Effect Of The Alkaline Extract Of Pericarp Of Citrus Reticulate On Epithelial-mesenchymal Transition Of A549 Cells Induced By TGF-?1

Objective:To observe the inhibitory effect of the alkaline extract of pericarp of citrus reticulate on the epithelial mesenchymal transformation of A549 cells induced by TGF-?1 in vitro,and its influence on the protein and mRNA expression of E-cad,a-SMA,Vimentin in A549 cells.It is beneficial to explore the new intervention mechanism of the alkaline extract of pericarp of citrus reticulate in pulmonary interstitial fibrosis,and provide new ideas for the treatment of pulmonary interstitial fibrosis with traditional Chinese medicine.Methods:The optimum cell planting density,the acting concentration of TGF-beta 1 and the acting time of TGF-beta 1 were screened by observing cell morphology under the microscope.CCK8 method was used to screen the concentration of the alkaline extract of pericarp of citrus reticulate.A549 cells growing in logarithmic phase were divided into 6 groups:blank control group,model group,the alkaline extract of pericarp of citrus reticulate groups(high,mddle and low dose group),and positive control group(Pirfenidone group).After starving with free-serum media over night,the model group was added TGF-?1(31,the final concentration was 5 ng/m1.The drug groups were mixed TGF-?1 with different concentration of alkaline extracts or Pirfenidone,lasting for 24h;The protein expression level of E-cad,a-SMA and Vimentin in A549 cells was detected by Western blot method,and the mRNA expression level of E-cad,a-SMA and Vimentin in A549 cells was detected by RT-PCR method.Results:The optimum cell planting density was 3,000 cells/hole,and the acting time of TGF-?1 was 24h,and the acting concentration was 5ng/ml by observing cell morphology under the microscope.In addition,the A549 cells induced by TGF-?1 were transformed from polygonal shape to long spindle pattern.The CCK8 test showed that the inhibitive effect of the alkaline extract of pericarp of citrus reticulate on EMT model in vitro was positively correlated with concentration,the inhibition rate of the concentration of 750 ?g/ml was 60.18%,so the concentration of 750 ?g/ml was selected as high dose group,and 500 ?g/ml as middle dose group,250 ?g/ml as low dose group.The LDH test showed that there was no statistical difference in the activity of LDH between the drug groups and the blank group.Western blot test indicated that compared with the blank group,the protein expression of E-cad in model group and drug groups was down-regulated(p<0.01).The expression level of E-cad in high and middle dose groups as significantly higher than that in model group(p<0.01).Compared with the blank group,the expression of a-SMA protein in the other groups except high does group was significantly higher(p<0.01).Compared with the model group,the expression of a-SMA protein in drug groups showed a significant downward(p<0.01).Compared with the blank group,the expression of Vimentin protein of model group and drug groups was up-regulated(p<0.01).Compared with the model group,Vimentin protein expression was significantly reduced in drug groups(p<0.01).RT-PCR results showed that compared with the blank group,the mRNA expression of E-cad was significantly reduced in model group and drug groups(p<0.05).Compared with the model group,the mRNA expression of E-cad in the drug groups exclusive of the low-dose group were up-regulated(p<0.05).In terms of the mRNA expression of a-SMA,the expression of model group and drug groups was significantly higher than that of the blank group(p<0.05),and the expression level of the model group was the highest.Compared with the model group,a-SMA mRNA expression of drug groups showed a significant downward(p<0.05).The middle dose group and the low-dose group showed higher than the positive control group(p<0.05).The expression of Vimentin mRNA in model group and drug groups was significantly higher than that in the blank groups(p<0.05).Compared with the model group,the expression of Vimentin mRNA in the drug groups was significantly reduced(p<0.05).Conclusion:?TGF-?1 may induce epithelial mesenchymal transformation of A549 cells.?The EMT model in vitro was interfered by the activity of the alkaline extract of pericarp of citrus reticulate rather than its toxicity.?In vitro model of EMT,the alkaline extract of pericarp of citrus reticulate down-regulated the protein and mRNA expression of a-SMA and Vimentin,up-regulated the protein and mRNA expression level of E-cad in A549 cells.It suggests that the alkaline extract inhibits the EMT process of A549 cells.?Pirfenidone may not increase the expression level of E-cad in EMT in vitro.