| Ginsenosides are the main active ingredients of traditional Chinese medicine ginseng.The study of the biological activity of ginsenosides has important clinical significance for the development and utilization of ginseng.Ginsenosides can be divided into diol and three glycol type saponins according to its structure.Studies showed that ginseng saponin has anti-cancer,immune regulation,anti-inflammatory,anti-allergic,anti-stress,antitumor activities.GLXXV is a type of diol saponins,and there are few studies on its biological activity.This paper studies the activity GLXXV in the inhibition of colitis of and its mechanism.Macrophages,as an important part of the immune system,can promote the activation of inflammatory signaling pathways after activation and then release various inflammatory cytokines.We selected mouse macrophage cell line RAW264.7as the experimental subject to study the anti-inflammatory activity of GLXXV.First,the cytotoxicity of GLXXV on RAW264.7 was tested by MTT assay.The results showed that GLXXV had no effect on cell survival at 50 μM and 100 μM concentrations.Then,ELISA assay was used to detect the release of inflammatory cytokines TNF-α,IL-6 and NO in RAW264.7 cells treated with GLXXV and LPS alone or together for 24 h.The results showed that GLXXV treatment had no significant change compared with the blank control group,indicating that GLXXV had no proinflammatory activity.TNF-α,IL-6 and NO were significantly upregulated in the LPS group.Under the cotreatment of GLXXV and LPS,TNF-α,IL-6 and NO were significantly down-regulated.These results indicated that ginsenoside GLXXV could inhibit the inflammatory response of macrophages induced by LPS.Next,the mechanism of GLXXV in inhibiting inflammatory response was further studied.We found that LPS can cause the phosphorylation of ERK,JNK and p38 in RAW264.7 cells,while GLXXV can significantly inhibit the phosphorylation of the above proteins in RAW264.7 cells.LPS treatment could induce both the degradation of IκBα protein and the phosphorylation of NF-κB,while GLXXV could inhibit the change of these two proteins induced by LPS.The distribution of NF-κB in cells was detected by immunofluorescence.The results showed that GLXXV could inhibit the nuclear translocation of NF-κB induced by LPS.The above results suggest that GLXXV inhibits the inflammatory response of macrophages induced by LPS by inhibiting the phosphorylation of ERK,JNK,p38 and NF-κB.The structure of GLXXV and glucocorticoid are similar.The effect of GLXXV on the expression of inflammatory cytokines was detected after knock down of the expression of glucocorticoid receptor(GR).The results showed that with GR knockdown,the inhibitory effect of GLXXV on the phosphorylation of ERK,JNK,p38 and NF-κB and thereby the release of TNF-α and IL-6 induced by LPS was significantly reduced.Therefore,GR might be the receptor of GLXXV in inhibiting inflammatory response.In addition,we established a DSS-induced mouse colitis model to detect the activity of GLXXV in inhibiting colitis.Blank control group,DSS model group,DSS+10 mg/kg GLXXV gavage group and DSS+20 mg/kg GLXXV gavage group were set respectively.The results showed that compared with the blank control group,the colonic length of the mice in the DSS model group was significantly shortened,and the colonic tissue was severely damaged.The expression of intestinal protein was detected by western-blot.The results showed that the phosphorylation of ERK,JNK,p38 and NF-κB was enhanced and the expression of Occludin,ZO-1 and Claudin were down-regulated.GLXXV inhibited the above intestinal tissue changes induced by DSS.Blood cell analyzer was used to detect the blood changes in mice.The results showed that in the DSS model group,the release of TNF-α and IL-6 were significantly up-regulated and the proportion of white blood cells were up-regulated,while GLXXV could inhibit the changes in the blood.In addition,the mouse thymus,spleen,kidney and liver were weighed.It was found that the spleen weight was up-regulated and the thymus weight was down-regulated in the DSS model group.GLXXV could inhibit these changes.There was no significant changes in kidney or liver weight.In conclusion,ginsenoside GLXXV plays anti-inflammatory effect by inhibiting the activation of MAPK and NF-κB through glucocorticoid receptors. |
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