香加皮三萜类化合物对实验性大鼠食管癌的阻断作用及机制

Inhibitory Effects of Triterpenes Compound of Cortex Periplocae on N-nitrosomethyl-benzylamine-induced Rat Esophageal Tumorigenesis

目的探讨中药香加皮提取物三萜类化合物(TCCP)对甲基苄基亚硝胺(NMBA)诱导F344大鼠食管癌前病变形成的阻断作用及其机制。方法健康雄性F344大鼠90只,随机分为模型组、TCCP干预组、大豆油对照组和正常对照组。模型组大鼠皮下注射0.5 mg/kg NMBA,TCCP干预组大鼠同时给予0.5 mg/kg NMBA皮下注射及香加皮三萜类化合物20 mg/kg肌肉注射,每周给药3次,连续5周;大豆油对照组大鼠肌注大豆油1 ml/kg,正常对照组大鼠常规饲养。分别在给药后第9、15和25周,麻醉后解剖大鼠,HE染色后光镜下观察食管上皮组织病理学变化;用Western blot法检测GSK-3β和β-cate-nin蛋白表达水平;用RT-PCR法检测c-myc mRNA表达水平。结果 (1)正常对照组及大豆油对照组在整个实验过程中未发现食管异常变化,模型组大鼠随诱癌时间延长食管病变逐渐加重。TCCP干预可缓解食管上皮的病变。(2)正常大鼠食管上皮中有少量β-catenin蛋白表达,模型组大鼠食管上皮β-catenin蛋白表达显著增强(P<0.05);正常大鼠食管上皮GSK-3β的蛋白表达丰富,随着诱癌时间延长表达水平逐渐降低;与模型组相比,在诱癌9、15和25周时TCCP干预组大鼠食管上皮组织中β-catenin蛋白表达水平均显著下降(P<0.05);而GSK3β蛋白表达显著升高(P<0.05)。(3)与正常对照组相比,各时间点模型组大鼠食管上皮c-mycmRNA表达水平均显著升高。与模型组相比,在诱癌9、15周时TCCP干预组大鼠食管上皮组织中c-myc mRNA表达水平均显著下降,而诱癌25周时差异无统计学意义(P>0.05)。结论 TCCP可能通过促进Wnt信号分子GSK-3β的表达,抑制β-catenin蛋白的表达,进而下调下游靶基因c-myc的转录,干扰细胞周期转化,逆转细胞的分化,可能是其抑制食管癌变细胞生长机制之一。

Objective To investigate the inhibitory effects of triterpenes compound of cortex periplocae （TCCP） on N-nitrosomethylhenzylamine （NMBA） -induced esophageal tumorigenesis in F344 rats and its mechanism. Methods Ninety male F344 rats （5-6 weeks of age） were randomly divided into four groups：rats were treated with 0.5 mg/kg NMBA only as model group and rats received NMBA 0.5 mg/ kg s. c. plus TCCP 20 mg/kg i. m. as TCCP treatment groups and those treated with soya oil 1 ml/kg intramuscularily（i, m. ） acted as soya oil control and normal control groups. The administration of drugs was scheduled as below：three times a week for 5 weeks. At 9th,15th and 25th week,5 rats from normal group and soya oil control group and 10 rats from model group and TCCP treatment group were euthanized by pentobarbital sodium and subjected to gross necropsy, respectively. The esophagus of each rat was excised and opened longitudinally. Then esophagus were fixed in 10% phosphate buffer formalin solution,and routinely embedded in paraffin for HE staining to observe the pathological changes of esopha geal tissues, while the expression of c-myc mRNA was detected by RT PCR. Western blot was used to investigate the protein expression of GSK-313 and 13 catenin in the esophageal epithelium. Results There were no abnormal change in normal and soya oil groups among bioassay. It was observed that the expres- sion of β-catenin was slightly present in normal mucosa of esophagi of the rats which were untreated with NMBA,and then was significantly increased by administering NMBA from week 9 to 25 （P〈0.05）. The up-regulated β-catenin expression was decreased significantly by TCCP treatment at each check point （P 〈0. 05）. The expression of GSK-3t3 was rich in normal mucosa of esophagus,but was decreased significantly by administering NMBA,and was lowest at 25th week （P〈20.05）. The decreased protein level of GSK313 was significantly elevated by TCCP treatment at each check point （P〈0.05）. The gene expression of c-myc of esophageal epithelium in NMBA control group was significantly increased at 9th, lSth, 25th week compared with normal control （P〈0.05）. TCCP suppressed the mRNA expression of c-myc at both 9th and 15th week （P〈0.05） ,but not at 25th week. Conclusion TCCP inhibited NMBA-induced rat esophageal carcinogenesis probably via activating GSK-3β expression and suppressing β-catenin and c- myc expression.