四逆散不同配伍干预实验性溃疡性结肠炎的效用和机制研究

【作者】 卢健；

【导师】 马骥；

【作者基本信息】 辽宁中医药大学 ， 方剂学， 2011， 博士

【摘要】 目的:通过实验,验证四逆散及其不同配伍干预实验性溃疡性结肠炎(ulcerative colitis,UC)的效用,探讨四逆散不同配伍干预实验性UC的机制。材料与方法:1四逆散不同配伍干预实验性UC的效用研究将实验大鼠分为14组,采用免疫法造模,以实验大鼠一般状况、结肠组织大体情况、结肠组织病理变化,胸腺指数、脾脏指数和结肠组织NF-κB作为观察与检测指标,用Q-PCR检测核因子-κB(nuclear factor-kappa B,NF-κB)在组织的基因表达,用免疫组化法检测NF-κB的活性。2四逆散干预实验性UC的机制研究在“四逆散不同配伍干预实验性UC的效用研究”的基础上,将实验动物分为5组,采用免疫法造模,以实验大鼠结肠组织大体情况、结肠组织病理变化和白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-4(interleukin-4,IL-4)、白细胞介素-10(interleukin-10,IL-10)作为观察与检测指标,用Q-PCR检测上述细胞因子在组织的基因表达,用ELISA法检测细胞因子活性。结果:1四逆散不同配伍干预实验性UC的效用研究1.1四逆散不同配伍对实验性UC大鼠一般状况的影响造模第l4天,大鼠多出现食量减少、拱背、聚堆、懒动;第21天,均出现饮食明显减少,稀便、毛发不泽,拱背、聚堆、懒动。各给药组造模大鼠上述症状有不同程度改善。正常组大鼠饮食、大小便、活动正常。给药前后正常组实验大鼠体重平均增长54%,而其他各组实验大鼠体重平均增长范围在32%～38%。1.2四逆散不同配伍对实验性UC大鼠结肠组织损伤的影响肉眼观察,模型组大鼠病变结肠肠壁增厚,肠黏膜糜烂,黏膜表面弥漫性充血、水肿,可见出血点和溃疡。各药物治疗组大鼠结肠黏膜表现不同程度充血、水肿,可见轻度糜烂和溃疡。结肠组织损伤程度评分结果显示,造模组大鼠的结肠损伤程度明显高于正常组(P<0.05)。与模型组比较,各药组大鼠的结肠损伤程度显著降低。光镜下观察,模型组黏膜表面可见溃疡形成,个别深达肌层,炎症反应较重,有充血、糜烂,中性粒细胞浸润,黏膜上皮部分缺失,局部固有层腺体减少,黏膜下层血管增生,黏膜肌层不明显。其余各组均有不同程度的病变。结肠病理组织学评分结果显示,造模组大鼠的结肠病理组织学评分结果明显高于正常组(P<0.05)。与模型组比较,芍甘组和枳甘组无明显差异,其余各组显著降低(P<0.05)。1.3四逆散不同配伍对实验性UC大鼠胸腺指数与脾脏指数的影响造模后,模型组和柴芍组大鼠胸腺指数显著低于正常组,以柴芍组降低最为显著(P<0.05),其余各组与正常组无显著性差异;与模型组比较,对照组、四逆散组、柴芍甘组、芍枳甘组、柴芍组、柴甘组、芍甘组和枳甘组大鼠胸腺指数均存在显著差异。造模后大鼠脾脏指数均显著高于正常组(P<0.05);与模型组相比,对照组、四逆散组和柴枳甘组的脾脏指数显著增高(P<0.05)。1.4四逆散不同配伍对实验性UC大鼠结肠组织NF-κB基因表达及活性的影响模型组实验大鼠结肠黏膜NF-κB mRNA表达水平明显高于正常组(P<0.05);各药物组实验大鼠结肠黏膜NF-κB mRNA表达水平与模型组比较均存在显著性差异,其中,枳甘组显著高于模型组(P<0.05)。模型组实验大鼠结肠黏膜NF-κB活性明显高于正常组(P<0.05);四逆散组与正常组比较,无显著差异;除柴甘组和枳甘组外,其余各药物组均显著低于模型组(P<0.05);与四逆散组比较,各药物组显著高于四逆散组(P<0.05)。2四逆散干预实验性UC的机制研究2.1四逆散对实验性UC大鼠IL-1β的影响模型组实验大鼠结肠黏膜IL-1βmRNA表达水平明显高于正常组(P<0.05);四逆散组、柴芍枳组和柴芍组与正常组比较无显著性差异;与模型组比较,四逆散组、柴芍枳组和柴芍组均显著降低(P<0.05)。模型组和四逆散组实验大鼠血清IL-1β含量明显高于正常组(P<0.05),而柴芍枳组和柴芍组与正常组比较无统计学意义;四逆散组与模型组比较无显著性差异;柴芍枳组和柴芍组显著低于模型组和四逆散组(P<0.05)。2.2四逆散对实验性UC大鼠TNF-α的影响模型组实验大鼠结肠黏膜TNF-αm RNA表达水平明显高于正常组(P<0.05);四逆散组、柴芍枳组和柴芍组与正常组比较无显著性差异;与模型组比较,四逆散组、柴芍枳组和柴芍组均显著降低(P<0.05);柴芍枳组明显高于四逆散组(P<0.05),而柴芍组明显低于四逆散组(P<0.05)。与正常组比较,模型组、四逆散组、柴芍枳组和柴芍组实验大鼠血清TNF-α的含量均明显高于正常组;与模型组比较,四逆散组、柴芍枳组和柴芍组显著降低(P<0.05)。2.3四逆散对实验性UC大鼠IL-4的影响模型组实验大鼠结肠黏膜IL-4 mRNA表达水平明显低于正常组(P<0.05);四逆散组和柴芍枳组与正常组比较无显著性差异,而柴芍组则显著高于正常组(P<0.05);与模型组比较,四逆散组、柴芍枳组和柴芍组均显著升高(P<0.05);柴芍枳组和柴芍组均显著高于四逆散组(P<0.05)。与正常组比较,模型组、四逆散组、柴芍枳组和柴芍组实验大鼠血清IL-4的含量均明显降低(P<0.05);给药后,柴芍枳组与模型组比较无显著性差异,而四逆散组和柴芍组大鼠明显高于模型组(P<0.05),其中,柴芍组明显低于四逆散组(P<0.05)。2.4四逆散对实验性UC大鼠IL-10的影响模型组实验大鼠结肠黏膜IL-10 mRNA表达水平明显低于正常组(P<0.05);四逆散组、柴芍枳组和柴芍组与正常组比较无显著性差异;与模型组比较,四逆散组、柴芍枳组和柴芍组均显著升高(P<0.05)。与正常组比较,模型组、四逆散组、柴芍枳组和柴芍组实验大鼠血清IL-10的含量均明显降低(P<0.05);给药后,柴芍枳组与模型组比较无显著性差异,而四逆散组和柴芍组大鼠明显高于模型组(P<0.05),柴芍组明显低于四逆散组(P<0.05)。结论:四逆散能够干预实验性UC,其作用可能是通过刺激免疫器官分泌免疫细胞,抑制NF-κB的激活来实现的;四逆散对实验性UC作用以全方作用最为理想。四逆散能够干预实验性UC,其作用机理可能为:①抑制促炎因子IL-1β的移行和TNF-α的分泌,促进抗炎因子IL-4和IL-10的分泌,调节促炎因子和抗炎因子之间的平衡,干预实验性UC;②降低实验性UC大鼠结肠黏膜NF-κB的表达水平,调控UC细胞因子网络。更多还原

【Abstract】 Purpose:The study of utility and mechanism is the Sini powders and the different compatibilities in intervening experimental ulcerative colitis.Material and method:1 Study of Utility on the Sini Powders and the Different Compatibilities in Intervening Experimental Ulcerative ColitisThe experimental animals were divided into 14 groups and modeled with immunization. The states, colon organization and its pathological changes varying, thymus index, spleen index and activity of NF-κB of rats were treated as observation and measurement indicators. NF-κB mRNA was detected by Q-PCR technique. And it’s protein activity was detected by immunohistochemistry.2 Study of Mechanism on the Sini Powders in Intervening Experimental Ulcerative Colitis Based on the first study, the experimental animals were divided into 5 groups and modeled with immunization. The states, colon organization and its pathological changes varying, IL-1β, TNF-α, IL-4, IL-10 of rats were treated as observation and measurement indicators. And we detected those cell factors mRNA with Q-PCR and activities with ELISA.Results:1 Study of Utility on the Sini Powders and the Different Compatibilities in Intervening Experimental Ulcerative Colitis1.1 The statesAnimalpatterns appeared diet reducedg, arch, polyethylene reactor and lazy action at the 14th day, and diet significantly reduced, diarrhea, hair dryed, arch, polyethylene reactor and lazy action at the 21st after modeling. Administrated orally, above symptoms the rats showed improvement with different degree.The increasing healthy rats’ weight was 54%, but the modeling rats’weight increased averagely the range within 32%～38% before and after drug administration.1.2 Colon Organization and its Pathological ChangesWith visualinspection, we found the intestinal wall thicken, mucous membrane erosion, suiface of mucous membrane diffuse congestion and edema of the rates colons of model group. And blutpunkte and ulcer cound be observed. But all treatment groups were improved inordinately. The experimental result showed the model group was significant difference with normal group (P<0.05). Compared with model group, the degrees of colonic injury of all drug groups were significantly lower (P<0.05).Under light microscope, we observed there were ulceration, neutrophilicgranulocyte infiltration, congestion, erosion, epithelium deletion, lamina propria gland lessen, vessel hyperplasia of the rates colons of model group. The experimental result showed the model group was significant difference with normal group (P<0.05).Compared with model group, besides Chaishao group and Zhigan group, the rest groups were all significantly lower.1.3 Thymus Index and Spleen IndexThe different compatibilities can improve significantly thymus index and spleen index. After modeling the rates’thymus indexes of Sini powders group and Chaishao group were significantly lower than normal group, especially Chaishao group (P<0.05). Compared with model group, besides Chaishaozhi group ,Chaizhigan group, Chaizhi group and Shaozhi group, the rest groups were significant difference. the rates’spleen indexes of modeled rates (P<0.05). Compared with model group, the rates’spleen indexes of control group, Sini powders group and Chaizhigan group increased up significantly.1.4 NF-κBThe mRNA and activities of NF-κB of all drug groups decrease significantly, especially the Sini powders (P<0.05). The colon mucous membrane NF-κB mRNA of the rates of modle group were obviously higher than normal group’s (P<0.05). Compared with model group, all drug groups were significant difference. In those, Zhigan group was significantly higher than model group (P<0.05).The colon mucous membrane NF-κB activity of the rates of modle group were obviously higher than normal group’s (P<0.05). Sini powders group was no significant difference with noemal group. Compared with model group, besides Chaigan group and Zhigan group, the rest drug groups were significantly lower (P<0.05). Compared with Sini powders group, the rest drug groups increased significantly (P<0.05).2 Study of Mechanism on the Sini Powders in Intervening Experimental Ulcerative Colitis 2.1 IL-1βIL-1βmRNA levels of model group was higher than normal group (P<0.05). Compared with model group, all drug groups were significantly lower (P<0.05). IL-1βprotein activity of model group were obviously higher than normal group (P<0.05). After feeding Chaishaozhi and Chaishao, IL-1βprotein activity of UC rats obviously reduce (P<0.05). But after feeding Sini Powders, there rased significantly (P<0.05).2.2 TNF-αTNF-αmRNA and protein levels of model group are higher than normal group (P<0.05). After feeding experimental herbal medicine, TNF-αlevels of UC rats obviously decrease (P<0.05), especially Sini Powders (P<0.05). Compared with Sini powders group, Chaishaozhi group increased significantly (P<0.05), but Chaishao group lowered (P<0.05).2.3 IL-4IL-4 mRNA levels of model group are lower than normal group (P<0.05). After feeding Sini Powders and Chaishao, IL-4 levels of UC rats obviously increased (P<0.05), especially Sini Powders (P<0.05). Compared with normal group, IL-4 protein activities of modeled rates were obviously decrease (P<0.05). After feeding experimental herbal medicine, Sini powders group and Chaishao group were obviously higher than model group (P<0.05). And Chaishao group was lowerer than Sini powders group (P<0.05).2.4 IL-10IL-10 mRNA levels of model group are lower than normal group (P<0.05). After feeding Sini Powders and Chaishao, IL-10 levels of UC rats obviously increased (P<0.05), especially Sini Powders (P<0.05). Compared with normal group, IL-10 protein activities of modeled rates were obviously decrease (P<0.05). After feeding experimental herbal medicine, Sini powders group and Chaishao group were obviously higher than model group (P<0.05). And Chaishao group was lowerer than Sini powders group (P<0.05).Conclusion:The Sini powders can intervene in experimental UC with stimulating the immune organs to excrete the immune cells and inhibit NF-κB activated.The Sini powders can intervene in experimental UC through adjusting cell factors levels. The mechanism of action chould include:①the Sini powders regulates the balance with inflammatory and anti-inflammatory cytokines through suppressing IL-1βmove and TNF-αsecret.and promoting IL-4 and IL-10 to secret;②the Sini powders can reduce the NF-κB levels of the experimental UC rats, and then regulat and control cytokine network.更多还原