Study On The Intervention Mechanism Of Feiwei Granules On Epithelial-mesenchymal Transition In Interstitial Lung Disease

Background:Interstitial lung disease(ILD)is a group of heterogeneous diseases involving the alveolar wall and alveolar tissue,mainly in the interstitial lung,and involves epithelial cells,lung endothelial cells,and pulmonary arteries and veins.Because ILD lesions are not limited to interstitial lungs,they are often associated with pulmonary parenchymal involvement,also known as diffuse interstitial lung disease(DPLD).The mechanism of ILD formation has been controversial so far,but it is recognized by many scientists as closely related to interstitial lung disease.It is the epithelial-mesenchymal transition(EMT).EMT refers to the passage under certain physiological or pathological conditions.Fibroblasts are produced to repair tissue damage caused by trauma and inflammation.At present,the research on the mechanism of action of TCM compound on EMT is still insufficiently supported.In order to understand the role of traditional Chinese medicine from the molecular level,this study is specially carried out.Objective:To study the mechanism of Pulmonary Fibrosis in the treatment of EMT induced by transforming growth factor(TGF-β1)by using A549 cells in vitro as a model.To investigate the intervention effect and mechanism of Fei wei presription on EMT in TGF-β1-induced lung adenocarcinoma A549 cells.Method:A549 cells were routinely cultured in F-12 medium(containing 100 IU/mL penicillin,100 mg/L streptomycin and 10%fetal bovine serum)at 37° C in a 5%C02 incubator.The cells were confluent to 70-80.After%,digested with 0.25%trypsin,passaged or related experiments;grouping and intervention methods,6-well culture plates were added,2.5mL of 8X 104 cells were added to each well,and starved for 10h.The A549 cells cultured in vitro were divided into 5 groups:blank group,model group,low concentration Chinese medicine group,medium concentration Chinese medicine group and high concentration Chinese medicine group.The lung sputum granules are composed of Xi Yang Shen5g,Sanqi 3g,Shan Yu Rou 10g,Wu Wei Zi6g,Zi Wan 10g,Mai Dong 10g,Bai Guo 10g,and Gan Cao 6g.The drug is provided by the Pharmacy of China-Japan Friendship Hospital.MTT assay was used to detect the effect of Fei wei presription on the proliferation of A549cells.A549cells were added to96-well plates and cultured for24hours.The culture solution was aspirated,and drugs containing different concentrations(3.962,7.8125,15.625,31.25,62.5,125,250 a g/mL)were added,and the F-12complete medium was used as a normal control,and the culture was continued for24hours,48hours,and 72hours.After adding 10 μ L of MTT,the cells were incubated in an incubator for 4 hours,and the absorbance was measured at a wavelength of 490 nm.The morphology of the cells was observed by an optical microscope.The cells of each well were observed under an inverted microscope with a 6-well plate under an inverted microscope.Five samples were randomly selected from each group,and each sample was randomly photographed with 5 fields of view.Real-time quantitative PCR(q-PCR)was used to detect mRNA expression.Trizol was added to 150 ml per well of 6-well plate,and total RNA was extracted 72 h after cell intervention.Blow the cells,use complete dissolution,and transfer to a 1.5ml EP tube.Then,centrifugation was carried out at 12,000 rpm for5 min,and the supernatant in2 tubes was taken to a 1.5 ml EP tube,and the precipitate was discarded.Chloroform was added to 0.2 ml of chloroform/ml Trizol,and the mixture was shaken and allowed to stand at room temperature for 15 minutes.Centrifuge at 12000 rpm for 1 min at 4° C,pipette the upper aqueous phase and transfer to another 1.5 ml EP tube.After the RNA purity was identified,the cDNA was reverse transcribed and cDNA was used as a template.The primer of the target gene is added to the reaction system,the reaction parameters are set,and the amplification of the target gene is performed,and the Ct value of the template and the initial copy number of the template are linearly related;the protein expression is detected by Western blot,and the protein is expressed in the well.The medium was aspirated,washed twice with PBS,and dispensed with 1000ml(NP)+ 10 μl(PNSF0.1M),100 μl per well,and the cells were brushed with a small brush and placed in an EP tube.The cells were placed in an ultrasound system for 1 minute,then centrifuged 12,000 rpm for 10 minutes,and the supernatant was taken.The primary antibody was incubated overnight.Primary antibody and primary antibody dilution ratio.The secondary antibody was incubated for 30 minutes.Wash with TBST three times(5 minutes X3),boot exposure and development;immunofluorescence technology,six-well plate per well with cold PBS soak for no more than3minutes.4%paraformaldehyde was added to each well and fixed on ice for14 minutes.Note that to be level,cover evenly.The PBS was slowly washed with a PBS for5 minutes,and the PBS-Triton was permeated for5 minutes.Wipe the edges of the slides,add 10%normal blocking serum to each slide with a micropipette,and block for 30 min in a room temperature wet box.Incubation of primary antibody:Add the diluted primary antibody,carefully add the primary antibody with a micropipette,place in a humidified box,and store in a4o refrigerator overnight.In the dark place,add a secondary antibody and cover it evenly and incubate for 1 hour at room temperature.Add 50 μL of secondary antibody to each well.The secondary antibody can be added together with the fluorescent material.(Rabbit secondary antibody 1:150),then washed 3 times with PBS-Tween for x5 min.In a dark place,a 6-well plate was inverted and the cells were observed under a200-fold inverted microscope.Five samples were randomly selected from each group,and each sample was randomly photographed with 5 fields of view.Statistical methods were used to analyze the data with SPSS 19.0statistical software.The data conforms to the normal distribution,and the measurement data is expressed as mean ± standard deviation(x±s).The variance of multiple sets of data is uniform,so one-way ANOVA is used,and the LSD method is used for pairwise comparison.P<0.05 was considered statistically significant.Results:The effect of FWCJ on the proliferation of A549cells was first tested by MTT assay.The results showed that FWCJ from 3.9062~250 a g/ml could significantly inhibit the proliferation of A549cells.The morphological changes of EMT induced by TGF-β 1 in A549 cells were observed by optical inverted microscope.Results Compared with the model group,the cells in the medium-concentration Chinese medicine group could maintain the original morphology and the cell circularity value increased.The expression levels of FN,Vim and E-Cad mRNA were detected by PCR.Results Compared with the model group,the expression of FN mRNA and the expression of E-Cad mRNA were decreased in the Chinese medicine group.The expression levels of EMT markers FN,Vim and E-Cad were detected by Western blot.Results Compared with the model group,the expression of FN in the low,medium and high concentration Chinese medicine groups was significantly reduced.Compared with the model group,the expression of Vim was decreased in the high concentration Chinese medicine group.E-Cad protein expression was significantly elevated compared to the model group.The immunofluorescence assay showed that the fluorescence expression of FN and Vim was up-regulated and the expression of E-Cad was down-regulated in the model group compared with the blank group.Compared with the model group,the fluorescence expression of FN and Vim in the high concentration Chinese medicine group was down-regulated,and the fluorescence expression of E-Cad was up-regulated.The expression of p38,pp38,Erk,p-Erk,JNK and p-JNK proteins in the MAPK signaling pathway was detected by Western blot.Level.Results Compared with the model group,the expression of pp38 was decreased in the high concentration Chinese medicine group.The expression levels of pSmad 3,TWSIT1 and Snail proteins in Smad signaling pathway were detected by Western blot.Results Compared with the model group,the expression of Snail in the high concentration Chinese medicine group was decreased,and the expression of TWIST 1 in the medium concentration and high concentration Chinese medicine group was decreased.The immunofluorescence assay was performed by immunofluorescence.The results showed that the expression of pSmad3,TWSIT1 and Snail was up-regulated in the model group compared with the blank group.Compared with the model group,the fluorescence expression of TWSIT1 and Snail in the high concentration Chinese medicine group was down-regulated.Conclusion:In vitro intervention of Fei wei presription can increase the expression of E-Cad and inhibit the expression of stromal cell markers FN and Vim during EMT,and inhibit the occurrence of EMT by TGF-β 1 through MAPK pathway and Smad pathway.Especially at high concentrations,the effect is remarkable.