水苏碱对葡聚糖硫酸钠诱导的炎性肠病的保护作用及其机制

Protective Effect and Mechanism of Stachydrine on Inflammatory Bowel Disease Induced by Dextran Sulphate Sodium

为了研究水苏碱(stachydrine,Sta)对葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的炎性肠病(inflammatory bowel disease,IBD)的保护作用及其机制.将42只同一批次、体重无明显差异的雄性C57BL/6J小鼠随机分为7组,即对照组(control)、模型组(model)、地塞米松(dexamethasone,DXM)组、水苏碱低中高剂量组(Low-Sta,Mid-Sta,High-Sta)以及水苏碱毒性组(Sta toxicity),每组6只.模型组、地塞米松组和水苏碱低中高剂量组采用DSS水溶液(0.03 g/mL)代替正常饮水进行造模,对照组和水苏碱毒性组采用正常饮水喂养.同时DXM组每天灌胃地塞米松水溶液(1 mg/kg),水苏碱低中高剂量组每天分别灌胃水苏碱水溶液(5、10和20 mg/kg),模型组和对照组每日灌胃等量的生理盐水,水苏碱毒性组每天灌胃高剂量(20 mg/kg)的水苏碱水溶液.7 d后处死小鼠,取各组小鼠结肠测量长度并进行苏木精-伊红染色(hematoxylin-eosin,HE)和高碘酸希夫氏染色(periodic acid schiff reagent staining,PAS),观察结肠组织的病理改变以及对照组和水苏碱毒性组肝肾的病理变化;采用蛋白印迹技术(Western Blotting,WB)检测结肠组织中炎症相关蛋白白介素-6(IL-6),核因子-κB(NF-κB)和氧化应激相关蛋白如核转录因子2(NRF2)、血红加氧酶1(HMOX1)、醌氧化还原酶1(NQO1)和闭合蛋白(occludin)及膜周蛋白(zonula occludens,ZO)的表达;采用酶联免疫吸附法(ELISA)检测小鼠血清中IL-6,IL-17a和肿瘤坏死因子-α(TNF-α)的含量;采用流式细胞术检测结肠细胞中活性氧(ROS)阳性细胞的比例.结果显示:水苏碱毒性组小鼠的肝肾及小肠的HE染色结果与对照组相比未见灶性坏死,未见明显纤维化,未见细胞结构破坏及炎症,没有明显差别;与对照组比较,模型组小鼠的结肠长度明显缩短,并且HE结果显示模型组小鼠的肠隐窝结构破坏严重,黏膜下层出现明显的水肿以及炎细胞的浸润,PAS结果显示出模型组的糖蛋白含量显著降低,而在地塞米松和水苏碱处理后上述症状出现了明显的好转;ELISA结果显示,相较于对照组,模型组小鼠血清中的IL-6,IL-17a和TNF-α等促炎因子的含量显著增高(P<0.001),相较于模型组,地塞米松组和低中高剂量水苏碱组的促炎因子的含量显著降低(P<0.05).WB结果显示,水苏碱可显著缓解因DSS诱导所致的结肠中IL-6和NF-κB蛋白含量升高,并同时提高了NRF2,HMOX1,NQO1,Occludin和ZO-1蛋白的表达量;流式细胞术检测ROS结果显示,相较于对照组,模型组的ROS阳性细胞比例显著增高,但在地塞米松和低中高剂量的水苏碱处理后,ROS阳性细胞的比例均明显降低(P<0.05),具统计学意义.以上结果表明,水苏碱可能通过抑制炎症因子的表达和抗氧化应激来缓解DSS诱导的小鼠炎性肠病.

The purpose of this experiment was to study the protective effect of stachydrine(Sta)on dextran sulphate sodium(DSS)induced inflammatory bowel disease(IBD)and its mechanism.42 C57 BL/6 J mice with no significant difference in body weight in the same batch were randomly divided into 7 groups:control group,model group,dexamethasone(DXM)group,the low,medium and high dose group of Sta(Low-Sta,Mid-Sta,High-Sta)and Sta toxicity group,six mice every group.Inducing acute inflammatory bowel disease by feeding the mice with DSS solution(0.03 g/mL)for 7 days to the model group,DXM group and the Low-Sta,Mid-Sta and High-Sta groups.The control group and the toxicity group were fed with normal drinking water.At the same time,DXM group was given dexamethasone solution(1 mg/kg)every day.The low,medium and high dose groups were gavage with low(5 mg/kg),medium(10 mg/kg)and high(20 mg/kg)dose of sulfonate solution every day.The model group and the control group received the same amount of saline daily.The toxicity group received high dose(20 mg/kg)of salicylate solution every day.After 7 days of treatment,the mice were sacrificed and the length of colon in each group was measured.The pathological changes of colon tissues and liver and kidney in control group and toxicity group were observed through hematoxylin-eosin(HE)staining and periodic acid schiff reagent staining(PAS).Expression of inflammation-related proteins interleukin-6(IL-6),nuclear factor kappa-B(NF-κB),oxidative stress-related proteins such as NRF2,HMOX1,NQO1 and tight-junction proteins including occludin and zonula occludens(ZO)in colon tissues were detected by Western Blotting(WB).The levels of IL-6,IL-17 a and TNF-αin serum of mice were detected by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to determine the proportion of reactive oxygen species(ROS)-positive cells in colon cells.Compared with the control group,there was no focal necrosis,fibrosis,damage of cell structure and inflammation in the HE staining results of liver,kidney and small intestine in the hydroquinone toxicity group.At the same time,the colon length of mice in the model group was significantly shortened.Moreover,HE results showed that the intestinal crypt structure of the model group was seriously damaged,and the submucosa showed obvious edema and infiltration of inflammatory cells.PAS staining results showed that the glycoprotein content of the model group was significantly reduced,and the treatment with salicylate showed a significant improvement.ELISA results showed that,compared with the control group,the expression of pro-inflammatory factors such as IL-6,IL-17 a and TNF-αin the model group was significantly increased(P<0.001),while were significantly decreased(P<0.05)in DXM and low,medium and high dose salicylate groups.Immunoblot results showed that stachydrine significantly alleviated DSS induced increases of IL-6 and NF-κB proteins in the colon,and increased the expression of NRF2,HMOX1 and NQO1 proteins.Flow cytometry of ROS showed that compared with the control group,the percentage of ROS positive cells in the model group was significantly increased,but decreased after the treatment with different dose of stachydrine.Stachydrine can alleviate inflammatory bowel disease induced by DSS in mice by inhibiting the expression of inflammatory cytokines and antioxidant stress.