ABSTRACT
Objective To determine the inhibitory effect of a synthetic hexa-peptide GGWSHW (G peptide) derived from thrombospondin-1 (TSP1) on TGF-? activation induced by angiotensin Ⅱ (Ang Ⅱ) 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 Ang Ⅱ for 1-24 hours (Ang Ⅱ stimulation group),so that optimal dosage and duration could be chosen.One hour prior to induction,HK-2 cells were pretreated with 10 ?mol/L peptide G (G peptide treated group)or losartan (losartan treated group), the blocker of I type receptor of Ang Ⅱ was acted as inhibitory control.The mRNA and protein levels of TSP1,TGF-?1,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-?1 and co-positive expression of two protein, respectively.The concentrations of total and active TGF-?1 as well as FN and PAI-1 in cell culture supematants were measured by ELISA. Additionally, the expression of Smad2 and p-Smad2 was also examined for the bioactivity of TGF-?1 signaling protein. Results Ang Ⅱ enhanced the expression of TSP1 and TGF-?1 in a temporal-spatial dependent manner. The optimal dosage and duration were 1 ?mol/L and 6 hours,for TSP1,and 0.1 ?mol/L and 12 hours for TGF-?1 respectively.Comparing with untreated HK-2,the co-expression of TSP1 and TGF-?1 induced by A Ⅱ 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-?1,whereas it significantly reduced the secretion of active TGF-?1,though total level of TGF-?1 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-?1 activation through a competitive mechanism and also reduces the secretion of FN and PAI-1 associated with fibrosis.