补阳还五汤防治周围神经损伤导致运动障碍的机理研究

发布时间：2018-07-27 11:39

【摘要】：目的通过观察补阳还五汤对大鼠腓总神经损伤后腓总神经及胫前肌形态与功能、神经生长因子(nerve growth factor, NGF)及mRNA表达的影响,探讨补阳还五汤治疗周围神经损伤导致运动障碍的部分机理,为临床应用和进一步研究补阳还五汤治疗周围神经损伤导致运动障碍提供客观的理论依据。方法1.补阳还五汤治疗周围神经损伤导致运动障碍的理论探讨收集、整理中医学有关痿证及西医学对周围神经损伤的文献研究,探讨防治周围神经损伤导致运动障碍的研究概况并总结近年来有关补阳还五汤组方、配伍、单味药及复方的现代药理研究等文献资料。2.补阳还五汤治疗腓总神经损伤导致运动障碍的实验研究SPF级雄性大鼠48只,随机分为假手术组、弥可保组(剂量为625mg/g)、模型组、补阳还五汤(Buyang huanwu decoction, BYHWD)高、中、低剂量组(剂量分别为25.92mg/g、12.96mg/g、6.48mg/g生药)。建立大鼠腓总神经夹伤模型,每日灌胃给药。21d后测量足趾宽度,记录展趾功能；进行肌电图检测,测量肌电位衰减率；检测胫前肌的湿重比；胫前肌HE染色,测定胫前肌肌纤维横截面积；运用透射电镜组织学技术观察得出髓鞘形态与厚度；运用免疫组化法检测大鼠NGF的分泌情况；PCR检测腓总神经中神经生长因子mRNA的表达,以此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。结果1.周围神经损伤导致的运动障碍相当于祖国医学的“痿证”范畴。发病的主要病理机制之一为元气大伤、血脉不通。病性为本虚标实。元气大伤为本,血脉瘀阻为标。主要治法为补气活血。补阳还五汤作为补气活血法的代表方,治疗本病,切合病机。2.展趾功能：术后第2d患侧出现足趾蜷缩和跛行。7d后BYHWD中、高剂量组和弥可保组足趾逐渐伸展,而模型组恢复则很慢。14d后情况继续好转。21d后各组足趾明显伸展,步态基本恢复正常。但是除假手术组外,其余各组患侧均出现不同程度的运动障碍。展趾功能结果显示,与假手术组比,模型组差异显著(P0.01)。BYHWD高剂量组、弥可保组与模型组相比差异显著(P0.01或P0.05)。BYHWD各组与弥可保组比较无统计学差异(P0.05)。3.重复神经电刺激检测衰减反应：假手术组除外,其余五组组大鼠RNS均出现不同程度的衰减,差异显著(P0.01)。BYHWD高剂量、弥可保组与模型组比较差异显著(P0.01)。BYHWD高剂量与弥可保组相比不具备统计学差异。4.胫前肌湿重比：与假手术组相比,各组大鼠患侧胫前肌的湿重比都呈现不同程度的降低(P0.01或P0.05)。与模型组相比,BYHWD高剂量、弥可保组均有显著增高(P0.05),而弥可保组与BYHWD组间无统计学差异(P0.05)。5.胫前肌横截面积：假手术组胫前肌横截面形态较规则,结缔组织很少。与假手术组相比,模型组肌纤维结构紊乱、细胞间隙增宽、结缔组织增生、肌纤维萎缩变性明显。BYHWD各组和弥可保组细胞间隙增宽,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肌纤维横截面积统计分析提示：其余五组与假手术组相比差异显著(P0.05)。BYHWD各组与弥可保组相比无明显差异(P0.05)。6.腓总神经电镜形态学观察：假手术组轴突内在轴突内可见到横断的微管、神经丝及轴系膜。部分髓鞘神经横切面形态规则,髓鞘板层结构排列整齐、均匀,无离散现象。BYHWD和弥可保组髓鞘厚度改变不明显,只有少量变性的神经纤维髓鞘板层离散。BYHWD中、低剂量组髓鞘厚度进一步变薄、板层离散情况更一步加重。模型组髓鞘厚度进一步减薄,部分髓鞘板层离散非常严重,形成大量髓鞘球。各组大鼠脱髓鞘厚度统计结果可见：其它五组与假手术组相比差异显著(P0.05),与模型组相比,BYHWD各组与弥可保组差异显著(P0.05)。与弥可保组相比,BYHWD高剂量组差异显著(P0.05)。7.NGF蛋白表达：假手术组、BYHWD各组和弥可保组可见较多NGF免疫反应阳性颗粒,呈浅棕色。模型组大鼠NGF阳性颗粒数目减少,着色浅淡。假手术组与模型组比较具有统计学意义(P0.05)。BYHWD各组弥可保组与模型组比差异显著(P0.01或P0.05)。8.NGF-mRNA的表达：6组的NGF-mRNA基因相对表达量比较,差异有统计学意义(P0.05),假手术组、弥可保组与BYHWD高、中、低剂量组NGF-mRNA基因相对表达量明显增高,分别是模型组的4.79倍、2.16倍、3.41倍、1.83倍、1.31倍；其中BYHWD中剂量及高剂量组、弥可保组NGF-mRNA相对表达量明显强于模型组(P0.05),BYHWD高剂量组NGF-mRNA相对表达量明显强于弥可保组(P0.05)。结论1.补阳还五汤能够有效促进大鼠夹伤神经及其靶器官骨骼肌形态与功能恢复,对大鼠周围神经损伤导致的运动障碍有较好的防治作用。2.补阳还五汤对周围神经再生的保护作用与它对NGF蛋白和腓总神经中NGF-mRNA的表达有关。其机理可能与该方大补元气,活血化瘀的功效相联。该方能够有效促进轴突和髓鞘生长来促进周围神经再生,其机理有待进一步的研究。
[Abstract]:Objective To observe the effect of Buyang Huanhui Five Decoction on the morphology and function of the peroneal nerve and the anterior tibial muscle, nerve growth factor (NGF) and the expression of mRNA after the injury of the peroneal nerve in rats, and to explore the partial mechanism of the treatment of the peripheral nerve injury caused by the tonifying Yang and the five soup, and further study the clinical application and further study of the tonifying yang five soup. The treatment of peripheral nerve injury can provide an objective theoretical basis. Method 1. the theoretical exploration of the treatment of peripheral nerve injury caused by peripheral nerve injury is collected, the literature of Chinese medicine related to flaccid syndrome and Western Medicine on peripheral nerve injury is studied, and the research situation of the prevention and treatment of peripheral nerve injury resulting in dyskinesia is discussed. In recent years, we summarize the literature of Buyang Huanhui Five Decoction, compatibility, single flavor medicine and compound medicine and other literature data,.2. Buyang Huanhui Five Decoction in the treatment of 48 SPF male rats in the treatment of general peroneal nerve injury, which are randomly divided into sham operation group, mica group (625mg/g), model group, Yang Yang five Decoction (Buyang Huanwu) Decoction, BYHWD) high, medium, low dose group (dose of 25.92mg/g, 12.96mg/g, 6.48mg/g raw medicine respectively). Establish the rat peroneal nerve clamp injury model, measure the width of the toe, record the function of the toe of the toes after the daily perfusion of the stomach for.21d, detect the electromyography, measure the decline rate of the muscle potential, detect the wet weight ratio of the tibial muscle, and determine the tibia by HE staining of the tibial muscle and determine the tibia. The transversal area of the anterior muscle fibers was observed and the morphology and thickness of the myelin sheath were observed by transmission electron microscopy, and the secretion of NGF in rats was detected by immunohistochemistry; PCR was used to detect the expression of nerve growth factor mRNA in the general peroneal nerve, so as to observe the regenerative and functional recovery of the total peroneal nerve of the rats after the five decoction. Results 1. The dyskinesia caused by peripheral nerve injury is equivalent to the category of "flaccid syndrome" in the Chinese medicine. One of the main pathological mechanisms of the disease is massive trauma, unconnected blood. The disease is a virtual standard. The main treatment is tonifying qi and activating blood. The main treatment is to make up the Qi and activating blood. The five decoction is the representative of the method of Invigorating Qi and activating blood. The.2. spread function of the disease machine: the toe curling and the claudication.7d after the operation on the 2D side were found in BYHWD, the high dose group and the Michal group's toes gradually extended, while the model group resumed after the slow.14d and continued to improve after.21d, and the gait was basically restored to normal. But the other groups were different from the sham operation group. The result of the degree of motor dysfunction showed that the difference between the model group and the model group was significant (P0.01).BYHWD high dose group compared with the sham operation group, and the difference was significant (P0.01 or P0.05) compared with the model group (P0.01 or P0.05), there was no statistical difference between the group.BYHWD and the mass group (P0.05).3. repetitive nerve electrical stimulation detection attenuation response, except the sham operation group. The RNS of the five groups had different degrees of attenuation, the difference was significant (P0.01).BYHWD high dose, and the difference was significant (P0.01) with the model group (P0.01), the high dose of.BYHWD was not statistically different from that of the mass group, and the wet weight ratio of the anterior tibial muscle was not statistically different: compared with the sham operation group, the wet weight ratio of the anterior tibial muscle of the rats in each group were different degrees. Lower (P0.01 or P0.05). Compared with the model group, the high dose of BYHWD was significantly higher (P0.05), but there was no statistical difference between the group and the BYHWD group (P0.05) the transverse section of the anterior tibial muscle of the.5.: the cross section of the tibial muscle in the sham operation group was more regular and the connective tissue was very few. The widening of intercellular space, connective tissue hyperplasia, muscle fiber atrophy and degeneration obviously widened in.BYHWD and myocutaneous fibers, and slight atrophy of muscle fibers, but the shape is more regular, the structure is clear, the staining is more uniform. The statistical analysis of the unidentified muscle fiber cross section of the connective tissue shows that the other five groups are different from the sham operation group. (P0.05) there was no significant difference in (P0.05).BYHWD in the peroneal peroneal nerve of all groups (P0.05). The morphological observation of the common peroneal nerve in.6.: the transverse microtubules, the nerve fibers and the axonal membrane in the axon of the sham operation group were seen. The shape of the transverse section of the myelin sheath was regular, and the myelin lamellar structure was arranged neatly and evenly, without the discrete phenomenon of.BYHWD and the mass group. Medullary sheath thickness changes were not obvious. Only a small amount of denatured nerve fiber myelin lamina dispersed.BYHWD, the myelin thickness was further thinner in the low dose group, and the lamellar dispersion was further aggravated. The medullary sheath thickness of the model group was further thinned and the medullary sheaths were dispersed very seriously and formed a large number of myelin spheres. The demyelination thickness statistics of rats in each group were statistical results. The difference between the other five groups was significantly different from the sham group (P0.05). Compared with the model group, the difference between the BYHWD and the mass groups was significant (P0.05). Compared with the mass group, the BYHWD high dose group was significantly different (P0.05).7.NGF protein expression: the sham operation group, the BYHWD groups and the mass groups showed more NGF immunoreactive particles, which were light brown. The number of NGF positive particles in the model group was reduced and the coloring was light. The comparison of the sham operation group and the model group was statistically significant (P0.05) the difference was significant (P0.01 or P0.05).8.NGF-mRNA in each group of.BYHWD groups (P0.01 or P0.05) in each group: the relative expression of NGF-mRNA gene in the 6 groups was statistically significant (P0.05), sham operation group, and mass The relative expression of NGF-mRNA gene in the group and BYHWD high, middle and low dose group was significantly higher, which was 4.79 times, 2.16 times, 3.41 times, 1.83 times, 1.31 times, respectively. The relative expression of NGF-mRNA in BYHWD and high dose groups was significantly stronger than that in the model group (P0.05), and the relative expression of NGF-mRNA in the BYHWD high dose group was obviously stronger than that of the mass group. Conclusion (P0.05). Conclusion 1. Buyang Huanhui Five Decoction can effectively promote the recovery of skeletal muscle morphology and function of the injured nerve and its target organs, and has a good preventive effect on the dyskinesia caused by peripheral nerve injury in rats. The protective effect of.2. Buyang huanfive Decoction on peripheral nerve regeneration and its expression of NGF-mRNA in NGF protein and peroneal nerve. The mechanism may be associated with the effect of this prescription, which can promote the growth of the axon and myelin sheath to promote the regeneration of the peripheral nerve. The mechanism needs further study.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R277.7

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