| Sepsis is a common clinical critical illness, leaving characteristics with high incidence and mortality. Lipopolysaccharide (LPS), also known as endotoxin, one of the major components that presents in the cell membrane of Gram-negative bacteria, is the main initiation factor for the occurrence of sepsis. Thus, it is one of effective approach to prevent sepsis from the source by arresting LPS-triggered inflammatory cascade.In our pervious studies, we have been carrying out an extensive study on limulus anti-lipopolysaccharide factor (LALF) deriving from an amoebocyte of blood cells in horseshoe crab. After analyzing the structural characteristics of LALF functional area, we obtained a cyclic limulus mimic peptide CLP-19 by using solid-phase synthesis, terminal amidation and rostrocaudal cyclization. CLP-19 has a high affinity with LPS. Unlike LALF, CLP-19 has no haemolyticus and immunogenicity. Both in vitro and in vivo studies displayed a good security of CLP-19. We found that an administration of CLP-19 prior to 20 hours could efficiently protect mice from acute peritonitis caused by Escherichia coli.bacteria. Furthermore, CLP-19 significantly suppressed elevated TNF-αlevel stimulated by IL-1β, a cytokine with different structures with LPS, which hinted that CLP-19 regulated the immune reaction. In this study, we preliminarily investigated the immune regulation and anti-LPS immune response of CLP-19. We also assayed the regulatory role of CLP-19 in LPS-induced mitogen-activated protein kinase (MAPK) pathways and cytokines in peripheral blood mononuclear cells (PBMCs). In addition, we investigated the binding of CLP-19 and mononuclear macrophages and the effects of CLP-19 pretreating inflammatory cytokines and chemokines induced by LPS. We also studied the protective effect of CLP-19 in LPS-induced sepsis in mice.Methods1. The effects of CLP-19 suppressing inflammatory signal pathways in PBMCs after LPS stimulation1.1. CLP-19 effect on MAPK phosphorylation pathways after LPS stimulation The MAPK phosphorylation pathways, including p-ERK1/2, p-JNK1/2 and p-P38 in PBMCs, were assayed after LPS stimulation at 0, 10, 20, 30, 60 minutes, respectively. Different concentrations of CLP (25μg/ml, 50μg/ml and 100μg/ml) were employed to explore the suppression of the signal pathway induced by LPS. Meanwhile, the corresponding concentrations of polymyxin B (PMB) were served as positive controls.1.2. CLP-19 effect on mRNA expressions of LPS-induced inflammatory cytokines and chemokines in PBMCs Respectively, PBMCs were treated with various concentrations of CLP-19 (25μg/ml, 50μg/ml and 100μg/ml) and LPS (100ng/ml) for 4 hours. Then, the mRNA levels of the cytokines, including TNF-α, IL-10, and IL-6, chemokines such as MCP-1 and CXCR4, and adhesion molecule ICAM-1, were measured with reverse transcription polymerase chain reaction (RT-PCR).2. CLP-19 effect on immune response in mononuclear macrophage induced by LPS2.1. 1. CLP-19 effect on LPS and RAW264.7 binding by FCM In the absence of serum, rhodamine labeled CLP-19 (R-CLP-19, 10μg/ml) and FITC-LPS (1μg/ml) were successively incubated or co-cultured. Subsequently, FACs was used to analyze CLP-19 or LPS binding to RAW264.7. In the presence of serum, the bindings of R-CLP-19, FITC-LPS and RAW264.7 were assayed.2. 1. 2. Assay of CLP-19 on the bindings of LPS and RAW264.7 by laser scanning confocal microscope In the serum-free condition, CLP-19 effect (10μg/ml, 100μg/ml) on the binding of FITC-LPS (1μg/ml) and RAW264.7 cells was observed. The serum condition was selected as a positive control.2. 2. CLP-19 suppressing LPS-induced immune response in RAW264.7 cells2. 2. 1. RT-PCR assays of mRNA expressions of proinflammatory cytokines and chemokines in RAW264.7 cells after CLP-19 The CLP-19 (50μg/ml)was used to treat RAW264.7 for different interval (0.5h,1h,2.5h,5h,10h,20h), or different concentrations (1μg/ml, 10μg/ml, 50μg/ml, 100μg/ml, 200μg/ml) of CLP-19 was used to treat RAW264.7 for 20 hours, respectively. And the cells were pretreated with CLP-19 for different interval (5h,10h,20h), then the cell were washed with PBS and treated with LPS for another 4 h. Then the total RNAs were extracted. The mRNA expressions of TNF-α, MCP-1, IL-10 and ICAM-1 were measured with RT-PCR.2. 2. 2. Real-Time PCR assays of mRNA levels of LPS-stimulated inflammatory cytokines and chemokines in RAW264.7 after CLP-19 treatments RAW264.7 were treated by different concentrations of CLP-19 (1μg/ml, 10μg/ml, 50μg/ml, 100μg/ml and 200μg/ml) or CLP-19 mixture with LPS (100ng/ml) for 4 hours respectively, followed by RNA extractions. Then real-time PCR assays were employed to detect mRNA levels of TNF-α, MCP-1, and IL-10. Meanwhile, the corresponding concentrations of limulus anti-lipopolysaccharide factor (cLALF) were served as positive controls.2. 2. 3. ELISA analysis of MCP-1 releasing in RAW264.7 after CLP-19 treatment RAW264.7 cells were treated with CLP-19 (100μg/ml) and cLALF (100μg/ml) for 0, 6, 12, and 24 hours, respectively. The MCP-1 release at the specific time phase was measured using ELISA, respectively. The effects on MCP-1 releasing in the absence or presence of LPS were assayed with different concentrations of CLP-19 (25, 50, 100μg/ml). Meanwhile, the corresponding concentrations of limulus anti-lipopolysaccharide factor (cLALF) were served as positive controls.2. 2. 4. CLP-19 effect on RAW264.7 Chemotaxis by Transwell method Different concentrations of CLP-19 (1μg/ml, 10μg/ml, 100μg/ml) were used to assay the CLP-19 effect on the directional movement of RAW264.7 cells. The serum was used as positive control.3. CLP-19 effect on LPS-induced immune response of sepsis in mice CLP-19 (2mg/kg) or cLALF (2mg/kg) was administrated prior to 20, 10, 5 hours in mice, followed by a sublethal LPS stimulation, respectively. The sera were isolated at 0 and 4 hours after LPS stimulation, respectively. The expressions of MCP-1, TNF-αand IL-10 were measured using ELISA. The LPS mixture being combined with CLP-19 or cLALF were served as controls. The histopathological changes of important organs such as liver, lung, kidney, small intestine, and heart were also evaluated.Results1. CLP-19 suppressing LPS-induced inflammatory signal pathways and cytokines in PBMCs1.1. CLP-19 significantly suppressing LPS-induced phosphorylation of MAPK pathway The phosphorylations of MAPK pathway (p-ERK1/2, p-JNK1/2, p-P38) were significantly enhanced after LPS stimulation for 30 minutes (P<0.01). CLP-19 significantly suppressed LPS-induced activation of MAPK pathway. The suppressing effect of CLP-19 was equal to the positive control PMB.1. 2. CLP-19 suppressing mRNA expression of inflammatory cytokines and chemokines CLP-19 significantly suppressed mRNA expression of TNF-α, IL-10, and IL-10, MCP-1 and CXCR4 and ICAM-1 in a dose-dependent manner (P<0.01).2. CLP-19 effect on LPS-induced immune response in mononuclear macrophage 2. 1. CLP-19 effect on the binding of LPS to RAW264.7 2. 1. 1. CLP-19 effect on the bindings of LPS to RAW264.7 by FCMS In the absence of serum, LPS could not be directly bind to RAW264.7, while CLP-19 could be directly bind to RAW264.7 and mediated the bindings of LPS to RAW264.7. Both CLP-19 and LPS could be bind to RAW264.7 in the presence of the two agents. In the presence of serum, single LPS, CLP-19 or LPS mixture with CLP-19 could be bind to RAW264.7. 2. 1. 2. CLP-19 effect on the binding of LPS and RAW264.7 by laser scanning confocal microscope In serum-free condition, CLP-19 mediated LPS binding to RAW264.7, particularly on the cell membrane. And this binding was in a dose-dependent manner.2. 2. CLP-19 suppressing LPS-induced immune response in RAW264.7 2. 2. 2. CLP-19 effects on mRNA expression of inflammatory cytokines and chemokines in RAW264.7 Single CLP-19 had no significant effect on the expression of TNF-αand IL-10mRNA, but significantly upregulated the mRNA expression of MCP-1, which was in a dose-dependent manner. In the presence of LPS, CLP-19 significantly downregulated mRNA expression of inflammatory cytokines and chemokines. 2. 2. 2. CLP-19 effect on MCP-1 releasing in RAW264.7 Single CLP-19 or cLALF significantly upregulated MCP-1 expression. Compared with cLALF, CLP-19 more significantly upregulated MCP-1 expression. The upregulating effect was in a dose-dependent manner. 2. 2. 4. CLP-19 promoting the chemotaxis of RAW264.7 CLP-19 (10μg/ml and 100μg/ml) had some chemotaxis effect on RAW264.7, which induced the directional movements of RAW264.7.3. CLP-19 effect on LPS-induced immune response of sepsis3.1. CLP-19 or cLALF pretreatment possessing no obvious proinflammatory effect There were no significant difference in TNF-αand IL-10 among groups after CLP-19 or cLALF administration in the mice 20, and 10 hours in advance. However, the administration of CLP-19 or cLALF prior to 5 hours significantly elevated TNF-αexpression(P<0.01). The MCP-1 expression was significantly upregulated after the administration of CLP-19 or cLALF prior to 20, 10, and 5 hours. Compared with cLALF, CLP more significantly upregulated MCP-1 at the same dose.3.2. CLP-19 or cLALF inhibiting LPS immune response The administration of single CLP-19 or the mixture of CLP-19 and cLALF prior to 20, 10, and 5 hours significantly downregulated the expressions of TNF-α, IL-10, and MCP-1. Except for slight pathological changes in liver, other organs had no significant pathological changes after CLP-19 and cLALF were administered. In LPS-induced mice, inflammatory pathological changes were observed in lung, liver, kidney, especially in the lung and liver. In the contrast, the pathological changes were attenuated after CLP-19 was administered in advance.Conclusions:1. CLP-19 significantly suppresses LPS-induced immune response in PBMCs.2. CLP-19 suppresses LPS-induced immune response in RAW264.7.2. 1. CLP-19 can bind to RAW264.7 and mediate the binding of LPS and the cells;2. 2. CLP-19 possesses the function of immunoloregulation in RAW264.7, especially induces chemokine MCP-1;2.3. CLP-19 can change the cellular immune status and suppress LPS-induced immune response;3. CLP-19 can significantly increase the expression of MCP-1 in a time-dependent manner, suppress LPS-induced immune response in organism, thereby reduce the expression of inflammatory cytokines.
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