摘要
背景:肠壁纤维化是克罗恩病的常见表现。转化生长因子β1是炎症性肠病中控制胶原合成和介导肠纤维化复杂调节机制中的重要生长因子,结缔组织生长因子是转化生长因子β特异性下游效应分子。目的:实验拟观察隔药灸与电针对克罗恩病大鼠结肠转化生长因子β1、结缔组织生长因子、Ⅰ型胶原及纤维连接蛋白表达的影响。设计、时间及地点:随机对照动物实验,于2007-07/12在上海中医药大学实验动物中心和国家中医药管理局针灸免疫三级实验室、临床免疫三级实验室完成。材料:健康雄性SD大鼠60只,48只用于制备大鼠克罗恩病模型;三硝基苯磺酸由Sigma公司提供。方法:60只大鼠随机数字表法分为5组,模型组:不作任何治疗,作与隔药灸组相同的固定。隔药灸组:取天枢(双)、气海穴,每次每穴灸2壮,每日隔药饼灸1次,连续10d。电针组:取天枢(双)、气海穴,采用LD202H型韩氏神经穴位刺激仪电针刺激,频率2/50Hz,20min/次,1次/d,连续10d。药物组:美沙拉嗪灌胃治疗,2次/d,连续10d。正常组:无任何干预措施,作与隔药灸组相同的固定。主要观察指标:苏木精-伊红染色后光镜下观察结肠病理组织学变化;免疫组织化学方法检测结肠转化生长因子β1、结缔组织生长因子、Ⅰ型胶原及纤维连接蛋白的表达;实时荧光定量聚合酶链反应检测结缔组织生长因子mRNA的表达。结果:纳入SD大鼠60只,每组各取1只用于模型鉴定。模型组4只大鼠死亡,7只进入结果分析;其他4组随机取8只进入结果分析。①苏木精-伊红染色正常组可见正常结肠壁组织,黏膜完好;模型组溃疡形成,黏膜下层可见大量纤维组织增生而明显增宽,肉芽组织增生。隔药灸组、电针组和药物组病理组织学均较模型组改善。②与正常组比较,模型组大鼠Ⅰ型胶原、纤维连接蛋白、转化生长因子β1、结缔组织生长因子蛋白表达以及结肠结缔组织生长因子mRNA表达均升高(P<0.01)。经治疗后,隔药灸组、电针组大鼠结肠Ⅰ型胶原、纤维连接蛋白、转化生长因子β1、结缔组织生长因子蛋白表达均降低(P<0.05或P<0.01),但结缔组织生长因子mRNA变化不显著(P均>0.05);隔药灸组对结缔组织生长因子蛋白的下调作用较电针组更显著(P<0.01);药物组Ⅰ型胶原、纤维连接蛋白、转化生长因子β1蛋白表达也显著降低(P<0.05或P<0.01),但结缔组织生长因子蛋白及mRNA变化不显著(P均>0.05)。结论:隔药灸和电针治疗能够下调克罗恩病大鼠结肠Ⅰ型胶原、纤维连接蛋白、转化生长因子β1、结缔组织生长因子蛋白表达,隔药灸对结缔组织生长因子蛋白的调节作用优于电针。
BACKGROUND： Fibrosis of intestine wall is common in Crohn＇ s disease. Transforming growth factor-beta 1 （TGF- β 1） is an important growth factor to control the collagen synthesis and mediate the intestine fibrosis in the complicated regulatory mechanism of inflammatoy bowel disease. Connective tissue growth factor （CTGF） is a specific lower effector molecule of TGF- β 1. OBJECTIVE： To explore the effects of electro-acupuncture and herbs-partitioned moxibustion on expression of TGF- β 1, CTGF, collagen type Ⅰ and fibronectin in Crohn＇s disease rats. DESIGN, TIME AND SETTING： The randomized control animal experiment was performed at the Animal Laboratory Center of Shanghai University of Traditional Chinese Medicine, the Third Class Acupuncture ＆ Moxibustion Immune Laboratory and the Third Class Clinical Immune Laboratory of State Administration of Traditional Chinese Medicine from July to December 2007.
MATERIALS： Of 60 healthy male SD rats, 48 rats were used to prepare for Crohn＇s disease model. Trinitro-benzene-sulfonic acid was purchased from Sigma, USA.
METHODS： A total of 60 rats were randomly divided into 5 groups. When the Crohn＇s disease models were created, rats in the model group were fixed the same as the herbs-partitioned moxibustion group, but no other treatment. Rat models in the herbs-partitioned moxibustion group were treated once daily on Tianshu （ST 25） （bilateral） and Qihai （CV 6） two cones each point, for ten days continuously. Rats in the electro-acupuncture group were acupunctured at Tianshu （ST 25） （bilateral） and Qihai （CV 6） points were stimulated by Hanshi LD202H neuron-points stimulator, with 2/50 Hz frequency for 20 minutes, once daily and lasting for 10 days continuously. Rats in the medication group were given mesalazine by intragastric administration, twice a day for 10 days continuously. Rats in the normal group were intact, but the same fixation as the herbs-partitioned moxibustion group.
MAIN OUTCOME MEASURES： Pathologic histology changes in the colon were observed under a light microscope by Hematoxylin and Eosin Staining. TGF- β 1, CTGF, collagen type Ⅰ and fibronectin expression were detected by immunohistochemical method and CTGF mRNA by real-time fluorescence quantitative polymerase chain reaction in the colon.
RESULTS： One rat from each group was identified for model. Four rats died and 7 were analyzed in the model group. Eight from the rest 4 groups each were analyzed for the results. Hematoxylin and Eosin Staining shows a normal intestine wall and good mucosa in normal group; ulceration formed and a widen mucosa under layer with a large amounts of fibroplasia and hyperplasy of granulation tissue in model group. Pathologic histology was improved in the herbs-partitioned group, electro-acupuncture group and medication group compared with the model group. Compared with the normal group, the expression of collagen type Ⅰ , fibronectin, TGF- β 1, CTGF protein and CTGF mRNA in the colon were significantly higher in the model group （P 〈 0.01）. After treatment, the expression of collagen type Ⅰ , fibronectin, TGF- β 1 and CTGF proteins decreased significantly in the herbs-partitioned moxibustion group and electro-acupuncture group （P 〈 0.05 or P 〈 0.01）, whereas the expression of CTGF mRNA changed insignificantly （P 〉 0.05） The effect of down regulation of CTGF protein was much significant in the herbs-partitioned moxibustion group compared with the electro-acupuncture group （P 〈 0.01）. The protein expression of collagen type Ⅰ, fibronectin and TGF- β 1 decreased significantly in the medication group （P 〈 0.05 or P 〈 0.01）, whereas the expression of CTGF protein and mRNA had no significant changes （P 〉 0.05）.
CONCLUSION： Herbs-partitioned moxibustion and electro-acupuncture can down-regulate the expression of collagen type Ⅰ, fibronectin, TGF- β 1 and CTGF protein in Crohn＇ s disease rats. Herbs-partitioned moxibustion is better than electro-acupuncture in the regulation of CTGF protein.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第20期3853-3858,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30400609)
上海市重点学科建设资助项目(T0302)~~
