Anti-pulmonary Fibrosis Effects And Mechanism Of Geniposide

Objectives:To study the anti-pulmonary fibrosis effects and mechanism of geniposide(Gen)in animal model and cells cultured in vitro.Methods:1.On the first day of the experiment,a pulmonary fibrosis(PF)model in mice was induced by intratracheal instillation of BLM,and then Gen(25,50,100 mg/kg body weight)was given by intraperitoneal injection every day.On the 14th and 28th day of the experiment,a batch of mice were sacrificed,and the lung tissues and bronchoalveolar lavage fluid(BALF)of the mice were collected.2.We recorded the lung wet weight of the mice and calculated the lung coefficient.Afrer routine HE staining and Masson staining of the lung tissue,we observed the pathological and morphological changes of the lung tissue of the mice under a microscope,and evaluated the degree of inflammation and fibrosis.3.We counted the total number of cells in BALF and the number of three sorts of inflammatory cells(macrophages,granulocytes,and lymphocytes),determined the total protein concentration in BALF by BCA method,and detectedcontent of TNF-? and MIP-1? in BALF by ELISA(enzyme linked immunosorbent assay).4.The hydroxyproline(Hyp)kit was used to determine the Hyp content in lung tissues;Western blot was used to detect the expression of TGF-?1,CTGF,Smad2/3 and p38 proteins in lung tissues.5.Non-invasive whole body plethysmography(WBP)was used to measure the lung function of mice on the last day of each week for 4 consecutive weeks.6.A549 cells were cultured in vitro,and an epithelial-mesenchymal transition(EMT)model was induced by TGF-?1.The cytotoxicity of Gen to A549 cells was detected by MTT method.7.Western blot and immunocytochemistry were used to detect the expression of E-cadherin,a surface marker of A549 cells.8.Western blot was used to detect the expression of CTGF,AKT/p-AKT and autophagy related proteins(LC3Band Beclin-1)in A549 cells9.Western blot and immunofluorescence were used to detect the expression of the PI3K/AKT/mTOR signaling pathwayrelated proteins and p-ERK1/2 protein in A549 cells.Results:1.From the growth curve of mice,the weight gain of the mice in the the sham operation(normal saline,NS)groupwas the fastest while that of the DXM group was the slowest.The growth rates of the other groups were similar.Pathological observations showed that a large number of inflammatory reactions were seen in the lung tissue of BLM-induced mice,and a large amount of fibrous tissue proliferation and collagen deposition were found in the lung interstitium and tissues around the bronchi.Pathological scores showed that the alveolitis and interstitial fibrosis scores in the model group were significantly increased(P<0.01)compared with NS,and Gen significantly reduced the alveolar inflammation and interstitial fibrosis scores in the model group(P<0.01).0.01).2.The lung coefficient,tissue Hyp content,and Western blot tests showed that compared with the sham operation(normal saline,NS)group,the lung coefficient,Hyp content,and TGF-?1,CTGF,and Smad2 in lung tissue/3 and p38 protein expressions were significantly increased in the model group(P<0.05).Compared with the model group,high dose of Gen(100mg/kg)significantly reduced lung coefficient,Hyp content,and expression of TGF-?1,CTGF,Smad2/3 and p38 protein in lung tissue(P<0.01).In addition,with the exception of lung coefficient,other indicators were comparable to those in the the positive drug(PFD)group(P>0.05).3.BALF total protein concentration,cell count and classification count,TNF-? and MIP-1? content detection results showed that compared with the NS group,the total protein concentration,the number of three types of inflammatory cells and total cells,and the content of TNF-? and MIP-1? in BALF were significantly increased in model group(P<0.05);compared with the model group,Gen at high dose significantly reduced the total protein concentration in BALF,the number of three types of inflammatory cells and total cells,and thecontent of TNF-? and MIP-1?(P<0.05),and the efficacy was comparable to the PFD group(P>0.05).4.Pulmonary function of mice:Compared with the NS group,the Ti,Te and Penh values in the model group were significantly increased(P<0.01),and the PIF,PEF,TV,EV,MV and EF50 values were significantly reduced(P<0.01);Compared with the model group,Gen at high dose or PFD treatment significantly improved the changes of the above pulmonary function parameter values(P<0.01),and the efficacy of Gen is equivalent to that of the PFD group(P>0.05).5.The in vitro cytotoxicity showed that Gen had low toxicity on A549 cells wiht IC50=9.85mg/ml.Cellular immunochemistry and western blot showed that compared with NS group,TGF-?1 significantly reduced the expression of E-cadherin protein in A549 cells(P<0.01),and the expression of E-cadherin protein significantly increased after Gen intervention(P<0.01).6.Western blot showed that compared with the NS group,the expression of CTGF protein in cells induced by TGF-?1 was significantly increased(P<0.01),while the expression of AKT protein was significantly decreased in the TGF-?1 group(P<0.01);compared with the TGF-?1 group,Gen significantly downregulated the expression of CTGF protein and upregulated the expression of autophagy related LC3B and Beclin-1(P<0.05),but had no significant effect on the expression of AKT protein(P>0.05).7.Detection of p-AKT,mTOR,and p-ERK1/2 proteins showed that compared with the NS group,the expression of p-AKT protein in TGF-?1-induced cells was significantly reduced(P<0.01),while p-ERK1/2 and mTOR protein expression was significantly increased(P<0.01);compared with the TGF-?1 group,Gen significantly decreased p-ERK 1/2 and mTOR protein expression(P<0.01),but had no significant effect on the expression of p-AKT protein(P>0.05).Conclusions:1.Intracheal administration of BLM can induce pulmonary fibrosis in mice,which is mainly characterized by an early severe inflammatory response and subsequent conversion of lung tissue to fibrotic lesions in the chronic inflammatory phase,which reduces lung function.2.Geniposide can significantly inhibit lung inflammation induced by BLM in mice,reduce fibrotic lesions in lung tissue,and improve lung function in PF mice.3.The anti-PF effect of geniposideis mainly through inhibiting the expression of inflammatory cytokines(NF-? and MIP-1?),reducing the vascular permeability and the exudation of inflammatory cells in lung tissues;reducing the collagen depositionin lung tissues,downregulating the expression of TGF-?1,CTGF,Smad2/3 and p38 protein in lung tissue,and improving the fibrotic lesions in the lungs of PF mice.4.Geniposide has low cytotoxicity on A549 cells in vitro with IC50=9.85mg/ml.5.Geniposide can significantly inhibit EMT in A549 cells induced by TGF-?1,and increase the expression of E-cadherin protein;the mechanism may be by downregulating the expression of CTGF,p-ERK 1/2 and mTOR proteins and enhancing autophagy in cells.