Background This study was to research the cytokines and phenotype of macrophages pre-treated with class A1 scavenger receptor (SR-A1) antibody in vitro as well as the influence on apoptotic pathway of colonic epithelial cells, also to explore the role of SR-A1 mediated macrophages in impaired intestinal barrier of inflammatory bowel diseases (IBDs). in the current presence of SR-A1 antibody. The induction of cleaved claudin-1 and caspase-3 in Caco-2 cells was also suppressed when SR-A1 antibody pre-treatment. Conclusions Pre-treatment with SR-A1 antibody can inhibit inflammatory response in LPS-induced macrophages inside a NF-B reliant way. Pre-treatment with SR-A1 antibody inhibits M1 phenotype manifestation of macrophages also, and attenuates the pro-apoptotic influence on colonic epithelial disruption and cells of intestinal hurdle integrity induced by macrophages. LPS group) (NF-B p-p56 manifestation was mainly within the cell nuclei, and NF-B p-p56 manifestation was upregulated after LPS excitement (P<0.05), which however was significantly inhibited in the presence of SR-A1 antibody pre-treatment. These findings indicate pre-treatment with SR-A1 antibody may up-regulate anti-inflammatory cytokines and down-regulate inflammatory cytokines secreted by RAW264.7 cells in a NF-B pathway dependent manner. Open in Rabbit Polyclonal to p50 Dynamitin a separate window Figure Dofetilide 2 NF-B p-p65 expression in RAW264.7 cells with different treatments. NF-B p-p65 expression was determined by immunofluorescence staining. Nuclei were stained with DAPI, and green signal represented NF-B p-p65 (100). Data are Dofetilide presented as the mean SEM (*, P<0.05 the expression of p-ERK1/2, JNK and p-JNK increased significantly after LPS stimulation (P<0.05). However, only the increased p-JNK expression could be inhibited in the presence of SR-A1 antibody (P<0.05 LPS group). We also detected the expression of SR-A1 by Western blotting, which indicated that SR-A1 was present in RAW 264.7 cells. Open in a separate window Figure 3 Expression of proliferation related protein in RAW264.7 cells with different treatments. Protein expression was detected by Western blotting. Data are presented as mean SEM (*, P<0.05 LPS group) (untreated cells had homogeneous nuclei, but Caco-2 cells co-cultured with RAW264.7 cells with LPS and IFN- treatment exhibited more condensed nuclei and apoptotic bodies after DAPI staining. SR-A1 antibody pre-treatment could attenuate Dofetilide the pro-apoptotic effect of macrophages on Caco-2 cells, which was demonstrated by less condensed nuclei and less apoptotic bodies in cells. Open in a separate window Figure 6 SR-A1 antibody pre-treatment attenuates the pro-apoptotic effect of RAW264.7 cells on Caco-2 cells in the co-culture system. Caco-2 in different groups were stained with DAPI and photographed under a ?uorescence microscope (100). Red arrow: condensed nuclei and apoptotic bodies in Caco-2 cells. Data are presented as mean SEM (*, P<0.05 LPS + IFN- group). SEM, standard error of the mean; LPS, lipopolysaccharide; SR-A1, A1 scavenger receptor. Discussion As the first-line defense in the lamina propria of mucosa, intestinal macrophages contribute to the integrity of intestinal barrier. On one hand, intestinal macrophages can eliminate invading pathogens though secreting cytokines and chemokines; on the other hand, intestinal macrophages are tolerant toward commensal microbiota though down-regulating recognition receptors (20,21). Macrophages have different subtypes that can orchestrate or counteract inflammation, including pro-in?ammatory M1 macrophages and anti-in?ammatory M2 macrophages. Macrophages of different phenotypes have been investigated in many inflammation-associated diseases, including IBD. In CD patients, intestinal macrophages, a large number of CD68+ macrophages especially, migrate through the thickened mucosa towards the submucosa usually. In UC individuals, macrophages generally infiltrate Dofetilide the intestinal mucous coating (22,23). Nevertheless, the exact system underlying the part of human being macrophages in the pathogenesis of Dofetilide IBD continues to be poorly understood. To be able to guard against the invasion of pathogens along the way of mucosal immune system, macrophages secrete a number of cytokines and exert solid anti-bactericidal impact via PRRs. SR-A1, as you of PRRs, can be a sort II membrane glycoprotein and may form homotrimers, concerning in the development of innate immunity, cell apoptosis and proliferation (24). SR-A1 can be indicated in macrophages primarily, monocytes, dendritic cells, soft muscle tissue cells and endothelial cells, and may type complexes with additional PRRS, exerting synergistic results for the inflammatory response (25,26). Early research reveal SR-A1 implicates in the pathogenesis of atherosclerosis, by mediating endocytotic aftereffect of foam cells on low-density lipoprotein (27). Upon the cytomegalovirus publicity, SR-A1, coordinating.