Effect Of Sanwubai Powder On Epithelial-mesenchymal Transition Of A549 Cells Induced By TGF-β1

Objective:Observe the anti-pro lifer at ive effect of Sanwubai Powder to A549 cell in vitro via induction of TGF-01, the affects of different time points of Sanwubai Powder to the level of secretive IFN-r, IL-4, VEGF on its cell supernatant, and the influence on protein expression of a-SMA, E-cad, MAPK family of P38MAPK and P-P38, and the mechanism of action to TGFβ1-EMT progress under the intervention of Sanwubai Powder is explored, so as to study the relationship between Sanwubai Powder and PF, and provide new ideas and breakthrough points for PF clinical treatment of traditional Chinese medicine.Method:After hungering serum-free medium and over night, A549 cells with good growth are randomly divided into 7 groups:select one group as a blank control group and is added with 6 ml complete culture solution, one group is added TGF-βto complete culture solution, (the final concentration of TGF-β1 is 5ng/ml) as a model group, the rest five drug groups, are added respectively extreme low, low, medium and high dose of SWB group (SWB final solution is 10-6, 10-5,10-4,10-3g/ml respectively) and Western medicine group. ①detect the anti-proliferative effects of different concentrations of Sanwubai Powder to TGF-β1 induced A549 cell in vitro at 24,48, and 72h with MTT method, choose suitable drug concentrations, drug action time. ② by means of microscope to observe A549 cell morphological changes of each group at 24 and 48 h. ③ELISA method is applied at two time points of 24,48 h to test the secreted expression level of groups of VEGF, IL-4, and IFN-r in cell supernatant of each group. ④ chose western blot method to detect the protein expression level of E-cad, a-SMA, P38MAPK and P-P38MAPK of each group at 24 h,48 h.Result:the anti-proliferative effect of Sanwubai Powder to A549 cell in vitro via induction of TGF-β1:at 24 h, the high, medium dosage of Sanwubai Powder have inhibitory effects on A549 cells induced by the TGF, the drug shows proportional relationship of cell inhibition rate and drug concentration, high dose group of the inhibition rate of high dose group is 23.68%; at 48 h, compared with model group, four concentrations set by Sanwubai Powder have obvious inhibitory effects for cell inhibition rate, appears a parabolic trend, among them, the highest inhibition ratio of medium dosage group can up to 33.4%; at 72h, the inhibition trend of each concentration of Sanwubai Powder to cell inhibition rate is consistent with the tend at 48h but both lower than the corresponding group at 48h.To observe A549 under the action of TGF-β1 with microscope, cell morphology is converted from the original cobble shape to the spindle radial pattern, and spindle transformation of A549 cell at 48h is more apparent than that at 24 h.ELISA results showed that the expression of VEGF and IL-4 in cell supernatant in model are significantly increased than blank control group, IFN-r expression is decreased obviously, the content of VEGF and IL-4 in cell supernatant in drug group, compared with the corresponding model group, have varying degrees of reduction, IFN-r expression quantity in cell supernatant of drug group, compared with the content of model group at the same time point, is significantly higher, the difference has statistical significance. The drug action of high, medium dosage is slightly better than low dosage group, but the inter-group difference between high and medium dosage group is not obvious.The detection results of western-blot method show that:after A549 stimulated by TGF-β1、for 24h, and 48h (compared with blank control group), the expression of a-SMA, P-P38MAPK is markedly increased; E-cad expression is reduced; the change of P38MAPK protein expression is not obvious. After the intervention of Sanwubai Powder, compared with model group, the expression quantity of both a-SMA and P-P38MAPK in drug group have different degrees of reduction, E-cad protein expression increases accordingly, its difference is statistically significant; compared with model group, P38MAPK protein expression in drug group has certain reducing trend high dose group, but only the protein reducing quantity of high dosage group at 24h, and medium dosage group at 48h are statistical significant.Conclusion: ①TGF-β1 can induce epithelial-mesenchymal transition of A549 cells. ②At 48h, the anti-proliferative effect of Sanwubai Powder in medium dosage group to A549 cell in vitro via induction of TGF-β1 is the most obvious. ③Sanwubai Powder may play the role of inhibition of pulmonary fibrosis disease process by regulating the concentration of IFN-r, IL-4, and VEGF of cytokines in A549 cell EMT in vitro model. ④Sanwubai Powder, by reducing the protein expression of a-SMA, P-P38MAPK, increasing E-cad protein, and giving play to an inhibition of alveolar epithelial cells EMT transdifferentiation process, has a certain effect of anti fibrosis.