G肽对血管紧张素Ⅱ诱导人肾小管上皮细胞转化生长因子β1活化的影响

目的观察根据血小板反应蛋白1（TSP1）分子WSHW序列人工合成的六肽（GGWSHW，G肽）对由血管紧张素Ⅱ（AngⅡ）诱导的肾小管上皮细胞TGF-β1过度活化的抑制作用。方法体外培养人近端肾小管上皮细胞系（HK-2），以未处理HK-2细胞为正常对照，以AⅡ刺激HK-2细胞（AngⅡ组），选择合成TSP1和TGF-β1最佳刺激浓度和时间。刺激前加入10μmol／LG肽进行干预（G肽组），并以AngⅡ受体阻断剂洛沙坦（losartan）为抑制对照（ losartan组）。分别用RT-PCR和Western印迹检测TSP1和TGF-β1以及细胞外基质纤连蛋白（FN）和纤溶酶原活化物抑制剂-1（PAI-1）mRNA转录和蛋白表达。共聚焦显微镜和流式细胞仪观察TSP1和TGF-β1的表达以及两者共表达。ELISA法检测培养细胞上清液中活性TGF-β1、总TGF-β1、FN和PAI-1的分泌含量。Western印迹检测TGF-β1信号转导蛋白Smad2及磷酸化Smad2（p-Smad2）表达水平。结果AngⅡ以时间和剂量依赖方式促进HK-2细胞合成TSP1和TGF-β1（优化后分别为6h的1μmol／L，12h的0．1μmol／L）。与正常对照组比较，AngⅡ组TSP1与TGF-β1阳性共表达的细胞数上调5．4倍；总TGF-β1和活性TGF-β1水平分别增加1．8和2．0倍；p-Smad2蛋白水平明显上调；FN和PAI-1 mRNA转录水平分别上调3和1．5倍，且细胞上清液中两者含量分别增加2和1．9倍。G肽对TSP1和TGF-β1 mRNA转录和蛋白表达水平均无显著性影响，且对总TGF-β1分泌无明显抑制作用，但可显著抑制活性TGF-β1的水平。此外与losartan组比较，G肽组p-Smad2蛋白表达减少28．9％。FN和PAI-1 mRNA转录水平分别下调34．5％和26％，且细胞上清液中两者含量分别减少11％和8．9％。结论TSP1肽抑制物体外能通过有效竞争抑制TSP1致HK-2细胞的TGF-β1活化作用，并可相应下调FN和PAI-1的合成分泌。

Inhibitory peptide GGWSHW influences the activation of TGF-β1 induced by angiotensin Ⅱ in human renal tubular epithelial cell

Objective To determine the inhibitory effect of a synthetic hexa-peptide GGWSHW (G peptide) derived from thrombospondin-1(TSP1) on TGF-β activation induced by angiotensin Ⅱ (AngⅡ) in cultured human renal tubular epithelial cells. Methods Human proximal tubular epithelial cell line(HK-2) was cultured in vitro， untreated HK-2 cells were acted as normal control group. HK-2 cells were then stimulated by AngⅡ for 1~24 hours (AngⅡ stimulation group)， so that optimal dosage and duration could be chosen. One hour prior to induction， HK-2 cells were pretreated with 10 μmol/L peptide G(G peptide treated group)or losartan (losartan treated group)， the blocker of Ⅰ type receptor of AngⅡ was acted as inhibitory control. The mRNA and protein levels of TSP1， TGF-β1， FN and PAI-1 were measured by RT-PCR and Western blot. Confocal microscopy and flow cytometry were performed to detect the presence of TSP1， TGF-β1 and co-positive expression of two protein， respectively. The concentrations of total and active TGF-β1 as well as FN and PAI-1 in cell culture supernatants were measured by ELISA. Additionally， the expression of Smad2 and p-Smad2 was also examined for the bioactivity of TGF-β1 signaling protein.Results AngⅡ enhanced the expression of TSP1 and TGF-β1 in a temporal-spatial dependent manner. The optimal dosage and duration were 1 μmol/L and 6 hours， for TSP1， and 0.1 μmol/L and 12 hours for TGF-β1 respectively. Comparing with untreated HK-2，the co-expression of TSP1 and TGF-β1 induced by AⅡ showed a increase of 5.4 folds. In addition， the protein level of p-Smad2 was elevated remarkedly， the mRNA level of FN and PAI-1 was up-regulated by 3 and 1.5 folds， and the concentration was increased by 2.0 and 1.9 folds respectively. Peptide G had less effect on the expression of TSP1 and TGF-β1， whereas it significantly reduced the secretion of active TGF-β1， though total level of TGF-β1 remained up-regulated. Furthermore， comparing with losartan treated group， p-Smad2 expression was reduced by 28.9%， the mRNA level of FN and PAI-1 was decreased by 34.5% and 26% respectively， and the protein levels were reduced by 11.0% and 8.9% respectively. Conclusion The inhibitory peptide derived from TSP1 effectively suppresses TGF-β1 activation through a competitive mechanism and also reduces the secretion of FN and PAI-1 associated with fibrosis.