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

Purpose:To observe relevant protein of GSK-3?/Snail signaling pathway on cisplatin-resistant cell line A549/DDP mediated by TGF-?₁,attempt to explore GSK-3?/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-3?/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-?₁ 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-?₁.Detect the protein and mRNA expressions of p-GSK-3??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-?₁was 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-?₁,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-?₁+10%blank rat serum?;A549/DDP+T+Z group?A549/DDP+TGF-?₁+10%Buzhong Yiqi Decoction rat serum??A549/DDP+T+Snailsi group?A549/DDP+TGF-?₁+Snail siRNA?.Protein expressions and localizations of p-GSK-3??ser9?and Snail were detected by immunocytochemical method,protein expressions of p-GSK-3??ser9?and Snail were detected by Western Botting,the mRNA expressions of p-GSK-3??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-3??Snail:protein expressions of p-GSK-3?and 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-3??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-?₁ 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-3??ser9?and Snail:conpare with A549/DDP+T+K groups,protein and mRNA expressions of p-GSK-3?^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-3?/Snail signaling pathway activating by TGF-?₁.2.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-3?/Snail.