| ObjectiveTo research the effect of Buyang Huanwu Decoction on mTOR/ULK1 pathway in the formation and progression of pulmonary fibrosis in mice with idiopathic pulmonary fibrosis.To clarify the mechanism of action of Buyang Huanwu Decoction on pulmonary fibrosis.Method144 8-week-old C57/BL6 male mice weighing 20±2g were randomly divided into sham-operation group,model group,prednisone group,high-dose Buyang Huanwu Decoction group,medium-dose Buyang Huanwu Decoction group and low-dose Buyang Huanwu Decoction group,24 mice in each group.Except for the sham-operation group,each group received a one-time airway instillation of bleomycin sulfate to establish a mouse model of pulmonary fibrosis.After successful modeling,each group was given intragastric administration once a day.The sham-operation group and model group were given 0.9%normal saline 10ml·kg-1,the prednisone group was given prednisone acetate tablets 6mg·kg-1,the Buyanghuanwutang high,middle and low dose groups were 28.08g·Kg-1,14.04g·kg-1,7.02 g·kg-1.Continuous administration for 28 days.8 mice were randomly assigned to be sacrificed on the 7th,14th,and 28th day in each group.Lung tissue specimens were collected.HE and Masson staining were used to evaluate alveolitis and pulmonary fibrosis in the mice at different time points.The expression of p-MTOR,ULK1,andα-SMA in lung tissue were measured by immunohistochemical method.The expression ofα-SMA protein was detected by Western Blot method.The number of positive cells of autophagic corpuscle was observed under electron microscope.Results1.In general terms:The mice in the sham-operated group were vigorous at various time points,had alertness,gained weight,had bright hair,and had normal diet and defecation.The mice in the model group had the worst mental state in 7 days,inactive,slow reaction,slow movement,reduced food and drinking water.As time went on,the body weight decreased first and then slowly increased.The Buyang Huanwu Decoction and the prednisone group mice had good spirits,quick response,gradual weight gain,hair luster,and normal diet and defecation.But slightly worse than the sham-operation group,better than the model group.2.Alveolitis progress:The alveolar structure was clear at each time point in the sham-operation group,and no obvious inflammatory reaction was observed.In the model group,the alveolar structure was slightly destroyed and the alveolar wall was destroyed on the 7th day,and a large number of inflammatory cell infiltrations were observed.On the 14th day,the alveolar wall was thickened,the interval was widened,accompanied by a small amount of inflammatory cells infiltrated.More fibroblasts were seen.On the 28th day of the model group,lung tissue was extensively materialized,and a small amount of inflammatory cells infiltrated.A large number of fibroblasts were seen.In the 7-day prednisone group,alveolar structure was slightly damaged,and more inflammatory cells were seen in the alveolar cavity.The alveolar wall thickened on the 14th and 28th days.A small amount of inflammatory cells and fibroblasts were seen.Fibroblasts increased in 28 days compared with 14 days.A small amount of inflammatory cells were seen in each dose group of Buyang Huanwu Decoction for 7 days,and the localized small parenchyma of lung tissue were seen in 14 days and 28 days,and there was a different degree of improvement compared with the model group.3.Pulmonary fibrosis process:In the sham-operation group,alveolar structure was clear at each time point and no fibrosis was seen.In the model group,the alveolar cavity became smaller in 7 days and the local alveolar wall thickened.Only a small amount of blue stained collagen was seen to proliferate.In 14 days,more blue stained collagen was proliferated.In 28days,a large area of lung consolidation was found,and a large number of blue staining collagen proliferation was seen in the fibrous foci.In the 7-day prednisone group,the alveolar walls thickened but only a very small amount of blue stained collagen proliferated.The 14-day and 28-day interval widened with more blue stained collagen hyperplasia.There was no obvious collagen proliferation in 7 days of each dose group of Buyang Huanwu Decoction.The alveolar wall thickened on the 14th and 28th days.The staining of blue collagen deepened and the area of fiber lesions was obvious.4.p-mTOR protein expression:Compared with the sham-operation group,the P-mTOR of the model group increased at each time point（P<0.05）,and reached the peak on the 28 day as time went on.Compared with the model group,p-mTOR expression was decreased in each dose group of Buyang Huanwu Decoction and prednisone group at each time point（P<0.05）.Compared with the prednisone group,p-mTOR levels in the high-dose group of Buyang Huanwu Decoction decreased at 14 days and 28 days（P<0.05）.5.ULK1 protein expression:Compared with the sham-operated group,ULK1 content in the model group increased on the 7th,14th,and 28th days（P<0.01）.Compared with the model group,ULK1 content in the prednisone group increased on the 7th,14th,and 28th days（P<0.05）;Compared with the model group,the high-dose group of Buyang Huanwu Decoction increased on the 14th and 28th day（P<0.05）,the contents of Buyang Huanwu Decoction in the middle and low dose groups increased on the 28th day（P<0.05）.Compared with the prednisone group,the ULK1 content in the middle and low dose groups of Buyang Huanwu Decoction increased at 28 days（P<0.05）.The ULK1 content of high dose group of Buyang Huanwu Decoction increased on the14th and 28th days（P<0.05）.6.α-SMA protein expression:In immunohistochemistry,compared with sham-operation group,the expression ofα-SMA protein in the model group was increased on days 7,14 and 28,and reached the highest level in 28 days（P<0.05）.Compared with the model group,the expression ofα-SMA protein was decreased in each dose group and prednisone group of Buyang Huanwu Decoction at each time point（P<0.05）.Compared with the prednisone group,the contents of Buyang Huanwu Decoction in each dose group decreased at 14 days and 28 days（P<0.05）,and there was no significant difference in the expression of Buyang Huanwu Decoction between each dose group and prednisone group at 7 days（P>0.05）.In WB,compared with the sham-operation group,the expression ofα-SMA protein in lung tissue of model group was significantly up-regulated at 7th,14th,and28th day at each time point（P<0.01）.Compared with the model group,α-SMA in the lung tissues of Buyang Huanwu Decoction each dose group and Prednisone group decreased significantly at 7,14,and 28 days（P<0.01）.Compared with the prednisone group,there was no significant difference in the expression ofα-SMA protein in each dose group of Buyang Huanwu Decoction（P>0.05）.7.Electron microscopy autophagosome expression:The 28-day sham-operation group had no autophagy expression,and the high-dose and middle-dose groups of Buyang Huanwu Decoction expressed the most autophagy.Number of autophagic bolus of high-dose Buyang Huanwu Decoction=middle-dose group of Buyang Huanwu Decoction>low dose of Buyang Huanwu Decoction=prednisone group=model group>sham-operation group.Conclusion1.A single animal model of bleomycin can be replicated in mice,which is similar to the pathological process of human pulmonary interstitial fibrosis.2.Buyang Huanwu Decoction can better improve the mental status,food intake,drinking water,fur,reaction sensitivity and other general conditions of mice with pulmonary fibrosis caused by BLM.3.High and middle doses of Buyang Huanwu Decoction can effectively inhibit the early inflammatory infiltration and pulmonary fibrosis in mice with pulmonary fibrosis.4.In the mouse model of bleomycin-induced pulmonary fibrosis,alpha-SMA,a marker of myofibroblasts,increased significantly.It suggests that the proliferation of alpha-SMA can be used as a marker for the active pulmonary fibrosis.5.BuYang Huanwu Decoction can inhibit the expression of alpha-SMA,a positive marker of myofibroblasts,and its mechanism may be related to the inhibition of p-mTOR,up-regulation of ULK1 protein expression,and promotion of autophagy activation.It is suggested that treatment of pulmonary fibrosis with Buyang Huanwu Decoction may be related to inhibiting activation of mTOR pathway and then inducing autophagy. |
PDF Full Text Request