Role Of Nogo-B In The Epithelial-mesenchymal Transition Of Acute Lung Injury Fibroproliferation

Backgrounds Acute lung injury(ALI)/ Acute respiratory distress syndrome(ARDS)is a severe respiratory disorder.The mortality of ALI has remained stubbornly high with the comprehensive treatments.But even when patients have passed the acute stage steadily,Chances are high that lung fibrosis will happen as a result of the fibroproliferation phrase characterized by accumulation of collagen and fibroblasts,which is closely related to ventilator dependence,reduced quality of life and grave prognosis.Three cell population seem to contribute to this phrase: lung epithelial cells transforming into myofibrocytes via epithelial–mesenchymal transition(EMT),fibrocytes from bone marrow and resident lung fibrocytes.Reticulon4B(RTB4B),also called Nogo-B,is located in the endoplasmic reticulum as a number of Reticulon family.It has reported that Nogo-B played an important role in regulating inflammation and tissue repair.It participates in liver fibrosis and the EMT of Hela cells according to recent studies.And we have already proved that Nogo-B is expressed on lung epithelial cells and regulates the inflammation phrase of ALI.We are now wondering whether it is involved in fibroproliferation phrase of ALI..This study aims to clarify the position and role of Nogo-B in fibroproliferation phrase of ALI to help with the further understanding of the pathogenesis of ARDS and provide new therapy for its treatments.Methods1.In vivo experiment:(1)Objects: West type C57BL/6 murine models and Nogo-B knockout murine models,6-8weeks,male.(2)Molding and Grouping: We built the ALI murine models by intratracheal instillation of lipopolysaccharide(LPS),or phosphate buffer saline(PBS)as control.Mice were were randomly divided into Day0(control group),Day 1,Day4,Day7 and Day10 groups.(3)Testing Methods: HE stain,Masson Stain,Immunohistochemistry(IHC),real-time quantitative PCR(rt-q PCR)and Western Blot(WB)were used to evaluate the expression of Nogo-B and whether it participates in fibroproliferation of lung tissue.We also tested the expression of epithelial cell marker E-cadherin and mesenchymal cell markers α-SMA and Vimentin,as well as the main inducing factor of fibrosis,TGFβ,to determine whether lung fibrosis happened in ALI.2.In vivo experiment:(1)Objects: Lung epithelial cells MLE-12(2)Molding and Grouping: In the experimental group,we down-regulated the expression of Nogo-B in MLE with the help of si RNA.And negative si RNA was used in negative control group while no si RNA in blank control group.TGFβ1 was used to induce the EMT of both normal MLEs and MLEs with Nogo-B down-regulation.RNA and proteins were collected for further tests.(3)Testing Methods: rt-q PCR and WB were used to test the expression of Nogo-B in TGFβ1 induced MLEs and compare the EMT markers of two groups to find out the role of Nogo-B in EMT.Results1.The expressiong of Nogo-B and its connection with lung fibrosis in LPS induced ALI murine models Accoring to the HE stain,the inflammation reaction was severe in Day 1.As time went by,fibroproliferation happened.On Day 7 and Day 10,the absorption of inflammation was basically completed but a significant degree of pulmonary fibrosis was observed.And Nogo-B was expressed even in control group,then its expression decreased in Day 1 but increased after some while.Its high expression lasted in Day7 and Day10,which has paralleled the change of lung fibrosis.Besides,the release of TGFβ increased and the expression of E-cadherin went down on day 1 and α-SMA and Vimentin went higher then.TGFβ,α-SMA and Vimentin was kept highly expression while E-cadherin was low on Day 7 and Day 10.2.Regulation of the expression of Nogo-B and its influence in inflammation and fibroproliferation phrase of ALI murine models.Nogo-B knockdown group(KO group)was compared with normal wild type group(WT group).The HE stains of the control of both groups were almost same.But on Day 1,the inflammation was worse in KO group.On Day 7,there was still some inflammation in KO group.But on Day 10,the degree of fibrosis was milder in KO group rather then WT group.With same expression of TGFβ in two groups,the expression of α-SMA and Vimentin was lower in KO group.It seemed that worse inflammation but milder fibrosis were happened in KO group.3.The possible mechanism of Nogo-B regulating lung fibrosis in ALI murine models.It is reported that EMT played an important role in lung fibrosis of ALI.We usesi RNA to down-regulate the expression of Nogo-B in MLE and TGFβ to induce EMT in it.With TGFβ1 inducing EMT in MLE-12,Nogo-B expressed higher in a time dependent way.But with down-regulation of Nogo-B using si RNA(si RNA group),E-cadherin kept a higher expression and α-SMA and Vimentin lower induced by TGF β 1 than control group.Besides,the down-regulation of Nogo-B interfered the phosphorylation of Smad2 induced by TGFβ1,which means Nogo-B regulated TGFβ1 induced EMT in MLE via TGFβ/Smad signals.Conclusion1.In LPS induced ALI murine moedels,lung fibrosis was happened and Nogo-B played a role in both inflammation phrase and fibroproliferation phrase.2.Compared with WT group,Nogo-B KO group suffered worse inflammation reaction but milder fibroproliferation reaction,which showed the importance of Nogo-B in protection of ARDS.3.Nogo-B down-regulation could reduce EMT of MLE induced by TGFβ1 via TGFβ/Smad signal.It suggested that Nogo-B might take a part in fibroproliferation of ALI with its participation in EMT.