| Objective: Ulcerative colitis(UC)can seriously affect the quality of life of patients.At present,the pathogenesis of UC is unclear,which may be related to the interaction of many factors,including peroxisome proliferator-activated receptor γ(PPAR-γ),immune cells,cytokines and their receptors.Long-term use of existing drugs to treat UC has high cost and many side effects.Therefore,it is imperative to find new therapeutic drugs.Phenylamine A(4-HPR)is a synthetic retinoid,which has the characteristics of low toxicity and good tissue distribution in vivo.Studies have shown that 4-HPR has anti-inflammatory effect and can regulate the expression of inflammatory factors in macrophages.Macrophages play an important role in the pathogenesis of UC.In UC patients,macrophages shift to M1 phenotype,showing high secretion of pro-inflammatory cytokines and enhancing cytotoxicity and phagocytosis.The activated macrophages expressed PPAR-γ significantly,and the activation of PPAR-γ could induce the phenotype polarization of macrophages from M1 to M2.It is also reported that4-HPR can inhibit the expression of pro-inflammatory cytokines by activating PPAR-γ,thus lowering blood pressure.Therefore,we speculate that the anti-inflammatory activity of 4-HPR may be related to PPAR-γ regulating the polarization of macrophages to M2.However,few studies have evaluated the effect of 4-HPR on UC,and whether 4-HPR can alleviate inflammatory bowel disease has not been reported.This study designed in vivo and in vitro experiments to explore whether 4-HPR can play a therapeutic role in UC through PPAR-γ regulating macrophage phenotype,and explore its mechanism.Methods:1.Male C57BL/6 mice were randomly divided into 6 groups(12 mice in each group):Control group,4-HPR group,UC group,UC+4-HPR-L group,UC+4-HPR-H group and UC+4-HPR-H+GW9662 group.Mice in Control group received routine feeding without added drinking water;100 mg/kg 4-HPR was injected intraperitoneally every day.Ulcerative colitis was induced by dextran sulfate sodium(DSS)intervention in other groups of mice.UC+4-HPR-L group and UC+4-HPR-H group were given intraperitoneal injection of 50 mg/kg and 100 mg/kg of 4-HPR,respectively.UC+4-HPR-H+GW9662group: 100 mg/kg PPAR-γ specific antagonist GW9662 was injected intraperitoneally on the premise that 4-HPR was given at the same time after the model was induced.The body weight of mice in each group was measured every day,the fecal consistency and bleeding were recorded,and the disease activity index(DAI)was scored.After the mice were killed,the colon tissue was taken to record the colon length,and the histopathological changes were detected by hematoxylin and eosin(HE)staining.Biochemical kit was used to detect Myeloperoxidase(MPO)activity in tissues.Detection of prostaglandin E2(PGE2)content in tissues by enzyme-linked immuno sorbent assay(ELISA);The m RNA expression of inflammatory mediators interleukin 1β(IL-1β),interleukin 6(IL-6),tumor necrosis factor-α(TNF-α),M1 related cytokines interleukin12(IL-12),inducible nitric oxide synthase(i NOS),M2 related cytokines chitinase 3 like1(Ym1),arginase 1(Arg1)and mannose receptor C-type 1(MRC1)were detected by Real-time PCR.The expression of PPAR-γ protein in tissue nuclei was detected by Western blot.The expression of cyclooxygenase 2(COX-2)protein was detected by immunohistochemistry.2.The mouse RAW264.7 macrophages were randomly divided into five groups: Control group,LPS group,LPS+4-HPR-L group,LPS+4-HPR-H group and LPS+4-HPR-H+GW9662 group.Control group was treated with dimethylsulfoxide(DMSO)as control for the same time;LPS group treated cells with lipopolysaccharide(LPS)to induce inflammation;LPS+4-HPR-L group and LPS+4-HPR-H group were treated with 1 μM and 10 μM 4-HPR respectively after inducing inflammation.LPS+4-HPR+GW9662 group pretreated cells with 5 μM GW9662 to induce inflammation,and then continued to use 10 μM 4-HPR intervention;After the intervention,the cells were collected and the expression of PPAR-γ protein in nucleus was detected by Western blot.The m RNA expression of IL-1β,IL-6,TNF-α,M1 related cytokines IL-12,i NOS,M2 related cytokines Ym1,Arg1 and mrc1 were detected by Real-time PCR.The expression of T-lymphocyte activation antigen CD86(M1 surface marker)and macrophage mannose receptor 1(CD206,M2 surface marker)was detected by flow cytometry to evaluate the number of macrophages.Results:1.Compared with Control group,the body weight of UC group was significantly reduced.On the 7th day,UC+4-HPR-L group significantly increased the body weight of UC mice.On the 5 th,6 th and 7 th day,UC+4-HPR-H group significantly increased the body weight of UC mice;Compared with UC+4-HPR-H group,the average body weight of UC+4-HPR-H+GW9662 group decreased significantly on the 4 th,5 th,6 th and 7 th day.In addition,compared with Control,the body weight of mice in 4-HPR(100 mg/kg)group had no obvious change(all P < 0.01).2.Compared with Control group,DAI score in UC group was significantly higher.Compared with UC group,UC+4-HPR-H group significantly reduced DAI;GW9662reduced the effect of 4-HPR-H on UC clinical symptoms in DSS-administered mice.Compared with Control group,DAI of mice in 4-HPR(100 mg/kg)group had no significant change(all P < 0.01).3.Compared with Control,the colon length in UC group was significantly shortened.Compared with UC group,both UC+4-HPR-L and UC+4-HPR-H groups increased colon length,and the effect of UC+4-HPR-H group was more significant.Compared with UC+4-HPR-H group,the colon length of UC+4-HPR-H+GW9662 group was significantly shortened.Compared with Control,the colon length of mice in 4-HPR group had no obvious change(all P < 0.05).4.Compared with the Control group,the colon wall of UC group was thickened,goblet cells were lost and inflammatory cells infiltrated.Compared with UC group,these characteristics were obviously reduced after 4-HPR treatment,and the effect of UC+4-HPR-H group was more obvious than UC+4-HPR-L group.Compared with UC+4-HPR-H group,GW9662 significantly reduced the effect of 4-HPR-H on colonic histopathological changes.In addition,the colon tissue of mice treated with 4-HPR alone was similar to that of Control group,and there was no pathological change.5.Compared with Control group,MPO activity,PGE2 content,IL-1β,IL-6,TNF-α,IL-12,i NOS m RNA expression and COX-2 protein expression in colon tissue of UC group were significantly increased.Compared with UC group,UC+4-HPR-L group and UC+4-HPR-H group can significantly inhibit DSS-induced MPO activity,PGE2 content,IL-1β,IL-6,TNF-α,IL-12,i NOS m RNA expression,COX-2 protein expression increased,and the inhibition of 4-HPR-H was more significant Compared with UC+4-HPR-L group,the inhibitory effect of 4-HPR-H on MPO activity,PGE2 content,IL-1β,IL-6,TNF-α,IL-12,i NOS m RNA expression and COX-2 protein expression was significantly reduced after intervention with GW9662(all P < 0.05).6.Compared with Control group,the m RNA levels of Ym1,Arg1 and MRC1,and the nucleoprotein level of PPAR-γ in colon tissue of UC group were significantly decreased,while 4-HPR-L and 4-HPR-H increased the expression of Ym1,Arg1 and MRC1 m RNA and PPAR-γ in different degrees,and compared with 4-HPR-L,4-HPR-H.In UC+4-HPR-H+GW9662 group,the promotion of 4-HPR-H on the expression of Ym1,Arg1,MRC1 m RNA and PPAR-γ was partially reversed by GW9662(all P < 0.05).7.Compared with the Control group,the nuclear protein level of PPAR-γ decreased significantly after LPS stimulation.Compared with LPS group,4-HPR-L and 4-HPR-H can promote the level of PPAR-γ nucleoprotein in different degrees,and the effect of4-HPR-H is more obvious(all P < 0.05).8.Compared with the Control group,LPS stimulated the expression of IL-1β,IL-6,TNF-α,IL-12,i NOS m RNA and CD86 increased significantly.Compared with LPS group,4-HPR-L and 4-HPR-H can inhibit the expression of IL-1β,IL-6,TNF-α,IL-12,i NOS and CD86 in different degrees,and the effect of 4-HPR-H is more obvious.In addition,the expressions of IL-1β,IL-6,TNF-α,IL-12,i NOS and CD86 in LPS+4-HPR-H+gw9662 group were higher than those in LPS+4-HPR-h group(all P <0.05).9.Compared with Control group,LPS stimulation significantly decreased the expression of Ym1,Arg1,MRC1 m RNA,and increased the expression of CD206.Compared with LPS group,both 4-HPR-L and 4-HPR-H significantly upregulated the expressions of Ym1,Arg1,MRC1 m RNA and CD206,and the effect of 4-HPR-H was more obvious.Compared with LPS+4-HPR-H group,the expressions of Ym1,Arg1,MRC1 m RNA and CD206 in LPS+4-HPR-H+GW9662 group decreased(all P < 0.05).Conclusions:This study demonstrated that 4-HPR might be a potent anti-UC agent that works by regulating macrophage polarization via PPARγ. |
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