Experimental Study On Epithelial-mesenchymal Transition And Fibrosis Of Bladder Induced By Lipopolysaccharide

Objective:In the present study,we first established an inflammatory bladder fibrosis rat model by intravesical instillation of LPS,and then explored the the potential for establishment of LPS-induced bladder EMT model as well as prevention of EMT by inhibiting of TGF-β pathway.Methods:(1)In vivo experiments : Eighteen Sprague-Dawley rats were randomly distributed into 3 groups:the LPS group,PBS group and control group.In the LPS and PBS groups.The intravesical instillation of LPS(500 μg/500 μl)or same volume of PBS solution was conducted every other day for 4 consecutive weeks,while rats in the control group were fed normally.Paraffin-embedded bladder tissue samples were used for several kinds of histological examinations,including HE staining for inflammation,TUNEL staining for apoptosis,Masson staining & Sirius Red staining for fibrosis as well as immunostaining for the degree of EMT and activation level of TGF-β1/p-Smad3 signal pathway;(2)In vitro experiments:A concentration gradient(0、1、2、4、6、8、10 μg/ml)of LPS were added into SV-HUC-1 cells separately,and then proliferation capacity and migration capacity of SV-HUC-1 cells were detected by CCK-8 and Wound Healing respectively to select the optimum concentration of LPS for promoting cell proliferation and migration.Expression of E-cadherin and Vimentin in SV-HUC-1,with or without selectively inhibition of Tβ1R in advance,was detected by immunofluorescence after 48-hour incubation with 4 μg/ ml LPS.Results :(1)Chronic inflammatory changes,such as bladder epithelial hyperplasia,submucosal edema,lymphocyte infiltration,apoptosis as well as obvious fibrotic changes were observed after intravesical instillation of LPS;(2)The number of E-cadherin +/Vimentin+ cells and the expression levels of TGF-β1and p-Smad3 protein in the bladder tissues of rats were significantly up-regulated after intravesical instillation of LPS;(3)The optimal concentration of LPS was 4 μg/ml to promote the proliferation capacity and migration capacity of SV-HUC-1 cells.After 48-hour treatment with 4 μg/ ml LPS,higher expression of E-cadherin and lower expression of Vimentin were shown in SV-HUC-1,indicated successful establishment of LPS-induced cell culture model of EMT.Meanwhile,a lesser degree of EMT was shown in SV-HUC-1 when Tβ1R was inhibited before treatment of LPS.Conclusion: The intravesical instillation of LPS induced pathological changes such as cystitis,fibrosis and EMT in rats and activation of TGF-β signaling pathway,suggesting that chronic cystitis-EMT-fibrosis may be an important process of bladder fibrosis induced by LPS.In vitro,LPS can also induced EMT of SV-HUC-1 cells,which was under the regulation of TGF-β signaling pathway.It suggested that TGF-β signaling pathway may be an important target for the prevention of EMT and EMT-dependent bladder fibrosis,and LPS-induced EMT model can also serve as an important tool for the follow-up study.