Effect And Mechanism Of The N-terminal Domain Of Thrombomodulin In Hepatic Failure

Part I Construction and expression of prokaryotic expression plasmids of The N-terminal domain of thrombomodulin and HMGB1Objective:To obtain the mouse recombinant N-terminal domain of thrombomodulin and recombinant HMGB1.Methods:Total RNA was extracted from liver tissue of mice, as the template for polymerase chain reaction (PCR).According to the requirements of the target gene, two primers were synthesized. PCR products were ligated into a pMD18-T vector by DNA Ligation kit,and confirmed by restriction digestion and sequencing. Construction of pET-28a-TM-N and pET-28a-HMGB1by DNA Ligation kit, and confirmed by restriction digestion and sequencing.Protein expressions were induced by adding different concentrations of isopropyl-1-thio-p-D-galactopyranoside (IPTG), Purification of two proteins was achieved by affinity chromatography using a Ni-NTA column. The molecular weight was tested by SDS/polyacrylamide-gel electrophoresis (SDS-PAGE) and the concentration of protein was checked by BCA Protein Assay Kit.Results:PET-28a-TM-N and PET-28a-HMGB1were successfully obtained, and confirmed by restriction digestion and sequencing. The molecular mass of the mouse recombinant N-terminal domain of thrombomodulin and recombinant HMGB1were confirmed by SDS/polyacrylamide-gel electrophoresis (SDS-PAGE),and the concentration of protein was checked by BCA Protein Assay Kit. Conclusion:PET-28a-TM-N and PET-28a-HMGB1were successfully constructed; the mouse recombinant N-terminal domain of thrombomodulin and recombinant HMGB1were successfully obtained. Part Ⅱ Effect of recombinant proteins in mouse liver failure model.Objective:To Study the effect of HMGB1on liver tissue in mice and study the N-terminal domain of thrombomodulin in mouse liver failure model.Methods:1. HMGB1or D-galactosamine and lipopolysaccharide were intraperitoneally injected into TLR4knock-out mice and normal mice, the expression of HMGB1in liver tissues was detected by immunohistochemisty and RT-PCR, and the serum ALT and HMGB1were quantified by ELISA at four time points:2、6、12、24h after intraperitoneal injections.2. Construction of mouse hepatic failure model, intraperitoneal injection of recombinant TM-N, the following steps according to the preceding method.Results:There were similar liver damages in the control group and the experimental group at each time point in common mice;There was no liver damage in the control group at each time point, but liver damage in the experimental group in TLR4knock-out mice.Conclusion:Liver failure can also induced by HMGB1.The process of liver damage induced by HMGB1was mainly associate with RAGE pathway; Liver damage induced by LPS can not happen without TLR4receptor. PartⅢ The anti-inflammatory effect of recombinant N-terminal lectin-like domain of thrombomodulin in mouse sinusoidal endothelial cellsObjective:To study the influence of N-terminal lectin-like domain of thrombomodulin(TM-N) on the expression of HMGB1in mouse sinusoidal endothelial cells by lipopolysaccharide(LPS) stimulation.Methods:Experimental group was treated with TM-N and LPS.Control group was delt with LPS.Different methods were used to detect cell viability and expressions of HMGB1,NF-κB,HMGB1-mRNA.Result:The recombinant prokaryotic expression vector PET-28a-TM-N was successfully constructed and mouse TM-N protein about16kD was successfully expressed. The cell viability of experimental group at48h (92.825%±2.42%) was significantly higher than that in control group (65.24%±3.79%)(P<0.05); The expression of HMGB1in experimental group at48h(IOD:0.124±0.034) was significantly lower than that in the control group (0.180±0.025)(P<0.05); The peak of HMGB1-mRNA in the experimental group was24.41±4.35, significantly lower than that in the control group (51.34±7.26).Conclusion:The TM-N could significantly reduce expression and secretion of HMGB1in SECs by LPS stimulation.