【作者】 张会芳；

【导师】 王玲玲；

【作者基本信息】 南京中医药大学 ， 针灸推拿学， 2013， 博士

【摘要】 研究目的：本课题理论研究部分,通过对古今文献的研究,探讨灸温是影响灸效的关键因素,适宜灸温是取得最佳灸效的重要前提,为下一步灸法“温通”效应机制研究提供理论基础；临床试验部分通过观察不同灸温对高脂血症患者血脂及血清Ox-LDL、ET-1、NO的影响,探讨不同灸温对高脂血症的“温通”效应,观察其能否通过调整血脂组分、抗氧化、保护血管内皮、改善血管舒缩功能、预防AS等不同病理环节体现；动物实验通过观察温度感受器TRPV1的激动剂capsaicin和拮抗剂capsazepine对高脂血症C57BL/6小鼠血脂及背根节TRPV1mRNA表达的影响,从阴性对照和阳性对照两个方面证实TRPV1介导了灸法以“温”促“通”效应机制。方法：理论研究：检索与本课题相关的古今文献资料,包括灸温、灸感、灸效、灸法作用机制、高脂血症、TRPV1,综合分析,归纳总结上述资料之间及与本课题之间的相关性,从不同方面为灸法“温通”效应机制研究奠定理论基础。临床试验：将42例原发性高脂血症患者随机分为45℃组和38℃组,用不同的艾灸温度艾灸患者神阙穴及足三里穴,每次每穴10分钟,隔日一次,治疗12周,肘静脉抽血观察治疗前后患者血脂、血清Ox-LDL、ET-1、NO的变化及组间疗效的差异。动物实验：分题一,将32只清洁级健康成年雄性C57BL/6小鼠随机分为正常组、模型组、46℃组、38℃组4组,每组8只。自制75%蛋黄乳剂25m1/kg腹腔注射,建立急性高脂血症模型。两次治疗结束后即刻取材,全自动生化分析仪检测血脂,荧光定量PCR法检测小鼠背根节TRPV1mRNA的表达量；分题二,将40只清洁级健康成年雄性C57BL/6小鼠随机分为正常组、模型组、46℃组、46+CPZ组、46+溶媒组5组,每组8只。造模方法同分题一,46+CPZ组在治疗前需涂抹capsazepine,46+溶媒组在治疗前涂抹溶媒剂,其它组处理同分题一,两次治疗结束后即刻取材,检测指标及方法同分题一；分题三,将48只清洁级健康成年雄性C57BL/6小鼠随机分为正常组、模型组、46℃组、38℃组、CAP组、溶媒组6组,每组8只。造模及治疗方法同分题一,CAP组只涂抹capsaicin不治疗,溶媒组只涂抹不治疗,其它组处理同分题一,两次治疗结束后即刻取材,检测指标及方法同分题一。结果：临床试验：1温、热、灼、痛四种不同灸感对应的温度范围有别,“温”的温度范围大概在32.8℃～35.8℃；“热”的温度范围大概在36.8℃～39.9℃；“灼”的温度范围大概在40.9℃～43.5℃；“痛”的温度大概大于等于44.5℃。2治疗后两组组间比较,45℃组降低了高脂血症患者血清中TC、TG、Ox-LDL的含量及ET-1/NO的比值,明显优于38℃组(P<0.05或者P<0.001)。同时,45℃组升高了高脂血症患者血清中NO的含量,明显优于38℃组(P<0.05)。345℃和38℃对于血清中Ox-LDL的含量均有明显的降低作用,但38℃组对NO、ET-1、ET-1/NO无明显影响,而45℃组作用明显,说明38℃艾灸对也具有一定的治疗作用,但其作用途径与45℃可能存在着区别。动物实验：4与模型组比较,46℃组TC降低明显,差异具有显著性统计学意义(P<0.05),与46℃组比较,38℃组TC含量明显较高,差异具有显著性统计学意义(P<0.05)。46℃组TRPV1mRNA的表达量是模型组的2.62倍,38℃组TRPV1mRNA的表达量是模型组的1.78倍,46℃组TRPV1mRNA的表达量是38℃组的1.47倍。5与46℃组相比,46+CPZ组TC的含量与明显高于46度组(P<0.05),说明capsazepine拮抗了46℃艾灸的降脂效应。46℃组TRPV1mRNA的表达量是模型组的2.62倍,46+CPZ组TRPV1mRNA的表达量是模型组的1.59倍,说明capsazepine拮抗了46℃艾灸产生的TRPV1mRNA的表达。6与模型组比较,46℃组、CAP组TC的含量明显降低,且差异有显著统计学意义(P<0.05),CAP组降TC的效应与46℃组降TC的效应从统计学上讲无差异(P>0.05)。46℃组TRPV1mRNA的表达量与CAP组TRPV1mRNA的表达量相同,是模型组的2.62倍,优于38℃组。理论研究：7灸感是艾灸起效的关键；温、热、灼、痛是灸感的主要表现形式；适宜的灸量是灸感、灸效产生的重要条件；灸温是不同施灸强度产生的综合灸量,适宜灸温是取得最佳灸效的重要前提。艾灸的作用机理与燃艾时所产生的理化因子有关,如穴位局部感受器、热休克蛋白、肥大细胞脱颗粒等,这些因子都会受到温度的影响,进而产生相应的生物学变化。结论：临床研究：1“温、热、灼、痛”四种不同的灸感对应不同的温度范围,分别是32.8℃～35.8℃.36.8℃～39.9℃.40.9℃～43.5℃.大于等于44.5℃,灸感与温度密切相关。245℃艾灸刺激具有调脂作用,不同程度地降低了血清中的TC.TG.ApoB.LDL-c的含量,同时明显升高了HDL-C的含量；显著降低了高脂血症患者血清中Ox-LDL的含量,具有抗氧化作用；明显降低了高脂血症患者血清中ET-1含量及降低ET-1/NO比值,同时升高了NO含量,具有保护血管内皮功能作用。338℃艾灸刺激调脂作用不明显；但可以显著降低高脂血症患者血清中Ox-LDL的含量,具有抗氧化作用；对ET-1、NO的含量及ET-1/NO比值影响不大,保护血管内皮功能作用不明显。4两组比较,45℃艾灸刺激调脂效应、抗氧化效应及保护血管内皮作用明显较佳。艾灸具有温通调脂作用,“温”是条件,即需达到一定的温度条件,45℃较38℃好,“通”是效应,艾灸刺激达到条件后才会产生较好的调脂通脉效应。动物研究：146℃艾灸刺激在降低TC的同时,明显增加了TRPV1mRNA的表达量,不同于38℃。2局部应用Capsazepine竞争性的拮抗了46℃艾灸热刺激产生的调脂作用,同时也抑制了艾灸热刺激产生的小鼠背根节TRPV1mRNA表达量。346℃艾灸刺激同Capsaicin一样,均可以激活TRPV1,并产生同样的调脂作用。4TRPVl介导了灸法以“温”促“通”效应机制,适宜的灸量既要激活TRPV1,同时又可使病人能耐受,即是43℃-45℃。理论研究：1灸感是艾灸起效的关键。2温热灼痛感是灸感的主要表现形式。3适宜的灸量是灸感、灸效产生的重要条件。4灸温是不同施灸强度产生的综合灸量,适宜灸温是取得最佳灸效的重要前提。更多还原

【Abstract】 Objective:The theoretical study of the subject through the study of ancient and modern literature, investigate that the moxibustion temperature is a key factor to the efficiency of moxibustion, that suitable temperature of the moxibustion is an important prerequisite to obtain the best moxibustion effective, which provide a theoretical basis for the next step moxibustion Warming effect mechanism; Clinical trials through the observation the impact of different moxibustion temperature on the content of lipids and Ox-LDL, ET-1, NO in serum of patients with hyperlipidemia, explore the warming and promoting effect mechanism of moxibustion, to see whether it can be reflect by different aspects of the pathology, including adjusting blood lipid components, antioxidant, protecting vascular endothelial improving vasomotor function, preventing AS and so on.The animal experiments observed the impact of temperature sensors TRPV1agonist capsaicin and antagonist capsazepine on the blood lipids and TRPV1mRNA expression of dorsal root ganglion in C57BL/6mice with hyperlipidemia. it is confirmed that TRPV1mediated mechanism of moxibustion regulating blood lipids and clearing the blood by negative control study and positive control study.Method:Theory research:search related ancient and modern literature, including temperature, feeling, effection, mechanism of moxibustion, hyperlipidemia, TRPV1and then summarize, analysis the correlation between the above information and the this topic.Method:Clinical trials:part one:test Li4of20healthy volunteers with laser doppler temperature probe to observe their sense of different temperature; part two:42patients with primary hyperlipidemia were randomly divided into45℃group and38℃, using different temperature moxibustion on CV8and ST36,10minutes for each acupoint one time, every other day at a time, after12weeks of treatment, draw elbow vein blood to observe the changes of index before and after treatment and the curative effect of the differences between groups. Animal experiment:part one:32SPF level healthy adult male C57BL/6mice were randomly divided into4groups:the normal group, the model group, the46℃group as well as the38℃group, eight for each group. Use selfmade75%egg yolk emulsion as25ml/kg by intraperitoneal injection at one time to foster acute hyperlipidemia model.on9p.m. of the first day, take food away and water left, the next day at13p.m to build mold, after the success of model making, start treatment at the third day. Draw materials immediately on the fourth day after treatment, then detect serum lipids with automatic biochemical analyzer and detect TRPV1mRNA expression amount of dorsal root ganglion by quantitative PCR. part two:40SPF level healthy adult male C57BL/6mice were randomly divided into5groups:46℃group, normal group, model group,46+CPZ group,46+soluble enzyme group, eight for each group.model making and treatment is the same as the part one,46+CPZ group with capsazepine daubed on the acupoints before treatment,46+enzyme group daub soluble enzyme agent before treatment, Draw materials immediately on the fourth day after treatment, then detect serum lipids with automatic biochemical analyzer and detect TRPV1mRNA expression amount of dorsal root ganglion by quantitative PCR; part three,68SPF level healthy adult C57BL/6male mice were randomly divided into6groups, the normal group, model group, the46℃group, the38℃group, the CAP group and the soluble enzyme group, each group of eight mice. Model making and treatment is the same as the part one. The CAP group daub capsaicin without other treatment, as well as the enzyme group. Draw materials immediately on the fourth day after treatment to detect, and then detect serum lipids in mice with automatic biochemical analyzer and detect TRPV1mRNA expression amount of dorsal root ganglion by quantitative PCR.Result:Clinical Research:1there are four moxibustion sense:warm, hot, burning, pain, they have different temperature range, the temperature range of warm is about32.8℃-35.8℃; the temperature range of heat is about36.8℃-39.9℃; the temperature range of hot is about40.9℃-43.5℃; the temperature range of pain is probably greater than or equal to44.5℃。2Comparison between the two groups after treatment, the45℃group reduce the content of TC, TG, Ox-LDL and ET-1/NO the ratio in the serum of patients with hyperlipidemia, significantly better than the38℃group (P<0.05or P<0.001). In the same time, the45℃group increased the content of NO in the serum of patients with hyperlipidemia, which is significantly better than the38℃group (P<0.05).3moxibustion stimulate of45℃and38℃can significantly reduce the levels of Ox-LDL in the serum, but the38℃group had no significant impact on the content of NO, ET-1, ET-1/NO, while45℃group had obvious impact. That indicate that moxibustion stimulate of38℃also has a therapeutic effect, but its pathway may be different from45℃moxibustion stimulate.Experimental Research:4Compared with the model group, the group of46℃decreased the content of TC significantly(P<0.05); Compared with the46℃group, the content of TC in38℃group is significantly more higher (P<0.05); expression of TRPV1mRNA in the46℃group is2.62times of the model group, the expression of TRPV1mRNA in the38℃group was1.78times of the model group, the amount is38℃group at46℃TRPV1mRNA expression, expression of TRPV1mRNA in the46℃group is1.47times of the38℃group.5Compared with the46℃group, the content of TC in46+CPZ group is significantly more higher (P<0.05), which indicate that capsazepine antagonized the lipid-lowering effect of46℃group moxibustion. The expression of TRPV1mRNA in the46℃group was2.62times of the model group, The expression of TRPV1mRNA in the46+CPZ group was1.59times of the model groupd, The expression of TRPV1mRNA in the46+CPZ group was1.64times of the46℃groupd, The expression of TRPV1mRNA in the46+soluble enzyme group was2.49times of the model groupd.6Compared with the model group, the content of TC in46℃group and CAP group was significantly reduced(P<0.05); the CAP group and46℃group statistically speaking had no differences in lowering effect of TC. The expression of TRPV1mRNA in the46℃group was2.62times of the model group. The expression of TRPV1mRNA in the CAP group was2.62times of the model group, the expression of TRPV1mRNA in the38℃group was1.78times of the model group, The expression of TRPV1mRNA in the soluble enzyme group was1.68times of the model groupd.Theoretical research:7The moxibustion sense is key in moxibustion onset; warm, hot, burning, pain feeling were the main manifestations of the moxibustion sense; suitable amount of moxibustion was the important condition to get moxibustion sense and the moxibustion efficiency; Moxibustion temperature was comprehensive moxibustion amount of different moxibustion strength, and the suitable moxibustion temperature is an important prerequisite to obtain the best moxibustion effective; moxibustion mechanism is associated with many physical and chemical factors during the action of burning, such as the acupoint local receptors, heat shock proteins, mast cell degranulation, etc, and these factors are affected by temperature and produce biological changes.Conclusion:Clinical research:1warm, hot, buring, pain corespound to four kind of different sense of moxibustion for different temperature range, which is32.8℃to35.8℃,35.8℃to39.9℃,40.9℃to43.5℃, and more than44.5℃. Sense of moxibustion is closely related with temperature.245℃moxibustion stimulating can regulate the lipid metablison,, reduce the serum TC, TG, ApoB, LDL-c content in different degrees, significantly increase the content of HDL-C;45℃moxibustion stimulation greately reduces the Ox-LDL content in serum of hyperlipidemia patients, proved to have antioxidant effects;45℃moxibustion stimulation can reduce ET-1in serum content, increase NO content and lower ratio of ET-1by NO, hyperlipidemia patients. Thus45℃moxibustion stimulation can protect the vascular endothelial function.338℃moxibustion stimulation has less effect in regulating the lipid levels;38℃moxibustion stimulation can largely reduce the content of Ox-LDL in serum of hyperlipemia patients, exert antioxidant effects;38℃moxibustion stimulation has little impact on the content of NO and ET-1, ratio of ET-1by NO,which means it can not protect the vascular endothelial function obivosly.4By contrast,45℃group ofmoxibustion shows better impact on regulating lipid index, exerting antioxidant effect and protecting the vascular endothelial function; moxibustion has the function of warming and promoting to regulate lipid,"warm" is one condition, and it need to reach a certain temperature,45℃is better than38℃,"tong" is the effect of moxibustion stimulation,Only when reaching the certain condition,will it exert better effect of regulating the lipid and unblocking the meridiansAnimal studies:146℃moxibustion stimulation reduced the content of TC, while significantly increase the expression level of TRPV1mRNA, which in different from38℃.2The local application capsazepine competition antagonised lipid metabolism function of the46℃the moxibustion thermal stimulation, as well as suppress of TRPV1mRNA in the mice dorsal root ganglion induced by moxibustion heat stimuli.3The same as Capsaicin,46℃moxibustion stimulus can activate TRPV1, and produce the same role in regulate lipid.4TRPV1mediates moxibustion warming and promoting effect mechanism, and a suitable amount of moxibustion is necessary to activate TRPV1amount. The suitable temperature can enable the patient tolerate and can also activate enough amounts of TRPV1.which is43℃-45℃.Theoretical studies:1A moxibustion sense moxibustion onset.2warm, hot, burning, pain feeling were the main manifestations of the moxibustion sense3suitable amount of moxibustion was the important condition to get moxibustion sense and the moxibustion efficiency.4Moxibustion temperature was comprehensive moxibustion amount of different moxibustion strength, and the suitable moxibustion temperature is an important prerequisite to obtain the best moxibustion effective更多还原