Effects Of Smad7 Ubiquitination On Pulmonary Fibrosis Induced By Bleomycin In Mice

Objective: To observe the expression changes in Smad7,Smad7 ubiquitination,Smad7 acetylation and related fibrosis indexes in pulmonary fibrosis caused by bleomycin(BLM)in mice,and explore the effect of TGF-?1 / Smads signaling pathway mediated by Smad7 ubiquitination on the occurrence and development of pulmonary fibrosis,and the relationship between Smad7 ubiquitination and Smad7 acetylation,so as to provide theoretical and experimental basis for finding therapeutic targets for pulmonary fibrosis.Methods: 30 C57 mice with 18-20 g in body weight were selected,and randomly divided into blank group,BLM group and BLM + MG132(ubiquitin-proteasome inhibitor)group,with 10 mice in each group.The pulmonary fibrosis model of BLM group and BLM+MG132 group was constructed with BLM(5mg/kg)by dropping it into trachea at once.The mice in BLM + MG132 group were intraperitoneally injected with MG132(0.1mg / kg)once a day from the 1st day after modeling until the 28 th day.At 28 th day after modeling,the lungs of mice were taken for further detection.Pathological sections were made for the inferior lobe of left lung in mice;HE staining was used to observe the morphological changes of the lung tissue under light microscope;Masson staining was used to observe the collagen content;Hydroxyproline content was determined to evaluate the degree of pulmonary fibrosis;Westernblot was used to detect the protein levels of Smad7,?-SMA and Col-I;q RT-PCR was used to detect m RNA expression changes of Smad7,Col1a1(Col-I),TGF-?1 and ?-SMA in each group;Immunoprecipitation(IP)of Smad7 protein and immunoblotting(IB)were used to detect the ubiquitination and acetylation levels of Smad7 in lung tissues of mice in each group.Results: 1.At 28 th day after BLM modeling,the lung tissue of C57 mice with HE staining showed stenosis and collapse of alveolar space,thickening of alveoli septum,infiltration of neutrophils and macrophages in the blood vessels around the alveoli and trachea,and destruction of normal lung tissue structure under light microscope;Masson staining showed that large areas of stained blue purple collagen fibers were found in pulmonary interstitium and alveoli septum under light microscope;the content of hydroxyproline in lung tissue of group M was higher than that in the blank group;the above results indicated that the pulmonary fibrosis model was successfully constructed in C57 mice at 28 th day after BLM was dropped into the trachea.After the intervention with MG132(mice in BLM + MG132 group),HE staining showed that the degree of alveolar stenosis,alveolar septum thickening,inflammatory cell infiltration was less than those in BLM group;Masson staining showed that the blue purple collagen fiber was reduced;meanwhile,the content of hydroxyproline in lung tissue was reduced,suggesting that the degree of pulmonary fibrosis was relieved.2.After BLM treatment,compared with the blank group,the expression of ?-SMA,Col-I protein increased,the expression of Smad7 protein decreased,and the difference was statistically significant(P< 0.05);the m RNA expression of Col1a1,?-SMA,TGF-? 1 and Smad7 was up-regulated,and the difference was statistically significant(P < 0.05);the level of Smad7 ubiquitination increased,the level of Smad7 acetylation decreased.3.After MG132 treatment,compared with BLM group,the levels of ?-SMA,Col-I protein in lung tissue decreased,Smad7 protein increased,and the difference was statistically significant(P < 0.05);the m RNA expression of COL1A1,?-SMA,TGF-? 1,Smad7 was down-regulated,and the difference was statistically significant(P < 0.05);the level of Smad7 ubiquitination decreased,the level of Smad7 acetylation increased.Conclusion: 1.the decreased expression of Smad7 in the pulmonary fibrosis caused by BLM in mice,and over-activation of TGF-?1/Smads signaling pathway promote the occurrence and development of pulmonary fibrosis.2 MG132 can inhibit the level of Smad7 ubiquitination,increase the level of Smad7 acetylation,up-regulate the protein level of Smad7,enhance the negative regulatory effect of Smad7 on TGF-? 1 / Smads signaling pathway,and alleviate the pulmonary fibrosis.In addition,there may be competitive relationship between ubiquitination and acetylation of Smad7.