The Regulation Of Pueraria Extract On Wnt/β-catenin Signal Pathway In Steroid-induced BMSCs

Purpose:To observe the regulation of herbal medicine pueraria active ingredient puerarin in Steroid-induced BMSCs and explore the mechanisms on Wnt/β-catenin signal pathway, provide a theoretical basis for the prevention and treatment of SANFH with puerarin.Method:1.BMSCs of SD rats were isolated and purified by using whole bone marrow adherent method,then identified by morphologic characteristics, detected OD to analysis the growth curve characteristics and surface antigens detected by flow cytometer.2.Get the optimal concentration of puerarin through the MTT method for BMSCs,with the best concentration of puerarin in the dose group,2times the optimal concentration of puerarin as the high dose group,and1/2times as low dose groups. Then BMSCs Were divided into5groups:the blank group was to DMEM containing15%FBS culture medium;Control group:15%FBS100ml+0.1ml*10’3mol/1dexamethasone stock solution; Control group:15%FBS100ml+0.1ml*10-3mol/1dexamethasone stock solution; The low-dose group:15%FBS99.5ml+0.1ml*10-3mol/1dexamethasone storage solution+0.5ml*10*mol/1puerarin storage solution; Middle dose group:15%FBS99ml+0.1ml*10-3mol/1dexamethasone stock solution+1ml*10-3mol/1puerarin storage solution; High dose group:15%FBS98ml+0.1ml*10"3mol/1dexamethasone stock solution+2ml*10-3mol/1puerarin storage solution.3.The5groups of above for6days for Line transmission electron microscopy, oil red O staining and triglyceride test, PCR assay the expression of Wnt/β-catenin signaling pathway members WntlOb,Fzl,GSK3β,β-catenin,TCF4mRNA and PPARγ mRNA�'�C/EBP a mRNA detection, By Western blot assay and analysis expression of β-catenin the key factor protein of Wnt/β-catenin signaling pathway.Result:1.Cell identification:Morphology light microscope observation of rat BMSCs, Cells showing a fibrous or spindle-shaped into,Adherent growth, cell growth are closely attach-ed,Gradual integration into the film, ordered along the long axis of the cell body, was swirling.Purified by passage BMSC,BMSCs form of three generations of a single uniform, was typical of the polar fusion,whirlpool growth;By flow cytometry for CD45and CD90antigen positive sign expression detection test results showed:The adherent cells grew well in the first three generations in the surface markers of the line.3rd generation cell CD45positive rate of about8.24%, CD90positive rate was99.98%.It Can explain the present study, bone marrow adherent cells were isolated and cultured in vitro for the BMSCs with high purity.2.Oil red O staining showed:The no control group was stained fat cells;The control group, fat cells staining most, Showed a cluster distribution.High, medium and low-dose group were found in varying amounts of staining of fat cells,Are all less than the control group.3.Electron microscopy observed:Control group, high, medium and low dose group did not find fat cells,and there is no fat particlesin cells,Cell morphology was normal,The control group,see a lot of fat particles of intracellular accumulation.4.Triglyceride test:control group,the triglyceride content of cells was significantly higher than the control group,The puerarin each group were significantly decreased compared with the control group,medication was no significant difference among the three groups.Compared with the control group, puerarin low, medium and high dose group was significantly lower than the control group.5.RT-PCR:compared with the blank group, the Wnt10b、β-catenin、TCF4mRNA expression was significantly lower in different condition, in puerarin dose group it becomes significantly higher;although GSK3β、PPAR γ、 C/EBP a mRNA expression in control group was significantly higher than blank group, puerarin group were significantly higher than the contral group.6.Western blot:in the contral group,β-catenin protein appeared significantly down regulated,and puerarin group were significantly up regulated blank group,of all the higher and middle dose’s regulation are better. Conclusion:1.puerarin As interferes,Intracellular triglyceride content, PPARγyand C/EBPamRNA expression were significantly lower than the control group,Oil red O staining, electron microscopy, see more fat cells and particles was seen after a simple intervention hormone fat cells and particles less, suggesting that puerarin can inhibit hormone induced BMSCs intoadipogenic differentiation.2.Puerarin on hormone-induced BMSCs inhibition of adipogenic differentiation probably through activation of Wnt/β-catenin signal pathway, regulate the key factor Wnt10b、 GSK3β、β-catenin、TCF4mRNA expression.3.Puerarin adipogenic differentiation inhibition of hormone-induced BMSCs may regulator the amount of β-catenin protein expression.