针刺对肥胖大鼠下丘脑神经肽Y基因表达的影响

Effect of acupuncture on expression of hypothalamic neuropeptide Y gene in obese rats

目的:观察针刺对肥胖大鼠下丘脑和血浆食欲刺激因子神经肽Y含量及下丘脑神经肽YmRNA表达的影响。方法:实验于2001-03/2002-08在南京中医药大学针灸实验室进行。①选用刚断乳SD雄性大鼠100只。将大鼠100只分2种饲料饲养:20只用普通饲料(实验鼠全价饲料,子卯牌,江苏省卫生厅药事干部培训中心、江浦实验动物饲料厂配制,江苏省药检所科协监制)喂养16周,另80只用自制高脂饲料(用改进后的高脂饲料,其配方质量分数为:普通饲料0.60,猪油0.12,蔗糖0.05,奶粉0.05,花生0.05,鸡蛋0.1,麻油0.01,食盐0.02)喂养16周。②选用以普通饲料喂养大鼠中的12只作为正常组;选用24只肥胖大鼠,按随机抽签法分为2组:模型组和针刺组,各12只。针刺组:针刺治疗前每日将所有大鼠放入自制的大鼠固定器中适应10min,连续3d。3d后,进行针刺治疗。取穴:后三里,内庭,用32号长1cm的美容针刺入约0.5cm,后三里用平补平泻法捻转运针1min,内庭用泻法捻转运针1min,然后接通G6805型电针治疗仪,采用频率为10Hz、强度为1.5V的连续波,以大鼠下肢出现轻微节律的抽搐为度。针刺每次10min,1次/d,两侧穴位交替使用。连续治疗14d。正常对照组和模型组大鼠每日同时放入固定器中适应10min,持续14d。③实验结束后,测定各组大鼠体质量、计算Lee’s指数[3√体质量g/体长(cm)×103];称量各组大鼠附睾、肾、心包周围脂肪及肝、肾质量;采用放射免疫法检测各组大鼠外周血和下丘脑神经肽Y含量;用反转录聚合酶链反应、RNA印迹试验测定下丘脑神经肽YmRNA表达水平。④组间比较用独立样本T检验,多组两两比较用方差分析。结果:根据实验需要,进入结果分析大鼠36只。①血浆神经肽Y:模型组和针刺组明显高于正常组(P<0.01,0.05),针刺组明显低于模型组(P<0.01)。②下丘脑神经肽Y,脑与血神经肽Y之比:模型组明显高于正常组(P<0.01),针刺组明显低于模型组(P<0.05,0.05)。③下丘脑神经肽Y基因表达:模型组显著高于正常组(1.45±0.57,0.40±0.10,P<0.01),针刺组明显低于模型组(0.35±0.12,P<0.01),针刺组与正常组相近(P>0.05)。④RNA印迹试验与反转录聚合酶链反应法测定的结果是一致的,下丘脑神经肽YmRNA表达:模型组明显高于正常组(P<0.01),针刺组明显低于模型组(P<0.05),针刺组与正常组差异不明显(P>0.05)。⑤体质量和Lee’s指数:模型组明显高于正常组(P<0.01),针刺组明显低于模型组(P<0.01)。⑥附睾周围、肾周、心包周围脂肪组织量、肝肾质量:肥胖组和针刺组明显高于正常组(P<0.05～0.01);针刺组明显低于模型组(P<0.05～0.01)。结论:针刺可显著降低肥胖大鼠体质量及外周血和中枢神经肽Y的含量,抑制下丘脑神经肽Y的基因表达,这可能是针灸减肥的重要作用机制之一。

AIM： To observe the effect of acupuncture on content of appetite stimulating factors neuropeptide Y （NPY） and expression of hypothalamic NPY mRNA of obese rats. METHODS： The experiment was completed in the Laboratory of Acupuncture and Moxibustion, Nanjing University of Traditional Chinese Medicine from March 2001 to August 2002. ① Totally 100 SD male rats of newly break milk were selected in this study. All rats were fed with 2 kinds of feeds. Twenty rats were fed with ordinary feed （primary price of feed, zimao brand, provided by Pharmacist Cadre Training Center of Jiangsu Administration of Public Health Bureau, Jiangpu Experimental Animal Feed Factory, Jiangsu Pharmaceutical Laboratory Bureau） for 16 weeks, and other 80 rats were fed with high fat feed （mass volume： ordinary feed 0.60, lard 0.12, saccharo 0.05, milk powder 0.05, peanut 0.05, egg 0.1, sesame oil 0.01, and salt 0.02） for 16 weeks. ② Twelve rats fed with ordinary feed were regarded as normal group, and 24 obesity rats were divided into model group and acupuncture group with 12 in each group according to randomized picking method. Acupuncture group： Rats were put into fixing apparatus for 10 minutes before acupuncture. Acupuncture was performed after 3 days. Points selection： housanli and neiting points, which were acupunctured into 0.5 cm with 32-size cosmetological needle with 1 cm length. Therapeutic method for mild tonification and mild purgation was peifformed at zusanli for 1 minute, and purgation was used at neiting for 1 minute. Then G6805 type electric needle treatment apparatus was turned on, with frequence of 10 HZ and successive power of 1.5 V. Mild rhythm tic in lower limbs was regarded as the degree. Acupuncture was treated for 10 minutes once a day, and two points were acupunctured alternatively for 14 days. Rats in normal control group and model group were put into fixing apparatus for 10 minutes for 1,4 days.③After experment, body. mass of rats in each group was measured to calculate Lee＇s index [3√body mass （g）/body length （cm） ×10^3]. Epididymis, kidney, pericardiac fat and mass of liver and kidney were measured; contents of peripheral and hypothalamic neuropeptide Y （NPY） were measured with radioimnlunoassay; expression of hypothalamic NPY mRNA was determined with reverse transcription polymerase chain reaction （RT-PCR） and Northern Blot （RNA） techniques. ④ T test was used to compare differences in each group, and analysis of variance was used for comparison between two groups. RESULTS： Totally 36 rats entered the final analysis. ① Contents of serum NPY in model group and acupuncture group were higher than those in normal group （P 〈 0.01, 0.05）, but those in acupuncture group was lower than those in model group （P 〈 0.01）. ② Contents of hypothalamiche NPY and ratio between brain and NPY were higher in model group than those in normal group （P 〈 0.01）, but those in acupuncture group was lower than those in model group （P 〈 0.05,0.05）. ③ Expression of hypothalamic NPY gene in model group was higher than that in normal group （1.45±0.57, 0.40±0.10, P 〈 0.01）, but that in acupuncture group was lower than that in model group （0.35±0.12, P 〈 0,01）, and that in acupuncture group was similar to that in normal group （P 〉 0.05）. ④ Results tested by RNA techniques were similar to those tested by RT-PCR. Expression of hypothalamic NPY mRNA was higher in model group than that in normal group （P 〈 0.01）, but that in acupuncture group was lower than that in model group （P 〈 0.05）, and there was no significant difference between acupuncture group and normal group （P 〉 0.05）. ⑤ Body mass and Lee＇s index were higher in model group than those in normal group （P 〈 0.01）, but those in acupuncture group were lower than those in model group （P 〈 0,01）,⑥ Volume of fat tissue and mass of liver and kidney surrounding dpididymis, kidney and heart were higher in obesity group and acupuncture group than those in normal group （P 〈 0.05-0.01）, but those in acupuncture group were lower than those in model group （P 〈 0.05-0.01）. CONCLUSION： Acupuncture can significantly decreased NYP levels of rats peripheral blood and central nervous system, and can also inhibit the over-expression of hypothalamic NPY gene, which might be one of an important acupuncture anti-obesity mechanisms.