| Objective: To observe the effect of Chinese medicine Shenxiankang on EMT of NRK-52 E cells induced by TGF-?1 in vitro.And to explore the function and mechanism of shenxiankang on Renal interstitial fibrosis in molecular level.Methods:The SD rats were fed with shenxiankang and Benazepril orally,two times a day for a period of five days.Five days later,rat abdominal aorta blood serum was separated,that was the shenxiankang and Benazepril containing serum.The NRK-52 E cells were cultured in DMEM/High-glouse with 10% FBS.The growth morphology and proliferation of cells were used to evaluate if the the normal rat serum?shenxiankang and Benazepril containing serum had toxic effcet on NRK-52 E cells in vitro.Cells were divided into six different groups:Normal control group(group A:10% normal rat serum),Model group(group B),Low dosage group(group C:2.5% shenxiankang containing rat serum),Medium dosage group(group D:5% shenxiankang containing rat serum),High dosage group(group E:10% shenxiankang containing rat serum),Benazepril group(group F:10% Benazepri1 containing rat dserum).The cells of the last five groups were stimulated by TGF-?1(10ng/ml).After coincubation 72 h with drugs,?-SMA ? Smad3 and Smad7 expression were observed by Immunofluorescence assay,the mRNA expression of ?-SMA?TGF-?1?Smad3 and Smad7 were tested by Real-time PCR.Results: 1?The normal rat serum?shenxiankang and Benazepril containing serum had no toxic effcet on NRK-52 E cells in vitro.2?The changes of cells on shape:The cells of group A were polygonal or oval,showing the shape of paving stones on the culture dish;Most of the cells in group B lost their original shape,and they were spindle shaped,and the arrangement was not close;In group C,most of the cells were spindle shaped,with a small number of them paving stones;In group D,some of the cells were oval and the others were spindle shaped;Most of the cells in group E were still ovoid,and some of them changed into spindle shape and that was similar to group F.3 ? Cellular immunofluorescence assay:In group A,there was no expression of ?-SMA protein,a small amount of Smad3 protein was expressed in cytoplasm,and almost no Smad3 protein was expressed in the nucleus.A lot of Smad7 protein was expressed in cytoplasm and nucleus.The expression of ?-SMA protein in cytoplasm and the expression of Smad3 protein in cytoplasm and nucleus of cells in group B were significantly higher than that of group A,while the expression of Smad7 protein in group B was significantly decreased compared with group A.Compared with group B,the expression of ?-SMA and Smad3 in group C?group D and group E were decreased but the expression of Smad7 was increased with the increase of the dose of shenxiankang.A small number of ?-SMA and Smad3 protein expression were observed,and most of the Smad7 protein expressed in group F.The expression of ?-SMA,Smad3 and Smad7 in group F were similar to that in group E.4?Testing the mRNA expression of ?-SMA ?TGF-?1?Smad3 by Real-time PCR:A small amount of ?-SMA ?TGF-?1?Smad3 mRNA and a large number of Smad7 mRNA expressed in group A.The expression of ?-SMA ? TGF-?1 ? Smad3 mRNA in group B were increased(P<0.05)and Smad7 mRNA was decreased(P<0.05)compared with group A,but ?-SMA ? TGF-?1 ? Smad3 mRNA significantly decreased(P<0.05)and Smad7 mRNA increased(P<0.05)in group C?group D ? group E and group F compared with group B.And in shenxiankang groups the decrease in ?-SMA ?TGF-?1?Smad3 mRNA and the increase in Smad7 mRNA showed dose-dependent.Conclusion:1.Shenxiankang containing serum had no poisonous on NRK-52 E cells.2.TGF-?1 can stimulate NRK-52 E cells transdifferentiation,up regulate the expression of TGF-?1??-SMA and Smad3,down regulate the expression of Smad7,and activate the TGF-?1/Smads pathway.3.TGF-?1 can inhibit the differentiation of NRK-52 E cells,and its mechanism may be related to decreasing the expression of TGF-?1??-SMA and Smad3,and increasing the expression of Smad7. |
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