| Objective: cholecystokinin is one of the most abundant neuropeptides that is most widely distributed in many systems in the body, where it acts as a neurotransmitter. It was demonstrated that biologically active CCK peptides are involved in the regulation of feeding, behavioral expression of anxiety and the control of learning and memory and so on. Recent years, a series of studies showed that CCK-8 had the effect of anti-inflammation and immunoloregulation. A lot of data demonstrate that CCK-8 causes an in vitro inhibition of LPS-induced NF-κB activity in rat PIMs. Consistently, the production of pro-inflammatory cytokines induced by LPS including TNF-α,IL-1βand IL-6 was also inhibited by CCK-8.IL-12 was named as a new member of interleukins in 1989. It plays a central role in the regulation of cell-mediated immunity. IL-12 stimulates the proliferation of activated T lymphocytes and enhances IFNγsecretion by NK cells and T lymphocytes. The enhancing effects of IL-12 on cell-mediated cytotoxicity involve stimulation of NK cell-mediated cytotoxicity as well as increased generation of lymphokine-act- ivated killer cells and cytotoxic T lymphocytes. IL-12 can drive the differentiation of precursor Th cells to the Th1 phenotype and inhibit the differentiation of Th2 phenotype. Even it can reverse the established Th2 responses. IL-12 is a heterodimer cytokine composed of two covalently linked chains, a heavy chain of 40 kDa and a light chain of 35 kDa linked by a disulfide bond. However the expression of the p35 gene is constitutive in a wide variety of cells, the p40 gene is highly tissue-regulated, being restricted to phagocytic cells with antigen-presenting capability and is therefore considered to function as the regulatory component for IL-12 expression. The key role of IL-12 in the immune response and in inflammation and the importance of this cytokine in anti-tumor resistance have raised considerable interest in the mechanisms of IL-12 gene transcription. two functional promoter regions of the p40 gene that confer LPS inducibility and IFNγaugmentation have been identified, The region spanning from position -132 to -122 contains a novel NF-κB site, whereas the region from -211 to -207 contains an Ets-2 element. Although the IL-12p40 promoter contains a complex array of transactivating binding sites, NF-κB is essential for maximal IL-12p40 transcription after IFNγ/LPS stimulation. It has been shown VIP and PACAP inhibited interleukin-12 production in mouse peritoneal macrophages stimulated with LPS and IFNγ. However, there are no reports about whether CCK-8 which is also a neuropeptide regulates the expression of IL-12 and what is the mechanism of the regulation, The aim of the study is to investigate the effect of CCK-8 on IL-12 secretion in LPS-in- duced mice in order to elucidate that whether CCK-8 regulate Th1/Th2 development by regulating IL-12 secretion.Methods: One hundred and eight healthy female BALb/C mice were randomly assigned to six groups injected different agents via intraperitoneal injection. For group receiving LPS, a bolus dose of LPS was injected into abdominal cavity. For group of LPS+CCK-8, a bolus dose of CCK-8 was pre-injected 10 min before the injection of LPS. For group of LPS+CCK-8+CR1409 or LPS+CCK-8+CR2945, a bolus dose of CR1409 or CR2945 was pre-injected 30 min before the injection of CCK-8,Then a bolus dose of CCK-8 was pre-injected 10 min before the injection of LPS. Saline or CCK-8 was injected separately in control or CCK-8 group. Lung and spleen tissues were dissected and stored in liquid nitrogen.The serum was stored at -70℃; The contents of IL-12p40,p70 in the serum,lung,spleen tissues were measured with the method of Enzyme linked immunoabsorbant assay (ELISA). Changes of pIκB, p-p38 protein and the NF-κB /DNA binding avtivity were examined by Western blot and EMSA respectively.Results:1 Effects of CCK-8 on the expressions of IL-12p40, p70 in the serum,lung and spleen tissues of LPS-induced mice : The concentrations of IL-12p40, p70 in LPS group were higher than that of NS group(P<0.01).The concentrations of IL-12p40, p70 in LPS+CCK-8 group increased further (P<0.01), In contrast to LPS +CCK-8 group , The concentrations of IL-12p 40, p70 in CR1409 group and CR2945 group decreased (P<0.05).2 Effects of CCK-8 on IκB, p38 phosphorylation and NF-κB/DNA binding activity in lung and spleen of LPS-induced mice : The IκB, p38 expression of six groups was not significant difference compared with each other (P>0.05).The phosphorylated IκB (pIκB) expression in LPS group increased in comparision to NS group (P<0.01). The pIκB expression of LPS+CCK group decreased in comparision to LPS group (P<0.01). In contrast to LPS+CCK group, The pIκB expression of CR1409 group and CR2945group increased. The phosphorylated p38 (p-p38) expression in LPS group increased in comparision to NS group (P<0.01). The p-p38 expression of LPS+CCK group increased further (P<0.05). In contrast to LPS+CCK group, the p-p38 expression of CR1409 group and CR2945 group decreased, The inhibitive effect of CCK-8 by CR-2945 was stronger than that of CR-1409. The NF-κB/DNA binding activity of LPS group was higher than that of NS group(P<0.01), The NF-κB/DNA binding activity in LPS+CCK group decreased in comparision to LPS group (P<0.01). In contrast to LPS+CCK group, The NF-κB /DNA binding activity in CR1409 group and CR2945 group inecreased, Also The inhibitive effect of CCK-8 by CR-2945 was stronger than that of CR-1409.Conclusion: CCK-8 could increase the expressions of IL-12p40, p70 in the serum,lung and spleen tissues of LPS-induced mice, inhibit IκB phosphorylation and NF-κB /DNAbindingactivity, increase p38 phosphorylation.CCK-8 inecrease the production of IL-12 in the serum,lung and spleen tissues of LPS-induced mice by increasing the phosphorylation of p38.
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