Study on the Effect and Mechanism of Hederagenin on Dextran Sodium Sulfate-Induced Ulcerative Colitis in mice / 中药新药与临床药理

ABSTRACT

Objective To study the effect and its mechanism of hederagenin(hed)on dextran sulfate sodium(DSS)-induced ulcerative colitis(UC)in mice.Methods(1)In vitro experiments:after treating RAW264.7 cells with different concentrations(0,2.5,5,10,20,40 μmol·L-1)of hed for 24 hours,the cell survival rate was detected by MTT assay.RAW264.7 cells were divided into:blank group,lipopolysaccharide(LPS)group(1 μg·L-1),LPS+2.5 μmol·L-1 hed group,LPS+5 μmol·L-1 hed group and LPS+10 μmol·L-1 hed group;an in vitro cellular inflammation model was established using LPS intervention for 24 hours and co-incubated with hed for 24 hours.The levels of interleukin 1β(IL-1β),IL-6 and tumor necrosis factor α(TNF-α)in the cell supernatant were determined by ELISA;the expression levels of TLR4/NF-κB pathway-related proteins in the cells were detected by Western Blot.(2)In vivo experiments:C57BL/6 mice were randomly divided into a blank group,a model group,a Salazosulfapyridine group(200 mg·kg-1),and an hed low-,medium-,and high-dosage groups(12.5,25,and 50 mg·kg-1),with 5 mice in each group.Mice were induced to establish UC model by drinking 3%DSS solution freely for 7 days.The UC model was then established by gavage once a day for 7 days.At the end of the administration,the Disease Activity Index(DAI)was evaluated;pathological changes in the colonic tissues of mice were observed by HE staining;the levels of IL-1β,IL-6,and TNF-α in the colonic tissue were measured by ELISA;and the expression levels of proteins related to the TLR4/NF-κB pathway in the colonic tissue were detected by Western Blot.Results(1)In vitro experiments:compared with the blank group(0 μ mol·L-1 group),there was no significant change in the cell survival rate in the 2.5-10 μmol·L-1 hed group(P＞0.05),and there was no significant toxicity effect on RAW264.7 cells.Compared with the blank group,the expression levels of IL-1β,IL-6,and TNF-α in RAW264.7 cells in the LPS group were significantly increased(P＜0.01);and the protein expression levels of TLR4 and p-NF-κ B/NF-κ B were significantly increased(P＜0.01).Compared with the LPS group,the expression levels of IL-1β and TNF-α in RAW264.7 cells in the hed 2.5,5,and 10 μmol·L-1 concentration groups were significantly decreased(P＜0.05,P＜0.01),and the protein expression levels of TLR4,p-NF-κB/NF-κB were significantly decreased(P＜0.05,P＜0.01);the IL-6 expression level of RAW264.7 cells in the hed 5 and 10 μmol·L-1 concentration groups was significantly reduced(P＜0.05,P＜0.01).(2)In vivo experiments:compared with the blank group,the body mass of mice in the model group was consistently reduced(P＜0.01),the DAI score was significantly elevated(P＜0.01),and the length of the colon was significantly shortened(P＜0.01);the colonic tissue showed obvious epithelial cell damage,and the histopathological scores were significantly elevated(P＜0.01);and the expression levels of the pro-inflammatory cytokines IL-1β,IL-6 and TNF-α were significantly increased(P＜0.01);protein expression levels of TLR4 and p-NF-κB/NF-κB were significantly increased(P＜0.01)in colon tissue.Compared with the model group,the body mass of mice in the low-,medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),the DAI score was significantly decreased(P＜0.05,P＜0.01),the pathological damage of colon tissue improved to different degrees,and the protein expression levels of TLR4,p-NF-κB/NF-κB in the colonic tissue were significantly decreased(P＜0.05,P＜0.01);the colon length of mice in the medium-and high-dose groups of hed were significantly increased(P＜0.05,P＜0.01),and the expression levels and histopathological scores of IL-1β,IL-6,and TNF-α in colon tissue were significantly reduced(P＜0.05,P＜0.01).Conclusion Hed were able to effectively ameliorate colonic histopathological injury and reduce the levels of inflammatory factors in DSS-induced UC mice,and their mechanism of action may be related to the inhibition of the TLR4/NF-κB pathway.