Effect And Mechanism Of Alpinetin On Bleomycin-induced Pulmonary Fibrosis In Mice

Background:Pulmonary fibrosis is a common interstitial lung disease in the respiratory system,which is the final manifestation of various lung diseases.Its characteristic is excessive extracellular matrix deposition leading to the formation of fibrous scars in lung tissue,leading to ventilation dysfunction and progressive respiratory failure.At present,the treatment of pulmonary fibrosis is limited.Only nintedanib and pirfenidone can effectively improve the lung tissue function and delay the disease progression,but adverse drug reactions and expensive treatment costs significantly limit its application.Alpinetin,as one of the natural flavonoids,is widely present in nature and has various biological activities such as antioxidant,anti-inflammatory,anticancer,and antimicrobial,and can improve liver fibrosis.Therefore,alpinetin may become a new therapeutic drug for pulmonary fibrosis.The aim of this study is to investigate the therapeutic effect and possible mechanism of alpinetin on bleomycin-induced pulmonary fibrosis in mice.Methods: 1.The pulmonary fibrosis model of all the mice involved in this research was successfully established by slow intratracheal instillation of BLM(2U/kg).And they were randomly separated into five groups,with 10 mice in each group,including model group(BLM),sodium carboxymethylcellulose group(CMC),low-dose alpinetin group(Alp25),medium-dose alpinetin group(Alp50)and high-dose alpinetin group(Alp100).The control group(NS)was slowly injected with the equivalent normal saline into the trachea,with 10 mice.From the next day of modeling,the corresponding dose of alpinetin solution was given once every other day,a total of 10 times,and the material was taken after21 days.2.Measure the weight of mice every other day and observe the general living conditions of mice.3.After the treatment,weigh the lung tissue of mice in each group and calculate the lung coefficient,measure the content of hydroxyproline(HYP),and evaluate the level of lung inflammation and fibrosis of mice by H&E staining and Masson staining.4.We used immunohistochemistry to analyze the protein expression of TGF-β1,Smad2/3 and Smad7.And immunofluorescence was carried out to detect the protein expression of E-cad,α-SMA and Col-1.Western blot was carried out for the qualitative and quantitative assessment of the expression behavior of Smad2/3 and Smad7.And the expression of TGF-β1 was measured by fluorescence quantitative PCR.Results: In the course of treatment,the BLM group showed the ordinary circumstances of mice gradually become worse,the weight significantly decreased,and some mice even died of pulmonary fibrosis.In addition to the above,some inflammatory cells and collagen fibers can also be found in the pathological sections derived from lung tissue.At the same time,the lung coefficient and the content of hydroxyproline were increased to varying degrees.And furthermore,for the lung tissue,the various methods we used above all showed that a consistent trend,such as an increase of the expression of Col-1,α-SMA,Smad2/3 and TGF-β1 and a decrease of the expression of E-cad and Smad7.Thus,the above results suggested that the degree of pulmonary fibrosis was severe.However,all aspects of the mice gradually improved after the treatment of alpinetin.In particular,compared with the BLM group,the general condition of mice in Alp50 group was significantly improved and their weight also showed a gradual upward trend.For the pathological section of lung tissue,we observed that terrific amount of inflammatory cells and collagen fibers significantly decreased to that of the BLM group.Meanwhile,the lung coefficient and the content of hydroxyproline in lung tissue all also decreased.Additionally,the expression of Col-1,α-SMA,Smad2/3 and TGF-β1 in lung tissue was lower,on the contrary,the expression of E-cad and Smad7 increased,suggesting that pulmonary fibrosis was significantly improved.Conclusion:1.The pulmonary fibrosis model of mice was successfully established by slowly injecting BLM into the trachea;2.Alpinetin can alleviate the general condition,lung inflammation and lung collagen deposition of bleomycin-induced pulmonary fibrosis mice;3.Alpinetin can reduce the expression of Col-1 and α-SMA,increases the expression of E-cad,reduces the deposition of collagen fibers,reduces the production of extracellular matrix,and improves pulmonary fibrosis;4.Alpinetin can reduce the expression of TGF-β1and Smad2/3,enhances the expression of Smad7,improves pulmonary fibrosis by affecting TGF-β 1/Smad signal pathway.