Effects Of Dispelling Wind-dampness Herbs On TGF-β1-induced Podocyte Phenotype

Objective:In order to observe the effects of dispelling wind-dampness herbs on gene expression of P-cadherin, FSP-1and a-SMA gene in TGF-β1-induced podocytes, to investigate the protective effects of dispelling wind-dampness-drugs herbs (including Chinese herbal compound and Chinese medicine monomer) on podocyte injury, and to provide a scientific basis for clinical application of this kind of traditional Chinese medicine in the treatment of Renal Albuminuria.Methods:Experiment1:Podocytes passaged cultured in a petri dish, differentiation culture at37°C, divided into normal group, model group, Fangjihuangqi decoction intervention group, modified Fangjihuangqi decoction intervention group. The model group and intervention groups were treated with TGF-β1(final concentration of5ng/ml) stimulation, intervention groups on the basis of incubation medium respectively containing10%of Fangjihuangqi decoction, modified Fangjihuangqi Decoction drug-containing serum of rats with podocyte, model group and normal group were incubated with medium containing10%normal rat serum.Experiment2:Podocytes passaged cultured in a petri dish, differentiation culture at37°C, divided into normal group, model group, Triptolide low concentrations group and Triptolide high concentrations group. The model group and intervention group were treated with TGF-β1(final concentration of5ng/ml) stimulation, the intervention groups on the basis of respectively containing triptolide to5nM,10nM. The above two experiments were performed using the MTT test for the detection of cell culture of mortality, detecting the expression of mitochondrial membrane potential changes by JC-1reagent and early apoptosis rate by flow cytometry, using reverse transcriptase polymerase chain reaction (RT-PCR) technique for testing the expression of P-cadherin, FSP-1and a-SMA gene, and using western-blotting technique to test the expression of P-cadherin and a-SMA protein.Results:Experiment1:MTT assay showed that the cell survival rate of each group had no significant difference; JC-1reagent model group mitochondrial membrane potential lower than normal group, the Fangjihuangqi decotion group and modified Fangjihuangqi decotion group mitochondrial membrane potential compared with the model group increased, but no significant difference among the groups. The RT-PCR results indicate:Compared with normal group, P-cadherin gene expression of model group was significantly reduced (P<0.05), Compared with the model group, P-cadherin intervention group were significantly increased (P<0.01). There was no significant difference on FSP-1mRNA and alpha-SMA mRNA among the groups in the normal group and model group.Experiment2:Compared with the normal group, model group, P-cadherin gene and protein expression decreased significantly, after10nM triptolide intervention, P-cadherin protein level was obviously higher than model group (P<0.05), while the5nM of triptolide intervention after the gene and protein expression has no significant difference (P<0.05). In model group, α-SMA protein level was significantly higher than the normal group,10nM triptolide intervention after, a-SMA protein expression decreased obviously (P<0.05).5nM triptolide intervention after, the gene and protein were not significantly different expression.Conclusion:Fangji Huangqi decoction and modified Fangji Huangqi decoction can inhibit the TGF-(β1glomerular podocyte apoptosisinduction and maintain the expression of the phenotypic molecules P-cadherin mRNA. High concentrations of triptolide significantly up-regulated TGF-betal-induced podocyte slit diaphragm protein P-cadherin levels and downward myofibroblast marker protein a-SMA expression levels. Those above suggests that both Chinese medicine compound (Fangji Huangqi decoction or modified Fangji Huangqi Decoction) and monomer (triptolide) could repair and protect podocyte injury.