Protective Effect Of Inonotus Sanghuang Polyphenols Lung Injury And The Underlying Mechanism

Acute lung injury(ALI)and acute respiratory distress syndrome(ARDS)are caused by impaired alveolar capillary barrier and inflammatory cell infiltration.Release of proinflammatory cytokines and reactive oxygen species(ROS)may result in acute hypoxic respiratory failure and irreversible interstitial pulmonary fibrosis.ALI reduces the quality of life of patients and may be life-threatening by affecting their respiratory system.Albeit many treatments have been applied in clinics,there is no specific medicines that can reverse the formation and progression of interstitial pulmonary fibrosis.Thus,it is urgent to develop novel medicines to prevent and/or treat ALI.In general,ALI and idiopathic pulmonary fibrosis(IPF)are uncontrolled oxidative and inflammatory reactions in lung tissues and trachea.Bleomycin(BLM),a glycopeptide antifugal medicine,is most commonly used to simulate the progression of ALI and establish animal model by trachea administration.BLM aggravates the progression of ALI through irritating oxidative and inflammatory reaction.Thus,anti-oxidation and anti-inflammation are the key to cure ALI.Additionally,transforming growth factor-β(TGF-β)plays an important role in developing the pathogenesis of pulmonary fibrosis from lung injury through stimulating the production of collagen in desmocytes.Our previous study has found that Inonotus Sanghung extract(polyphenols)has anti-oxidative,antiproliferative,and anti-microbial activities.However,whether Inonotus Sanghung polyphenols could affect lung injury and fibrosis remains unknown.Current studies aim to determine whether wild Inonotus Sanghung extract(ISE)has the ability to attenuate inflammation in ALI and pulmonary interstitial fibrosis using BLMinduced ALI animal model and TGF-β-induced human lung carcinoma A549 cell model,repectively,and then to investigate the mechanism of action.Part 1.Inonotus Sanghung polyphenols may alleviate acute lung injury in Bleomycin-treated micePurposeThe aim of this study was to evaluate the lung injury protective effects of ISE in BLM-treated mice.MethodsFemale C57BL/6 mice were randomly divided into four groups: control group,BLM group,BLM + 0.15% ISE group and BLM +0.6% ISE group.All four groups were treated with diet with different concentration of ISE or normal diet for 4 weeks.The mice were euthanized day 3 after induced by BLM or normal saline.Following specimens will be collected:(1)lung tissue: HE staining was used to the histological pathological alteration,lung wet/dry weight was used to evaluate the formation of lung edema,Western blot assay was used to investigating the effect of ISE on MAPK and NF-kB signal pathways and biochemical methods were used to detect the MDA level,SOD,CAT,GSH-Px enzyme activity;(2)Bronchoalveolar lavage fluid(BALF)was collect to evaluate the inflammatory cells composition by flow cytometry(FCM),the protein content by biochemical method,and pro-inflammatory cytokines IL-1β,IL-6 and TNF-α by ELISA.(3)Spleen was collected and then splenocytes were isolated to evaluate the spleen cell composition by FCM or stimulated to by LPS to measure the IL-1β,IL-6 and TNF-α levels or ConA to detect the IL-2,IL-4 and IFN-γ levels by ELISA.Results(1)ISE attenuated pulmonary pathology,reduced the protein exudation(P < 0.05)and the levels of IL-1β(P < 0.05),IL-6(P < 0.05),TNF-α(P < 0.05)and inflammatory cell infiltration in the BALF in BLMinduced mice;(2)ISE reduced the production of MDA(P < 0.05),and improve the activity of SOD,CAT,GSH-Px(P < 0.05)in lung tissues from BLM-induced mice;(3)ISE supplementation inhibited the phosphorylation and degradation of IκB-α,a key inhibitor of NF-κB(P < 0.05)in lung tissues,suggesting that the protective effect of ISE on ALI might be mediated via inhibiting NF-κB signal.(4)ISE had less impact on the immune cells distribution subsets,producing inflammatory response in LPS-induced spleen cells and conA induced spleen’s T cell proliferation except IL-1β and IL-2.ConclusionDietary ISE ameliorates BLM-induced ALI in a dose-dependent manner by inhibiting inflammatory and oxidative stress responses through inhibiting NF-kB signaling pathway.Part 2.Effect of ISE on TGF-β-induced epithelial mesenchymal transformation(EMT)of A549 cellsPurposeThe aim of this study was to evaluate the effect of ISE on the epithelial mesenchymal transformation of A549 cells induced by TGF-β.MethodsA549 cells were divided into four groups: control,TGF-β,TGF-β + ISE(2μg/mL),and TGF-β + ISE(4μg/mL).After starving without free-serum media over night,cells were treated with different concentration of ISE for 1 hour,and then stimulated with TGF-β at 5 ng/ml.After 24 h,the cell morphology was evaluated by Microscope,the protein expression of E-Cadherin,Vimentin,Snail,α-Smooth muscle actin(α-SMA),pSmad2/3 and Smad2/3 in A549 cells was detecting by western blot and the toxicity effect of ISE on A549 cells was evaluated by cell counting kit-8(CCK-8)assay.Results(1)ISE suppressed TGF-β-induced A549 cells morphological alteration,migration and invasion abilities;(2)ISE reversed the TGF-β-induced A549 cells epithelial–mesenchymal transition;(3)ISE inhibited EMT through inhibiting TGF-β/Smad2/3 signaling pathway,and EMT-associated transcription factors in TGF-β-treated A549 cells.ConclusionISE suppressed EMT phenotype via inhibiting TGF-β/Smad2/3 signaling pathway.SummaryCurrent study demonstrated the protective effect of dietary ISE on BLM-induced ALI in mice in a dosedependent manor and TGF-β-induced EMT in A549 cells,which might be mediated via inhibiting NF-κB signal and modulating TGF-β/Smad2/3 signaling pathway,respectively.