隔药灸对溃疡性结肠炎大鼠中性粒细胞胞外诱捕网的调节作用研究OA

Objective To investigate the immunomodulatory mechanism of herb-partitioned moxibustion in inhibiting inflammation in ulcerative colitis(UC)from the perspective of neutrophil extracellular trap(NET)formation.Method Male Sprague-Dawley(SD)rats were selected and randomized into a normal group(NG),a model group(MG),an herb-partitioned moxibustion group(HPM),and a medicine group(MED),with 8 rats per group.The UC model was induced using Dextran sulfate sodium(DSS).After successful model establishment,the HPM received herb-partitioned moxibustion,and the MED was intervened with Sulfasalazine enteric-coated tablets via gavage.Rats in each group were observed for their general condition,gross morphological damage of the colon,disease activity index(DAI),and colonic histopathology.Enzyme-linked immunosorbent assay(ELISA)was adopted to detect the concentrations of NET-associated complexes[cell-free DNA(cf-DNA)and myeloperoxidase(MPO)-DNA]in peripheral blood neutrophil culture supernatants and plasma,as well as the protein concentrations of pro-inflammatory cytokines[interleukin-6(IL-6)and interferon-gamma(IFN-γ)]and anti-inflammatory cytokines[interleukin-4(IL-4)]in plasma.Immunofluorescence was used to determine the co-expression of the NET's characteristic marker proteins[MPO,neutrophil elastase(NE),and citrullinated histone H3(CitH3)]in the colonic tissues of each group.Result Compared to the NG,the MG presented incomplete colonic epithelium,notable ulcer formation,significant inflammatory cell infiltration,and significantly increased DAI,colonic gross morphological damage scores,and plasma IL-6 and IFN-γ concentrations(P＜0.05),as well as markedly decreased plasma IL-4 concentrations(P＜0.05);in the MG,the concentrations of cf-DNA and MPO-DNA complexes in peripheral blood neutrophil culture supernatant and plasma were significantly elevated(P＜0.05),and the expression levels of MPO,CitH3,and NE proteins were increased at colonic ulcers infiltrated by massive neutrophils(P＜0.05),presenting obvious co-expression.Compared to the MG,the HPM and MED showed colonic mucosa covered by regenerated epithelium,healed ulcers,and decreased DAI,colonic gross morphological damage scores,and plasma IL-6 and IFN-γ concentrations(P＜0.05),as well as notably increased plasma IL-4 concentrations(P＜0.05);the two intervention groups also exhibited significant decreases in the concentrations of cf-DNA and MPO-DNA complexes in peripheral blood neutrophil culture supernatant and plasma(P＜0.05),along with reduced expression levels of MPO,CitH3,and NE proteins at colonic ulcers infiltrated by neutrophils(P＜0.05),with only a small amount of co-expression.Conclusion Herb-partitioned moxibustion can regulate the expression of colonic MPO,CitH3,and NE proteins,down-regulate the concentrations of cf-DNA and MPO-DNA complexes in peripheral blood,and inhibit excessive clustering of NETs in colonic tissues,which may be one of the main mechanisms of herb-partitioned moxibustion alleviating colonic inflammation in UC rats.