The Mechanism Of Shenkangling Treating Epithelial-Mesenchymal Transition In Renal Tubular Epithelial Cells In Rat Through P38MAPK Signal Pathway

Objective:To observe the effects of Shenkangling (a traditional Chinese medicine with the effect of benefiting the kidney and promoting the blood circulation) on the rat renal tubular epithelial cells and the difference of expression of p38mitogen-activated protein kinase (p38MAPK). MAPK Kinas3/6(MKK3/6) and cAMP-responsive element binding protein (CREB) in renal tubular epithelial cells before and after transforming growth factor-betal (TGF-βl) induced, besides, the influence of a-smooth muscle actin (a-SMA) and fibronectin (FN). In order to clarify the possible mechanism of Shenkangling in treatment of primary nephritic syndrome (PNS) through preventing renal interstitial fibrosis.Methods:Rat renal tubular epithelial cells induced by TGF-β1were cultured in vitro. The effect of different concentration shenkangling on cells were evaluated by MTT at24h and48h, and choose the most proper concentration for subsequent experiment. Cells were divided into3groups:control groups, TGF-β1groups and Shenkangling groups. Western blotting was used to measure the expression of a-SMA. Cells were divided into4groups:control group、 TGF-β1group, Shenkangling group and SB203580group. Expression of p38MAPK. MKK3/6, CREB and their phosphorylated proteins were determined by western blotting. RT-PCR was performed to detect the expression of FNmRNA. All tests were repeated with more than three times and the data were analyzed by one-way ANOVA and T-Test.Results:(1) MTT result showed that there were not significant change of cells proliferation rate in all groups after the treatment of different concentration Shenkangling (compared with control group, all groups-P=0.231>0.05) at24h. Compared with control group, the proliferation rate of all groups change manifestly after48h (P-0.000<0.01); between6.25ug/ml、12.5ug/ml、25ug/ml, the concentration of Shenkangling increased (the three groups P<0.01). the growth of cell was evidently raise, although there still were raise, compared with25ug/ml group, the rate of50μg/ml group and100μg/ml group had no obvious change (the three groups P=0.900>0.05).(2) Western blot results showed that:1> compared with control group,expression of a-SMA was up-regulated in TGF-β1group(.P=0.01<0.05), while was down-regulated in Shenkangling group(P=0.018<0.05).2) compared with control group, the expression of MKK3/6, p38, CREB phosphorylation protein was significantly increased in TGF-β1group(all groups P=<0.05), and inhibited in Shenkangling group(PMKK=0.012<0.05、Pp38=0.015<0.05、PCREB=0.011<0.05), it was returned to normal by SB203580(PMkk=0.001<0.01、Pp38=0.004<0.01、PCREB=0.0061<0.01).(3) RT-PCR result showed that: compared with control group, the expression of FN mRNA were significant increased(P=0.016<0.05), while it could be decreased by Shenkangling,and had similar role with SB203580group(P=0.048<0.05和P=0.005<0.01).Conclusion:Shenkangling, a traditional Chinese medicine with the effect of benefiting the kidney and promoting the blood circulation, could hinder the process of epithelial-mesenchymal transition after TGF-β1induced by hampering the MKK3/6-p38-CREB signal pathway,the MKK3/6、p38、CREB is crucial working spot.