| Objectives Glomerular sclerosis is the same common pathway which the chronici renal disease is to develop into renal failure,therefore ,it is significant for us to investigate how to prevent kidney from fibrosis .Transforming growth facto(rTGF)-β1 is considered to have been playing an important role during the glomerular sclerosis.Leflunomide is a kind of immunodepressant.Different density leflunomide was choosen to observe their effects on leflunomide (LEF )on the expression of collagenⅣand Smad2/3 of glomerularmesangial cell induced by transforming growth factor(TGF)-β1 in vitro,Further to investigate its role in TGF-β1/Smads signaling transduction pathway ,and provide the theoretical evidence for the renal protection of leflunomide.Methods After the rat GMC was treated to let it resuscitated,bred and multiplied with classical method. Passages 3~10 ofcells were used in the experiment after identification.The experiment included 4 groups:control group(Ctrl group),transforming growth factorβ1 group(TGF-β1 group) ,transforming growth factorβ1 plus leflunomide (LEF )5μg/ml group,transforming growth factorβ1 plus leflunomide (LEF )50μg/ml group.The changes of quantitative were observed at following time at the 15minute ,30minute,1hour,2hour,6hour:①The expression of P-Smad2/3 by Indirect immunof Iuorescence.②the change of of collagenⅣsecretion was detected by the enzyme-linked immunosorbent-assay (ELISA).③The change of Smad2 mRNA expression was detected by semi- quantitative real-time(RT)-PCR.Results1. Results of Indirect immunof Iuorescence:The expressions of p-Smad2/3 was significantly increased at 30min after stimulation ,lasted to two hours.Meanwhile the fluorescence was seen concentrated in both the mucelus and perimeter.After two hours ,the expression of protein deline, the fluorescence turn to weaken and disperse to cytoplasm.On the contrary, it is descendent that the P-Smad2/3 protein expression of the groups LEF , the fluorescence is poor with the longer time and the higher density.2. Test result of supernatant fluid of cells Only tiny amount of basic secretion was seen in the control group ColⅣ.Comparing with the control group,the secretion of ColⅣin the TGF-β1 stimulus group had increased remarkably (P<0.05) at 30min,1h. Comparing with the TGF-β1 induced ,LEF groups(5μg/ml,50μg/ml), deceased the protein expression of ColⅣat 30 min,1h. it has some statistics meaning(P<0.05). But ,the change of the amount expression of ColⅣprotein in LEF groups occurred between 2h-6h are not obvious。The inhibitory rate was observed increasing along with the time elapsing ,and the inhibitory effect of LEF on ColⅣprotein secretion in mesangial cell has the time dependent effect.3. The change of Smad2 mRNA expression was detected by semi-quantitative real-time(RT)-PCR. The TGF-β1(5ng/ml)group was compared with the control group:The mRNA expression of TGF-β1(5ng/ml)group were higher significantly in control group ,but lower significantly in all of LEF (5μg/ml),(50μg/ml)groups than those in TGF-β1 group(F=11.836 P<.0001).Conclusion1. Human recombinant TGF-β1(5ng/ml) can notablely up-regulate P-Smads protein expession of primary cultured rat GMC.2. TGF-β1/Smads signaling transduction pathway in rat GMC can cause the incrasing of secretive of ColⅣand inhibit ,The affection is time-dependent to some degree.3. The inhibitory effect of LEF is showed in the TGF-β1/Smads signaling transduction pathway in rat GMC .LEF not only strain the secretive of ColⅣ,but also decrease the secretion of Smad2/3 protein and expression of Smad2 mRNA.The inhibitory effect is time-dependent to some degree. |
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