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Gut-derived LPS Promotes Concanavalin A-induced Hepatitis in Mice

Author: LinYan
Tutor: WangHongYang
School: Second Military Medical University
Course: Oncology
Keywords: Hepatitis ConA LPS Toll-like receptor 4 T cells Th1
CLC: R-332
Type: Master's thesis
Year: 2011
Downloads: 109
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Abstract

[Background and Objective】 hepatitis is one of the higher incidence of liver disease worldwide, from a variety of causes, such as viral infections, autoimmune disorders, alcoholism, drug toxicity, malnutrition and cholestasis, China hepatitis high incidence countries, most of the hepatitis virus infection, especially in HBV infection mainly. The main pathogenesis of viral hepatitis immune response caused by the virus antigen into the body to cause damage to the liver cells, cellular immune response, the main effects of cell-specific cytotoxic T lymphocytes. The patient's immune status lead to different clinical manifestations of hepatitis. Acute liver injury (Acute liver failure) is a highly lethal liver disease caused by acute severe hepatitis, the fatality rate as high as 40-80%. Majority of chronic viral hepatitis patients receiving long-term drug treatment, it will eventually develop cirrhosis, liver cancer, liver chronic inflammation is an important reason to cause liver cancer, viral hepatitis research has important clinical significance. Autoimmune hepatitis (AIH) is an unknown etiology of the liver and chronic inflammatory diseases, a higher incidence in Western countries, that the cellular immune response and humoral immune responses are involved in the pathogenesis of cytotoxic T cells is mainly caused disease factors, the majority of patients eventually develop cirrhosis or liver cancer. Concanavalin A (ConA) is an extract of a vegetable protein composition, T lymphocyte surface receptor, TCR, CD3, including the sugar residues of the glycoprotein, the liver is one of the ConA target organs. Intrahepatic presence of Kupffer cells, T cells and other immune cells and therefore the tail vein after ConA able specificity via the blood circulation in conjunction with the surface of T cells in the hepatic sinusoids receptor and cause the activation of T cells, leading to liver inflammation and injury . ConA-induced mouse hepatitis mainly transaminase levels and severe liver pathological damage can be well simulated viral hepatitis, autoimmune hepatitis and other liver diseases mainly mediated by the immune factors. Due to anatomical, liver long-term exposure to lower concentrations of intestinal bacteria and bacterial products to stimulate the next, and the liver is the main filtering organ of the intestinal flora. A large number of immune cells such as macrophages, dendritic cells, T lymphocytes and B lymphocytes in the liver, normal liver can be quickly and effectively clear the gut-derived bacteria and bacterial products. However, when impaired liver function, intestinal bacterial products via the portal vein or collateral circulation into the formation of blood endotoxemia. Intestinal endotoxemia potent proinflammatory cytokines can cause a of intrahepatic large number of immune cells activated which cause intrahepatic inflammatory cytokine secretion, these inflammatory factors aggravate liver has inflammation of the occurrence and development. Endotoxin (LPS) through its receptor Toll-like receptor 4 (TLR4) play a role. TLR4 and LPS binding by MyD88 pathways and the TRIF pathway activation downstream signal, the former can cause the release of various inflammatory mediators, which can cause the release of type I and type II interferons. In the liver, TLR4 expression in Kupffer cells, stellate cells, biliary epithelial cells, endothelial cells, and dendritic cells in the development of a variety of liver diseases, may play an important role. This topic is intended to be applied, ConA-induced mouse hepatitis model within the research enterogenic of toxins in the immune factors mediated hepatitis, while establishing TLR4 knockout mice ConA hepatitis model to explore of intestinal endotoxemia LPS in this model in the mechanism of action, factors for viral hepatitis, autoimmune hepatitis, immune-mediated hepatitis prevention and treatment of potential clinical value of experimental data. 【Methods】 1. Establish ConA-induced hepatitis model in mice, LPS levels detected in mice after injection of ConA; 2. Use of antibiotics to clear the intestinal endotoxemia, compare antibiotic treatment group and the control group of mice ConA-induced liver injury extent and LPS level changes; clear the endotoxin in mice enterogenous, comparing the antibiotic treated group and the control group mice injected with ConA after infiltration of T cells in the liver, and the activation of CD4 ~ T cells with antibiotics; 4. antibiotic treatment group and a control group of mice injected with ConA detected by ELISA in serum Th1 and Th2 cytokine expression; 5. using Western Blot method to detect liver tissue after antibiotic treatment and control groups of mice injected with ConA Related to promote compare LPS receptor Toll-like receptor 4 (TLR4) knockout mice and control mice ConA caused liver damage; using flow cytometry TLR4 knockout molecule expression and inhibition of apoptosis; participation in the liver of mice and control mice ConA hepatitis caused by T cell infiltration as well as the CD4 ~ T cell activation; 8. separation TLR4 knock out mice and control mice spleen cells, and CD4 ~ T cells purified by separation, vitro validation ConA on CD4 ~ T cell activation; 9 ELISA was used to detect TLR4 knock after serum addition to the mice and the control mice injected with ConA Th1 and Th2 cytokine expression; Real-time PCR detection Th1 cell differentiation molecule expression of T-bet; 10 to detect TLR4 knockout mice and control mice injected with ConA liver tissue to promote and to inhibit the expression of apoptotic molecules; Real-time PCR Western Blot method method detection perforin Perforin and Granzyme B expression. 【Results】 1. Elevated serum LPS levels in ConA-induced mouse hepatitis model; antibiotic treated mice after ConA injection serum LPS was elevated significantly lower than the control group, and the use of antibiotics cleared gut-derived LPS can be significantly reduced ConA-induced liver injury and hepatocyte apoptosis; 3. clear the gut-derived LPS significantly reduced the ConA induced intrahepatic T cell infiltration and activation of CD4 ~ T cells; 4. clear enterogenic LPS can inhibit ConA induced Th1 cytokine production; 5. clear the gut-derived LPS significantly inhibited ConA-induced hepatic apoptosis; 6 of TLR4 deletion significantly reduced ConA-induced liver injury and liver cell apoptosis; 7 intrahepatic deletion of TLR4 inhibition of T cell infiltration, and can effectively inhibit the activation of CD4 ~ T cells; the 8 of TLR4 missing ConA induced Th1 cytokine production can be suppressed, and promote the production of Th2 cytokines IL-10; 9 . TLR4 deletion can significantly inhibit ConA induced hepatocyte apoptosis; 10. perforin / granzyme B pathway may be involved in TLR4 missing the protective effect of hepatocyte apoptosis. Conclusion This study demonstrates that gut-derived LPS ConA-induced immune factors mediated played an important role in the development of hepatitis. Combination of gut-derived LPS via its receptor TLR4 promote ConA induced intrahepatic T cell infiltration and activation of CD4 ~ T cells in the liver, which in turn promote the generation of Th1 cytokines, thereby increasing the ConA induced mouse liver injury; clear LPS of enterogenic and TLR4-deficient mice after ConA treatment less liver damage and intrahepatic T cell infiltration significantly reduced the CD4 ~~ T cell activation and in vivo Th1 cytokine levels significantly lower than the control group, while antagonism of Th1 cytokines Th2 cytokines IL-10 were significantly increased. Clear gut-derived LPS and TLR4 missing upregulated expression of Mcl-1 down the expression of Bax, and can enhance Erk1 / 2 phosphorylation, thereby reducing the ConA-induced hepatic apoptosis, perforin / granzyme B pathway may be involved the TLR4 lack of a protective effect on the liver cell apoptosis. Our results suggest that inhibition of intestinal bacteria the abnormal translocation clear gut-derived LPS may effectively improve liver function in patients with hepatitis, and to reduce the development of liver inflammation.

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