The Protective Role And Mechanism Of Gentiopicroside Against Acute Lung Injury And Chronic Pulmonary Fibrosis

Background and objectives:Acute lung injury(ALI)is wide-ranging and excessive inflammation induced by many causes such as serious infection,trauma and poisoning[1-3].ALI may be aggravated as acute respiratory distress syndrome(ARDS),threatening to life.Its pathogenesis is complex with high case fatality.Regulation disorder of inflammatory cells and inflammatory medium is one of the most important pathogenesis of ALI.Although,to drug therapies of ALI,some studies had shown that the anti-inflammatory therapies had the potential to reduce lung injury,there is still no convincing evidence that drugs,such as granulocyte macrophage colony stimulating factor,glucocorticoid,antioxidarnts and others,can significantly reduce the mortality of patients with ALI in clinical trials[4-6].Several studies had shown that microRNAs(miRNAs)involved in the pathogenesis of ALI regulation17,8],and had the potential to regulate inflammation and immune reaction.Therefore,research on miRNAs expression changes in ALI models or patients with drug treatment,is one important way to search potential drug targets.ALI patients were so lucky to be pull through acute crisis,but the survivors are still possibly suffered with some long-term sequelaes,including development of chronic pulmonary fibrosis diseases inducing pulmonary dysfunction and affecting the quality of life.Pulmonary fibrosis(PF)is a chronic interstitial lung disease induced by a variety of causes,with only 20%5 years survival rate[9]The mechanism of PF is not fully clear.The pirfenidone and Nintedanib is main new drugs for the treatment of PF[10,11],but their price is too expensive to patients with PF for long-term administration.Unfortunately,in China,there is still no anti-PF drug with independent intellectual property rights.Therefore,it's very necessary to research and develop economic,high efficiency,and low toxicity of anti-ALI and anti-PF drugs with Chinese independent intellectual property rights.As reported,the gentiopicroside(GPS)as the main effective components of gall grass has many pharmacological effects such as anti-infection,anti-ALI and so on[12-15].But the mechanism of GPS anti-ALI is still unclear,the study on PF treated with GPS is lacking.Hence,in this study,the formation process of ALI and PF in lungs of model mice were intervened by GPS,miRNA expression gene chip technology was used to detect the change of miRNAs expression in mice with lipopolysaccharide(LPS)-induced ALI before and after the mice were treated with GPS.The pathological morphology,protein and gene expression level were studied to explore the effects of GPS on prevention and treatment to ALI and PF.The objects were to further clarify the mechanism of action of GPS anti-ALI,and explore whether GPS take anti-PF effects and its mechanism of the action.This research was done to provide experimental basis for GPS to prevent and treat the diseases of ALI and chronic PF.Methods:1.To copy the mice models of LPS-induced ALI and bleomycin(BLM)-induced PF.2.The staining of HE,Masson's trichrome and immunohistochemistry,and electron microscopy examination were done to evaluation of histopathological morphology of inflammatory,PF,and NF-?B expression,respectively.3.The contents of total proteins,cell counts,and the contents of TNF-a and IL-1? in bronchoalveolar lavage fluid(BALF)were tested by bicinchoninic acid(BCA)method,cells smears,and enzyme linked immunosorbent assay(ELISA),respectively.4.To detect the effects of GPS on ALI and PF mice,the contents of hydroxyproline(Hyp),superoxide dismutase(SOD),myeloperoxidase(MPO)and malonaldehyde(MDA)were determinated by respective test fits.5.The proteins expression of AQP1,AQP5,TGF-?1 and CTGF were tested by western blot,respectively.6.The gene expression of AQP1 and AQP5 were tested by qPCR,respectively.7.Using the miRNAs gene chip array technology to analyze the change of miRNAs expression levels in lung tissues of LPS-induced ALI mice by treated with GPS.8.In vitro and immunocytochemistry(ICC),A549 cells stimulated by TGF-?1 were treated with increas:ing concentration of GPS and positive staining cells of E-cathrin and ?-SMA were counts to analyze the change of epithelial-mesenchymal transition process.Results:1.In the evaluation of histopathological,diffuse alveolar inflammation and severe pulmonary edema were found in lungs of LPS-induced ALI mice,while pulmonary fibrosis and inflammation presented in lungs of BLM-induced PF mice.2.In the evaluation of cells smears,qPCR,western blot,ELISA and immunohistochemistry,the contents of total proteins,granulocytes,TNF-? and IL-1?in BALF,and NF-?B expression in lung tissues were significantly increased in ALI model group(P<0.05,compared with normal saline group),while significantly decreased in GPS treated groups(P<0.05,compared with ALI model group),and more significantly in high dose of GPS group,respectively.The genes and proteins expression of AQP1 and AQP5 were significantly decreased by LPS(P<0.05,compared with normal saline group)and increased by GPS(P<0.05,compared with LPS group),respectively.3.The contents of MPO,MDA and SOD in lung tissues were significantly increased and deceased in ALI model group(P<0.05,compared with normal saline control group),while significantly decreased and increased in dose-dependence in GPS treatment groups(P<0.05,compared with ALI model group).4.In the evaluation of cells smears,Hpy test,western blot and ELISA,at the same stage,the contents of total proteins,TNF-??IL-1? and ratios of macrophages in BALF,and contents of Hyp,TGF-?1 and CTGF in lungs were significantly increased in PF model group(P<0.05,compared with normal saline group),While significantly decreased in dose-dependence in GPS treatment groups(P<0.05,compared with PF model group),respectively.5.In immunocytochemistry(ICC),the positive cells ratios of E-cadherin and a-SMA in TGF-? group were significantly decreased and increased(P<0.05,compared with control group),while significantly increased and decrease in TGF-?1 group(P<0.05,compared with TGF-? group),respectively.GPS took effects similar with DXM(P>0.05)in dose-dependence.6.In miRNAs gene chip testing in mice lung tissues,60 miRNAs were found to be closed relative with pathogenesis of ALI,and mainly involved in 36 significant signaling pathways,including 6 up-regulated and 30 down-regulated,respectively.The regulation of inflammation and immune response were involved by 6 and 3 main signaling pathways,respectively.7.Go-pathway analysis shown that,for the treatment of ALI mice,GPS and DXM commonly regulated 18 effective targeted miRNAs regulating 885 predicted target genes.The target genes participated in regulating 5 significant signaling pathways.8.The miRNA-Gene-Net analysis shown that in the process of mice LPS-induced ALI,the miRNAs and genes with the highest network degrees were mmu-miR-124-3p and mmu-miR-6394,Amot and B3gat2 genes,respectively.Conclusions:1.GPS can significantly inhibit the ALI and inflammatory responses induced by LPS in the lungs of mice by decreasing the levels of granulocytes percentage,TNF-a and IL-1? in BALF,down-regulating the expression of NF-?B,MDA,and MPO,and up-regulating the expression of AQP1,AQP5 and SOD in lungs of PF mice.2.In the process of LPS-induced ALI in mice,mmu-miR-124-3p and mmu-miR-6394 were the key regulatory miRNAs.The targeted genes such as Amot and B3gat2 were regulated by relatively more miRNAs.3.In the treatment of LPS-induced ALI mice,GPS and DXM common regulated 18 target miRNAs including 12 down-regulated miRNAs.4.GPS can continuously and significantly inhibit both inflammatory and fibrotic responses in lungs of BLM-induced PF mice by decreasing the levels of macrophages percentage,TNF-a and IL-1? in BALF and down-regulating the expression of TGF-?1 and CTGF in lungs of PF mice.5.GPS inhibited epithelial-mesenchymal transition of A549 cells induced by TGF-?1.The alveolar epithelial cells and TGF-?1 may be the key target cells and molecular in the anti-PF process of GPS,respectively.