| BackgroundP.aeruginosa is one of the most frequent opportunistic pathogens responsible for serious lower respiratory tract infection,which is becoming difficult to treat because of complex drug-resistant mechanism and limited therapeutic options.It has been reported that P.Raeruginosa infection is associated with airway fibrotic remodeling which can lead to irreversible functional decline and poor prognosis,how to alleviate this pathologic change remains unknown.Epithelial cells could transdifferentiate into fibroblast phenotype via epithelial-mesenchymal transition(EMT),and then activated fibroblast cells would undergo a differentiation into myofibroblasts and oversecret MMPs,such as MMP-2 and MMP-9,all of this can contribute to airway fibrosis process,thus,we hypothesize that bronchial epithelial cells may play an important role within P.aeruginosa-caused airway fibrotic remodeling via EMT.Epithelial-restricted integrin avβ6 can up-regulate TGF-β1 function locally by binding to the latency peptide.Integrin avβ6 could regulate fibrosis in lung and kidney as well as EMT via activation of TGF-β1-Smad2/3 signaling pathway,which has been regarded as a critical mechanism of fibrotic process.Therefore,we guess that integrin avβ6 could mediate EMT in bronchial epithelial cells induced by P.aeruginosa via TGF-β1-Smad2/3 signaling pathway and might be a promising therapeutic target for airway fibrotic remodeling associated with P.aeruginosa.There are two parts in this paper.We will investigate whether EMT is involved in P.aeruginosa-caused airway fibrotic remodeling and the associated signaling pathway.Then,we will explore the effect of integrin avβ6 on P.aeruginosa ’s LPS-induced EMT in BEAS-2B cells and therapeutic value of integrin avβ6 neutralizing antibody in P.aeruginosa-associated airway fibrotic remodeling.Part 1Epithelial-mesenchymal transition in BEAS-2B cells induced by P.aeruginosa’s LPSObjectiveThe present study aims at evaluating the effects of EMT on P.aeruginosa-associated airway fibrotic remodeling and the potential signaling pathway.MethodsBEAS-2B cells were incubated with P.aeruginosa ’s LPS to build a vitro EMT model.Epithelial marker E-Cadherin(E-Cad)and mesenchymal marker vimentin(Vi)were detected using western blotting and immunofluorescence.Cell morphology was observed with an inverted microscope.Myofibroblast marker,a-smooth muscle actin(α-SMA),was examined using western blotting.The secretion of MMP-2 and MMP-9 in cell medium were measured by ELISA.The activation of TGF-β1-Smad2/3 signaling pathway was examined.Results(1)P.aeruginosa’s LPS induced EMT in BEAS-2B cells.After incubation with LPS at a dose of 2μg/ml for 24,48 and 72 hours respectively,decreased epithelial marker E-Cad(p<0.05)and increased mesenchymal marker Vi(p<0.05)were detected in a time-dependent manner by both western blotting and immunofluorenscence.Meanwhile,BEAS-2B cells stimulated by LPS changed cobblestone-shape into mesenchymal-like spindle appearance;(2)Western blotting illustrated that the expression of myofibroblast marker a-SMA was significantly up-regulated in BEAS-2B cells after incubation with P.aeruginosa’s LPS for 72h(p<0.05);(3)ELISA assays revealed that the secretion of MMP-2 and MMP-9 were significantly increased in cell medium stimulated by LPS(p<0.05).(4)Western blotting showed that p-Smad2/3/T-Smad2/3 was significantly elevated in LPS-incubated BEAS-2B cells(p<0.05).ELISA showed that the secretion of TGF-β1 was significantly increased by P.aeruginosa’s LPS(p<0.05).TGF-β1-Smad2/3 signaling pathway was activated during P.aeruginosa’s LPS-induced EMT in BEAS-2B cells.Conclusions(1)P.aeruginosa’s LPS is sufficient to induce BEAS-2B cells to undergo EMT process;(2)Accompanied with EMT induced by P.aeruginosa’s LPS,BEAS-2B cells could transdifferentiate into myofibroblast phenotype and oversecret MMP-2 and MMP-9;(3)EMT induced by P.aeruginosa’s LPS in BEAS-2B cells is associated with the activation of TGF-β1-Smad2/3 signaling pathway.Part 2The effect of integrin avβ6 on EMT in BEAS-2B cells induced by P.aeruginosa’s LPS and the regulatory mechanismObjectiveThis study aims at evaluating the effects of integrin av(36 on P.aeruginosas LPS-induced EMT in BEAS-2B cells and exploring the regulatory mechanism to provide therapeutic option for preventing P.aeruginosa-associated airway fibrotic remodeling.Methods(1)The expression of integrin avP6 in BEAS-2B cells stimulated by P.aeruginosa’s LPS for 24,48,or 72 hours respectively were detected using western blotting.(2)BEAS-2B cells were divided into 5 groups:Control group,LPS group,LPS+10D5 group,LPS+SB431542 group,and 10D5 group.Epithelial marker E-Cad and mesenchymal marker Vi were detected using western blotting and immunofluorescence.Cell morphology was observed with an inverted microscope.(3)Myofibroblast marker a-SMA was examined using western blotting.The secretion of MMP-2 and MMP-9 were measured by ELISA.(4)The activation of TGF-β1-Smad2/3 signaling pathway was detected.Results(1)Accompanied with EMT process,P.aeruginosa’s LPS increased the expression of integrin avβ6 in BEAS-2B cells in a time-dependent manner(p<0.05);(2)Compared with the control group,decreased epithelial marker E-Cad(p<0.05)and increased mesenchymal marker Vi(p<0.05)were detected by both western blotting and immunofluorenscence in BEAS-2B cells of the LPS group,which was attenuated by integrin avβ6 blocking antibody 10D5 as well as TGF-β1 receptor antagonist SB431542.Meanwhile,BEAS-2B cells in LPS group changed original cobblestone-shape into mesenchymal-like spindle appearance,which could be abrogated by integrin avβ6 blocking antibody 10D5 and TGF-β1 receptor antagonist SB431542;(3)Western blotting showed that compared with the control group,the expression of myofibroblast marker a-SMA was significantly up-regulated in BEAS-2B cells of the LPS group(p<0.05);both integrin avβ6 blocking antibody 10D5 and TGF-β1 receptor antagonist SB431542 could abrogated the increase of myofibroblast marker a-SMA ecpression(p<0.05);ELISA assay revealed that,compared with the control group,the secretion of MMP-2 and MMP-9 were significantly increased in cell medium of the LPS group(p<0.05),which could be inhibited by integrin avB6 blocking antibody 10D5 as well as TGF-β1 receptor antagonist SB431542.(4)Western blotting illustrated that,compared with the control group,p-Smad2/3/T-Smad2/3 was significantly elevated in LPS group cells(p<0.05),while the ratio mentioned above was remarkably declined by both 10D5 and SB431542(p<0.05).ELISA showed that the increased secretion of TGF-β1 induced by LPS was significantly reversed by integrin avβ6 blocking antibody 10D5 but not TGF-β1 receptor antagonist SB431542(p<0.05).These data suggested that Integrin avβ6 mediated P.aeruginosa’s LPS-induced EMT in BEAS-2B cells via TGF-β1-Smad2/3 signaling pathway.Conclusions(1)Accompanied with EMT induced by P.aeruginosa’s LPS,the expression of integrin avβ6 in BEAS-2B cells is increased;(2)Blocking of integrin avβ6 could prevent EMT in BEAS-2B cells induced by P.aeruginosa’s LPS and attenuate the increased expression of a-SMA as well as oversecretion of MMP-2 and MMP-9;(3)Integrin avβ6 mediates EMT in BEAS-2B cells induced by P.aeruginosa’s LPS via activation of TGF-β1-Smad2/3 signaling pathway.Integrin avβ6 might be a promising therapeutic target to prevent P.aeruginosa-associated airway fibrotic remodeling. |
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