健脾清热活血方介导β-catenin, TCF-4表达防治溃疡性结肠炎相关癌变研究

Effect of Jianpi Qingre Huoxue Decoction on the Expression of β-catenin and TCF-4 in Ulcerative Colitis Associated Carcinogenesis

摘要:

目的: 观察溃疡性结肠炎相关癌变(ulcerative colitis associated carcinogenesis,UCAC,简称溃结相关癌变)小鼠组织形态、超微结构以及结肠黏膜β-catenin、 T细胞因子4(T cell factor-4,TCF-4)的表达变化,探讨健脾清热活血方药防治溃结相关癌变的可能机制。方法: 120只清洁级BALB/c小鼠随机分6组,每组20只,设正常组、模型组、美沙拉嗪0.4 g·kg^-1阳性对照组、健脾清热活血方低、中、高剂量治疗组(4.29,8.58,17.16 g·kg^-1);除外正常组,其余各组采用1,2-二甲肼(DMH)/葡聚糖硫酸钠(DSS)复合法制备溃结相关癌变模型,模型组予等剂量生理盐水,对照组予美沙拉嗪,治疗组予不同剂量健脾清热活血方药水提液,连续干预16周。应用光镜、透射电镜观察溃结相关癌变小鼠结肠组织形态学以及超微结构变化;免疫组化、Western-bolt及实时荧光定量PCR(Real-time PCR)检测结肠黏膜β-catenin、TCF-4蛋白及其mRNA表达变化。结果: 模型组小鼠结肠光镜下可见黏膜上皮脱落,腺体萎缩、高级别上皮内瘤变,部分癌变,电镜下可见大、小不等、排列紊乱的上皮细胞,胞核不规则、核仁增大并上移;治疗组光镜下见黏膜充血肿胀,个别轻度不典型增生和淋巴滤泡出现,未见溃疡及癌变,电镜下可见上皮细胞体积明显变小,核染色质边集、核固缩或碎裂,核膜断裂,凋亡小体形成;与模型组比较,治疗组结肠黏膜损伤有不同程度改善(P<0.05)。模型组结肠β-catenin,TCF-4蛋白及其mRNA 呈高表达特点;治疗组结肠β-catenin、TCF-4蛋白及其mRNA 表达均呈下降趋势,与模型组比较,有统计学意义(P<0.05)。结论: 健脾清热活血方药可能通过调控β-catenin,TCF-4表达,促进炎症缓解,发挥防治溃疡性结肠炎相关癌变的效应。

Abstract:

Objective: The mechanism of preventing ulcerative colitis associated carcinogenesis (UCAC) by Jianpi Qingre Huoxue decoction was discussed by observing the changes of β-catenin and T cell factor-4(TCF-4)in colon mucosa. Method: One hundred and twenty BALB/c rats were randomly divided into six groups:normal group, model group, positive control group,low dosage, mild dosage and high dosage groups of Jianpi Qingre Huoxue,20 rats in each group. Except normal group, the other groups were intraperitoneal injected with 0.4% dimethylhydrazine (DMH) 20 mg·kg^-1 by skin and peritoneal once a week. After intraperitoneal injection of DMH, those groups were given orally 3% dextran sulfacte sodium(DSS) for one week. The model group was treated with normal saline by intragastric administration for sixteen weeks. The positive control group was treated with mesalazine decoction for sixteen weeks. The intervention group was treated with different dosage of Jianpi Qingre Huoxuo decoction by intragastric administration for sixteen weeks. The colon histomorphology was assayed by naked eyes and light microscope. The ultra microstructure was checked by electron microscope. The expression of β-catenin and TCF-4 was detected by immunohistochemistry and western-blot and Real-time PCR. Result: The changes of high-grade intraepithelial neoplasia and partical carcinogenesis were found in model group by electron microscope,Meanwhile, a lot of epithelial cell of specimen size and arranged in disorder were found in colon mucosa of blank group, at same time,the irregular cell mucleus and the mucleolued of increasment and upward displacement appeared.However,the colon mucosa damage obviously improved in intervention group compared withmodel group. Colon mucosa is characterized by hyperemia and swelling by light microscope, small amout mild atypical hyperplasia and lymphoid follicle could be found, but the ulceration and carcinogenesis could not be found. By electron microscope,epithelial cell became smaller,at the same time,condensation of the nuclear chromatin,shrinkage and nucleus membrane and apoptofic bodies were found in intervention groups. The colonic mucosa damage was improved obiviously in intervention groups, compared with the black group (P<0.05).The expression of β-catenin and TCF-4 was increased in model group, meanwhile, the expression of β-catenin and TCF-4 was declined in intervention groups, compared with the model group (P<0.05). Conclusion: The Jianpi Qingre Huoxue decoction may exert a preventing effect on ulcerative colitis associated carcinogenesis through promoting inflammation laxation,inducing cell apoptosis, regulating the expression of β-catenin and TCF-4.