TGF-β Regulation Of Acrolein-mediated Pulmonary Epithelial Cell Toxicity And Rhythmic Protein

BackgroundAcrolein(Acr)is a widespread air pollutant,and its volatility makes it more likely to enter the body through the lung.Lung injury is one of the important hazards caused by acrolein to the body.The mechanism may be that it causes oxidative stress,leading to the disorder of cell redox,and further causing the decrease of cell activity or death.Rhythmic gene Per 1(Period1,Per1)and transforming growth factor β(TGF-β)have important regulatory effects on the proliferation of multiple cells and repair after tissue injury.By studying the effect of acrolein on the proliferative activity of lung epithelial cells and its effect on the expression of Per1 gene,our study intends to explore the damage of lung epithelium cell caused by acrolein and its repair mechanism,providing evidence for the study of respiratory diseases caused by acrolein exposure and its treatment.ObjectiveThe aim of this study was to investigate the effects of acrolein on proliferation of pulmonary epithelial cells,and on the expression of Per 1 rhythmic protein.At the same time,the study tried to explore TGF-β regulation of pulmonary epithelial cell proliferation and Per 1 protein expression after acrolein stimulation.MethodsTwo pulmonary epithelial cell lines,A549 and MLE15,were selected as the study model.When the cell confluence reached 80%,the cells were treated with phosphate buffer PBS(phosphate buffer salt)containing or without acrolein(80 μMol/L)for 30 minutes,and recorded as A549-Acr group,A549-PBS group,MLE15-Acr group and MLE15-PBS group,respectively.The culture medium was discarded and cultured for another 24 hours.Cell proliferation was detected by CCK-8 kit(every 12 hours)and EdU Cell Proliferation kit.Then the expression level of Per 1 protein in pulmonary epithelial cells was detected by Western blot(24 hours after treatment).Then,we select A549 and MLE15 as the study model to explore whether TGF-β intervention can improve the injury of pulmonary epithelial cells stimulated by acrolein.When the cell confluence reached 80%,the cells were treated with phosphate buffer PBS containing or without acrolein(80 μMol/L)for 30 minutes,then with or without TGF-β(10 mg/L)to establish a model of acrolein and TGF-β co-treatment of lung epithelial cells,and they were recorded as A549-Acr-TGF-β,A549-PBS-TGF-β,A549-Acr-PBS,A549-PBSPBS,MLE15-PBS-TGF-β,MLE15-Acr-PBS and MLE15-PBS-PBS groups.Similar methods were used to detect the proliferation and Per 1 expression level.The results were analyzed by Analysis of Variance,Comparisons between the two groups were done with a T test.P<0.05 was considered significant in all analyses.Results 1.With the microscope we found that in A549 cells,the cells floated more in A549-Acrgroup,the cell confluence decreased,and the cell morphology changed.In MLE15 cells,after acrolein treatment the cells floated and the cell confluence decreased,too.2.In A549 cells,compared with PBS control group,the experimental absorbance valueCCK-8 acrolein treatment group was lower than that of PBS control group at the sametime after treatment,and the difference was statistically significant(F=211.782,P<0.001).In MLE15 cells,compared with PBS control group,the experimentalabsorbance value CCK-8 acrolein treatment group was lower than that of PBS controlgroup at the same time after treatment,and the difference was statistically significant(F=22.495,P=0.003 which indicated that the proliferation of cells in different treatmentgroups was different,and the absorbance of the same treatment mode was different atdifferent time,indicating that the same treatment mode of the same cell was different atdifferent time.In A549 cells,the proliferation rate of cells in the A549-Acr group wassignificantly reduced(T=7.625,P<0.001).In MLE15 cells,the proliferation rate of cellsin the MLE15-Acr group was significantly reduced compared with that in the MLE15-PBS group(T = 20.757,P<0.001).At 24 hours after acrolein stimulation,the expressionof Per 1 protein in the A549-Acr group was lower than that in the A549-PBS group(T=4.26,P=0.013),and the expression of Per 1 protein in the MLE15-Acr group waslower than that in the MLE15-PBS group(T=8.347,P=0.001).3.The difference was statistically significant(P<0.001)after adding TGF-β in the processof cell recovery culture after acrolein treatment,and PBS-TGF-β cells,the experimentalabsorbance value was lower than that of the PBS-TGF-β group(P<0.001);theexperimental absorbance value of Acr-TGF-β group was decreased compared with thatof the PBS-PBS group and the difference was statistically significant(P<0.001);Theexperimental absorbance values CCK-8 Acr-PBS group were lower than those of PBS-TGF-β group,and the difference was statistically significant(P<0.001);the experimentalabsorbance values CCK-8 Acr-PBS group were lower than those of the PBS-PBS group,and the difference was statistically significant(P<0.001).the experimental absorbancewas also different at different time points in the same treatment mode of the same cell.EdU assay found that A549-Acr-TGF-β group had a larger and statistically significantdifference EdU positive cell ratio compared with A549-Acr-PBS group(P=0.001);however,addition of acrolein after treatment in MLE15 cells did not increase the cellEdU positive cell ratio.A549-Acr-TGF-β group increased Per1 protein expression(P<0.05)compared to the MLE15-Acr-PBS group,and the MLE15-Acr-TGF-β groupincreased(P<0.05).Conclusion 1.Acrolein treatment can reduce or even lose the proliferation ability of pulmonaryepithelial cells,at the same time,it can down regulate the expression level of rhythmicprotein per 1.2.After treatment with acrolein,TGF-β did not significantly improve the cell proliferationinduced by acrolein,but significantly increased the expression level of per 1 protein.