The Role And Mechanism Of ResolvinD1 In Airway Barrier Damage Induced By Influenza A Virus

Objective: Influenza A virus(IAV)can cause seasonal burden and even pandemics,leading to considerable morbidity and mortality throughout the world,it is a major public health event that endangers human health.Airway epithelial cells are the first physical barrier in lung tissue and play a crucial role in defense against inhaled allergens,toxins,and infectious pathogens such as influenza viruses.As important components of airway epithelial cell barrier,tight junction proteins(such as ZO-1)and adhesion junction proteins(such as E-cad)maintain the structural and functional integrity of airway barrier.Studies have shown that during influenza virus infection,a large number of inflammatory cytokines and ROS are produced in lung tissue,and these mediators are inducers of airway barrier injury.Nuclear factor erythrocyte 2-associated factor 2(Nrf2)is a key antioxidant transcription factor.Under oxidative conditions,Nrf2 enters the nucleus and initiates the transcription of antioxidant genes(such as HO-1)and regulates antioxidant enzymes,thereby reducing oxidative damage.Resolvin D1(RvD1)is a lipid medium derived from omega-3 polyunsaturated fatty acids and has anti-inflammatory and antioxidant activities.Studies have shown that RvD1 protects the integrity of intercellular adhesion junctions and barrier function by inhibiting ROS-mediated SHP2 inactivation.Airway barrier injury and inflammatory induced by IAV are thought to be associated with disease progression and prognosis.As a potential lipid mediator,RvD1 has an important protective effect on lung infection.Whether RvD1 has a protective effect on injury induced by IAV,and the mechanism remains unclear.Therefore,we hypothesized that RvD1 could protect the airway barrier and inhibit the inflammatory response,aiming to explore the role and mechanism of RvD1 in the airway barrier injury caused by IAV.Methods:1.Isolation and culture primary normal human bronchial epithelial cells(pNHBE);Different concentrations of H3N2 infected pNHBE cells and Western blot was used to detect the expression of E-cad and ZO-1 to find out the best working concentration.Western blot was used to detect the expression of E-cad and ZO-1 in pNHBE cells infected with H3N2(MOI 50)for different times,and the optimal action time was explored.pNHBE cells infected with H3N2 were treated with different concentrations of RvD1,and the expressions of E-cad and ZO-1 were detected by Western blot to find out the best working concentration of RvD1.2.pNHBE cells were treated with IAV(MOI 50),RVD1(200n M),IAV(MOI 50)+RvD1(200n M)or control solvent,the expression levels of E-cad and ZO-1 were detected by PCR,Western blot analysis and immunofluorescence staining.3.pNHBE cells were treated with IAV(MOI 50),RvD1(200n M),IAV(MOI 50)+RvD1(200n M)or control solvent,Phospho-NF-κB p65,NF-κB p65,IKBα and p-IKBα were detected by Western blot,IL-8 and TNF-α were detected by PCR.4.pNHBE cells were treated with IAV(MOI 50),RvD1(200n M),IAV(MOI 50)+RvD1(200n M)or control solvent,ROS levels in different treatment groups were detected by DCFH-DA probe.5.pNHBE cells were treated with IAV(MOI 50),RvD1(200n M),IAV(MOI 50)+RvD1(200n M)or control solvent,the levels of Nrf2 and HO-1 were analyzed by Western blot.The nucleoprotein extraction kits were used to extract the nucleus and cytoplasm of pNHBE cells in different treatment groups.The levels of Nrf2 in the nucleus and cytoplasm of pNHBE cells in different treatment groups were analyzed by Western blot,then quantified by comparing with histone in the nucleus and β-actin in the cytoplasm,respectively.The expression level and distribution of Nrf2 in different treatment groups were detected by immunofluorescence staining.6.The pNHBE cells were transfected with recombinant lentivirus HBLVH-NRF2 sh RNA-Puro,then pNHBE cells were treated with RvD1(200n M),IAV(MOI 50),IAV(MOI 50)+ RvD1(200n M)or control solvent.ZO-1,E-cad,Nrf2,HO-1,Phospho-NF-κB p65,NF-κB p65,IKBα,and p-IKBα were detected and analyzed by Western blot.IL-8 and TNF-α levels were detected by PCR.DCFH-DA probe was used to detect intracellular ROS levels in different treatment groups.7.Mice were inhaled with 100μl 0.9% normal saline or H3N2 via airway and given RvD1(100 ng)on day 4 and 6,the body weight changes and survival of each group were recorded;After mice were treated with normal saline or H3N2 for 7 days,BALF,lung tissues and serum were collected,and the levels of IL-8 and TNF-α in BALF were detected by ELISA.The total number of BALF cells were counted and the number of cells were further classified by Wright-Giemsa staining.The lung tissues were fixed,and HE staining and immunohistochemistry were performed.Results:1.pNHBE cells were infected with H3N2(MOI 50)for 24 h,and expressions of E-cad and ZO-1 in airway epithelial cells were down-regulated;RvD1(200n M)upregulated the expressions of E-cad and ZO-1 in airway epithelial cells infected with H3N2.2.After H3N2 infection,the m RNA and protein expression levels of E-cad and ZO-1 in airway epithelial cells were significantly lower than those in the control group(P <0.05).RvD1 treatment inhibited the decrease of E-cad and ZO-1 m RNA and protein expression in H3N2-induced pNHBE cells(P < 0.05).Immunofluorescence staining further confirmed the effect of RvD1.E-cad staining of cell membrane was obvious and complete in the control group.After H3N2 infection,E-cad staining around cells was significantly weakened and discontinuous.RvD1 treatment inhibited the damage of E-cad in pNHBE cells induced by H3N2,with significant difference(P < 0.05).3.Infection pNHBE cells with H3N2 significantly increased intracellular NF κB p65 and IKBα phosphorylation and increased the levels of IL-8 and TNF-α m RNA.RvD1 treatment effectively inhibited the level of inflammatory response in airway epithelial cells induced by H3N2,with significant difference(P < 0.05).4.After pNHBE cells were infected with H3N2,the intracellular ROS level was significantly increased,and RvD1 inhibited increased level of ROS in airway epithelial cells induced by H3N2,with significant difference(P < 0.05).5.H3N2 infection down-regulated the expression levels of Nrf2 and HO-1 in pNHBE cells,while RvD1 treatment inhibited them and promoted Nrf2 nuclear translocation.6.RvD1 inhibits the down-regulated expression of E-cad and ZO-1,and the up-regulated expression of IL-8 and TNF-α in pNHBE cells induced by H3N2.However,when Sh-Nrf2 was transfected simultaneously,the inhibitory effect of RvD1 was weakened.The expression of phosphorylated NF κB p65 and p IKBα,the levels of IL-8 and TNF-α and the production of ROS were significantly increased.7.In mice infected with H3N2 for 7 days,bronchial and alveolar cavities were infiltrated by inflammatory cells,and neutrophils,lymphocytes and cytokines in BALF were also significantly increased.After inhalation of H3N2,RvD1 was given on day 4and 6,respectively,and inflammation in lung tissue was reduced.The neutrophils,lymphocytes and cytokines in BALF were significantly reduced after treatment with RvD1(P < 0.05).However,the inhibition effect of RvD1 on virus proliferation was not obvious.RvD1 alleviated weight loss in mice infected with H3N2,but there was no significant difference in mortality in mice infected with H3N2(P < 0.05).RvD1 had a protective effect on airway injury induced by H3N2.Immunohistochemical results showed that bronchial epithelial cells were damaged in mice infected with H3N2,and E-cad staining was weaker than that in the control group.RvD1 significantly inhibited the damage of airway epithelial cells in mice infected with H3N2.In addition,changes in barrier function were further observed by detecting FD4 leakage after H3N2 and/or RvD1 intervention.The ratio of BALF to serum FD4 increased from 0.743 to 1.371 after H3N2 infection(P < 0.05),then decreased to 0.951 after RvD1 treatment(P < 0.05).These results suggest that RvD1 effectively protects bronchial epithelium from H3N2-induced permeability changes.Conclusion:RvD1 can alleviate H3N2-induced airway epithelial barrier injury and airway inflammation through the Nrf2 pathway,which may provide a better treatment strategy for influenza A virus infection.