常春藤皂苷元对葡聚糖硫酸钠诱导小鼠溃疡性结肠炎的作用及机制研究

Study on the Effect and Mechanism of Hederagenin on Dextran Sodium Sulfate-Induced Ulcerative Colitis in mice

目的研究常春藤皂苷元对葡聚糖硫酸钠(DSS)诱导小鼠溃疡性结肠炎(UC)的作用及其机制。方法(1)体外实验:用不同浓度(0、2.5、5、10、20、40μmol·L^(-1))常春藤皂苷元处理RAW264.7细胞24 h后,采用MTT法检测细胞存活率。将RAW264.7细胞分为:空白组、脂多糖(LPS)组(1μg·L^(-1))、LPS+2.5μmol·L^(-1)常春藤皂苷元组、LPS+5μmol·L^(-1)常春藤皂苷元组和LPS+10μmol·L^(-1)常春藤皂苷元组;采用LPS干预24 h建立体外细胞炎症模型,并用常春藤皂苷元共孵育24 h进行干预。采用ELISA法测定细胞上清液中白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)水平;Western Blot法检测细胞中TLR4/NF-κB通路相关蛋白的表达水平。(2)体内实验:将C57BL/6小鼠随机分为空白组、模型组、柳氮磺吡啶组(200 mg·kg^(-1))及常春藤皂苷元低、中、高剂量组(12.5、25、50 mg·kg^(-1)),每组5只。小鼠自由饮用3%DSS溶液7 d,诱导建立UC模型。造模同时灌胃给药,每日1次,连续7 d。给药结束后,进行疾病活动指数(DAI)评价;采用HE染色法观察小鼠结肠组织病理变化;ELISA法测定结肠组织中IL-1β、IL-6、TNF-α水平;Western Blot法检测结肠组织中TLR4/NF-κB通路相关蛋白的表达水平。结果(1)体外实验:与空白组(0μmol·L^(-1)组)比较,2.5~10μmol·L^(-1)常春藤皂苷元组的细胞存活率无明显变化(P>0.05),对RAW264.7细胞无明显毒性作用。与空白组比较,LPS组RAW264.7细胞的IL-1β、IL-6、TNF-α表达水平显著升高(P<0.01);TLR4、p-NF-κB/NF-κB蛋白表达水平显著升高(P<0.01)。与LPS组比较,常春藤皂苷元2.5、5、10μmol·L^(-1)浓度组RAW264.7细胞的IL-1β、TNF-α表达水平明显降低(P<0.05,P<0.01),TLR4、p-NF-κB/NF-κB蛋白表达水平明显降低(P<0.05,P<0.01);常春藤皂苷元5、10μmol·L^(-1)浓度组RAW264.7细胞的IL-6表达水平明显降低(P<0.05,P<0.01)。(2)体内实验:与空白组比较,模型组小鼠的体质量持续降低(P<0.01),DAI评分显著升高(P<0.01),结肠长度显著缩短(P<0.01);结肠组织出现明显的上皮细胞损伤,组织病理学评分显著升高(P<0.01);结肠组织中的促炎细胞因子IL-1β、IL-6、TNF-α表达水平及TLR4、p-NF-κB/NF-κB蛋白表达水平均显著升高(P<0.01)。与模型组比较,常春藤皂苷元低、中、高剂量组小鼠的体质量明显增加(P<0.05,P<0.01),DAI评分明显降低(P<0.05,P<0.01),结肠组织病理损伤有不同程度的改善,结肠组织中TLR4、p-NF-κB/NF-κB蛋白表达水平明显降低(P<0.05,P<0.01);常春藤皂苷元中、高剂量组小鼠的结肠长度明显增加(P<0.05,P<0.01),结肠组织中IL-1β、IL-6、TNF-α表达水平及病理学评分显著降低(P<0.05,P<0.01)。结论常春藤皂苷元能够有效改善DSS诱导UC小鼠的结肠组织病理损伤,降低炎症因子水平,其作用机制可能与抑制TLR4/NF-κB通路有关。

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 RAW264.7cells with different concentrations(0,2.5,5,10,20,40μmol·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(1μg·L^(-1)),LPS+2.5μmol·L^(-1)hed group,LPS+5μmol·L^(-1)hed group and LPS+10μmol·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 factorα(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(0μmol·L^(-1)group),there was no significant change in the cell survival rate in the 2.5-10μmol·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-κB/NF-κB were significantly increased(P<0.01).Compared with the LPS group,the expression levels of IL-1βand TNF-αin RAW264.7 cells in the hed2.5,5,and 10μmol·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-6expression level of RAW264.7 cells in the hed 5 and 10μmol·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-αwere 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 mediumand 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.