| Toll-like receptors (TLRs) are important pattern recognition receptors(PRRs) and it involve in the regulation of innate immune responses via activating NF-κB that induce the expression pro-inflammatory,induce T lymphocyte activation and regulation of adaptive immune to maintain immune balance. Liver failure is associated with severe endotoxemia,LPS active TLR4signal transduction via binding TLR4expressed on the surface of liver cells and non-parenchymal cells, inducing pro-inflammatory factor released,leading to liver damage. Recent studies have shown that ACLF display ’sepsis-like’ immune paralysis, and the altered immune status have been associated with increased infectious morbidity and mortality.The mechanisms on TLR4signaling abnormal imbalance leading to liver injury remains to be further studied.TLR signaling and functions are to be necessary to be under tight negative regulation attenuating TLR signaling to maintain this immunological balance. So far,knowledge about the role of these negative regulators of TLR signaling pathway in ACLF is highly limited.In the present study, we studied the transcription feactures of the four molecular involved in negative control of TLR signaling cascade,as a result, the levels of TNF-α and IL-10in plasma were detected.To the end, Moreover,correlation analysis were carried out to explore their dynamic role in the progress of chronic liver disease.Objective To explore the role of negative regulators of toll-like receptor signal pathways in immunological pathogenesis of acute-on-chronic liver failure by investigating the gene expression of toll-like receptor4(TLR4) and the negative regulators of toll-like receptor signal pathways,such as myeloid-differentiation-88-short(MyD88s),interleukin-1R-associated-kinas e(IRAK)-M,single-immunoglobulin-interleukin-1R-related-molecule(SIGI RR),A-20mRNA in peripheral blood mononuclear cells(PBMCs) and their revalant to the levels of serum inflammatory cytokine levels in patients with liver failure.Methods The subjects enrolled in the study were mainly divided into two groups:20patients with CHB,26patients with HBV-related ACLF,and the patients were admitted to the First Affiliated Hospital of Chongqing University of Medical Science from Febrary2011to July2011. Patients with ACLF were divided17cases early group and9cases late group according to the2006guideline of liver failure. In addition,18healthy controls(HC),gender and age matched with CHB and ACLF patients,were recruited.Blood samples from healthy donors were from Health Examination Center at the First Affiliated Hospital of Chongqing University of Medical College. Human peripheral blood mo no nuclear cells (PBMCs) were isolated via density gradient centrifugation using Ficoll-Paque Plus. Total RNA was extracted using TRIzol reagent following the manufacturer’s instructions.The concentration of RNA was determined by spectrophoto metrically at OD260nm,and the quality of RNA was evaluated by measuring OD (260/280) and by gel electrophoresis. Reverse transcription reaction was implemented with olige dT Primers and random primers using PrimeScript RT reagent kit. The cDNA samples were stored at-20℃. Real-time PCR was performed using the iCycler iQ Real-Time Detection System using the SYBR Premix EX Taq.CT values were analysised by Bio-Rad Gene Expression MacroTM Version1.1.The serum levels of TNF-α and IL-10were detected by ELISA.All statistical analyses were performed with SPSS17.0. The significance of the difference between groups were analyzed by one-way analysis of variance (ANOVA).Correlations were determined using Spearman’s correlation test. P<0.05was considered statistically significant difference.Results Compared with the healthy controls,the expression of MyD88s,IRAK-M,A20mRNA was upregulated in the active stage of chronic hepatitis B(CHB) patients and liver failure patients (both early stage and intermediate-end stage)[(3.51±2.03vs6.23±3.85vs10.96±4.12vs15.28±5.93,F=23.261,P<0.05),(15.04±14.22vs43.46±35.13vs69.45±38.61vs125.34±64.71,F=18.854,P<0.05)and(1.73±0.50vs15.67±8.65vs33.52±31.09vs51.90±36.17,F=13.516,P<0.05)]; the levels of TNF-α and IL-10increased in CHB and liver failure[(24.291±5.870vs56.335±11.390vs95.415±17.465vs110.869±23.063, F=108.293,P<0.01)and(12.991±3.939vs32.402±9.328vs44.656±8.969vs57.577±6.859,F=84.118,P<0.01)].TLR4mRNA expression were gradually increased with the desease exacerbations, interestly, in the late stage of ACLF, TLR4mRNA expression were lower than that in the early stage, in liver failure patients in intermediate-end stage was lower than that in the early stage (5.01±3.24vs20.46±9.20vs109.04±42.01vs41.50±19.94, F=66.424,P<0.05);Multiple comparisons among four groups,no significance were found in SIGIRR mRNA expression(11.840±20.024vs16.386±13.428vs9.097±6.559vs6.018±7.574,F=1.484,P>0.05). IL-10mainly increased in late stage ACLF,no significant difference was found in early stage ACLF compared with CHB group(P>0.05).Correlation analysis showed TLR4mRNA expression is positively correlation with Tbil(r=0.547vs r=0.635,P<0.05), the level of TNF-α (r=0.881vs r=0.842,P<0.01),and negatively correlation with PTA(r=-0.658vs r=-0.724,P<0.05) in patients with CHB and ACLF-E.Conclusion The alteration of the immune status may associated with increased expression of negative regulators in TLRs signaling of PBMCs in different stages of patients in ACLF. |
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