补肾方药归经与实验性骨质疏松靶器官信号转导分子Smad2的表达

Meridian tropism of prescription for tonifying kidney and the expression of signal transduction protein in target organs following experimental osteoporosis

目的:观察补肾方药归经与实验性骨质疏松靶器官信号转导分子Smad2的基因表达,从基因水平研究中医归经理论,为靶向给药提供依据。方法:实验于2004-06/2006-11在河北医科大学中西医结合基础实验室完成。实验分组:选择3个月龄健康雌性SD大鼠(未曾交配)90只,体质量(300±20)g。常规喂养1周后,随机数字表法分成7组:正常对照组、骨质疏松模型组、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,其中正常对照组和骨质疏松模型组每组20只,其余每组各10只。实验处理:除正常对照组喂正常饲料,自由饮水外,其余各组均喂食低钙饲料,饮用蒸馏水,每周2次在大鼠大腿内侧肌肉注射地塞米松1mg/kg体质量,5周后建立骨质疏松模型。实验评估:造模后,用抗骨松穴位贴剂分别外贴肾经和膀胱经穴位、非经非穴位、并与口服补肾方药比较治疗骨质疏松,以双能X线骨密度仪检测连续给药16周后大鼠离体股骨骨密度,采用逆转录-聚合酶链反应、蛋白免疫印迹杂交方法分别检测Smad2的mRNA、蛋白表达,观察其治疗效果。结果:纳入90只大鼠,在实验第5周时,正常对照组、骨质疏松模型组分别取10只,用于验证造模成功,70只进入结果分析。①给药16周后,与骨质疏松模型组比较,补肾方药口服组、肾经外贴组、膀胱经外贴组、依普拉封口服组的股骨骨密度明显增加[(0.161±0.016),(0.206±0.028),(0.196±0.023),(0.202±0.015),(0.205±0.023)g/cm2,P均<0.01]。②应用补肾中药防治16周后,与骨质疏松模型组比较,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Smad2mRNA的表达明显上调(0.517±0.031,0.524±0.033,0.596±0.033,0.592±0.021,0.583±0.032,P<0.01);Smad2蛋白表达亦明显增强,差异显著(50.901±2.205,71.802±2.100,72.352±2.306,74.012±2.145,73.802±2.203,P<0.01)。结论:①补肾方药通过口服和外贴穴位两种不同途径均发挥“归经”作用,引起靶器官骨组织上调Smad2的表达而有效改善骨密度。②Smad2mRNA表达及蛋白水平的下调可能是原发性骨质疏松症发生的重要机制之一。

AIM： To explore the correlation between the channel tropism of prescription for tonifying kidney and signal transduction protein （Smad2） of target organ in experimental osteoporosis, and study the meridian tropism in traditional Chinese medicine at gene levels, so as to provide evidence for target administration. METHODS： The experiment was conducted in the Basic Laboratory of Integrated Chinese Medicine and Western Medicine, Hebei Medical University between June 2004 and November 2006. Ninety healthy female SD rats of 3 months old and （300±20） g were selected and randomly divided into 7 groups after routine feeding for 1 week： control group, model group, prescription group, kidney meridian sticking group, urinary bladder meridian sticking group, ipriflavone group, non-meridian or acupoint sticking group with 20 animals in the control group and model group and 10 in other groups. Except the control group was given common diet and drinking freely, all the others were fed with low calcium diet and distilled water, and femoribus internus muscle of rats was injected with 1 mg/kg body mass dexamethasone twice every week to establish the models of osteoporosis after 5 weeks. Then, the Chinese herb plaster was stuck at the acupoints of kidney meridian, urinary bladder meridian, and non-meridian or acupoint, and the curative effects were compared with ：the group with taken of prescription for tortifying kidney. After 16 weeks of administration, the ex vivo femoral bone density was detected by dual energy X-ray absorptiometry, and the expressions of Smad2 mRNA and its proteins were measured by RT-PCR, Western blot to observe the curative effects. RESULTS： Among the 90 rats, 10 from the control group and model group respectively were selected for modeling, and 70 were involved in the result analysis. ①Compared with the model group, the bone mass density of the prescription group, kidney meridian sticking group, urinary bladder meridian sticking group, and ipriflavone group was significantly increased after 16 weeks of administration [（0.161±0.016）, （0.206±0.028）, （0.196±0.023）, （0.202±0.015）, （0.205±0.023） g/cm^2, P 〈 0.01]. ②Compared with the model group, the expressions of Smad2 mRNA of the prescription group, kidney meridian sticking group, urinary bladder meridian sticking group, and ipriflavone group was significantly up-regulated after 16 weeks of administration （0.517±0.031, 0.524±0.033, 0.596±0.033, 0.592±0.021, 0.583±0.032, P 〈 0.01）; the expressions of Smad 2 protein were obviously increased, which had significantly differences （50.901 ±2.205, 71.802±2.100, 72.352±2.306, 74.012±2.145, 73.802±2.203, P〈 0.01）. CONCLUSION： ①The prescription for tonifying kidney displays meridian tropism by oral taken and sticking approaches, which induce the target organs to up-regulate the expression of Smad2 and increase the bone density. ②The down-regulation of the expression of Smad2 mRNA and its protein in bone tissues may be one of the important mechanisms for primary osteoporosis.