陕产七叶一枝花对小鼠溃疡性结肠炎的治疗作用及机制实验研究

Therapeutic effect and mechanism of Paris polyphylla Sm.from Shaanxi on ulcerative colitis in mice

目的:探讨陕产七叶一枝花对小鼠溃疡性结肠炎的治疗作用,分析可能的机制。方法:利用葡聚糖硫酸钠(DSS)建立溃疡性结肠炎小鼠模型。将50只小鼠分为Con组、DSS组、陕产七叶一枝花低剂量组(L-RPTS+DSS组,干浸膏5 mg/kg)与高剂量组(H-RPTS+DSS组,干浸膏15 mg/kg)、阳性对照5-氨基水杨酸组(5-ASA组,50 mg/kg),每组10只。Con组自由饮用蒸馏水,其余各组小鼠自由饮用每日新鲜配制的3%DSS溶液,持续7 d;陕产七叶一枝花低、高剂量组及5-ASA组除自由饮用DSS溶液外,第1天开始通过灌胃给予不同剂量药物,持续14 d。每日观察小鼠生活状态并记录相应的疾病活动指数(DAI)评分。14 d后进行相关表型测定,酶联免疫吸附法(ELISA)检测小鼠结肠组织炎症因子肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6、IL-1β的水平变化;HE染色和透射电镜评估结肠组织病理学变化。最后采用Western blot检测TNF-α、IL-6、紧密连接闭合蛋白(Occludin)、Claudin-7蛋白表达水平。结果:与Con组比较,DSS组小鼠结肠组织出现严重溃疡反应,炎症细胞浸润,肠上皮细胞严重坏死,结肠组织炎症因子含量及TNF-α、IL-6蛋白表达升高,而Occludin、Claudin-7蛋白表达降低(均P<0.05)。与DSS组比较,L-RPTS+DSS与H-RPTS+DSS组及5-ASA组能够明显改善小鼠溃疡症状,其DAI评分降低(均P<0.05),炎症浸润现象减轻,炎症因子水平减少,肠道黏膜屏障损伤减轻。同时,陕产七叶一枝花给药组结肠组织TNF-α、IL-6蛋白表达降低,而Occludin、Claudin-7蛋白表达增加(均P<0.05)。H-RPTS+DSS组溃疡指标的改善优于L-RPTS+DSS(均P<0.05)。结论:陕产七叶一枝花能够改善DSS诱导的小鼠溃疡性结肠炎症状,其可能机制与调节炎症因子释放及调控肠道紧密连接蛋白、改善肠黏膜屏障损伤有关。

Objective:To investigate the therapeutic effect of Paris polyphylla Sm.from Shaanxi on ulcerative colitis in mice and explore the possible mechanism.Methods:A mouse model of ulcerative colitis was established using dextran sulfate sodium(DSS).Fifty mice were divided into normal group(Con group),DSS group,Paris polyphylla Sm.from Shaanxi low-dose and high-dose groups(L-RPTS+DSS group and H-RPTS+DSS group,dry extract 5,15 mg/kg),Positive control 5-aminosalicylic acid group(5-ASA group,50 mg/kg),with 10 mice in each group.The Con group drank distilled water freely,and the mice in the other groups drank 3%DSS solution prepared freshly every day for 7 days.The L-RPTS+DSS group,H-RPTS+DSS group and the 5-ASA group drank DSS solution freely.In addition,different doses of drugs were given by gavage starting from the first day and continued for 14 days.The living conditions of the mice were observed daily and the corresponding Disease Activity Index(DAI)scores were recorded.14 days later,the phenotype was determined,and the ELISA was used to detect the inflammatory factors TNF-α,IL-6 and IL-1βin the colon tissue of the mice,and HE staining and transmission electron microscopy were used to evaluate colon histopathological changes.Finally,Western blot was used to detect the protein expression levels of TNF-α,IL-6,Occludin,and Claudin-7.Results:Compared with the Con group,the colon tissue of mice in the DSS group showed severe ulceration reaction,inflammatory cell infiltration,severe necrosis of intestinal epithelial cells,and the content of inflammatory factors and the expression of TNF-αand IL-6 proteins in the colon tissue were significantly increased(all P<0.05),while the expression of Occludin and Claudin-7 proteins decreased(all P<0.05).Compared with the DSS group,the L-RPTS+DSS group,H-RPTS+DSS group and 5-ASA group could significantly improve the intestinal of mice with ulcer symptoms.The DAI scores were significantly reduced(all P<0.05).HE staining and transmission electron microscopy results showed that the inflammatory infiltration phenomenon was reduced,the content of inflammatory factors was reduced,and the intestinal mucosal barrier damage was reduced.At the same time,the protein expressions of TNF-αand IL-6 in the colon tissue of Paris polyphylla Sm.from Shaanxi group were significantly reduced(all P<0.05),while the expression of Occludin and Claudin-7 were increased(all P<0.05).The improvement of ulcer indicators in the H-RPTS+DSS group was better than that of the L-RPTS+DSS group(all P<0.05).Conclusion:Paris polyphylla Sm.from Shaanxi can improve the symptoms of ulcerative colitis induced by DSS in mice.The possible mechanism is related to regulating the release of inflammatory factors and regulating intestinal tight junction proteins,and improving intestinal mucosal barrier damage.