三仁汤通过抑制Th17细胞分化改善DSS诱导的小鼠炎症性肠病OA

OBJECTIVE To investigate the therapeutic effects of Sanren decoction(SRD)against inflammatory bowel disease(IBD)and the underlying mechanisms.METHODS Male C57BL/6J mice were randomly divided into five groups:the control group,dextran sulfate sodium(DSS)group,and DSS+SRD 0.36,0.72 and 1.44 g·kg-1 groups.Except for the control group,all the mice received 2.5%DSS in drinking water for 7 days to induce colitis.During this experiment,mice of treatment groups were intragastrically administered with SRD at respective dosages once daily.Body weight was recorded daily.All the mice were euthanized on day 8 to measure colon length.HE staining was used to quantify the percentage of colonic tissue areas infiltrated with inflammatory cells.Alcian blue-periodic acid-schiff(AB-PAS)staining was adopted to count goblet cells in colonic tissue.Immunofluorescence staining was performed to observe the morphology of goblet cells.Network pharmacology was used to predict the core therapeutic targets.Western blotting was performed to detect the protein expression levels of matrix metalloproteinase 3(MMP3),MMP13 and interleukin-17A(IL-17A)in colonic tissue.The mRNA expression levels of IL-17A,IL-6,MMP3,MMP10,MMP13,IL-1α and IL-1β were detected with RT-qPCR.The proportion of T helper 17 cells(Th17)in peripheral blood mononuclear cells(PBMCs)and spleen cells was calculated via flow cytometry,and immunofluorescence staining was conducted to visualize Th17 cell infiltration inside colonic tissue.RESULTS Mice in the DSS group showed obvious body mass loss and shortened colons compared to controls,accompanied by increased inflammatory cell infiltration and a marked reduction of goblet cells in colonic tissue.Compared with mice in the DSS group,administration of SRD at 0.72 and 1.44 g·kg-1 significantly increased body mass and colon lengths,markedly reduced the area of inflammatory cell infiltration in colonic tissue,and substantially elevated goblet cell counts.Network pharmacology analysis indicated that targets linked to the IL-17 signaling pathway might serve as potential core targets of SRD against IBD.Western blotting and RT-qPCR suggested that in the DSS group,the protein expression levels of MMP3,MMP13 and IL-17A in colonic tissue were significantly increased compared with the control group.The mRNA levels of IL-17A,IL-6,MMP3,MMP10,MMP13,IL-1α and IL-1β were notably increased.Relative to the DSS group,the protein expression level of IL-17A in colonic tissue was markedly reduced in the DSS+SRD 1.44 g·kg-1 group.The protein expression levels of MMP3 and MMP13,together with the mRNA levels of IL-17A,MMP3 and MMP10,were significantly decreased in the mice treated with DSS+SRD 0.36,0.72,and 1.44 g·kg-1.Additionally,administration of SRD at the doses of 0.72 and 1.44 g·kg-1 signifi-cantly suppressed the mRNA expressions of IL-6,MMP13,IL-1α,and IL-1β.Flow cytometry and immu-nofluorescence staining found that the proportion of Th17 cells in PBMCs and spleen cells was much higher in the mice of the DSS group than in the control group.Administration of SRD at 1.44 g·kg-1 significantly reduced the proportion of Th17 cells in PBMCs and spleen cells,and alleviated Th17 cell infiltration in colonic tissue.CONCLUSION SRD can alleviate DSS-induced IBD in mice,which is likely related to suppressed differentiation and infiltration of Th17 cells and reduced levels of Th17-associated pro-inflammatory cytokines.