Effect Of Fisetin On Pulmonary Fibrosis In Mice And Its Mechanism

Objective: To observe the therapeutic effect of Fisetin on pulmonary fibrosis(PF) in mice,a bleomycin(BLM)-induced model of pulmonary fibrosis(PF) was established,and Fisetin(Fis) was intraperitoneally injected as intervention.Human pulmonary fibroblasts(HPFs) were treated with Fisetin to explore the relationship between Fisetin and the proliferation and migration of HPFs induced by human transforming growth factor-β1(TGF-β1) and the possible signaling pathways involved in this process,so as to provide experimental basis for the discovery of new drugs for pulmonary fibrosis treatment.Methods: Animal experiments part: 1.In this study,a mouse model of pulmonary fibrosis was established and intraperitoneal injection of Fisetin was used to intervene.Mice were randomly divided into four groups :(1)PBS group (n=10);(2)DMSO group(n=10);(3)BLM+DMSO group (n=10);(4)BLM+Fis(2 mg/kg) group (n=10),the body weight of mice was monitored regularly.2.Serum concentrations of alanine aminotransferase(ALT),ascorbic aminotransferase(AST),creatinine(Cre) and uric acid(UA) were determined to evaluate the liver and kidney function of mice.3.The lung tissue pathology of mice was detected by H&E,Masson and Sirius red staining.4.Collagen content in lung tissue was determined by hydroxyproline acid hydrolysis method.5.The expression of phenotypic factors(Fibronectin,Collagen 1,α-SMA)of pulmonary fibrosis in mouse lung tissue was detected by Western Blot,RT-PCR and immunofluorescence.Cell Experiment Part: 1.In this study,human pulmonary fibroblasts(HPFs) were cultured in vitro,and HPFs was induced by Fisetin treatment and TGF-β1.All the cells experiments were divided into three groups :(1)DMSO(2‰) group;(2)DMSO(2‰)+ TGF-β1(10 ng/mL) group;(3)TGF-β1(10ng/mL)+ Fis(10μM)group.2.Western Blot,RT-PCR and immunofluorescence results showed that the expressions of Fibronectin,Collagen1 and α-SMA in TGF-β1 induced group were increased to varying degrees compared with DMSO control group,but the expressions of these phenotypic factors were decreased to varying degrees in TGF-β1+ Fis group compared with DMSO+ TGF-β1 group.3.Cell Proliferation/Toxicity Assay Kit(CCK-8)assays the cell viability of HPFs treated with different concentrations of Fisetin for different periods of time.4.The proliferation and migration of cells were detected by Ed U proliferation kit and Transwell cell migration assay.5.The expression of PI3K/Akt/m TOR signaling pathway was detected by Western Blot.Results: By single airway injecting bleomycin,pulmonary fibrosis in mice model was established successfully,pathological dyeing results show that,compared with control group,BLM can be obviously lung inflammation and collagen deposition made module,the structure of the lung damage,but the BLM+ Fis group area of pulmonary fibrosis and pulmonary structure damage degree was lower than that in group BLM + DMSO(P<0.001),the collagen content of BLM + Fis group also significantly lower than the BLM + DMSO group(P <0.001).The body weight monitoring results showed that the body weight of the model group decreased significantly compared with the control group within 7days after BLM airway injection(P<0.05),after 7 days,the body weight of the model group began to rise slowly,but the degree of weight gain in the BLM+Fis group was significantly higher than that in the BLM+DMSO group(P<0.01).The evaluation results of liver and kidney function in mice showed that the intervention of Fisetin did not lead to abnormal liver and kidney function in mice.Western Blot,RT-PCR and immunofluorescence results showed that the expression of phenotypic factors(Fibronectin,Collagen 1,α-SMA)of pulmonary fibrosis in the BLM model group was significantly increased compared with the control group,but the expression of these factors were significantly decreased in the BLM+Fis group compared with the BLM+DMSO group(P<0.001);In the experiment of human lung fibroblasts cultured in vitro,the results of CCK-8showed that the cell viability of different concentrations(0μM,5μM,25μM,50μM,100μM,200μM)of Fisetin on HPFs was more than 50% at different time periods(12h,24h,48h).Western Blot,RT-PCR and immunofluorescence results showed that the expression of Fibronectin,Collagen 1 and α-SMA were increased in TGF-β1-induced group compared with DMSO control group,but these phenotypic factors were decreased in TGF-β1+ Fis group compared with DMSO+TGF-β1 group to different degrees.Ed U results showed that TGF-β1 stimulated the proliferation of HPFS significantly,but the treatment of Fisetin significantly reduced the proliferation of HPFs(P<0.001).Transwell cell migration assay showed that TGF-β1 significantly stimulated the migration of HPFs,but the degree of migration of HPFs was significantly reduced by the treatment of Fisetin(P<0.001).Western Blot analysis of the expression of PI3K/Akt/m TOR signaling pathway showed that the phosphorylation level of PI3K/Akt/m TOR signaling pathway was significantly reduced by Fisetin treatment,and the inhibition effect was most significant at the time point of 3h(P<0.01,P<0.001,P<0.001).Conclusions: 1.Fisetin can effectively improve the degree of pulmonary fibrosis induced by bleomycin in mice,reduce lung inflammation and reduce lung structural damage 2.Fisetin can inhibit the differentiation,proliferation and migration of HPFS cells induced by TGF-β1 3.Fisetin inhibits the phosphorylation of PI3K/Akt/m TOR signaling pathway.