Zhang Xiao-lan, Jiang Hui-qing, Liu Li, Bai Yun, Song Mei
Department of Gastroenterology, Second Hospital of Hebei Medical University, Shijiazhuang 050000, China.
Zhonghua Gan Zang Bing Za Zhi. 2003 Aug;11(8):479-82.
To investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on integrin signaling and apoptosis in fibronectin (FN) -stimulated hepatic stellate cells (HSCs).
3H-thymidine incorporation, annexin-V/propidium iodide double-labeled flow cytometry (FCM) and transmission electron microscopy were employed to estimate the influence of RGDS on the proliferation and apoptosis of HSCs. And the adhesion rates were observed by toluidine blue colorimetric assay. The expression of focal adhesion kinase (FAK) mRNA and protein in HSCs was detected using RT-PCR and western blotting analysis, respectively.
RGDS tetrapeptide at the concentrations of 25 microg/ml, 50 microg/ml and 100 microg/ml inhibited the proliferation of HSCs and induced HSCs apoptosis in dose-dependent and time-dependent manners, with the apoptotic rates of 9.49%, 27.67%, 31.59%, and the necrotic rates of 3.47%, 5.38%, 9.10%, respectively. Both the rates were higher than those in FN group (apoptotic rate: 3.44%; necrotic rate: 2.39%), F=8.02, P<0.05. After adding RGDS tetrapeptide to HSCs for 2 hours, the adhesive inhibition rates were 8.82%, 29.41% and 45.59%, respectively, which were higher than that in FN group (F=20.58, P<0.01). After exposure of HSCs to RGDS tetrapeptide for 24 hours, FAK protein decreased, and FAK mRNA was down-regulated earlier, about 2 hours after exposure to RGDS tetrapeptide.
These results suggest that RGDS tetrapeptide may induce apoptosis of HSC in both dose-dependent and time-dependent manners in vitro, which may be related to the disruption of cell matrix adhesion and down-regulation of FAK expression.
研究精氨酸 - 甘氨酸 - 天冬氨酸 - 丝氨酸（RGDS）四肽对纤连蛋白（FN）刺激的肝星状细胞（HSCs）中整合素信号传导及细胞凋亡的影响。
采用³H - 胸腺嘧啶核苷掺入法、膜联蛋白V/碘化丙啶双标记流式细胞术（FCM）及透射电子显微镜观察RGDS对HSCs增殖和凋亡的影响。通过甲苯胺蓝比色法观察黏附率。分别采用逆转录 - 聚合酶链反应（RT - PCR）和蛋白质免疫印迹分析检测HSCs中黏着斑激酶（FAK）mRNA和蛋白的表达。
浓度为25μg/ml、50μg/ml和100μg/ml的RGDS四肽以剂量和时间依赖性方式抑制HSCs增殖并诱导其凋亡，凋亡率分别为9.49%、27.67%、31.59%，坏死率分别为3.47%、5.38%、9.10%。两者均高于FN组（凋亡率：3.44%；坏死率：2.39%），F = 8.02，P < 0.05。向HSCs中加入RGDS四肽2小时后，黏附抑制率分别为8.82%、29.41%和45.59%，高于FN组（F = 20.58，P < 0.01）。HSCs暴露于RGDS四肽24小时后，FAK蛋白减少，FAK mRNA在暴露于RGDS四肽约2小时后较早下调。
这些结果表明，RGDS四肽在体外可能以剂量和时间依赖性方式诱导HSC凋亡，这可能与细胞 - 基质黏附的破坏及FAK表达下调有关。
