论原花青素通过促进p27的表达,使细胞周期阻滞于G0/G1期,从而抑制CFb增殖和胶原分泌量的增加。

Objective To investigate the effect of procyanidin from Vaccinium vitisidaea on the proli-feration of cardiac fibroblast(CFb) induced by angiotensin Ⅱ(AngⅡ),and to explore its mechanism.Methods The CFb proliferation of cultured neonatal rat was induced by AngⅡ and detected by MTT assay.The levels of collagen Ⅰ,collagen Ⅲ,and TGF-β1 were measured by ELISA.The change of NO content and iNOS activity were measured by nitric acid reductase method and spectrophotometry.Cell cycle was assessed via flow cytometry(FCM). The expression of cell cycle regulatory protein p27 was determined by the combination of immunocytochemical staining and image analysis software.Results CFb Proliferation,collagen content,and TGF-β1 levels in culture medium were markedly inhibited when CFb were treated with procyanidin at concentration of 25,50,and 100 mg/L(P0.05 and 0.01).And iNOS-NO system activities in CFb were incerased(P0.05 and 0.01).In the FCM analysis,it was found that procyanidin could block CFb in the G0/G1 phase from entering the S phase,resulting in more cells in the G0/G1 phase and fewer in the S phase.The percentage of the cells in the G0/G1 phase and the S phase at the dosage of 25,50,and 100 mg/L were significantly different in comparison to the model group(P0.05 and 0.01).Procyanidin induced p27 expression with statistic significance compared to model group(P0.01).Conclusion The inhibition of procyanidin on CFb proliferation might be due to the stimulation of p27 expression and arresting of cells in G0/G1 phase.

吉林省科技厅发展计划项目资助课题(200705405)