华蟾素对溃疡性结肠炎大鼠的作用及机制

Effect and the mechanism of Huachansu on ulcerative colitis in rats

目的研究华蟾素对溃疡性结肠炎(UC)大鼠的作用及其机制。方法用2,4,6-三硝基苯磺酸(TNBS)结肠灌注建立UC大鼠模型。按照大鼠体重随机分为对照组、模型组、阳性对照组、华蟾素低剂量组(HCS-L)、华蟾素高剂量组(HCS-H),每组6只。对照组、模型组予以生理盐水灌肠,阳性对照组给予美沙拉嗪灌肠液1.5 g·kg^(-1)灌肠,HCS-L、HCS-H分别予以华蟾素200、400 mg·kg^(-1)灌肠,每日1次,连续14 d。用疾病活动指数(DAI)、结肠黏膜损伤指数(CMDI)进行评分,用苏木素-伊红(HE)染色观察结肠病理变化,用酶联免疫吸附(ELISA)法检测血清中炎性因子水平,用免疫组化法检测结肠组织中NOD样受体蛋白3(NLRP3)、胱天蛋白酶-1(caspase-1)、咬合蛋白(Occludin)、带状闭合蛋白(ZO-1)的表达情况。结果对照组、模型组、阳性对照组、HCS-L、HCS-H的DAI评分分别为0.11±0.27,2.44±0.54,1.22±0.78,1.33±0.73,1.06±0.61;上述5组的CMDI评分分别为0.17±0.41,2.83±1.17,1.00±0.89,1.33±0.52,0.83±0.75;与正常组比较,模型组DAI评分、CMDI评分均显著升高,与模型组比较,阳性对照组、HCS-L、HCS-H的DAI评分、CMDI评分均显著降低(均P<0.05)。与正常组比较,模型组血清中白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)均显著升高,与模型组比较,阳性对照组、HCS-L、HCS-H血清中IL-1β、IL-18、TNF-α均显著降低(均P<0.01);与正常组比较,模型组结肠组织中NLRP3、caspase-1的平均光密度值均显著升高,Occludin、ZO-1的平均光密度值均显著降低(均P<0.01),与模型组比较,阳性对照组、HCS-L、HCS-H结肠组织中NLRP3、casepase-1的平均光密度值均显著降低,Occludin、ZO-1的平均光密度值均显著升高(均P<0.01)。结论华蟾素可明显缓解UC大鼠症状,改善UC大鼠肠黏膜病变,其机制可能与抑制NLRP3的过度激活、降低炎症反应、增强黏膜屏障的完整性有关。

Objective To investigate the effect and underlying mechanism of Huachansu on ulcerative colitis(UC)in rats.Methods UC rat model was induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS)intra-colonic adminisrataion.According to the weight of the rats,they were randomly divided into five groups:control group,model group,positive control group,Huachansu low-dose group(HCS-L),and Huachansu high-dose group(HCS-H),with 6 rats in each group.The control group and model group were given normal saline via enema administration,and the positive control group was given mesalazine enema 1.5 g·kg^(-1),HCS-L and HCS-H were given Huachansu 200 and 400 mg·kg^(-1).All the drugs were administered once a day for consecutive 14 days.The disease activity index(DAI)and colonic mucosal injury index(CMDI)were evaluated.Hematoxylin-eosin(HE)staining was used to observe colon pathological changes.Serum inflammatory factor levels were detected by enzyme-linked immunosorbent assay(ELISA).Immunohistochemistry was used to detect the expression of NOD-like receptor protein 3(NLRP3),Cysteinyl aspartate specific proteinase-1(caspase-1),Occludin,and Zonula occludens-1(ZO-1)in colon tissue.Results The DAI scores of the control group,model group,positive control group,HCS-L,and HCS-H were 0.11±0.27,2.44±0.54,1.22±0.78,1.33±0.73,1.06±0.61,respectively;the CMDI scores of the above five groups were0.17±0.41,2.83±1.17,1.00±0.89,1.33±0.52,0.83±0.75;compared with control group,the DAI score and CMDI score of model group were significantly increased(P<0.01);compared with model group,the DAI score and CMDI score of positive control group,HCS-L and HCS-H were significantly decreased(all P<0.05).Compared with control group,the serum levels of interleukin-1β(IL-1β),interleukin-18(IL-18)and tumor necrosis factor-α(TNF-α)in model group were significantly increased;compared with model group,the serum levels of IL-1β,IL-18,and TNF-αin positive control group,HCS-L,and HCS-H were significantly reduced(all P<0.01).Compared with control group,the average optical density values of NLRP3 and caspase-1 in the colon tissue of model group were significantly increased,and the average optical density values of Occludin and ZO-1 were significantly decreased(all P<0.01);compared with model group,the average optical density values of NLRP3 and caspase-1 in positive control group,HCS-L and HCS-H colon tissues were significantly reduced,and the average optical density values of Occludin and ZO-1 were both significant increase(all P<0.01).Conclusion Huachansu can significantly relieve the symptoms of UC rats and improve the intestinal mucosal lesions of UC rats.The mechanism may be related to inhibiting the excessive activation of NLRP3,decreasing colonic inflammation,and enhancing the integrity of the mucosal barrier.