The Role Of TLR4 In LPS-induced EMT Of Intrahepatic Biliary Epithelial Cells Of Rats

Objective: This study investigated whether lipopolysaccharide(LPS),a cell wall constituent of gram-negative bacteria,can induce EMT of IBECs via activating Toll-like receptor 4(TLR4),and observe whether NF-κB/snail signaling pathway is involved in regulation of the process in SD rat.Methods: 1.Healthy Specific Pathogen-Free(SPF)grade Sprague Dawley(SD)male rats(n=32),weighted 270±10 g per rat,were used throughout the experiments and were stochastically divided into 4 groups(n=8 per group): a sham control with normal saline(NS)(SH group),LPS group,TLR4 sh RNA+LPS(KD group),negative control sh RNA+LPS(NC group).2.The rats in KD group and NC group were seriatim relaxedly injected TLR4 sh RNA adenovirus or negative control sh RNA(NC sh RNA)(1x109PFU per rat)into the caudal vein.After 96 h,1 mg/kg LPS were infused retrogradely into the common bile duct(CBD)per rat of LPS group,KD group and NC group while NS(1ml/kg)per rat of SH group were infused for 48 h,then intrahepatic biliary tissues was isolated and preserved.3.Hematoxylin Eosin(HE)staining observed histopathological changes of biliary epithelial cells;The m RNA and protein expression of epithelial markers(E-cadherin,Cytokine 7),mesenchymal markers(N-cadherin,MMP-2),TLR4 as well as nuclear transcription factor NF-κB p65,snail were assayed using real-time PCR and western bolt analysis.4.All data were presented as mean ± standard error of mean(SEM)and analyzed by SPSS 17.0 statistics software.The results were analyzed statistically by the one-way ANOVA,P<0.05 was considered to be statistically significant.Results:1.TLR4 m RNA expression of LPS group significantly increased compared with SH group(P<0.01).The expression of TLR4 m RNA remarkably reduced in the KD group compared with LPS group and NC group(P<0.01,P<0.01).In accordance with the protein expression of TLR4 detected by Western bolt.2.The expression of the epithelial markers(E-cadherin,CK7)of LPS group obviously lower than SH group(P<0.01;P<0.01).The expression of E-cadherin and CK7 in KD group notably increased compared to LPS group and NC group(P<0.01,P<0.01;P<0.01,P<0.01).The trends of E-cadherin and CK7 m RNA expression were consistent with the protein change levels of them.3.Compared to SH group,the m RNA expression of N-cadherin and MMP-2 in LPS group significantly increased(P<0.01;P<0.01),while the m RNA expression of N-cadherin and MMP-2 in the KD group significantly reduced compared with LPS group and NC group(P<0.01,P<0.01;P<0.01,P<0.01).There was no difference between the protein expression changes of N-cadherin and MMP-2.4.The level of NF-κB p65 phosphorylation and the expression of snail in LPS group higher than SH group significantly(P<0.05;P<0.01).After inhibiting TLR4 m RNA in KD group,the phosphorylation level of NF-κB p65 and the expression of snail lower than LPS group and NC group(P<0.05,P<0.05;P<0.01,P<0.01).Conclusion: LPS induced epithelial-mesenchymal transformation(EMT)of intrahepatic biliary epithelial cells(IBECs)in rats by stimulating TLR4 and TLR4-mediated the activation of NF-κB/snail signaling pathway may play an important role in LPS-induced EMT.