Study On The Effect And Mechanism Of Demethylzeylasteral In Bleomycin-induced Pulmonary Fibrosis In Mice

Objectives:Pulmonary fibrosis（PF）is a chronic inflammatory lung disease,and the fibroblast-to-myofibroblast transition（FMT）is the key step in its pathogenesis.Demethylzeylasteral（T96）is a natural compound which has anti-inflammatory and immunosuppressive actions.At present,the effects and its mechanism of T96 on PF have not been clarified.In this study,the bleomycin（BLM）-induced PF mice model were established to observe the effects of T96on pulmonary fibrosis.Human fetal lung fibroblast 1（HFL1）stimulated by TGF-β₁ were treated with T96 to observe the effects and the related mechanisms of T96on the proliferation,migration and differentiation of HFL1.Based on this study,we hope to provide a new experimental basis for the clinical treatment of PF.Methods:1.For in vivo study:（1）A single airway injection of BLM were used to establish the mice PF model,and T96 were injected intraperitoneally.（2）The body weight,behavioral score and survival rate of mice were observed to evaluate the general condition of mice.（3）The safety of drug was evaluated by detecting liver and kidney function in mice.（4）The severity of pulmonary fibrosis was estimated by H&E staining,Masson staining and Sirius red staining,and the collagen content of lung tissue was detected by hydroxyproline（HYP）content assay kit.（5）The expression levels of fibrosis-related markers in lung tissue of mice were detected by q RT-PCR and Western Blot to evaluate the effect of T96on PF.2.For in vitro study:（1）CCK-8 kit was used to detect the activity of HFL1cells and determine the optimal concentration of T96.（2）The proliferation,migration and differentiation of cells were determined by Ed U kit staining,Transwell,scratch and immunofluorescence experiment respectively.（3）The m RNA and protein expression levels of fibrosis related markers in HFL1 cells were detected by q RT-PCR and Western Blot.（4）The effects of T96 on TGF-β/Smad signaling pathway were investigated by detecting the expressions of protein its phosphorylation level for related factors.Results:1.In vivo results:（1）After T96 intervention,there was no obvious abnormality of liver and kidney function in BLM-induced pulmonary fibrosis mice.（2）The pathological staining and HYP content measurement of lung tissue of mice indicated that T96 could alleviate BLM-induced pulmonary inflammation and fibrosis in mice.（3）T96 can significantly reduce the protein and m RNA levels of PF related factors,and inhibit the FMT.2.In vitro results:（1）CCK-8assay confirmed that T96（10μM）had little effect on the activity of HFL1 cells.（2）The levels of related fibrosis factors secreted by HFL1 cells after T96pretreatment were significantly down-regulated compared with TGF-β₁ group.（3）T96 inhibited Smad2/3 phosphorylation induced by TGF-β₁ in vitro.Conclusions:1.T96 can reduce BLM-induced lung inflammation and fibrosis in mice with PF in vivo.2.T96 inhibited the proliferation,migration and myofibroblast differentiation of HFL1 induced by TGF-β₁ in vitro.3.T96 may play a therapeutic role in pulmonary fibrosis by inhibiting TGF-β/Smad signaling pathway.