Regulation Of PTPRO On LPS / TLR4 - Mediated Liver Injury

Part 1 Role of PTPRO/TLR4/NF-κB in live cancerBackgrounds: Inflammation always accompanies or promotes the development of cancer. It plays critical roles in tumor growth, infiltration, metastasis, escape and regression. Inflammatory response is a native defender against exogenous pathogens. It is also involved in repairing damaged tissues and their regeneration. Acute inflammation can contribute to tissue repair and remodeling rather than interfere with homeostasis if it is under proper control. Nonetheless, pathogens can escape from immune surveillance and break out immune tolerance and thus cause malignant tumors. Primary hepatic cancer is one of the ten common malignant tumors presented by the world health organization. For the past several years, the incidence and mortality of this cancer have been increasing. It is called “the king of cancer” because of its high morbidity, which ranks No.3 in high-morbidity cancers worldwide. Primary hepatic cancer often follows chronic hepatic injury including hepatitis and hepatic cirrhosis called precancerosis in liver. Long term and chronic inflammation in live leads to hepatic cirrhosis and promotes self repair of the liver by triggering its ability of regeneration. Shorter self repair is critical for reconstructing the damaged tissues in liver, while sustained activation of self repair may cause long term inflammation condition, which may induce cancer finally. Liver cancer is divided into three types including hepatocellular carcinoma(HCC), cholangiocarcinoma, and the mixed type. HCC is the most common type of liver cancer. The main cause of HCC is chronic and active hepatitis, such as HBV, HCV, alcoholic hepatitis and nonalcoholic fatty liver. HBV is the leading cause of HCC in China. Many studies have suggested that Toll-like receptor 4(TLR4) mediated innate immune plays important roles in the development and progression of liver cancer. However, the precise role of TLR4 needs further elucidation. Protein tyrosine phosphatase receptor type O(PTPRO) is a new tumor inhibitor, which is markedly reduced in HCC tissues. Little is known about the interaction between LPS/TLR4 and PTPRO in hepatic injury.Objective: To determine the influence of PTPRO on the activation of LPS/TLR4 signaling and their interaction in live cancer.Methods:(1) To assay the mRNA and protein of PTPRO and TLR4 in tissues of 84 HCC patients by Real-time PCR and Westernblot, respectively;(2) To construct PLV-PTPRO-GFP plasmid and transfect HuH7 and SNU423 cells by use of the constructed plasmid;(3) To detect the mRNA and protein of TLR4 in PTPRO-overexpressed HuH7 and SNU423 cells;(4) To assay the mRNA and protein of PTPRO and TLR4 in LPS-stimulated HCC cells and to estimate the activation of IκBα and NF-κB/P65 by Westernblot and laser confocal imaging analyses;(5) To evaluate the growth of LPS-stimulated PTPRO-overexpressed HuH7 cells by CCK-8 assay;(6) To determine the mRNA and protein of PTPRO in LPS-stimulated HuH7 cells in combination with Bay11-7082, a specific inhibitor of NF-κB signaling;(7) To assess the relative activity of WT(HuH7-PTPRO-WT) and mutated ptpro(HuH7-PTPRO-MUT) promoters in LPS-stimulated HuH7 cells by luciferase reporter assay.Results:(1) Reduced expression of TLR4/PTPRO in human HCC specimens; PTPRO and TLR4 was positively associated according to Pearson’s correlation analysis;(2) The PLV-PTPRO-GFP plasmid was constructed by use of Clone ExpressTM II One Step Cloning Kit according to the protocol. PLV-PTPRO-GFP plasmid was transfected to HuH7 and SNU423 HCC cells;(3) TLR4 was up-regulated rather than down-regulated when PTPRO was overexpressed in SNU423 cells and HuH7 cells;(4) LPS could elevate the expression of PTPRO by activating IκBα and NF-κB/P65 but did not affect the expression of TLR4 in HCC cells;(5) Cell proliferation was inhibited in PTPRO-overexpressed HuH7 cells;(6) Bay11-7082 could inhibit the up regulation of PTPTO by LPS stimulation in HuH7 cells;(7) LPS could not activate NF-κB in HuH7 cells due to its loss of NF-κB/Rel binding region for promoter mutation.Conclusion: There is a feedback loop between PTPRO and LPS/TLR4 signaling in HCC-associated inflammation dependent on activation of NF-κB signaling pathway.Part 2 Role of PTPRO/TLR4/NF-κB in Fulminant hepatitisBackgrounds: Fulminant hepatitis is also called acute necrotic hepatitis, acut e liver failure or fulminant liver failure, which is characteristic of necrosis o f considerable liver cells and severe hepatic damage. Its fatality rate is very high. Infection, excessive alcohol consumption, and drug abuse are the mai n causes for fulminant hepatitis. PTPRO in liver cells can prevent liver inju ry in hepatic ischemia reperfusion injury, while it can promote inflammation in macrophages by activating NF-κB. It can be concluded that PTPRO play s different roles in diverse immune microenvironment. TLR4, a transmembra ne protein, is a vital member of TLRs family, which is a specific recptor of LPS. Studies have implicated that the activation of TLR4 and its downstrea m signal can promote the development and progression of fulminant hepatiti s. We have previously found important effect of PTPRO and TLR4/NF-κB s ignaling in HCC-related inflammation by feedback loop. Little is koown abo ut the influence of PTPRO only or ineraction between PTPRO and TLR4/N F-κB signaling in fulminant hepatitis. We construct a mouse model by perito neal injection of LPS and D-Ga IN to observe the effect of PTPRO on LPStriggered inflammation in liver and demonstrate their modulatory mechanisms in fulminant hepatitis.Objective: We aim to explore the regulatory effects of PTPRO/LPS/TLR4 in fulminant hepatitis.Methods:(1) To observe the survival rate of mice after peritoneal injection of LPS/D-Ga IN;(2) To estimate liver injury by assaying hepatic enzymes, HE and TUNEL apoptosis;(3) To determine inflammatory cytokines in serum and liver by Real-time PCR and ELISA;(4) To meaure F4/80+TLR4+ cells in spleen by FACS and intrahepatic TLR4 by Real-time PCR and Western-blot assay;(5) To estimate T subtypes in mice spleen;(6) To estimate the phosphorylation of IκB-α and NF-κB/P65 by Western-blot and the nuclear translocation of NF-κB/P65 by IHC;(7)To investigate the effect of PTPRO on LPS/TLR4 triggered inflammatory response in RAW264.7 cells.Results:(1) The mortality of PTPRO KO mice is higher than WT mice after administration of LPS/D-Ga IN; All mice die after injection for 48h;(2) ALT and AST in PTPRO KO mice serum increase obveriouly after LPS/D-Ga IN administration; Necrosis and cell apoptosis in the liver of PTPRO KO mice are more severe than WT mice;(3) Seral and intrahepatic TNF-α and IL-6 of PTPRO KO mice are higher that those mice in blank control but lower than in WT mice;(4) F4/80+TLR4+ cells in spleen of PTPRO KO mice are less than WT mice; the expression of TLR4 in PTPRO KO mice is higher than blank control mice but is apparently lower than WT mice;(5) cells of CD3+IFN-γ+ and CD3+TNF-α+ in spleen of PTPRO KO mice are lower than those in WT mice;(6) the phosphorylation of IκB-α and NF-κB/P65 in the spleen of PTPRO KO mice decrease than WT mice; the nuclear translocation of p-NF-κB/P65 in liver of PTPRO KO mice is less apparent than WT mice, but Bay11-7082 can inhibit the activation of NF-κB;(7) PTPRO plays a critical role in LPS/TLR4 mediated acute inflammatory response dependent on the activation of NF-κB signaling.Conclusion: PTPRO plays a complicated role in LPS/TLR4-mediating liver injury, which probably palys opporsite roles in liver cells and inflammatory cells in microenvieonment by activating NF-κB signaling pathway. The interaction between PTPRO and TLR4/ NF-κB signaling is a brige linking innate immune and adaptive immune in liver. However, the molecular mechanisms need further elucidation.