Effects Of Carbon Monoxide Releasing Molecule-3 On Polarization Of Lipopolysaccharide-induced RAW264.7 Cells

BackgroundCarbon monoxide-releasing molecule-3(CORM-3)is a new type of water-soluble carbon monoxide releasing molecule.In recent years,the effect of CORM-3 in anti-inflammatory,anti-apoptosis and anti-proliferation has been confirmed.Macrophages,as resident cells or monocyte derived cells of inflammatory response,their phagocytic capacity is a key factor in the development of acquired immunity.Macrophages can polarize to two extremes of functional state after being stimulated by the external stimuli:M1 macrophage(pro-inflammatory macrophage)and M2 macrophage(anti-inflammatory macrophage).Periodontal disease is a chronic infectious disease caused by the invasion of bacteria in periodontal tissue.It is characterized by the destruction of periodontal supporting tissue,the formation of periodontal pocket,the attachment loss and the absorption of alveolar bone.ObjectiveIn this experiment,RAW264.7 cells were used as the research object in vitro and experimental periodontitis mice as the research object in vivo.This study was to explore whether CORM-3 can regulate the polarization of RAW264.7 cells and the possible mechanism of this regulatory effect,and whether CORM-3 can inhibit the progress of periodontitis,which will provide a new method and theoretical basis for the treatment of periodontitis.Methods1.In vitro experiments:?RAW264.7 cells were treated with different concentrations of CORM-3(0,100 ?M,200 ?M,400 ?M,800 ?M)for 24 hours,and the cell viability was measured by Cell Counting Kit(CCK-8)method;?RAW264.7 cells were divided into five groups:control group,Lipopolysaccharide(LPS)group,different concentrations of CORM-3 group(100 ?M,200 ?M,400 ?M).The proportion of M0 macrophage(undifferentiated),M1 macrophage(pro-inflammatory type)and M2 macrophage(anti-inflammatory type)in each group was detected by flow cytometry(FCM);?the total RNA was extracted and the mRNA expression of tumor necrosis factor-a(TNF-?),nitric oxide synthetase(iNOS),interleukin-1 beta(IL-1?),IL-6,IL-10 and arginine-1(Arg-1)were detected by the method of real-time quantitative polymerase chain reaction(PCR);?The concentrations of IL-1?,IL-6 and IL-10 in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA).2.Mechanism:Extracting the total protein of every group to detect the expression of nuclear transcription factor-B(NF-?B)signal pathway related protein p65 and phosphorylated p65(p-p65),p50/p105 and p-p50/p-p105,I?B and P-I?B.3.In vivo experiment:The amount of 45 male C57 mice about 8 weeks old were divided into three groups:normal group,periodontitis group(LPS group)and CORM-3 group.In the LPS group,the maxillary second molars of the mice were ligated bilaterally and LPS solution(1 ?g/?L)injected into the buccal and palatal gingival sulci,5 ?L per side,20 ?L per mouse.The mice were fed with sticky food and were injected with physiological saline every 2 days;the treatment of mice in the CORM-3 group was similar to the LPS group,instead of physiological saline,CORM-3 solution at a dose of 10 mg/kg was injected intraperitoneally into C57 mice.The mice in the normal group and LPS group were injected with normal saline intraperitoneally.The mice were killed on the 3rd,7th and 10th day after modeling,the maxilla were extracted,decalcifying,embedding and sectioning then observed by the method of immunofluorescence and hematoxylin eosin staining(HE).Some of the maxilla tissues were quickly stored in liquid nitrogen for the extraction of total RNA,and detected mRNA expression of IL-1?,IL-6,IL-10 and Arg-1 by PCR;The concentrations of IL-6,IL-1? and IL-10 in the serum of mice were detected by ELISA.Results1.In vitro experiments:?The RAW264.7 cells viability increased with the drug concentration increasing,the peak value at 400 ?M,when the concentration at 800?M the RAW264.7 cells viability was inhibited.?With the drug concentration increasing,the proportion of Ml macrophages decreased,the proportion of M2 and M0 macrophages increased.?With the drug concentration increasing,the mRNA expression of TNF-?,iNOS,IL-1? and IL-6 decreased,and the concentrations of IL-10 and Arg-1 increased with the concentration of CORM-3 increasing.?The concentrations of IL-6 and IL-1? decreased with the CORM-3 concentration increasing,the IL-10 concentration increasing and reached the peak value when the concentration was 200 ?M2.Mechanism:the result of Werstem Blot showed that,with the drug concentration increasing,the proportion of p-p65,p-p50,p-p105 and P-I?B in the total protein decreased.3.In vivo experiments:?CORM-3 inhibits the macrophage polarization in local inflammatory tissue of experimental periodontitis in mice.?HE staining showed that CORM-3 significantly inhibited the progress of periodontitis in mice.?PCR results showed that the expression of IL-1? and IL-6 mRNA in CORM-3 group was lower than LPS group and the expression of Arg-1 and IL-10 mRNA in serum of CORM-3 group was higher.?ELISA results showed that compared with LPS group,the concentrations of IL-1? and IL-6 in serum of CORM-3 group were significantly lower,and the concentrations of IL-10 in serum of CORM-3 group were higher.ConclusionsIn vitro experiments show that CORM-3 inhibits the polarization of RAW264.7 cells to M1 type and promote the polarization to M2 type.By regulating the polarization of macrophage,CORM-3 inhibits the mRNA expression and secretion of M1 macrophage related proinflammatory factors,promotes the mRNA expression and secretion of M2 macrophage related anti-inflammatory factors.The effects of CORM-3 on regulating macrophage polarization and inhibiting inflammation may be related to the effect on inhibiting the activation of NF-?B signaling pathway.In vivo experiments show that CORM-3 inhibits the polarization of macrophages to M1 type and promotes the polarization to M2 type.CORM-3 inhibits the inflammatory response of the local periodontal tissue of experimental periodontitis mice and reduces the absorption of alveolar bone.At the same time,CORM-3 inhibits the mRNA expression and secretion of M1 macrophage related proinflammatory factors in periodontal tissues and serum of periodontitis mice,promotes the gene expression and secretion of M2 macrophage related anti-inflammatory factors.This study shows that CORM-3 inhibits the polarization of LPS-induced RAW264.7 cells to M1 macrophages and promotes the polarization to M2 macrophage,and has the effect of inhibiting inflammatory response and this effect may be related to NF-?B signaling way.This experiment provides theoretical basis for the application of CORM-3 in the treatment of periodontitis.