| The liver is the largest digestive organ in the human body. It continuously receives blood from the gastrointestinal tract which contains a lot of extrinsic antigens. In order to against those extrinsic antigens, there are a large number of immune cells around the reticular liver sinusoid which gives birth to complex immune response. The live stays dynamic balance betewwn immune rejection and immune tolerance so that the liver cells are protected from the overactive immune system responding. The occurrence and development of viral hepatitis, autoimmune hepatitis and many other kinds of liver disease are related to the immunity imbalance of the liver. Therefore, studies targeting the immune regulation substancesare very important to the prevention and treatment of the liver desease.Augmenter of liver regeneration(ALR) is a small protein with no species specificity and a fine thermal stability. Previous studies have focused on promoting liver regeneration, inhibiting apoptosis and promoting the expression of mitochondrial gene and so on. Exogenous recombined ALR protein can exert therapeutic effect on rats with drug-induced liver injury, cirrhosis and experimental hepatic fibrosis. In many kinds of toxic liver damage animal model, ALR can promote liver regeneration, relieve acute liver damage, improve the liver function, extend animal survival time and increase animal survival rate. Recent studies show that ALR can inhibit the activation of T cells, NK cells and many other kinds of immune cells, exerting immune regulation effects. There is few vivo studies of ALR because of its poor stability and low bioavailability, and the mechanism involved in its immune regulatory effect remains undefined. The gene therapy method can offset the disadvantages of the recombined protein, this study aims to investigate the protective effects of ALR on Con A-induced liver damage by gene therapy and shed more light on the mechanism of its immune regulation function.In our laboratory the human ALR-minicircle and the recombinant plasmid of hALR have been constructed successfully before. Based on these research, a large amount of hALR minicircle were obtained. Hydrodynamic tail vein injection of mc-hALR into the rats significantly enhanced the expression of ALR in liver. With the administration of mc-ALR hydrodynamic injection, the survival rate and survival time were obviously improved in the ALR-treated group compared to the control group in the severe Con A-induced liver injury animal model (30 mg/kg). In the mild Con A-induced liver injury animal model (15 mg/kg), the glutamic-pyruvic transaminase and glutamic-oxalated transaminase in serum were decreased prominently in the ALR-treated group compared to the control group. Treatment with ALR significantly reduced the levels of TNF-a and IL-6 but failed to affect the level of IFN-y. These results indicate that the up-expression of ALR in the liver can reduce the Con A-induced liver injury. Therefore, we made further investigation to elucidate the mechanism of ALR’s immune regulatory effects.The qPCR results showed that the levels of TNF-a mRNA and IL-6 mRNA are much lower in the ALR-treated group compared to the control group, but the IFN-y mRNA showed no significant difference between the two groups, which is in accord with the previous serum protein results. These results confirm that ALR can reduced the levels of TNF-a and IL-6 but failed to affect the level of IFN-γ in the Con A-induced liver injury. Furthermore, we detected that the expression of iNOS mRNA, CXCL-10 mRNA and CCL-3 mRNA were reduced by ALR administration, the percentage of CD4+, CD8+T cells were calculated by flow cytometry, they were all increased strongly after Con A injection. Nevertheless, the percentage of infiltrating CD4+and CD8+cells went down significantly in ALR group. These data indicate that ALR could dramatically lessen CD4+and CD8+T cells recruiting into the liver. Moreover, western blot results showed that compared with Con A group, the phosphorylation of IxBa and p65 were inhibited markedly by ALR pretreatment, indicating that that ALR may inhibit Con A-induced hepatitis through the NF-xB pathway. The ALR pretreatment showed a strong inhibition of the phosphorylation of JNK and p38-MAPK. These results reveal that JNK and p38 MAPK are also inhibited by ALR.These results indicate that ALR can attenuate Con A-induced hepatitis injury by inhibiting the inflammatory cytokines, chemokine, T-cells and kinds of signaling pathways, demonstrating that ALR has effects on the immune regulation. |
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