补肾方药归经与实验性骨质疏松骨组织转化生长因子βmRNA的表达

Meridian tropism of prescription for tonifying kidney and the expression of transforming growth factor-beta 1 mRNA in target organs following experimental osteoporosis

背景:祖国医学中"肾主骨"的理论符合现代医学骨代谢调节机制,依此理论研制的补肾复方中药在骨质疏松症的治疗中虽取得较好的效果,但其归经作用途径需进行相关分子生物学方面的深入探讨。目的:观察补肾方药归经与实验性骨质疏松骨组织转化生长因子β 1mRNA表达的关系。设计、时间及地点:随机对照动物实验,于2004-01/2007-12在河北医科大学中西医结合基础实验室完成。材料:清洁级3个月龄健康雌性SD大鼠(未曾交配)70只,体质量(300±20)g,喂食低钙饲料,肌肉注射地塞米松建立骨质疏松模型。补肾方药由地黄、淫羊藿、山药、丹参、骨碎补、独活等药物组成。方法:将SD大鼠随机分成7组:正常对照组、病理模型组、补肾方药口服组、肾经外贴组、膀胱经外贴组、依普拉封口服组、非经非穴位外贴组,每组10只。正常对照组不造模,补肾方药口服组造模大鼠按8g/kg补肾方药灌胃给药;依普拉封口服组造模大鼠按10mg/kg灌胃给药;正常对照组及模型组大鼠每天灌胃等体积的生理盐水;膀胱经外贴组造模大鼠取肾俞、飞扬穴,肾经外贴组选太溪、大钟穴,非经非穴位外贴组选非经非穴位,于脱毛区贴上相应的膏剂,左右交替进行,1次/d。主要观察指标:检测连续给药16周后,造模各组和不造模正常对照组大鼠血清卵泡刺激素、黄体生成激素、促甲状腺素、孕酮水平,以双能X线骨密度仪检测腰椎骨密度,以反转录-聚合酶链反应检测骨组织转化生长因子β1mRNA的表达。结果:70只大鼠均进入结果分析。给药16周后,与模型组比较,补肾方药口服组、肾经外贴组、膀胱经外贴组、依普拉封口服组的血清卵泡刺激素、黄体生成激素、促甲状腺素、孕酮、骨密度明显增加(P均<0.01),转化生长因子β1mRNA的表达明显上调(P均<0.01)。结论:①补肾方药通过口服和外贴穴位两种不同途径给药均有效改善骨密度,发挥"归经"作用,该作用可能与靶器官骨组织上调转化生长因子β1mRNA的表达有关。

BACKGROUND： According to the theory of the kidney is in charge of the bones and the mechanism of bone metabolic regulation in modern medicine, the tonifying kidney prescription for osteoporosis has effective action, but its action mechanism of meridian tropism deserves further researches at molecular biology levels. OBJECTIVE： To investigate the correlation between the meridian distribution of prescription for tonifying kidney and the expression of transforming growth factor-beta 1 （TGF- β 1） mRNA of target organ in experimental osteoporosis. DESIGN, TIME AND SETTING： A randomized control animal experiment was carried out in the Basic Laboratory of Integrated Chinese Medicine and Western Medicine, Hebei Medical University （Shijiazhuang, Hebei, China） between January 2004 and December 2007. MATERIALS： Seventy healthy female SD non-copulated rats of 3 months old and clean grade, aged （300 ± 20） g, were selected and fed with low calcium forage. Osteoporosis models were established by intramuscular injection of dexamethasone. The prescription for tonifying kidney comprised rehmannia glutinosa, epimedium grandiflorum, dioscorea opposita, salvia miltiorrhiza, and drynaria pubescens Maxim. METHODS： SD rats were randomly divided into 7 groups： normal control group, pathological model group, prescription group, kidney meridian sticking group, urinary bladder meridian sticking group, ipriflavone administration group, and non-meridian or acupoint sticking group with 10 rats in each group. Except the rats of the normal control group were given common feed and drank freely, all the others were induced the models of osteoporosis. Then, rats in the prescription group were treated with 8 g/kg the prescription for tonifying kidney by gastric perfusion. Rats in the ipriflavone group were administrated with 10 mg/kg ipriflavone by gastric perfusion. Rats in the normal control group and pathological model group were treated with the saline of the same volume every day by gastric perfusion. The Chinese herb plaster was stuck at the Shenshu （BL 23） and Feiyang （BL 58） of urinary bladder meridian in the urinary bladder meridian sticking group, at the Taixi （KI 3） and Dazhong （KI 4） of kidney meridian in the kidney meridian sticking group, and at the non-meridian or acupoint of femoribus internus muscle in the non-meridian or acupoint sticking group, alternated from left to right, once a day. MAIN OUTCOME MEASURES： After 16 weeks of continual administration, serum follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone and progesterone levels were determined. The lumbar vertebra bone mineral density was detected by dual energy X-ray absorptiometry, and the expressions of TGF- β 1 mRNA by reverse transcription-polymerase chain reaction. RESULTS： All 70 rats were involved in the result analysis. Compared to the pathological model group, serum follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, progesterone levels and the bone mineral density of the prescription group, kidney meridian sticking group, urinary bladder meridian sticking group, and ipriflavone group were significantly increased after 16 weeks of the administration （P 〈 0.01）. Moreover, the expressions of TGF- β 1 mRNA were significantly up-regulated in those groups （P 〈 0.01）. CONCLUSION： The prescription for tonifying kidney displays meridian tropism by oral administration and sticking approaches. The mechanism may be related to the up-regulated expression of TGF- β 1 mRNA in the target organs.