The Study Of Buzhong Yiqi Decoction Containing Serum Through GSK-3β/Snail Pathway To Interfere With Epithelial-mesenchymal Transition And Apoptosis Of A549/DDP Cells

Purpose:To observe relevant protein of GSK-3β/Snail signaling pathway on cisplatin-resistant cell line A549/DDP mediated by TGF-β₁,attempt to explore GSK-3β/Snail signaling pathway associated with epithelial-mesenchymal transition（EMT）.Analyze the effects of Serum Containing Buzhong Yiqi Decoction in EMT and apoptosis by disturbing the signaling pathway of GSK-3β/Snail.The purpose is to provide experimental basis for Buzhong Yiqi Decoction in the therapy of lung cancer drug resistance reversal.Material and method:The first experiment contain two groups:A549/DDP group;A549/DDP+T group（TGF-β₁ 5ng/ml,48h）.Immumofluorescence、Western bloting and real time PCR method detect protein and mRNA expressions of E-cadherin and N-cadherin mediated by TGF-β₁.Detect the protein and mRNA expressions of p-GSK-3β（ser9）and Snail by immumofluorescence、Western bloting and real time PCR method.The second experiment contain five groups:A549 group、A549/DDP group、A549/DDP+T group、A549/DDP+NC group、A549/DDP+Snailsi group.The effect of cisplatin on cell growth mediated by TGF-β₁was determined by MTT assay.Resistance index was calculated by 50%growth inhibition（IC50）.To test the changs of P53、Bax、Bcl-2 in A549/DDP cell mediated by TGF-β₁,protein expressions of P53、Bax and Bcl-2 were detected by Western Botting,the mRNA expressions of P53、Bax and Bcl-2 were detected by real time PCR.To test the changs of P53、Bax、Bcl-2in A549/DDP cell mediated by Snail siRNA,protein expressions of P53、Bax and Bcl-2 were detected by Western Botting,the mRNA expressions of P53、Bax and Bcl-2 were detected by real time PCR.The third part,20 SD rats were divided into two groups:control group（2 ml salin irrigated）;Buzhong Yiqi Decoction group（6.57g/kg decoction irrigated）,accordance with the method of serum pharmacology,serum was obtained after 7 days.The experiment contain four groups:A549/DDP group;A549/DDP+T+K group（A549/DDP+TGF-β₁+10%blank rat serum）;A549/DDP+T+Z group（A549/DDP+TGF-β₁+10%Buzhong Yiqi Decoction rat serum）、A549/DDP+T+Snailsi group（A549/DDP+TGF-β₁+Snail siRNA）.Protein expressions and localizations of p-GSK-3β（ser9）and Snail were detected by immunocytochemical method,protein expressions of p-GSK-3β（ser9）and Snail were detected by Western Botting,the mRNA expressions of p-GSK-3β（ser9）and Snail were detected by real time PCR.Western bloting and real time PCR method detect protein and mRNA expressions of EMT and apoptosis mediated by E-cadherin and N-cadherin mediated by Serum Containing Buzhong Yiqi Decoction.Data was expressed as means±standard deviation,statistical significance was tested using one-way ANOVA,SPSS19.0 statistical software used for data analysis.P values<0.05 were considered significant.Results:1.Conpare with A549/DDP groups,A549/DDP+T groups cells were observed:cells disperse,assume a spindle shape and develop pseudopodia,a myofibroblast-like morphology.This transformation was conform to classic EMT markers.2.Immunocytochemical、Western Botting and real time PCR method detect protein and mRNA expressions of E-cadherin、N-cadherin:conpare with A549/DDP groups,protein and mRNA expressions of E-cadherin in A549/DDP+T groups were significantly lower（P<0.05）;protein and mRNA expressions of N-cadherin were significantly higher in A549/DDP+T groups（P<0.05）.3.Immunocytochemical、Western Botting and real time PCR method detect protein and mRNA expressions of p-GSK-3β、Snail:protein expressions of p-GSK-3βand Snail were significantly higher in A549/DDP+T groups（P<0.05）.conpare with A549/DDP groups,mRNA expression of Snail were significantly higher in A549/DDP+T groups（P<0.05）;the differences between p-GSK-3β（ser9）mRNA expression of A549/DDP groups and A549/DDP+T groups were not statistically significant（P>0.05）.4.The effect of cisplatin on cell growth mediated by TGF-β₁ was determined by MTT assay.Resistance index of A549/DDP groups was 7.62,A549/DDP+T groups resistance index up to12.60,and rise index was 1.65.5.Western Botting and real time PCR method detect:conpare with A549/DDP groups,protein and mRNA expressions of P53、Bax in A549/DDP+T groups were significantly lower（P<0.05）;protein and mRNA expressions of Bcl-2 were significantly higher in A549/DDP+T groups（P<0.05）.6.Immumofluorescence and Western Botting method detect:conpare with A549/DDP groups,protein expressions of Snail in A549/DDP+Snailsi groups were significantly lower（P<0.05）;The transfection efficiency of Snail siRNA in A549/DDP groups cell was 64%.7.Western Botting and real time PCR method detect:conpare with A549/DDP groups,protein and mRNA expressions of P53、Bax in A549/DDP+Snailsi groups were significantly higher（P<0.05）;protein and mRNA expressions of Bcl-2 in A549/DDP+Snailsi groups were significantly lower（P<0.05）.8.Immunocytochemical、Western Botting and real time PCR method detect protein and mRNA expressions of p-GSK-3β（ser9）and Snail:conpare with A549/DDP+T+K groups,protein and mRNA expressions of p-GSK-3β^ser9、Snail were significantly lower in A549/DDP+T+Z groups（P<0.05）.9.Western Botting and real time PCR method detect protein and mRNA expressions of E-cadherin、N-cadherin:conpare with A549/DDP+T+K groups,protein and mRNA expressions of E-cadherin were significantly higer in A549/DDP+T+Z groups（P<0.05）;N-cadherin were significantly lower in A549/DDP+T+Z groups（P<0.05）;10.Western Botting and real time PCR method detect protein and mRNA expressions of P53、Bax and Bcl-2:conpare with A549/DDP+T+K groups,protein and mRNA expressions of P53 and Bax were significantly higher（P<0.05）,and protein and mRNA expressions of Bcl-2 were significantly lower in A549/DDP+T+Z groups（P<0.05）.Conclusion:1.The mechanism of EMT in A549/DDP might be related to GSK-3β/Snail signaling pathway activating by TGF-β₁.2.Snail intervenes the apoptosis of A549/DDP by regulating the expression of P53、Bax and Bcl-2.3.Serum Containing Buzhong Yiqi Decoction intervenes EMT and apoptosis by disturbing the signaling pathway of GSK-3β/Snail.