The Effect Of TGF-?₁ On Autophagy And Collagen Degradation After Autophagy In Bronchial Fibroblasts Of Rats

Objective:To investigate the effects of transforming growth factor-?₁ on autophagy and autophagy related collagen degradation in rat bronchial fibroblasts.Methods:1.RBFS were cultured by enzyme combined digestion tissue block sticking method,the morphology of RBFs of 3-4 generations were observed under microscope,and RBFs marker protein vimentin were identified by immunofluorescence method.2.CCK8 assay were used to detect cell proliferation.3.Establish a model to induce autophagy in RBFs.Rapamycin and EBSS were used to induce RBFs autophagy,respectively.The optimal action concentration and time of TGF-?1 and rapamycin were measured by cck8.4.Grouping:Rapamycin-induced autophagy:control group,rapamycin group,TGF-?1group,TGF-?1+rapamycin group.EBSS starvation-induced autophagy:control group,EBSS induction 2 h group,EBSS induction 4 h group,EBSS induction 8 h group,EBSS induction 12 h group,EBSS induction 24 h group,EBSS induction 2 h+TGF-?1 group,EBSS Induced 4 h+TGF-?1 group,EBSS induced 8 h+TGF-?1 group,EBSS induced 12h+TGF-?1 group,EBSS induced 24 h+TGF-?1 group.5.Western blot detected the expression of LC3?,Beclin-1,P62 proteins in RBFs.6.The content of matrix metalloproteinase-1?MMP-1?and matrix metalloproteinase inhibitor-1?TIMP-1?in supernatant of RBFs were detected by Enzyme-linked immunosorbent assay?ELISA?.Results:1.The fibroblasts digested with enzyme digestion tissue adherence method could make cells migrate faster and produce more cells.The cells cultured by the tissue adherence method is relatively slow to climb out,the culture period is longer,and the number of cells obtained are relatively small.The proliferation curve of primary bronchial fibroblasts presented a typical"S"type.The fibroblasts were cultured for 48 hours,more than 60%adherent cells were obtained by collagen protease separation,and the cells grew rapidly.The cells were cultured for 6-7 days.The tissue adherence method cultured cells for 5 days,cells began to grow from the adherent wall at the edge of the tissue block,and then a large number of cells adhered to the surrounding of the tissue block,which could be passaged around 14 days.Immunofluorescence method was used to identify primary cells,fibroblasts expressed vimentin,and the cytoplasm was stained green,proving that the cells studied in this experiment were bronchial fibroblasts.2.CCK8 results showed that compared with the control group,different concentrations of TGF-?1 promoted the proliferation of RBFs.With the increasing of the concentration,the OD450 value also increased significantly?P<0.05?;rapamycin inhibit the survival rate of RBFs,compared with the control group.There were significant differences between the groups?P<0.05?.In this experiment,the optimal intervention concentration was TGF-?₁20ng/mL,and 2h as the optimal intervention time.10 nmol/mLwas used as the effective intervention concentration of rapamycin for 2 h as the optimal intervention time.3.WB results showed that after rapamycin induced RBFs autophagy,the levels of LC3?and Beclin1 protein increased compared with the control group?P<0.05?,while the expression of P62 was down-regulated compared with the control group?P<0.05?.Compared with the control group,the expression of LC3?and Beclin1 protein were down-regulated under the stimulation of TGF-?₁,and the expression of P62 protein was increased.After starvation-induced RBFs autophagy,the expression of LC3?and Beclin1began to increase at 2h,reached its peak at 12h,and the expression level decreased significantly at 24h;the expression level of P62 was down-regulated with the extension of starvation time.With the increase of starvation time,TGF-?₁ down-regulated the expression of LC3?and Beclin1,and up-regulated the expression level of P62.4.ELISA results showed that the addition of TGF-?₁ reduced MMP-1 protein levels and upregulated TIMP-1 protein levels.Conclusion:1.Enzymatic digestion+tissue block adhesion method is a primary culture method that can efficiently and quickly isolate and obtain bronchial fibroblasts.2.TGF-?1 can inhibit the autophagy activity of RBFs.3.Inhibition of collagen degradation is related to the inhibition of autophagy activity of RBFs by TGF-?1.