【作者】 周岚；

【导师】 梅晓云；

【作者基本信息】 南京中医药大学 ， 中医基础理论， 2011， 博士

【摘要】 目的通过观察补阳还五汤对损伤神经及其靶肌形态与功能的影响,并应用基因芯片技术寻找补阳还五汤防治失神经肌萎缩的分子靶点,探讨补气活血法防治失神经肌萎缩的生物学作用及其机理,为中医药防治失神经肌萎缩提供新的科学资料。方法1补气活血法防治失神经肌萎缩的理论探讨系统地整理古代有关痿证文献及现代中医学家对痿证的研究；探讨失神经肌萎缩的现代医学研究概况及补气活血法的防治机理；并收集近年来有关补阳还五汤的文献资料：包括组方、配伍、及其现代药理研究。2补阳还五汤延缓大鼠胫前肌失神经肌萎缩的实验研究SPF级雄性SD大鼠60只,随机分为6组：BYHWD高、中、低剂量组(剂量分别为25.92、12.96、6.48g生药/kg)、弥可保组(剂量为625μg/kg)、模型组、假手术组。采用大鼠腓总神经5mm夹伤模型,每日灌胃给药。术后18天行足迹实验测定展趾功能；应用电生理学技术测定神经传导速度与复合肌动作电位振幅；应用光电镜技术从形态学上测定再生有髓神经纤维数、髓鞘厚度、胫前肌截面积与湿重比；观察补阳还五汤给药后大鼠腓总神经再生与功能恢复情况及其对胫前肌失神经肌萎缩的防治作用。3补阳还五汤延缓大鼠胫前肌失神经肌萎缩的的分子机制研究SPF级雄性SD大鼠随机分为补阳还五汤高剂量组与模型组,每组10只动物。模型建立与术后给药同上。术后第7、18天取材进行芯片研究。并用实时荧光定量PCR验证部分差异表达基因,以期找出补阳还五汤对失神经肌萎缩起保护作用的可能分子靶点。结果1.失神经肌萎缩属于祖国医学的“痿证”范畴,病性为本虚标实。元气大伤为本,血脉瘀阻为标；元气大伤、血脉瘀阻是失神经肌萎缩发病的主要病理机制；补气活血是其主要治法。补阳还五汤是补气活血法的代表方剂,用以治疗本病,切合病机。2.展趾功能：各组大鼠TSF值均随术后时间的推移逐渐增加,与第3天的展趾功能比较,模型组、中、低剂量组从第15天开始出现显著性差异(P<0.05或P<0.01),而高剂量组与弥可保组第9天开始就出现显著性差异(P<0.05或P<0.01)。另外,术后12天开始BYHWD高剂量组、弥可保组TSF均显著高于模型组(P<0.05或P<0.01)；术后15天及18天,BYHWD高、中剂量组、弥可保组TSF与模型组比较有显著性差异(P<0.05或P<0.01)。3.大鼠术侧腓总神经近、远侧端CMAP波幅：与假手术组相比,其余各组波幅均明显降低(P<0.01)；而与模型组比较,BYHWD高、中剂量组和弥可保组波幅均显著增高(P<0.01)。4.腓总神经传导速度：与假手术组相比,其余各组神经传导速度均明显降低(P<0.01)；而与模型组相比,BYHWD中、高剂量组和弥可保组均显著增高(P<0.05或P<0.01)。5.胫前肌形态学观察：假手术组胫前肌横切面可见肌纤维截面积较大,形态较规则,且很少见到结缔组织；模型组与假手术组相比截面积明显减少,且表现为大小不一,形态不规则,肌纤维结构紊乱,细胞间隙明显增宽,结缔组织明显增生,肌纤维明显萎缩变性；BYHWD各组和弥可保组与假手术组相比截面积有所减小,细胞间隙增宽,肌纤维轻度萎缩变性,但形态较规则,结构尚清晰、染色较均一,结缔组织增生不明显。6.胫前肌湿重比：与假手术组相比,各组动物术侧胫前肌湿重比皆不同程度的降低(P<0.01)；而与模型组相比,BYHWD中、高剂量组和弥可保组均显著增高(P<0.05或P<0.01)。7.胫前肌横截面积：与假手术组相比,各组动物术侧胫前肌横截面积皆不同程度的减小(P<0.01)；与模型组相比,BYHWD各组和弥可保组明显增加(P<0.01)；弥可保组与BYHWD高剂量组相比差异有显著性(P<0.05)。8.运动终板：术后18天,大鼠胫前肌运动终板乙酰胆碱酯酶染色后观察,可见假手术组运动终板为棕黑色,纵切面上其形状为椭圆形或圆形,着色均匀。弥可保组与BYHWD高剂量组仅表现为运动终板边缘不如假手术组光滑；BYHWD中、低剂量组与模型组的运动终板表现为数量逐渐减少,形态逐渐不规则,边缘逐渐不光滑；终板内部着色也逐渐变浅。9.神经轴突NF免疫荧光组织化学染色观察：假手术组神经轴突排列密集,分布整齐；模型组轴突密度明显低于假手术组,且排列紊乱；而BYHWD组和弥可保组轴突密度较模型组高,分布也较整齐。其中,补阳还五汤高剂量组和弥可保组的再生神经轴突较多,而中、低剂量组的再生神经较少。10.电镜形态学观察：假手术组有髓神经纤维成熟。髓鞘厚。排列致密；BYHWD各组和弥可保组可见较成熟的、排列较致密的有髓纤维,髓鞘较厚；而模型组有髓神经纤维排列稀疏紊乱,髓鞘较薄,成熟度欠佳,尚可见较为明显的退变现象。11.有髓神经纤维计数、髓鞘厚度：与假手术组相比,模型组、BYHWD各组、弥可保组大鼠术侧腓总神经夹伤远端再生有髓神经纤维计数、髓鞘厚度明显降低(P<0.01)；与模型组比较,BYHWD高、中剂量组和弥可保组显著增高(P<0.01)。12.芯片结果：BYHWD作用胫前肌第7天,芯片分析结果表明BYHWD高剂量组与模型组相比,基因表达谱有显著差异,其中4条基因表达上调,3条基因下调。而BYHWD作用胫前肌18天后,差异表达基因中16条基因上调,13条基因下调。差异表达基因中变化最明显的是Angpt14,大约上调15倍,它与血管新生密切相关；其次就是Pik3c2g,它可活化Akt后参与多种信号转导而发挥广泛的生物学效应。这些差异表达基因具有的生物学功能可分为直接和间接延缓肌萎缩两大类。其中促进肌肉蛋白合成,抑制其分解,能直接延缓失神经肌萎缩的功能有：促进蛋白质合成,细胞周期调控,抗凋亡,抑制泛素化途径,促进肌细胞增殖与分化。而通过其他机制间接延缓失神经肌萎缩的功能有：血管重塑,血管新生,改善微循环,神经保护,胶原合成,能量合成增加,抗氧化损伤。以上功能按出现频率由多至少依次排列为：能量合成增加(8次)、血管新生(5次)、抗凋亡(5次)、抗氧化(4次)、神经保护(4次)；其他功能相对较少。另外,还有一些差异表达基因功能不明,或者目前还不能确定是否具有保护失神经肌萎缩的作用,有待进一步的深入研究。13. qRT-PCR检测Angpt14与Pik3c2g基因在补阳还五汤高剂量组与模型组的差异表达,结果与芯片结果一致,说明芯片结果可靠。结论1.补阳还五汤对大鼠胫前肌失神经肌萎缩有较好的直接防治作用；并有利于轴突生长和髓鞘形成,通过促进大鼠夹伤神经再生与功能恢复,间接延缓失神经肌萎缩的发生。2.补阳还五汤对失神经肌萎缩的保护作用与它对多种相关基因的调控有关。其机理可能通过大补元气,活血化瘀促进能量合成与血管新生、抑制细胞凋亡、抗氧化、保护神经等方面来防治失神经肌萎缩的发生,其机制有待进一步的研究。3.补阳还五汤还可能通过大补元气激活PI3K/Akt信号通路促进蛋白质合成、减少蛋白降解、促进血管新生、肌卫星细胞的增殖与分化、细胞周期调控等多种生理功能来延缓失神经肌萎缩的发生。4.补气活血法是是防治失神经肌萎缩的有效方法,它可能是通过调控基因表达来实现其保护作用。更多还原

【Abstract】 ObjectiveIn order to research the preventive and therapeutic effect and its mechanisms of buyang huanwu decoction on denervated tibial muscle atrophy of rats, this project aims to observe the morphology and functions of the injuried nerve and its target muscle by using the crushed common peroneal nerve as a model, and to explore the molecular mechanisms of Buyang Huanwu Decoction delaying denervation muscle atrophy by the GeneChip analysis. We hope that this study will contribute to the better prevention and cure denervated muscle atrophy by traditional Chinese medicine.Method1. Collect literature about flaccidity zheng systematically, investigate the etiology、pathogenesis、therapeutic principles and method from the qi-blood and zang-fu zheng differentiation. Assemble documents of the buyang huanwu decoction on the compose、compatibility、pharmacologic actions and clinical application on the peripheral nerve lesion and denervated muscle atrophy.2. The Experimental Study on the preventive and therapeutic effect of buyang huanwu decoction on denervated tibial muscle atrophy of ratsAfter 60 male Sprague-Dawley rats were subjected to Common Peroneal nerve crush model of 5 mm injury, they were randomly divided into 6 groups for daily intragastric administration of drugs:BYHWD high-dose(25.92 g/kg), medium-dose (12.96 g/kg), low-dose groups(6.48 g /kg), mecobalamin group (625μg/kg, positive control), model group, sham operation group. Normal saline was for model group and the sham operation group. The drug administration lasted for 18 days. Footprint test was performed at the 18th days after surgery to evaluate the toe spread function (TSF); Electrophysiology was also performed to determine the amplitude of compound muscle action potentials (CMAPs) and nerve conductive velocity(NCV). At the same time, the number of regenerated myelinated nerve fibers, thickness of myelin sheath and cross sectional area and wet weight ratio of tibial muscle were also evaluated by histomorphology. All these were to observe the regeneration and functional rehabilitation of crushed Common Peroneal nerve and the preventive and therapeutic effects of buyang huanwu decoction on denervated tibial muscle atrophy of rats.3. The study on the molecule mechanism of buyang huanwu decoction delaying denervated tibial muscle atrophy of rats After 20 male Sprague-Dawley rats were subjected to Common Peroneal nerve crush model of 5 mm injury, they were randomly divided into 2 groups for daily intragastric administration of drugs:BYHWD high-dose(25.92 g/kg) and model group. The method of model construction and administering drug were the same as that of experiment 1. At the7th、18th days after surgery, the GeneChip analysis were performed. At the same time, qRT-PCR was also used to validate some differential expression gene. All this is to explore the possible molecule mechanism of buyang huanwu decoction delaying denervated tibial muscle atrophy of rats.Results1. The denervated muscle atrophy belong to "flaccidity zheng". Ii is asthenia in origin and asthenia in superficiality. The main pathomechanism is qi and blood deficiency and stagnation of vessel of blood. The main therapeutic principle is reinforcing qi and activating blood circulation. Buyang huanwu decoction is the correct prescriptions.2. TSF:all groups increased gradually after operation. At the 12th day after operation, the TSF in BYHWD high-dose group (P<0.01) and in mecobalamin group(P<0.05) increased significantly compared with that of model group. At the 15th and 18th day after the operation, the TSF in BYHWD high-dose group、medium-dose group and mecobalamin group also increased significantly compared with that of model group. (P<0.05 or P<0.01).3. The proximum and distally amplitude of CMAP:Compared with the sham operation group, model group、BYHWD groups and mecobalamin group all descended obviously(P<0.01): compared with the model group, BYHWD high-dose、medium-dose groups and mecobalamin group heightened significantly (P<0.01)4. NCV:Compared with the sham operation group, the NCV of model group、BYHWD groups and mecobalamin group all descended obviously(P<0.01); the NCV in BYHWD medium-dose group (P<0.05)、high-dose group (P<0.01) and mecobalamin group (P<0.01) were also increased significantly compared with that of model group.5. The section area of tibial muscle in the sham operation group was comparatively large, morphous was rule, connective tissue was little; compared with the sham operation group, the section area in the model group reduced, morphous was not rule, fiber structure was chaos, intercellular space widened significantly, connective tissue hyperplasia was obviously, muscle fiber atropy was severity; compared with the sham operation group, the section area in the BYHWD group and mecobalamin group muscle fiber atropy was slightly; connective tissue hyperplasia was not obviously.6. The wet weight ratio:Compared with the sham operation group, the wet weight ratio of model group、BYHWD groups and mecobalamin group all descended obviously (P<0.01) compared with the model group, the wet weight ratio in BYHWD medium-dose group(P<0.05)、 high-dose group (P<0.01) and mecobalamin group (P<0.01) were also increased significantly.7. The section area of tibial muscle:Compared with the sham operation group, the section area of model group、BYHWD groups and mecobalamin group all descended obviously(P<0.01); the section area in BYHWD group and mecobalamin group all increased significantly compared with that of model group (P<0.01); there was also striking variation between model group and mecobalamin group (P<0.05)8. At the 18th day after operation, by cholinesterase dyeing in the sham operation group were Sepia、oval or round; the BYHWD high-dose group and mecobalamin group has little chage; the quantity of motor end plates in the BYHWD medium-dose group、low-dose group and the model group had lessen, shape was not rule gradually, the edge were rough; coloring were slight gradually.9. NF fluorescence histochemistry dyeing showed that:the neuritis in the sham operation group were dense and in good order; the neurites in the model group were raritas and chaos; the BYHWD high-dose group and the mecobalamin group had more neurite, and the BYHWD medium-dose group and low-dose group had lessen neurite.10. Electron microscope showed that nerve fibers in the sham operation group were mature、thick、dense; the nerve fibers in the BYHWD group and the mecobalamin group were comparatively mature、thick、dense; the nerve fibers in the model group were sparseness、chaos、thin and immature.11. The number of regenerated myelinated nerve fibers and thickness of myelin sheath Compared with the sham operation group, the model group、BYHWD groups and mecobalamin group all descended obviously (P<.01); BYHWD high-dose、medium-dose group and mecobalamin group were all increased significantly compared with the model group (P<0.01).12. Genechip:At the 7th day after operation, four genes had up-regulation expression of mRNA, three genes had down-regulation expression of mRNA; at the 18th day after operation, sixteen genes had up-regulation expression of mRNA, thirteen genes had down-regulation expression of mRNA. In the differential expression genes, Angptl4 had the most obviously change which were correlated with the blood neogenesis; the next was Pik3c2g which can activate AKt and had general biological effects. The biological functions of these differential expression gene can be divided into two type:the one can postpone denervated muscle atropy directly which promote the synthesis of protein and inhibit the decompose of protein, the other can delay denervated muscle atropy indirectly. The first functions are as foiiows:synthesis of protein、cell cycle、ani-apoptosis、polyubiquitinated pathway、cell proliferation and differentiation. The second functions are as foiiows:blood remodeling、blood neogenesis、improving microcirculati on、neuroprotection、collagen synthesis、energy synthesis、anti-oxidant damage. The sort by the frequency of occurrence were as foiiows:energy synthesis(8)、blood neogenesis(5、ani-apoptosis(5)、anti-oxidant damage(4)、neuroprotection(4). Otherwise, some gene has obscure function and need lucubrate. The mRNA expression of Angptl4 and Pik3c2g were validated by the qRT-PCR, the results were the same as that of genechips. So, the gengchip analysis were reliable.13. The results of qRT-PCR were the same as the Genechip, so, the conclusions of the Genechip were right.Conclusion1. BYHWD has a significant effect of prevention and therapy on denervated tibial muscle atrophy of rats. At the same time, it can accelerate the axon growth and myelinogenesis、promote nerve regeneration and functional rehabilitation which indirectly delay the denervated tibiai muscle atrophy of rats.2. The preventive and therapeutic effect of buyang huanwu decoction on denervated tibial muscle atrophy of rats are relevant with the regulation of many genes. The mechanism may be as follows:promoting energy synthesis and blood neogenesis、ani-apoptosis、anti-oxidant damage and neuroprotection, we will need the further research.3. The PI3K/Akt signal transduction pathway can accelerate synthesis of protein and inhibit decompose of protein、promote blood neogenesis and the hyperplasia and differentiation of muscle satellite cells. So, the PI3K/Akt signal transduction pathway probably play an important role in delaying the denervated muscle atropy which is one of the mechanisms.更多还原