| Liver is the largest metabolism and detoxification organ of human body, and is also a very important immune organ. Blood flows through the liver, and brings a large number of immune cells to resist the invasion of foreign antigens concomitantly. Large amounts of antigen in the blood is taken by antigen presenting cells and lymphocytes in hepatic sinus network, which start the complex process of immune response. Revealing and exploring the hepatic immune regulation network will be conducive to the establishment of regulating liver damage, fibrosis and regeneration strategies, and have great significance for the treatment of chronic liver disease.Polyunsaturated fatty acids (PUFAs) are the fatty acids which have two or more double bonds, and it is an essential fatty acids for human, because which must be taken from foreign food. PUFAs can be divided into n-6 and n-3 PUFAs according to the location of the unsaturated bonds. n-3 PUFAs are refer to the first one unsaturated bond between the third C atoms and the forth C atoms from the terminal methyl of polyunsaturated fatty acids, such as linolenic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), which usually exist in plants and deep-sea fishes. The fatty acid composition ratio of food, especially n-6 and n-3 PUFAs ratio has different effects on lipid metabolism and immune function, but the effects on lipid metabolism and inflammatory reaction of regulation in vivo are not clear. Liver is the main organ for lipid metabolism in our body, and a large number of free fatty acids can be circulation to it. Then the ratio of n-6/n-3 PUFA will inevitably affect the immune microenvironment of the liver tissue, so as to regulate the immune defense and immunological surveillance in liver.In this paper, we choose the fat-1 transgenic mice, which can endogenously transfer n-6 PUFAs into n-3 PUFAs, as an animal model to study the regulation of n-3 PUFAs on liver tissue injury. We aim at providing a new perspective for the auxiliary clinical treatment of liver injury and chronic hepatitis.Part I N-3 polyunsaturated fatty acids have a protective effect on Con A induced autoimmune liver injuryThe immunological liver injury model induced by Concanavalin A (Con A) can well simulate the human autoimmune liver disease and the pathogenesis of viral hepatitis. Mice intravenous injected with Con A to activate the intrahepatic T cells can be used to establish an experimental animal model of liver injury. The model presents the clinical and pathological characteristics of acute hepatitis, including the infiltration of leukocytes and lymphocytes in liver, necrosis of liver cells, elevation of ALT and AST levels in serum and the secretion of inflammatory cytokines. Now, this model has been widely applied to research the pathogenic mechanism of T cell-mediated hepatitis, which formed the process of liver injury we know at present.Fish oil which rich in n-3 PUFAs has been widely used as a prescription drug, it has many benefits for human health, such as anti-inflammatory and regulating blood fatty acid. However our understanding about fish oil is not enough, among them, the regulation of n-3 PUFAs on T cell mediated autoimmune hepatitis is unclear yet.Objective:Although there are a lot of studies about the process of liver injury, the exact cellular and molecular mechanism still remains unclear. More and more data show that the activation of T cells plays an important role in the process of chronic liver damage. In this chapter, we choose the fat-1 transgenic mice, which can endogenously transfer n-6 PUFAs into n-3 PUFAs, as an animal model to study the regulation of n-3 PUFAs on T cell mediated autoimmune hepatitis induced by Con A, aiming at providing a prevention and treatment for current clinical hepatitis.Methods:We choose thefat-1 transgenic mice as an animal model to study the regulation of n-3 PUFAs on autoimmune hepatitis induced by Con A.(1) The effects of endogenous n-3 PUFAs on Con A induced liver injury in mice. WT and fat-1 transgenic mice were injected via the tail vein with 35 mg/kg of Con A to observed the survival of mice, or injected with Con A by 15 mg/kg to induce liver injury. At different time points, collect the samples of serum and detect the GPT activity; HE staining method was used to observe the pathological changes of mice liver tissue, andTUNEL staining was used to analysis the necrosis of liver.(2) The regulation function of endogenous n-3 PUFAs on T cell activation in Con A induced mice liver injury. WT and fat-1 transgenic mice were injected with Con A via the tail vein, then using flow cytometry analysis the mouse liver mononuclear cells (MNCs), including the proportion of T cells, NK cells and NKT cells; the expression of CD69. Using Q-PCR method to measured the cytokines expression in liver tissue (such as:IFN-γ, TNF-α and IL-6) and the transcript factors of T cells(T-bet, GATA3, RORγt and Foxp3); ELISA method was taken to detect the cytokines levels of serum at different time points; Using flow cytometry method to detect the frequency of T cells expressing IFN-γ in liver; The STAT1 and STAT3 phosphorylation levels in liver tissue were detected by Western blot.(3) Analysis the regulation of n-3 PUFAs on T cell activation in vitro. WT mice and fat-1 transgenic mice liver mononuclear cells were separated, and stimulated with different doses of Con A (1,2.5,5 μg/ml) for 72 h. T cell proliferation and the expression level of IFN-γ were determined by 3H-thymidine incorporation and ELISA assays. STAT1 and STAT3 phosphorylation levels of liver mononuclear cells were measured at different time after Con A treated.(4) WT mice and fat-1 transgenic mice liver mononuclear cells were separated. CFSE (2.5 μM)-labeled hepatic MNCs were stimulated with different concentrations of Con A (1,2.5,5 μg/ml) and DHA (25,50 and 100μmol/L)for 4d. T cell proliferation was assessed by flow cytometry analysis of CD3+ T cells based on the dilution of CFSE intensity. Cell production of cytokines (IL-2 and IFN-γ) were determined from the 24h and 48h culture supernatant.Results:(1) Compared with fat-1 transgenic mice WT mice were more likely to induce autoimmune liver injury and endogenous n-3 PUFAs had a protective effect on Con A mediated liver injury. Intravenously given fat-1 mice and WT mice lethal dose of Con A (35 mg/kg). Survival rate of WT mice was significantly lower than that of fat-1 transgenic mice. The degree of liver damage in WT mice was heavier and more apoptosis or necrosis cells were observed in the liver of WT mice.(2) Endogenous n-3 PUFAs can inhibit the secretion of inflammatory cytokines induced by Con A. Attenuated liver damage in fat-1 transgenic mice was accompanied by highly pronounced reduction of pro-inflammatory cytokines (i.e. TNF-α, IFN-γ, and IL-6) and increased secretion of anti-inflammatory cytokine IL-10 in serum, while no significant difference in IL-4 was observed between Con A treated WT and fat-1 transgenic mice. Accordingly, intrahepatic mRNA expression of the pro-inflammatory cytokines was significantly suppressed in fat-1 transgenic mice compared with those in WT mice upon Con A administration Endogenous n-3 PUFAs can inhibit the T cell activation induced by Con A.(3) After Con A injection, the proportion of T cells, NK cells and NKT cells in the total mononuclear cells from WT and fat-1 mice liver tissue had no difference, but the activation of CD3+T cells in fat-1 transgenic mice liver was significantly lower than that of wild type mice (the expression of CD69); At the same time, the CD69 activation of CD3+NK1.1+ NKT cells and CD4+ T cell in fat-1 transgenic mice liver was significantly lower than that of wild type mice.(4) Endogenous n-3 PUFAs can inhibit the frequency of IFN-γ-producing T cells after Con A injection. After Con A treatment, the frequency of IFN-γ-producing CD4+T cells in fat-1 mice liver was lower than that of WT mice. In addition, after Con A injection the phosphorylation levels of STAT1 in fat-1 mice liver tissue was significantly weaker than that of WT mice liver. At the same time, the phosphorylation level of IL-6 related signal pathway STAT3 had a weak expression in liver tissue, and the phosphorylation level of WT mice liver was stronger than that of fat-1 mice at 6 h.(5) Endogenous n-3 PUFAs might direct the differentiation of T cells in Con A induced hepatitis. T-bet was markedly suppressed in the liver from fat-1 transgenic mice compared to their WT counterparts. By contrast, the Th2 transcript factor GATA3 was comparable in the two groups before and after Con A injection. Surprisingly, the Th17 transcript factor RORyt was lower while the Treg transcript factor Foxp3 was higher in liver from fat-1 transgenic mice than those from WT mice in the presence of Con A stimulation.(6) MNCs from fat-1 transgenic mice were not responsive to Con A stimulation. Mice MNCs were isolated from WT and fat-1 transgenic mice, then stimulated with different concentrations of Con A in vitro. The results show that, the proliferation of MNCs from fat-1 transgenic mice liver was inhibited, and secretion of IFN-γ was significantly lower than that of WT mice. The phosphorylation level of STAT1 in fat-1 mice MNCs was significantly weaker than that of WT mice. and the phosphorylation level of STAT3 in WT mice MNCs was stronger than that of fat-1 mice at 24h.(7) DHA could attenuate the response of MNCs from WT mice to Con A challenge. MNCs from WT mice liver were isolated, and cocultured with different concentrations of DHA for 4 hours. DHA can inhibit the proliferation of Con A-stimulated T cell and the secretion of cytokine IFN-y and IL-2 in a dose-dependent manner in CFSE proliferation assay. In addition, DHA can also inhibit the secretion of cytokines IFN-y and IL-2 after stimulated with different concentrations of Con A.Conclusion:n-3 PUFAs can inhibited T cell proliferation, activation and inflammatory cytokine secretion in vitro and in vivo. n-3 PUFAs may also retard the symptoms of autoimmune diseases.That is n-3 PUFAs had a protective effect on Con A-induced liver injury, which provide a new perspective for prevention and treatment of the autoimmune hepatitis in clinical.Part II N-3 PUFAs participate in the regulation of autoimmune liver injury by promoting the level of autophagy in the liverAutophagy is a process that the aggregated proteins or damaged intracellular organelles encapsulated into a double-membrane structure termed autophagosome, which fuses with lysosomes for protein and organelle degradation. When the cells in a adverse environment, autophagy is an important mechanism for self-protection to maintain cells survive and avoid cell apoptosis. Autophagy is increased in drugs or immune cell activation induced liver injury. Studies have reported that cell autophagy plays a protective role to liver injury, but the role of autophagy in the mechanism of liver disease remains further study, n-3 PUFAs can promote autophagy level of cells in the liver, however the significance in acute liver injury induced by Con A is not clear.Objective:To study the regulation of liver cell autophagy level on T cell activation induced liver injury, and to provide experimental basis for the treatment of liver diseases by regulating autophagy changes.Methods:(1) in vivo:WT mice and fat-1 transgenic mice liver MNCs were isolated after Con A injection, then the NF-κB activation and the autophagy related proteins LC3 and P62 change were detected by immunoblotting technique; The expression of LC3 in T lymphocytes of the mice and NF-κB activation in effector T cells were analysed by intracellular staining.(2) In vitro experiment:WT mice and fat-1 transgenic mice liver MNCs were isolated then treated with Con A. Autophagy related proteins LC3 and P62 change was detected by immunoblotting technique and the expression of LC3 in T lymphocytes of the mice was measured by intracellular staining. WT mice hepatic MNCs were pre-cultured with DHA, then stimulated with Con A. The expression of LC3 in MNCs was analysed by intracellular staining.(3) WT mice and fat-1 transgenic mice intraperitoneally given autophagy inhibitor CQ before intravenous injection of Con A,then the level of serum transaminase was measured and the pathological condition of liver were assessed; Analysis of serum cytokine secretion, T cells activation, intracellular IFN-y expression of T cells, STAT1 and STAT3 phosphorylation level in liver, all above methods reference to the first chapter.Results:(1) In the process of liver injury induced by Con A, endogenous n-3 PUFAs promoted autophagy level in the liver. When WT mice and fat-1 transgenic mice stimulated by Con A, and the expression of LC3 in fat-1 transgenic mice liver was higher than that in WT mice. In addition, corresponding to the expression of LC3, the P62 level in fat-1 transgenic mice liver was lower, besides, the expression of LC3 in CD3+T cells was obviously higher than that of WT mice. When mouse liver MNCs were co-culture with Con A in vitro, results showed that the expression of LC3 in CD3+T cells from fat-1 transgenic mice was significantly higher than that in WT mice.(2) The NF-κB activity was inhibited in the liver tissue and effector T cells of fat-1 transgenic mice upon Con A stimulation.(3) DHA can improve the level of autophagy in WT mice liver MNCs stimulated by Con A. The MNCs of WT mice liver were isolated, the level of LC3 in the DHA group was significantly higher than that group only using Con A to stimulate.(4) Chloroquine can block the protective role of endogenous n-3 PUFAs on the liver injury mediated by Con A. fat-1 transgenic mice were intraperitoneally injected with CQ 40 mg/kg and WT mice were injected with an equal volume of PBS, then two groups of mice were injected by tail vein with Con A 15 mg/kg lhour later. Data showed that the GPT level at each time point of the mice had no difference between the two groups and HE staining results also showed similar degree of injury in the two groups of mice.(5) CQ could block the inhibition effects that endogenous n-3 PUF As inhibiting the secretion of cytokines and the activation of T cells in mice autoimmune hepatitis fat-1 transgenic mice were intraperitoneally injected with CQ 40 mg/kg and WT mice were injected with an equal volume of PBS, then two groups of mice were injected by tail vein with Con A 15 mg/kg lhour later. The levels of inflammatory cytokine in WT mice and fat-1 transgenic mice had no obvious difference, such as IFN-y and TNF-a. After Con A injection, the proportion of T cells, NK cells and NKT cells in the total mononuclear cells from WT and fat-1 mice liver tissue had no difference, besides, the activation of CD3+T cells, the CD69 activation of CD3+NK1.1+ NKT cells and CD4+ T cell in WT and fat-1 mice had no obvious difference either.(6) CQ could block the inhibition effects that endogenous n-3 PUF As inhibiting the frequency of IFN-y-producing T cells in mice autoimmune hepatitis, fat-1 transgenic mice were intraperitoneally injected with CQ 40 mg/kg and WT mice were injected with an equal volume of PBS, then two groups of mice were injected by tail vein with Con A 15 mg/kg 1hour later. The frequency of IFN-y-producing CD4+T cells in WT and fat-1 mice was similar. In addition, the phosphorylation levels of STAT1 and STAT3 in WT and fat-1 mice were basically the same too.Conclusion:In the process of liver injury induced by Con A, endogenous n-3 PUF As promoting autophagy level in the liver, that is the level of autophagy in fat-1 transgenic mice was improved. The protective effect of endogenous n-3 PUF As on Con A induced liver injury was weakened or disappeared after autophagy inhibited by CQ. That is to say, endogenous n-3 PUFAs may play a protection role in autoimmune liver damage by promoting autophagy.In summary, n-3 PUFAs can inhibited T cell proliferation, activation and inflammatory cytokine secretion in vitro and in vivo. n-3 PUFAs may also slow down the symptoms of autoimmune diseases, the possible mechanism of which may be the increased level of autophagy. That is endogenous n-3 PUFAs may play a protection role in autoimmune liver damage by promoting autophagy, which provide a new perspective for prevention and treatment of the autoimmune hepatitis in clinical. |
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