ERM's Related Pathways In Influenza Virus-induced Permeability Increases In Pulmonary Microvascular Endothelial Cells And The Intervention Of Xijiaodihuang And Yinqiaosan Prescription

BackgroundViral pneumonia is featured by permeability increases and pulmonary microvascular leak.Ezrin/radixin/moesin family proteins,the linkers between plasma membrane and actin cyto skeleton,have been reported to modulate vascular permeability.It has been confirmed that ERM is involved in cell adhesion,motility and may modulate endothelial permeability in response to TNF-a and thrombin.In our previous study,we have confirmed that influenza virus can induce permeability increases in PMVEC.However,the possible role of ERM's related pathways in influenza virus-induced hyperpermeability is unclear and needs further investigation.According to the Traditional Chinese medicine,viral pneumonia is caused by the injury of lung collaterals.Thus,we should treat the disease by expurgating poison and unblocking the collaterals.Xijiaodihuang and Yinqiaosan formula,a classical prescription to treat febrile diseases,was shown to be effective in inhibitinginfluenza virus-induced pulmonary microvascular permeability increases by targeting the PKC-SSeCKS pathway.However,whether the XDY prescription can reverse the permeability increases in PMVEC by regulating ERM via p38MAPK,Rho/ROCK and PKC pathways is still unclear.ObjectiveTo explorethe role of ERM proteins and its upstreams in modulating the permeability increases in PMVEC induced by influenza virus as well as the possible mechanisms.To investigate the effect of XDY on ERM proteins and the pathways that may be involved in the process.Methods1Part ?1.1 The effect of influenza-virus on permeability increases in PMVEC:Influenza virus mouse lung-adapted strain A/FM1/47(H1N1)was propagated in embryonated eggs.Ultimate TCID50 was measured.PMVEC were infected with 100TCID50 influenza virus.At 2h,6h,12h,24h,28h,32h after the virus infection,PMVEC permeability was measured by Transmembrane Resistance Method.1.2 The effect of influenza virus on F-actin of PMVEC:At 24h after influenza virus infection,the expression level of F-actin was evaluated by flow cytometry.The distribution F-actin cytoskeleton was examined using confocal microscopy.1.3 The effect of influenza virus on ERM phosphorylation in PMVEC:At 24h after influenza virus infection,the expression level of p-ERM was evaluated by Western blot.The distribution of p-ERM was examined using confocal microscopy.1.4 The effect of influenza virus on p38MAPK,Rho/ROCK and PKC pathways:At 24h after influenza virus infection,expressions of p-p38,p-MKK,ROCK were evaluated by Western blot and PKC activity was evaluated by PKC activity assay.1.5 Maximum nontoxic concentrations of p38MAPK inhibitor(SB203580),Rho/ROCK inhibitor(Y27632),PKC inhibitor(LY317615)were measured by MTT.1.6 The effect of p38MAPK,Rho/ROCK and PKC pathways on permeability increases and F-actin rearrangement in PMVEC infected with influenza virus:PMVECs were incubated with SB203580,Y27632,LY317615 for 2h before virus infection.At 24h after infection,permeability in PMVEC was measured by TER.The distribution and expression of F-action were evaluated by confocal microscopy and flow cytometry.1.7 The effect of p38MAPK,Rho/ROCK and PKC pathways on virus-induced ERM phosphorylation:PMVECs were incubated with SB203580,Y27632,LY317615 for 2h before virus infection.At 24h after infection,the expression and distribution of p-ERM were measured by Western blot and confocal microscopy respectively.2 Part ?2.1 The production of serum containing XDY:rat serum containing XDY was produced equivalent to the medium concentration that is clinically applied.Toxicity test was performed to evaluate its toxicity at concentrations of 5%,10%and 20%on PMVEC.2.2 The effect of XDY on virus-induced permeability increases:After treatment of virus-infected PMVEC with XDY,PMVEC permeability was measured by TER at 2h,6h,12h,24h,28h,32h after the treatment.2.3 The effect of XDY on virus-induced F-actin rearrangement:At 24h after XDY treatment,the distribution and the expression of F-actin were examined using confocal microscopy and flow cytometry.2.4 The effect of XDY on virus-induced ERM changes:At 24h after XDY treatment,expressions of p-ERM was evaluated by Western blot,and ERM distribution was detected by confocal microscopy.2.5 The effect of XDY on p38MAPK,Rho/ROCK and PKC pathways:At 24h after XDY treatment,p-p38,p-MKK and ROCK were measured by Western blot.PKC activity was measured using PKC activity assay.Results1 Part I1.1 Influenza virus infection led to permeability increases in PMVEC at 6h,which peaked at 24h.1.2 At 24h,influenza virus induced the F-actin stress fiber aggregation in the cytoplasm as the confocal microscopy manifested.Flow cytometry showed that the F-actin expression in the virus group was higher than that in the control group(P<0.001).1.3 After the influenza virus exposure for 24h,p-ERM in the virus group was higher as compared to that in the control group(P<0.01),as Western blot suggested.As seen in confocal microscopy,influenza virus induced p-ERM increases in both membrane and cytoplasm.1.4 p-MKK and ROCK,as compared to the control group(P<0.01;P<0.05;P<0.001).Influenza virus also promoted PKC activity.1.5 Inhibitors of the p38MAPK,Rho/ROCK and PKC pathways abolished virus-induced permeability increases and F-actin rearrangement.1.6 Inhibitors of the p38MAPK,Rho/ROCK and PKC pathways downregulated p-ERM.The decreases in p-ERM were statistically insignificant as compared to the p-ERM in the virus group.2 Part ?2.1 TheXDY at concentrations of 20%,10%and 5%are all nontoxic to PMVEC.2.2 TER in the XDY group was higher than that in the virus group at 2h,6h,12h,24h,28h and 32h after the influenza virus infection(P<0.001).2.3 XDY prevented influenza virus-induced F-actin rearrangement,as featured by the decreases in stress fiber formation.As flow cytometry showed,the F-actin level decreased in the XDY group as compared to that in the virus group '(P<0.05).2.4 XDY suppressed virus-induced ERM phosphorylation in PMVEC as Western blot indicated.The amount of p-ERM in XDYgroup was lower,as compared to that in the virus group(P<0.05).Confocal microscopy suggested XDY downregulated p-ERM levels in PMVEC infected with virus.2.5 The expressions of p-p38,p-MKK and ROCK were lower in the XDY group as compared to those in the control group(p-p38:P<0.001,ROCK:P<0.001).PKC activity was weaker in the XDY group,compared to that in the virus group(P<0.01).Conclusion1 Influenza virus may increase transendothelial permeability in PMVEC,with the primary mechanism involving activation of p38MAPK,Rho/ROCK and PKC pathways,ultimately leading to ERM phosphorylation.The activated ERM proteins then localize primarily along the periphery of PMVEC where they may cross-link membrane with F-actin,leading to stress fiber formation and paracellular gap formation and eventually contributing to permeability increases.2 XDY prescription may ameliorate influenza virus-induced permeability increases by inhibiting ERM phosphorylation via targeting p38MAPK,Rho/ROCK and PKC pathways.