摘要
糖尿病周围神经病变(Diabetic Peripheral Neuropathy,DPN)为2型糖尿病(Type2Diabetes Mellitus,T2DM)患者最常见并发症之一,其病变的发展标志着T2DM本身的发展及多系统损害的加剧,对本病病理变化的探索及中医药治疗本病的机理研究是近年来本领域研究的热点之一。我们通过理论探索和临床实践,提出DPN的病机要点在于中焦热盛、流窜经络、阴液受损、经络失养,其病位在于经络；由于其基本病机与消渴基本一致,因此以“清热养阴”为基本治疗大法；以本法为组方原则的王灿晖降糖基本方为实验方剂,通过理论研究和动物实验,观察本法治疗DPN的疗效,并探讨其作用机理。
理论研究
①根据大量的文献记载,归纳古代医家对本病的描述,总结古代医家对本病的病因、病机、辨治规律的认识。②结合现代研究对本病的认识,阐述对本病病因、病理、治疗、预后的研究成果。③从DPN的基本病机出发,对本病的基本治法——清热养阴法进行阐述。④结合临床病例,阐述清热养阴法代表方剂——灿晖降糖基本方组方依据、用药特色、随证加减、及其在治疗DPN中的运用。
实验研究
目的：观察清热养阴法代表方剂——王灿晖降糖基本方对T2DM所致DPN大鼠模型的治疗作用及其对坐骨神经内相关性基因表达的影响,探讨DPN的发病机制,揭示清热养阴法治疗DPN的作用机理,初步探索药效作用靶点,为临床深入研究和推广运用提供科学依据。
方法：选用雄性SD大鼠,通过一次性小剂量注射链脲佐菌素(STZ)配合高糖高脂饮食复制DPN大鼠模型,运用生化、电生理、光镜、免疫组化、华联ROA1.1大鼠全基因组表达谱芯片、RT-PCR等检测手段和方法,观察王灿晖降糖基本方对DPN模型大鼠一般状况、空腹血浆C肽水平、热刺激反应闽值、坐骨神经传导速度、坐骨神经形态、NGF、Trk A表达的影响,并且通过聚类分析从坐骨神经组织mRNA表达谱中筛选出和DPN发病密切相关的PLA2G2A mRNA表达调控的影响。
结果：(1)DPN大鼠模型复制成功。(2)用药1月后,王氏降糖方能显著降低DPN大鼠血糖水平,但和模型组相比并未减少空腹血浆C肽水平。(3)和模型组相比,王氏降糖方能显著提高大鼠热刺激反应阈值(p<0.001)。(4)和模型组相比,王氏降糖方能显著提高大鼠SNCV(p<0.001),但各组MNCV无显著差异。(5)光镜检查HE染色显示：王氏降糖方减轻DPN大鼠坐骨神经干的病理损伤,改善神经功能,减轻DPN症状。(6)王氏降糖方能提高DPN大鼠坐骨神经干NGF表达,但对Trk A的表达无显著影响。(7)对DPN大鼠坐骨神经组织基因表达谱筛查结果显示：与模型组相比,王氏降糖方能治疗组的坐骨神经组织基因表达上调204条,下调104条；其中多条基因与花生四烯酸代谢、过氧化物酶体增殖物激活受体信号传导、抗氧化作用有关。(8)通过RT-PCR检测显示：DPN大鼠坐骨神经组织PLA2G2A mRNA基因在模型组表达上调,经王氏降糖方治疗后表达下调。
结论：DPN模型大鼠坐骨神经受损的分子调控机制和多条基因的表达调控有关,PLA2G2A mRNA的过度表达是其痛阈值降低、坐骨神经传导速度降低、坐骨神经形态改变的关键环节。以清热养阴法为主要治法能改善血糖水平、提高痛阈值、提高神经传导速度,其机制可能是：①促进胰岛素分泌,同时增加胰岛素敏感度；②增加NGF逆向运输,改善轴索内物质运输状况；③调控多条基因的表达,调低PLA2G2A mRNA的过度表达,以达到直接保护周围神经组织的目的。
Diabetic Peripheral Neuropathy(DPN) is one of the most common complications of Type2Diabetes Mellitus(T2DM) to the patients.During the deteriorate of the lesion, it will influenced the evolution of the T2DM and the multiple organs. Through those year, it became a popular and necessary subject which are pathological changes research and the treatment method of the Chinese medical. Base on the theoretical exploration and clinical experience, the pathogenesis of the DPN are include "excessive heat of spleen and stomach","Fleeing into meridians and collaterals"," Yin deficiency"," Dystrophy of meridians and collaterals". Clearing heat and nourishing yin"is the fundamental therapy of this disease which is because the same pathogenesis.Observing the clinical effect of this therapy on DPN and investigating the echanism of it by theoretical studies and animal experiments in the way of using Wang's Hypoglycemic Decoction.
Theoretical studies
①Concluding the cognitions of Pathogeny, Pathogenesis and Syndrome differentiation. Meanwhile, to analyze the treatment of DPN on the ancient medical experts by research of the journals.①Summarizing the Pathogeny, Pathology, Treatment and Prognosis of DPN on the base of the results of rational medical studies.③Analyzing the basical therapy(Clearing heat and nourishing yin)of DPN by discussing the basic pathogenesis of DPN.④Analyzing the representative decoction of this therapy(Wang's Hypoglycemic Decoction)which include the composition basis, medication characteristic, the card plus or minus,and the application in DPN.
Experimetal studies
Objective:To observe the thrapeutical effect of Wang's Hypoglycemic Decoction on DPN model rats and related gene expression of sciatic nerve. Discussed the mechanism of the treatment on DPN, discover the mechanism of "Clearing heat and nourishing yin", preliminary explore its pharmacodynamic targets,then provide scientific basis for thorough research and clinical practice.
Methods:the model rats male SD were established with disposable injection of low dose STZ and high caloie diet.Kinds of examinations were applied:ontaining biochemistry, electrophysiology,light microscope.immunohistochemical.Hua-lian rat gene expression profiling chip,and real time quantitative reverse transcription polymerase chain reaction technique to observe the effect of Wang's Hypoglycemic Decoction on indictors such as general conditions, fasting plasma C peptide level, thermal stimulus response threshold, the conduction velocity of sciatic nerve, sciatic nerve morphology,the expression of NGF and Trk A,especially gene expression profiling and PLA2G2A mRNA gene.
Results:(1)The model of DPN rat has been cloned successfully.(2) Wang's Hypoglycemic Decoction has significant effects on decreasing blood glucose after1month influced,However, it hasn't decrease the fasting plasma C peptide level in comparing with the model group.(3)The Increased thermal stimulus response threshold significantly(p<0.001)in comparing with the model group.(4)The increased SNCV significantly (p<0.001) in comparing with the model group,but each groups'MNCVs haven't any differences.(5)Protecting sciatic nerve from microstructure pathology damaging,proctecting nerve function and easing the symptoms of DPN.(6)Increasing the expression of NGF in sciatic nerve,however the significants are differences of the expression of Trk A.(7)By analyzing gene chip,we found that there were genes expression204up-regulation and genes expression104down-regulation in the treatment group in comparing with the DPN model group rat sciatic nerves,some of there genes related to metabolism of Arachidonic acid, peroxisome proliferators activated receptor pathway, anti-oxidative.(8)With real time quantitative reverse transcription polymerase chain reaction technique to study DPN,results show that,the PLA2G2A mRNA gene was up-regulation in the DPN model group rat sciatic nerves and down-regulation in the Wang's Hypoglycemic Decoction treatment group rat sciatic nerves.
Conclusions:DPN molecular mechanisns relation to genes expression regulations, over expression of PLA2G2A mRNA gene play an important role decreasing pain threshold and conduction velocity of sciatic nerve, microstructure pathology damages of sciatic nerve. The method of clearing heat and nourishing yin heat for primary principle is beneficial for controlling blood glucose,increasing pain threshold and conduction velocity of sciatic nerve through the machnism as follow:①Promoting insulin secretion,increasing Insulin sensitivity at the same time.②ncreasing the retrograde transport of NGF,improve the material transport status in axonal.③Protecting sciatic nerve directly by regulating expressions of several genes,especially down-regulating the over-expression of PLA2G2A mRNA.
引文
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