脉络通瘀汤治疗脱疽气虚血瘀证对气体信号分子的影响及疗效观察

【作者】 曹刚；

【导师】 庞鹤；

【作者基本信息】 北京中医药大学 ， 中医外科， 2014， 博士

【摘要】 闭塞性动脉硬化(atherosclerosis obliterans, ASO)是血管外科常见病、高发病,在中医外科属“脱疽”范畴。中医药对脱疽病诊疗有着悠久的历史,中药对脱疽病的治疗也在不断发展,但针对脱疽病中药干预与气体信号分子相关性的研究较少。本文主要包括脱疽与益气活血法及闭塞性动脉硬化与气体信号分子、脉络通瘀汤治疗脱疽气虚血瘀证疗效观察、脉络通瘀汤对闭塞性动脉硬化大鼠气体信号分调节作用三个部分。第一部分文献综述。阐述了脱疽病因病机及益气活血法治疗脱疽的相关文献研究；闭塞性动脉硬化的现代医学研究、气体信号分子与动脉硬化性疾病的相关文献研究。第二部分实验研究。研究脉络通瘀汤对脱疽气虚血瘀证大鼠模型干预作用与气体信号分子的调节机制。方法：50只大鼠以随机法分为空白对照组(A)10只、模型组(B)10只、低剂量治疗组(C)10只,中剂量治疗组(D)10只、高剂量治疗组(E)10只。采用高脂喂养、饥饿、疲劳复合因素复制大鼠脱疽气虚血瘀证模型。实验周期12周。观察气虚血瘀证模型大鼠形态、症候学变化。应用导师庞鹤教授临床验方脉络通瘀汤,治疗组分低(1g/Kg)、中(2g/Kg)、高(3g/Kg)剂量进行给药治疗。给药4周。监测一般指标：凝血功能、血脂、血糖、肝肾功能等,评价安全性。评价疗效。色谱法测定检测气体信号分子H2S、NO、iNOS含量。放射免疫分析法检测血管舒缩活性物质ET、VEGF的表达。观察组织病理学改变。结果：脉络通瘀汤对闭塞性动脉硬化症大鼠LDL、GLU影响,与正常对照组比较,模型组大鼠LDL、GLU显著升高,有差异；随治疗剂量增加,GLU呈递减趋势并最终恢复到正常水平,治疗高剂量组与模型组比较呈显著差异；LDL虽无明显递减趋势,但均恢复到对照组正常水平及以下,变化指标统计差异有显著性。脉络通瘀汤对闭塞性动脉硬化大鼠H2S的影响：与正常对照组比较,模型组大鼠H2S降低,有差异；随治疗剂量增加,H2S呈递曾趋势,动脉匀浆中相关指标也呈同样趋势；高剂量组与模型组比较呈差异显著。脉络通瘀汤对闭塞性动脉硬化大鼠ET、VEGF的影响,与正常对照组比较,模型组大鼠血浆中ET、VEGF、动脉血管匀浆中ET值具统计学意义；与模型组对比,治疗组随治疗剂量增加,血浆中ET、动脉血管匀浆中ET值均呈递减趋势,血浆中VEGF值呈递增趋势,各组数据变化统计差异有显著性。结论：通过高脂喂养、饥饿、疲劳的复合因素方法,可实现复制大鼠脱疽气虚血瘀证模型。脱疽气虚血瘀证模型大鼠,形态学表现出毛色变暗、枯干、体重增长率下降、运动能力减弱等；证候学表现进食少、反应迟缓、爪甲、尾色紫暗等。益气活血法治疗脱疽气虚血瘀证模型大鼠,改善脏腑功能,调节机体糖、脂代谢,可降低LDL、GLU,控制AS危险因素。闭塞性动脉硬化病变形成,H2S呈降低趋势；中药益气活血治疗后H2S呈升高表现,提示H2S生物学效应可能为益气活血中药治疗脱疽的内在机制。ET是较早发现的血管内皮细胞产生并分泌释放的长效血管收缩剂,VEGF作为一种广泛应用于临床治疗的内皮源性生长因子,能促进肢体侧枝循环的建立,本文研究表明,益气活血法治疗脱疽气虚血瘀证模型大鼠,可降低血浆ET含量、升高血浆VEGF表达。气虚血瘀为本病的始动因素、基础病机,益气活血法为治疗本病的主要立法。益气活血法是现代中医药治疗动脉硬化性疾病的重要立法,应用益气活血立法组方的脉络通瘀汤,体现了“气为血帅,血为气母,气行则血行,气滞则血瘀”的中医学经典理论。第三部分疗效观察。进行导师庞鹤教授经验方脉络通瘀汤对脱疽气虚血瘀证临床疗效观察。参考《中国中西医结合学会周围血管疾病专业委员会1995年制订的诊断标准》、《中医病证诊断疗效标准》(ZY/T001.2-94),观察对象为北京中医药大学附属东方医院2013年3月-12月诊疗的60名患者。证候诊断为气虚血瘀、脉络瘀阻证。治疗方案：基础治疗：注射用血塞通、内科病治疗；对照组治疗方案：仅予基础治疗；中药组治疗方案：基础治疗+中药治疗(方选脉络通瘀汤：黄芪60g、三七12g、牛膝10g、桂枝12g、泽兰20、地龙10g)。疗效评价参考脱疽临床路径验证方案中评价标准。结果：按中华全国中医学会脉管专业委员会制订的疗效标准判定。治愈：对照组3例(10.00%),中药组4例(13.33%)；显效：对照组12例(40.00%),中药组16例(53.33%)；有效：对照组14例(46.67%),中药组10例(33.33%)；无效：1例(3.33%)为对照组。对照组治疗评分与脉络通瘀汤组对比,在临床麻木、冷感改善方面有显著差异。讨论：早期诊断、早期治疗,应用益气活血法为指导的脉络通瘀汤对脱疽气虚血瘀证的治疗明确临床价值。更多还原

【Abstract】 Arteriosclerosis obliterans (ASO) is a common disease with high prevalence in vascular surgery. In traditional Chinese medicine (TCM) surgery, the disease is categorized under the area of "digital gangrene". TCM has a long history in diagnosis and treatment of digital gangrene, and its Chinese medicine treatment has been consistently developing over the ages. However, research targeted at digital gangrene TCM intervention and its correlation with gaseous signaling molecules is relatively few. In this study, our objectives can be concluded into three main sections:1) studying the correlation between digital gangrene and replenishing qi activating blood method, as well as the correlation between ASO and gaseous signaling molecules,2) observation of therapeutic efficacy in the treatment of qi deficiency blood stasis pattern using vessel collateral dredging stasis decoction,3) observation of the regulatory effects of vessel collateral dredging stasis decoction on gaseous signaling molecules in ASO rats.The first section: literature review. Literature related to digital gangrene’s disease cause and mechanism of disease, treatment of digi tal gangrene using qi replenishing blood activating method, modern medical research on ASO, relationship between gaseous signaling molecules and arteriosclerotic disease were reviewed.The second section: experimental research. Research on vessel collateral dredging stasis decoction’s intervening effects in qi deficiency blood stasis pattern rat models, and its regulatory mechanism on gaseous signaling molecules were conducted.Methods:50lab rats were randomly distributed into5groups: blank control group (A),10rats; model group (B),10rats; low dosage treatment group (C),10rats; middle dosage treatment group (D),10rats; high dosage treatment group (E),10rats. The rats were subjected to composite factors comprising high-fat diet, starvation and fatigue in order to construct qi deficiency blood stasis rat models. Experimental period was set as12weeks.Observation of morphology and symptomatology changes in qi deficiency blood stasis rat models.Using the vessel collateral dredging stasis decoction, a clinically proven formula by Professor Pang He, rats from the respective treatment groups were administered with low dosage (1g/Kg), middle dosage (2g/Kg) and high dosage (3g/Kg) drug treatments accordingly. Drug treatment was administered for a period of4weeks.Monitoring of general indicators:Blood coagulation function, blood lipid level, blood glucose (GLU) level, liver function and kidney function were monitored. The results were then used as indicators for safety evaluation. Therapeutic effect was also evaluated.Chromatography was used to detect content of gaseous signaling molecules hydrogen sulphide (H2S), nitric oxide (NO) and inducible nitric oxide synthase (iNOS).Radioimmunoassay analysis was used to detect expression of vasoactive substances endothelin (ET) and vegetative growth factor (VEGF)Histopathological changes were observed.Results:Effects of vessel collateral dredging stasis decoction on low density lipoprotein (LDL) and blood GLU levels in ASO rats:Compared to the blank control group, significant increase in LDL and blood GLU levels were observed in model group rats. Difference in LDL and blood GLU levels was statistically significant. As drug dosage increases, progressive decrease in blood GLU levels eventually leading to normal levels was observed. Significant difference was observed between the high dosage treatment group and model group. Although no significant progressive decrease was observed for LDL levels, LDL levels in all three treatment groups recovered to normal or lower than normal levels. Statistical analysis of its change indicator revealed significant difference.Effects of vessel collateral dredging stasis decoction on H2S levels in ASO rats:Compared to the blank control group, a significantly different decrease in H2S levels was observed in model group rats. As the drug dosage increases, progressive increase in H2S was observed, along with a similar increasing trend in related indicators from the artery homogenate. Difference between the high dosage treatment group and model group was statistically significant.Effects of vessel collateral dredging stasis decoction on BT and VEGF levels in ASO rats:Compared to the blank control group, in blood serum ET, blood serum VEGF and artery homogenate ET levels in model group rats were statistically significant. Compared to the model group, progressive decrease was observed in blood serum and artery homogenate ET levels as drug dosage increased. Blood serum VEGF values however, displayed progressive increase as drug dosage increased. Significant difference was observed for change indicators in the data from each group.Conclusion:Through composite factor method using high-fat diet, starvation and fatigue, digital gangrene qi deficiency blood stasis rat models can be reproduced.Digital gangrene qi deficiency blood stasis rat model:Morphology changes included darkening and drying up of fur, decrease in body weight increasing rate, weakening of exercise capacity etc. Symptomatology changes include decrease in food intake, slower response, and purplish-dark claw and tail coloration.Treatment of digital gangrene qi deficiency blood stasis rats using qi replenishing blood activating method improves its organ functions, regulates GLU and lipid metabolism, decreases LDL and blood GLU levels, and controls arteriosclerosis (AS) risk factors.In ASO formation, H2S displays a decreasing trend. After TCM qi replenishing blood activating treatment, H2S levels increased, thereby suggesting that biological effects of H2S may be the intrinsic mechanism in qi replenishing blood activating TCM treatment of digital gangrene.ET, a biological substance produced and secreted by vascular endothelial cells, is one of the early discovered long-acting vasoconstrictors. On the other hand, VEGF is a type of endothelial growth factor widely-used in clinical treatment, and is known to promote limb collateral circulation formation. In our study, we have shown that treatment of digital gangrene qi deficiency blood stasis rats using qi replenishing blood act ivat ing method can decrease blood serum ET levels, and increase blood serum VEGF expression.Qi deficiency blood stasis is the initiating factor and fundamental disease mechanism in digital gangrene. Hence, qi replenishing blood activating method is the main method in treating this disease. Qi replenishing blood activating method is an important method in modern TCM treatment of arteriosclerotic diseases. Vessel collateral dredging stasis decoction, formulated under the therapeutic method of qi replenishing blood activating, reflects the classical TCM theory "Qi is the commander of blood, blood is the mother of qi, if qi moves then blood moves, if qi stagnates then blood stasis".The third section:observation of therapeutic effect. Therapeutic effect of Professor Pang He’s empirical formula, vessel collateral dredging stasis decoction, on digital gangrene qi deficiency blood stasis pattern was observed.60patients from the Beijing university of Chinese medicine Dong Fang hospital that were admitted between March2013to December2013were diagnosed and observed under protocols referenced from "China Society of Chinese and Western integration medicine peripheral vascular disease Committee diagnostic standards set in1995" and "Standards of diagnosis and therapeutic effect for TCM diseases and patterns"(ZY/T001.2-94). The patients admitted into our study group had been diagnosed with either qi deficiency blood stasis pattern or pattern of vessel collateral static blood obstruction.Treatment plan: basic treatment: Xue Sai Tong (Notoginseng triterpenes) injection and internal medicine treatment; Control group treatment plan: only basic treatment was administered; Chinese medicine group treatment plan: basic treatment plan plus Chinese medicine treatment vessel collateral dredging stasis decoction was used: Milkvetch root60g, Sanqi12g, medicinal Cyathula root10g, Cassia twig12g, Hirsute shiny Bugleweed herb20g, Earthworm10g)."Digital gangrene clinical verification scheme" was used as reference for therapeutic efficacy evaluation standards. Results: Therapeutic efficacy was evaluated using evaluation standards set by "Chinese National traditional Chinese medicine association blood vessel Committee". Healed:control group:3cases (10%), Chinese medicine treatment group:4cases (13.33%); significant effect:control group:12cases (40%), Chinese medicine treatment group:16cases (53.33%); effective:control group:14cases (46.67%), Chinese medicine treatment group:10cases (33.33%); non-effective:control group:1cease (3.33%).Statistically significant difference was observed between treatment scores of the control group and vessel collateral dredging stasis decoction groups for improvements in clinical symptoms such as numbness and cold. Discussion: Early diagnosis, early treatment, and use of vessel collateral dredging stasis decoction, which uses qi replenishing blood activating method as a guideline, clearly has clinical value in treatment of digital gangrene qi deficiency blood stasis pattern.更多还原