Molecular Mechanism And The Role Of Thetumor Necrosis Factor (TNF) Family Member LIGHT In TLR3 Induced Acute Liver Injure

The tumor necrosis factor (TNF) family member LIGHT expressing on the surface of several immune cell including T cell, granulocytes, monocytes and dendritic cell is a recently identified type Ⅱ transmembrane glycoprotein, which participate in innate and adoptive immune responses in vivo. Recent studies suggest that LIGHT as a proinflammatory cytokine has important effects on Nonalcoholic Fatty Liver Disease, autoimmune acute hepatitis and asthmatic, but the precise role and molecular mechanism of LIGHT is unclear in TLR3 induced acute livers injure. TLR3 expressing in innate immue cell could induce liver injure by recogenizing the dsRNA from hepatitis viruse genesome or damaged tissues. In our study, mice were injected poly (I:C) and D-GalN to induced acute liver injure, which occurs similarly to TLR3-triggered acute liver injure in vivo. We explore the pathogenic role and molecular mechanism of enhanced expression of LIGHT in TLR3 induced acute liver injure. The main results of this study are as follows:1.Increased expression for LIGHT in poly (I:C) and D-GalN induced TLR3-tiggerd acute hepatitis. After administration poly (I:C) and D-GalN to induced mice acute liver injure, mice serum ALT level is significantly increased compared to the control at 8h,16h, 24h.Through hematoxylin and eosin (H&E) detected, serious injury was obvioused in the liver. LIGHT expression was investigated in mRNA and protein level by Real-Time PCR and western blot. The results indicate that an obviously enhanced expression of LIGHT was induced by activation of TLR3.2.Construction of HVEM prokaryotic expression vector and purification of HVEM protein. HVEM as a specific receptor of LIGHT was selected to block the function of LIGHT in vivo. We obtained the human HVEM extracellular domain region DNA 492bp by cloning PBMC cells cDNA to construct pET-28a-HVEM expression vector. E. Coli expression strain BL21 was transformed with pET-28a-HVEM plasmid and induced with IPTG to express HVEM protein. HVEM protein with 283 amino acid sequence and molecular size of 17kD was added His tag in the C-terminal. Purification of HVEM with NI-NTA was identificated by Coomassie blue staining and Western Blot.Expression of HVEM is correct and no hybrid band.3.Blockade of LIGHT moderate liver injure by TLR3-trrigered.(1) Mice were pretreated for one hour with HVEM protein before induction of acute liver injury. Twenty-four hours after injection of a lethal dose of poly (I:C) and D-GalN,100% of mice untreated with HVEM died of acute liver injure, but the survival rate of mice receiving HVEM were significantly increased. (2) Mice were induced hepatitis with a sublethal dose of poly (I:C) and D-GalN and tested serum ALT activity at 8h,16h and 24h. In contrast to increase of serum ALT levels in control mice, mice pretreated with HVEM profoundly inhibited serum ALT leve. Liver sections from HVEM treated and control BALB/c mice showed that blockade of LIGHT significantly reduced necrosis and apoptosis in the liver. Taken together, these findings suggest that LIGHT is an important event for the initiation and progression of TLR3-trrigered liver disease.4. NF-κB signaling pathway is crucial for TLR3-trrigered acute liver injure. By using the pharmacological p38, JNK and IκBαinhibitors, the MAPK, JNKand NF-κB of the TLR3-trrigered intracellular signal pathway were blocked. Mice were pretreated for one hour with respective relevant inhibitor before administration with poly (I:C) and D-GalN. Mice subjected to p38 and JNK inhibitor did not decrease serum ALT level as compared with control BALB/c mice. In contrast, mice treated with IκBα inhibitor had significantly decreased serum ALT levels. Thus, these results clearly indicate that NF-κB signaling pathway is essential for TLR3-trrigered acute liver injure.5. TLR3 regulates LIGHT expression by NF-κB in TLR3-tiggerd acute liver injure. Mice were subjected to IκBα inhibitor for one hour before being challenged with poly (I:C) and D-GalN. Treatment with the IκBα inhibitor profoundly decreased LIGHT mRNA expression in mice livers. These results suggest that TLR3 regulates LIGHT expression by NF-κB in TLR3-tiggerd acute liver injure.