Positve Feedback Regulation Between Tissue Transglutaminase And TLR4 Signaling In HSC In Liver Fibrosis Post Schistosoma Japonicum Infection

ObjectivesLiver fibrosis can be caused by viral infection,alcohol,bile duct ligation and carbon tetrachloride,ect.,while liver fibrosis caused by Schistosoma japonicum(Sj)infection is the main pathological changes of schistosomiasis japonica.Hepatic stellate cell(HSC)activation is the central link of liver fibrosis.Activation of Toll-like receptor 4(TLR4)signaling pathway especially in hepatic stellate cells plays an important role in hepatic fibrosis.However,the relationship between TLR4 signaling and hepatic fibrosis caused by Sj infection and its mechanisms are not very clear.We have reported that transglutaminase 2(TGM2)plays an important role in Sj infection-induced liver fibrosis.Therefore,the aim of this study is to identify the regulatory relationship between TGM2 and TLR4 signaling pathways in hepatic stellate cells and the effect of this regulation on liver fibrosis induced by Sj infection.Methods1.Establishment of animal model of liver fibrosis induced by Schistosoma japonicum infection: BALB / c mice(6-8 weeks old,healthy,SPF grade,female)were infected by Sj cercariae through the abdominal skin method.Tissue specimens were collected at week 0,5,6,8 and 12 after infection.2.Identification of the animal model of liver fibrosis caused by Schistosoma japonicum infection by Sirius red staining: using Abcam's Sirius Red Kit and follow its instructions.3.Schistosoma japonicum infected mice were treated by TLR4 signaling pathway inhibitor TAK242: 4 weeks after Sj infection,mice were intraperitoneally injected by TAK242(2 times / week,0.3mg/kg per mouse,)for 4 weeks.Specimens were collected at week 8 after infection.4.Schistosoma japonicum infected mice were treated TGM2 enzyme activity inhibitor cystamine(CTM): 3 days after Sj infection,mice were intraperitoneally injected by CTM(10-2mmol/L,100?L/mice,1 time/day)for 7 days.Samples were collected at week 8 post infection.5.Collection and treatment of mice livers: at each time point,the mice livers of each part were collected to extract total RNA,total protein and for paraffin embedding and other treatment respectively.6.Culture of hepatic stellate cell lines: Hepatic stellate cell lines were purchased in Shanghai Cells Institute.They were cultured in DMEM medium containing 10% fetal bovine serum.7.Determination of RNA expression level: Trizol was used to extract total RNA from tissues/cells,followed by reverse transcription to c DNA using the Prime Script RT-PCR Master Mix kit according to the manual.SYBR Green II PCR Master kit was used to detect?-smooth muscle actin(?-SMA),Collagen I,Collagen III,Transglutaminase 2(TGM2),CD14,tumor necrosis factor(TNF)-?,prointerleukin(pro-IL)-1?,and interferon-inducible protein(IP)-10,regulated upon activation normal T cell expressed and secreted(RANTES)RNA expression levels.Semi-quantitative analysis was then performed.8.The protein expression level was detected by Western blotting and immunohistochemical staining combined with H-score semi-quantitation.9.Cellular localization of protein: immunofluorescence assay was used together with laser confocal microscopy and subsequent Image J software analysis.Results:1.The level of representative molecules activated by TLR4 signaling pathway was consistent with the level of collagen expression and ?-SMA protein levels in liver tissue at week 5,6 and 8 after infection with Schistosoma japonicum.2.While TAK242 inhibited the activation of TLR4 signaling pathway,the expression level of collagen and ?-SMA were decreased in the liver tissues of mice after infection by Sj for 8 weeks.3.Sj-infected mice liver and in vitro cultured HSCs showed that TLR4 and ?-SMA could co-localize,and the level of localization was reduced with TAK242 treatment.4.Sj-infected mice liver and in vitro cultured HSCs showed down-regulated TGM2 level as TAK242 inhibited LPS-induced TLR4 signaling pathway activation.5.Sj-infected mice liver and in vitro cultured HSCs appeared low activation of TLR4 signaling pathway with the TGM2 activity inhibitor CTM treatment.6.With TGM2 agonist all trans retinoic acid or recombinant TGM2 protein treatment,TLR4 signaling pathway was activated,and alpha-SMA and collagen levels were upregulated,which can be suppressed by CTM or si RNA of TGM2.Conclusions:1.Activation of TLR4 signaling pathway promotes liver fibrosis induced by Schistosoma japonicum infection by promoting the activation of hepatic stellate cells.2.The positive feedback regulation between TGM2 and TLR4 signaling pathway promotes hepatic stellate cell activation followed by hepatic fibrosis.