| Pulmonary fibrosis?PF?is a fibrous dysplastic disease[1,2],which is caused by a variety of causes,and the normal structure of the alveoli is damaged and eventually leads to interstitial fibrosis of the lung.At present,the pathogenesis of pulmonary fibrosis is not yet clear,and there is no special effective treatment,which leads to the high morbidity and mortality of pulmonary fibrosis,which is a serious threat to human health.Pulmonary fibrosis as modern medicine Difficult miscellaneous diseases to clarify its pathogenesis,and prevention strategy has positive significance to human health.Many related studies have shown that in the process of pulmonary fibrosis,autophagy is not activated,autophagy,deficiency,PTEN,ULK1 content is very low,and pulmonary fibrosis is accelerated.Can pulmonary fibrosis be delayed by increasing PTEN and ULK1 content,activating or inducing autophagy?As a modern medical problem,pulmonary fibrosis is a difficult disease in modern medicine.During the study of lung fibrosis,TGF-beta 1 may inhibit autophagy through Smad and PI3K/Akt/m TOR signaling pathway during the study of lung fibrosis in the past.In our previous study,we found that Buyang Huanwu Decoction can initiate multiple signaling pathways related to pulmonary fibrosis,such as TGF-?1/Smad,MAPKs,NF kB and other related signaling pathways,and these signaling pathways are associated with cell autophagy[1-4].Based on this,we choose the mTOR signaling pathway,which is closely related to cell autophagy,to study autophagy and explore the relationship between autophagy and pulmonary fibrosis through this signal pathway,and to reveal the mechanism of the prevention and treatment of pulmonary fibrosis by Buyang Huanwu DecoctionObjective:To study the relationship between mTOR signaling pathway and pulmonary fibrosis by using human embryonic lung fibroblast?HFL1?and human embryo lung cells?MRC5?in vivo and in vitro,and to explore the relationship between autophagy and pulmonary fibrosis,and reveal the mechanism of the prevention and treatment of pulmonary fibrosis by Buyang Huanwu Decoction in this study.Methods:1,144 SPF healthy male C57BL/6 mice were randomly divided into 6groups:group NS,BLM group,prednisone group,Buyang Huanwu Decoction high,middle and low dose group,seventh day,fourteenth day and twenty-eighth day,and 8femoral arteries were randomly selected to take the lung tissue for HE,Masson pathological section and immunohistochemical detection mTOR.2,the HFL1 and MRC5 cells were divided into six groups:blank group,TGF-beta 1stimulation group,20%blank sera group,5%,10%,20%three group of Buyang Huanwu Decoction serum,and 5ng/ml TGF-?1 stimulated HFL and MRC5 cells.The serum was prepared by using the serum of supplementing Buyang Huanwu Decoction.After 24h,the cell culture liquid supernatant was extracted after 24h.ELISA method was used to detect the effect of serum containing Buyang Huantang five on the expression of?-SMA,p-S6 and other proteins in cell culture supernatant.Pressure2.the HFL1 and MRC5 cells were divided into six groups:blank group,TGF-?1stimulation group,20%blank sera group and 5%,10%,20%three group of Buyang Huanwu Decoction serum.After using 5ng/ml TGF-?1 to stimulate HFL1 and MRC5cells,the total protein of the cell was extracted by using 5%,10%and 20%Buyang Huanwu Decoction serum respectively.Western blot was used to detect the effect of serum containing Buyang Huantang Decoction on the expression of PTEN and ULK1proteins in different time.PressureResults:1.General situation:In the blank group,mice were vigorous,alert,sensitive,weight gain,bright hair,normal diet and defecation.Mice in the model group,after 7 days of lethargy,unresponsiveness,weight loss.After 14 days,mental retardation,slow response,weight loss,hair loss,diet and urine were more frequent.28 days of mental daze,standing unstable or difficult to move,depilatory and miscellaneous,light,physical and thin,poor diet,more urine,and rearing a number of wood in the cage was drowned in urine.Buyang Huanwu Decoction and prednisone group had better spirit,agility,weight gain,hair gloss,and normal diet and defecation in five groups.But it was a little worse than that in the blank group,and better than the model group.2.Pathomorphology:HE staining showed that the lung structure of NS group was basically intact on the 7 day,and the alveolar septum was not thickened.There was no obvious inflammatory infiltration,but 14 days and 28 days were almost normal.In group BLM,pulmonary nodules changed,septal edema,alveolar wall thickening,congestion and inflammatory infiltration occurred on 7 days.On the fourteenth day,hyperemia,edema,septal widening,inflammatory infiltration,alveolar wall fibrosis and alveolar atrophy were found in focal areas.On the twenty-eighth day,alveolar inflammation was relieved,alveolar destruction was observed,the interval was obviously widened and broken,a large number of fibroblasts,collagen deposits,and fiber scarring appeared.The different doses of Buyang Huanwu Decoction were improved in different degrees.The prednisone group was similar to the buyanghuan Five Decoction in each dose group.The alveoli were damaged,the wall of the alveoli was thickened and the edema was mild.Masson staining showed that there was no pale blue collagen fiber in the alveolar area of the NS group on the 7 day,but no expansion was observed on the 14and 28 days.Group BLM was widened at 7 day intervals,blue collagen fibers were seen in the wall and interval,blue collagen fibers were visible on the 14 day of blood vessel wall and alveolar septum,and a large number of blue collagen fibers were found in 28 days of bronchioles and fibrous areas.The prednisone group and the Buyang Huanwu Decoction group had wider 7 day intervals,and blue collagen fibers were observed.The blue expansion and deepening in 14 days were observed in five days.There was no obvious expansion and deepening in the 28 day.Compared with the BLM group,the color was shallow and the area was small.3.the expression of alpha-SMA,p-S6,ULK1 and mTOR protein:Compared with group NS,the level of alpha?-SMA in group BLM increased at 7,14 and 28 days,and increased gradually over time,reaching a peak value at 28d,*P<0.05.Compared with group BLM,the content of alpha?-SMA in prednisone group and Buyang Huanwu Decoction group decreased at 7,14,and 28 days,and P<0.05was also increased.Compared with prednisone group,the content of alpha?-SMA in high,medium and low groups in 14 days and 28 days was lower than that in prednisone group,and the content of P<0.05 was five.Compared with group NS,the p-S6 content in group BLM increased at 7,14 and28d,and gradually increased with time,reaching a peak value at 28d,*P<0.05.Compared with group BLM,the p-S6 content in prednisone group at 14 and 28d decreased,while p-S6 content in Buyang Huanwu Decoction at 14 and 28d decreased,#P<0.01.Compared with the prednisone group,the content of p-S6 in 7d and 14d decreased in the lower group of Buyang Huanwu Decoction,and the content of 7D in the middle group of Buyang Huanwu Decoction decreased,and the content of 28d in the group of tonifying Buyang Huanwu Decoction was reduced,P<0.05.Compared with group NS,the ULK1 content in group BLM at 7,14 and 28d increased,*P<0.01.Compared with the BLM group,the content of ULK1 in 7,14and 28d in the prednisone group increased,P<0.05,the tonifying yang and five soup was low,the ULK1 content of the middle group increased in 28d,and the ULK1content of 14 and 28d in the high group of Buyang Huanwu Decoction increased,P<0.05.Compared with prednisone group,the content of ULK1 in 28d increased in the middle and low groups of Buyang Huanwu Decoction,while the content of ULK1in 14d and 28d increased in the Buyang Huanwu Decoction group.Compared with group NS,the content of mTOR in group BLM increased,*P<0.01.Compared with group BLM,the content of mTOR in prednisone group and Buyang Huanwu Decoction group decreased at 7,14 and 28d,and P<0.05 was also observed.Compared with prednisone group,the content of mTOR in 7,14d and 28d decreased in the Buyang Huanwu Decoction group,P<0.05.4.The results of ELISA detection showed that the expression of alpha?-SMA and p-S6 protein in HFL1 and MRC5 cells with 5ng/ml's TGF-?1 stimulated HFL1 and MRC5 cells after the intervention of different concentrations of supplementing Buyang Huanwu decoction containing serum and TGF-beta 1 to HFL1 and MRC5cells,and the P<0.01 was compared with that of the blank control group.Using different concentrations of Buyang Huanwu Decoction serum to intervene HFL1 and MRC5 cells,the expression of alpha-SMA and p-S6 protein in two kinds of cells decreased.In HFL1 cells,the effect of 20%%containing serum group was obvious,compared with that of TGF-beta 1 stimulation group P<0.01.In MRC5 cells,the effect of 20%%containing serum group was obvious,compared with that of TGF-?1stimulation group P<0.01.5,The results of Western blot detection showed that after HFL1 cells stimulated by5ng/ml TGF-?1,the expression of PTEN and ULK1 in the cells decreased at 48h and72h,and compared with that of the blank control group P<0.01.The expression of PTEN and ULK1 in HFL1 cells was increased by the serum of Buyang Huanwu Decoction,and the expression of different serum concentrations in the tonifying yang five soup increased obviously at 48h and 72h,and P<0.01.As time and concentration progressively strengthened,Buyang Huanwu Decoction increased the expression of PTEN in HFL1 cells in a time and concentration dependent manner.SuchAfter stimulation of MRC5 cells with 5ng/ml TGF-beta 1,the expression of PTEN and ULK1 in 24h,48h and 72h decreased,compared with the control group,P<0.01.The expression of PTEN and ULK1 in MRC5 cells was increased by the serum of Buyang Huanwu Five Decoction,and the expression of different serum concentrations in the tonifying yang five soup increased obviously at 48h and 72h,and P<0.01.As time and concentration progressively strengthened,Buyang Huanwu Decoction increased the expression of PTEN in HFL1 cells in a time and concentration dependent manner.Conclusion:1,Buyang Huanwu Decoction can improve the degree of pulmonary fibrosis induced by bleomycin in mice induced by bleomycin,and the transformation of myofibroblast in pulmonary fibrosis is associated with the activation of mTOR signaling pathway.2,Buyang Huanwu Decoction can inhibit the expression of alpha-SMA and p-S6?mTOR protein in lung tissue of mice with pulmonary fibrosis,and promote the expression of ULK1 protein.3.The serum of Buyang Huanwu Decoction can effectively inhibit the expression of alpha-SMA and p-S6 in HFL1 and MRC5 cells in vitro,and can effectively activate the expression of PTEN and ULK1 in HFL1 and MRC5 cells.4,the serum of Buyang Huanwu Decoction may be mediated by blocking mTOR signaling pathway,effectively activating autophagy,reducing the production of extracellular matrix,and exerting anti pulmonary fibrosis. |
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