辣木叶总黄酮对小鼠溃疡性结肠炎防治作用的研究

Preventive and Therapeutic Effects of Moringa oleifera Leaf Flavonoids on Ulcerative Colitis in Mice

【目的】以葡聚糖硫酸钠(DSS)诱导的溃疡性结肠炎(UC)小鼠为模型,探究辣木叶总黄酮(MOLF)对UC的防治作用。【方法】将50只BALB/c小鼠随机分为5组:对照组、DSS组(模型组)和MOLF-L(25 mg/kg)、MOLF-M(50 mg/kg)、MOLF-H(100 mg/kg)处理组,每组10只。小鼠通过饮用4%DSS诱导UC模型,各MOLF处理组灌胃相应剂量药物0.2 mL,对照组和DSS组灌以等体积的生理盐水,每天1次,连续7 d。每天记录各组疾病活动指数(DAI),在末次给药的次日经眼眶静脉丛采血,分离血清测定白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)、高迁移率族蛋白B1(HMGB1)和内毒素(LPS)含量;小鼠脱颈处死后取结肠组织进行HE染色观察组织形态、PAS染色观察组织杯状细胞数量变化;实时荧光定量PCR法检测结肠组织中IL-1β、TNF-α、IL-10、HMGB1 mRNA表达水平;免疫荧光法检测肠黏膜闭锁连接蛋白-1(ZO-1)和闭合蛋白(Occludin)表达情况;Western blotting分析凋亡相关蛋白cleaved-caspase 3、Bcl-2、Bax的表达水平。【结果】UC小鼠造模成功,DSS组小鼠体内LPS、炎性因子含量、cleaved-caspase 3和Bax蛋白表达量与对照组相比差异极显著(P<0.01);不同剂量MOLF均可以改善UC小鼠体内炎症状况,尤以MOLF-H组效果最佳:与DSS组相比,DAI评分以及血清中LPS含量极显著降低(P<0.01),小鼠血清和结肠组织中促炎因子IL-1β、TNF-α、HMGB1含量和mRNA表达量均极显著降低(P<0.01),抗炎因子IL-10的表达量极显著升高(P<0.01),且cleaved-caspase 3和Bax的表达量极显著上调,Bcl-2的表达量极显著下调(P<0.01)。此外,MOLF可以改善结肠黏膜水肿、炎性细胞浸润情况,维持杯状细胞数量及其形态,增加ZO-1和Occludin蛋白表达量,且这些作用具有剂量依赖性。【结论】MOLF对DSS诱导的UC具有显著的防治作用,这可能与其抑制炎症并维护肠黏膜屏障的完整性有关。

【Objective】 The aim of this study was to investigate the preventive and therapeutic effects of Moringa oleifera leaf flavonoids(MOLF) on ulcerative colitis(UC) in mice induced by dextran sodium sulfate(DSS).【Method】 50 BALB/c mice were randomly divided into 5 groups: control group, DSS group(model group),MOLF-L(25 mg/kg),MOLF-M(50 mg/kg) and MOLF-H(100 mg/kg) groups.Each group contained 10 mice.Mice were induced UC by drinking 4% DSS, and MOLF-L,MOLF-M and MOLF-H groups were given the corresponding doses of MOLF 0.2 mL by gavaged, the control and DSS groups were gavaged with an equal volume of normal saline once a day for 7 d.The disease activity index(DAI) of each group was recorded daily. After 7 d of treatment, the blood was collected from the transorbital venous plexus, and the serum was separated from the blood samples for the determination of IL-1β,IL-10,TNF-α,HMGB1 and LPS.Mice were sacrificed and their colon tissues were harvested for HE staining to observe tissue morphology, and PAS staining to observe goblet cell numbers in tissues. The mRNA expression levels of IL-1 β,IL-10,TNF-α and HMGB1 in colonic tissue were respectively detected by Real-time quantitative PCR,the expressions of zonula occludens-1(ZO-1) and Occludin were detected by immunofluorescence assay, and the expression levels of apoptosis related proteins cleaved-caspase 3,Bcl-2 and Bax were analyzed by Western blotting.【Result】 UC was successfully established in mice.Compared with the control group, the levels of LPS and inflammatory factors, the expressions of cleaved-caspase 3 and Bax in the DSS group were extremely significantly different(P<0.01).Different doses of MOLF could improve the inflammation in UC mice, especially in the MOLF-H group: Compared with the DSS group, the DAI score and LPS content were extremely significantly reduced(P<0.01), the mRNA expression of pro-inflammatory factors IL-1β,TNF-α and HMGB1 in serum and colon tissues were extremely significantly reduced(P<0.01),the expression of anti-inflammatory factor IL-10 was extremely significantly increased(P<0.01),besides, cleaved-caspase 3 and Bax were up-regulated and Bcl-2 expression level was down-regulated extremely significantly(P<0.01).In addition, MOLF could improve colonic mucosal edema and inflammatory cell infiltration, protect the number and morphology of goblet cells, and increase the expression of ZO-1 and Occludin proteins, with a dose-effect relationship between MOLF.【Conclusion】 MOLF could protect against UC by regulating the levels of inflammatory factors, ameliorating DSS induced impairment of ZO-1 and Occludin expression and abnormal expression of apoptosis related proteins in the colon tissues of mice.