| ObjectiveTubulointerstitial fibrosis (RIF) is one of the important manifestations of the development end-stage chronic kidney disease. Fibrosis of the key cellular muscle into fiber cells (MF) generation, and its accumulation in the renal interstitium and subsequent tubular atrophy have been considered a key factor of chronic RIF. The source of MF has drawn much attention, and In the past10years, reports have found that tubular epithelial cells-mesenchymal cell transdifferentiation (EMT) may be the source of MF. The role of renal tubular EMT in the development of RIF has been paid more and more attention. There are many factors of renal tubular EMT including injury,inflammation and growth factors, which fibrogenic factor TGF-(3i of EMT in renal tubular epithelial cells, is paid more and more attention. Baicalin, the extract of Chinese medicine, has effect of anti-RIF, lung fibrosis and liver fibrosis but its mechanism of action of anti-RIF wether by inhibiting renal tubular EMT or not is unclear and further research is also needed. This topic discussed the method of cell culture observation of baicalin in human proximal tubular epithelial cells HK-2cell differentiation induced by TGF-(31in vitro and discuss the possible mechanisms of the antifibrosis of baicalin, which can provide a theoretical basis of clinical application of baicalin against RIF.Material and MethodsCultured HK-2cells at logarithmic phase of growth was choosed and were divided into6experimental groups:blank control group:only including the DMEM culture medium containing serum;5μg/L TGF-β1induced concentration group;4baicalin intervention groups:5μg/L TGF-β1+40μmol/L baicalin,5μg/L TGF-β1+80μmol/L baicalin,5μg/L TGF-β1+160μmol/L baicalin;5μg/L TGF-β1+320μmol/L baicalin. According to the preliminary studies of this study, after12h inducation by TGF-β1,the HK-2cell morphological changed, and with time, the more obvious did the cells change. In this study, after72h culture in selected cells, the changes of cell morphology was observed under an inverted microscope, and at24h,48h,72h respectively, cell proliferation were observated, and the expression of Smad3mRNA, Smad7mRNA were RT-PCR assay at72h.Results1.After72h cell culture, cell shape was changed a square or slightly polygonal, the cell volume did not increase, the cell gap was not widened, the cytoplasm was still transparent, with a typical cobblestone features. HK-2cells in TGF-β1stimulation group were elongated, hypertrophy, variable-length spindle cell gap was larger, decrease of cytoplasmic transparency, loss of cobblestone growth pattern. HK-2cells in baicalin each group, cell deformation degree were less than that of TGF-β1group, and with increasing concentration of baicalin, cells spindle change were better gradually, when baicalin concentration increased to320μmol/L, the morphology of HK-2cells remained normal.2. HK-2cell proliferation were induced significantly, and the proliferative capacity increased gradually in5μg/L TGF-β1-induced group compared to blank control group(P <0.05). After the intervention of baicalin, the role of promoting cell proliferation was significantly inhibited, and the inhibition in intervention groups was more obvious with increasing baicalin concentration, and the inhibition in40μmol/L baicalin group was weaker, while the inhibition in160μmol/L,320μmol/L baicalin group were basically the same.3. After cells culture of TGF-β1-induced HK-2for72h, the expression levels of Smad3mRNA increased significantly and Smad7mRNA was in obvious decline. After the intervention of different concentrations of baicalin, with the increasing concentration of baicalin, expression of Smad3mRNA reduced gradually, but the expression of Smad7 mRNA increased gradually. The expression of Smad3mRNA and Smad7mRNA of HK-2in40μmol/L baicalin group is closer to the TGF-β1-induced group. The expression of Smad3mRNA and Smad7mRNA in160μmol/L,320μmol/L baicalin group was closer to the blank control group, and there was no significant difference between these two groups.Conclusions1.5ug/L TGF-β1can become the long fusiform of HK-2cell in shape, but baicalin can inhibit it.2. Baicalin can inhibit HK-2cell proliferation incuded by TGF-β13. The transdifferentiation role of baicalin on HK-2cell is related to the higher expression of Smad7and the lower expression of Smad3.4.The inhibition of transdifferentiation role on HK-2cell is not increased untill the density of baicalin to160μmol/L. |
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