【作者】 王家有；

【导师】 赖新生；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2010， 博士

【摘要】 目的探讨针刺太冲与非经非穴对自发性高血压大鼠(SHR)血压的影响,并利用二维凝胶电泳和质谱技术分离并鉴定各组大鼠延髓内表达差异的蛋白,以期从蛋白质整体水平探讨针刺太冲穴降低SHR高血压的延髓内分子机制,并为寻找与经穴效应特异性相关的生物效应指标提供线索。方法选用9w、雄性SHR30只,随机分为模型组、太冲组、和非经非穴组(均n=10),并以同体重正常血压SD大鼠(n=10)作为正常组。太冲组为针刺大鼠双侧太冲穴(定位在大鼠后肢足背侧第1、2跖骨间),非经非穴组为针刺大鼠双侧非经非穴(定位在大鼠后肢足背侧第3、4跖骨间凹陷处)。针刺参数为大鼠清醒状态下,针刺1次／d,持续时间5min／次,捻转频率80±5次／min,捻转幅度180±5度,太冲组毫针斜向涌泉,不穿透、不触及足底皮肤,而非经非穴组毫针垂直于皮肤进针并穿透足底皮肤,于针刺结束5min后采用BP-6无创血压测量系统对大鼠进行尾动脉血压测定。模型组和正常组不给予针刺。连续针刺治疗到第7d后,对各组大鼠断头取脑,在冰上分离获取延髓。然后裂解、超声破碎延髓组织,经除盐处理,通过二维凝胶电泳分离蛋白,Typhoon9200扫描仪获得凝胶图像,Imagemaster 2D 6.0图像分析软件分析各组蛋白差异点,用MALDI-TOF-TOF串联质谱仪分析蛋白质,所得的质谱图通过GPS软件在MASCOT数据库中检索并鉴定蛋白质,再利用酶联免疫分析实验进一步验证差异蛋白的变化。结果1.针刺太冲与非经非穴对SHR血压的影响。组间比较结果：针刺前收缩压即原始收缩压的比较,太冲组、非经非穴组及模型组之间大鼠收缩压均无显著性差异(均P>0.05)。于针刺第1d,太冲组、非经非穴组及模型组之间大鼠收缩压均无显著性差异(均P>0.05)。于针刺第2d,与非经非穴组、模型组比较,太冲组大鼠收缩压显著降低(P<0.01),提示于针刺第2 d,太冲组大鼠收缩压开始降低。于针刺第3 d,与模型组比较,非经非穴组大鼠收缩压显著升高,提示针刺非经非穴可能是一种非良性刺激,导致大鼠收缩压显著升高。于针刺第4d,太冲组、非经非穴组及模型组之间大鼠收缩压均无显著性差异(均P>0.05)。于针刺第5d,与非经非穴组比较,太冲组大鼠收缩压显著降低(P<0.01)。于针刺第6、第7d,与非经非穴组、模型组比较,太冲组大鼠收缩压显著降低(均P<0.01),提示针刺太冲穴于第5d开始稳定的降压水平。与正常组比较,不同时间段太冲组、非经非穴组以及模型组大鼠收缩压均显著性升高(均P<0.01),提示本实验针刺太冲穴和非经非穴都未能使收缩压恢复至正常水平。组内比较结果：与第1d比较,太冲组大鼠收缩压于第2d开始显著降低(P<0.01),然而于第3、第4d收缩压有小幅度升高(P>0.05),然后又于第5d开始降低(P<0.01),直到第6d、第7d收缩压降低水平保持稳定(均P<0.01),提示针刺太冲穴于第5d开始稳定的降压水平。非经非穴组于不同时间段收缩压均无显著性差异(P>0.05)。与第3d相比,模型组大鼠收缩压于第6d显著升高(P<0.01),与第4d比较,模型组大鼠收缩压于第6d和第7d均显著升高(均P<0.01),提示模型组大鼠的收缩压呈上升趋势。正常组于不同时间段收缩压均无显著性差异(P>0.05)。2.各组大鼠延髓二维凝胶电泳图谱及其分析结果。本实验建立了正常SD大鼠、SHR以及对SHR针刺太冲与非经非穴后延髓的二维凝胶电泳图谱,共发现了SD大鼠延髓有621±30个蛋白点,SHR延髓有621±20个蛋白点,SHR经针刺太冲后延髓有621±40个蛋白点,SHR经针刺非经非穴后延髓有621±20个蛋白点,其中,与SHR组比较,SHR经针刺太冲后延髓表达有差异的蛋白点88个(大于2倍)；SHR经针刺非经非穴后延髓表达有差异的蛋白点91个(大于2倍),正常SD大鼠延髓表达有差异的蛋白点47个(大于2倍)。3.鉴定出的差异蛋白质在各组大鼠延髓表达的结果。(1)与模型组比较,太冲组大鼠延髓下调的蛋白有5个(大于2倍)：突触素蛋白1,NAD依赖的脱乙酰酶,蛋白激酶C抑制蛋白,电子转移黄素蛋白和髓磷脂碱蛋白；上调的蛋白有6个(大于2倍)：谷氨酸脱氢酶,谷胱甘肽S转移酶,醛脱氢酶2, Rho GDP解离抑制因子,DJ-1蛋白和过氧化物歧化酶。(2)与模型组比较,非经非穴组大鼠延髓下调的蛋白有1个(大于2倍)：电子转移黄素蛋白；上调的蛋白有3个(大于2倍)：热休克蛋白90,泛素水解酶和DJ-1蛋白。(3)与非经非穴组比较,太冲组大鼠延髓下调的蛋白有7个(大于2倍)：热休克蛋白90,突触素蛋白1,丙酮酸激酶,NAD依赖的脱乙酰酶,蛋白激酶C抑制蛋白1,泛素水解酶和髓磷脂碱蛋白；上调的蛋白有6个(大于2倍)：谷氨酸脱氢酶,醛脱氢酶2,谷胱甘肽S转移酶,Rho GDP解离抑制因子,DJ-1蛋白及过氧化物歧化酶。(4)与正常组比较,模型组大鼠延髓下调的蛋白6个(大于2倍)：丙酮酸激酶,谷氨酸脱氢酶,谷胱甘肽S转移酶,醛脱氢酶2,泛素水解酶和过氧化物歧化酶；上调的蛋白有7个(大于2倍)：热休克蛋白90,突触素蛋白1,NAD依赖的脱乙酰酶,电子转移黄素蛋白,蛋白激酶C抑制蛋白1,蛋白激酶C抑制蛋白1和髓磷脂碱蛋白。(5)与正常组比较,太冲组大鼠延髓下调的蛋白有3个(大于2倍)：过氧化物歧化酶,过氧化物歧化酶和髓磷脂碱蛋白；上调的蛋白有4个(大于2倍)：热休克蛋白90, Rho GDP解离抑制因子,DJ-1蛋白和过氧化物歧化酶。(6)与正常组比较,非经非穴组大鼠延髓下调的有4个(大于2倍)：谷氨酸脱氢酶,醛脱氢酶2,谷胱甘肽S转移酶和过氧化物歧化酶；上调的蛋白有7个(大于2倍)：热休克蛋白90,突触素蛋白,NAD依赖的脱乙酰酶,蛋白激酶C抑制蛋白1,泛素水解酶,DJ-1蛋白和髓磷脂碱蛋白。5.用酶联免疫分析结果。(1)各组大鼠延髓谷胱甘肽S转移酶表达的变化。模型组大鼠延髓谷胱甘肽S转移酶表达显著低于正常组(P<0.01)。经针刺治疗后,太冲组大鼠延髓谷胱甘肽S转移酶表达显著高于模型组、非经非穴组(均P<0.01),与正常组比较无统计学差异(P>0.01),而非经非穴组大鼠延髓谷胱甘肽S转移酶表达与模型组比较无统计学差异(P>0.01),且低于正常组(P<0.01)。(2)各组大鼠延髓醛脱氢酶表达的变化。模型组大鼠延髓醛脱氢酶表达显著低于正常组(P<0.01)。经针刺治疗后,太冲组大鼠延髓醛脱氢酶表达显著高于模型组、非经非穴组(均P<0.01),而非经非穴组大鼠延髓醛脱氢酶表达与模型组比较无统计学差异(P>0.01),且低于正常组(P<0.01)。(3)各组大鼠延髓蛋白激酶C表达的变化。模型组大鼠延髓蛋白激酶C表达与正常组无显著性差异(P>0.01)。经针刺治疗后,太冲组大鼠延髓蛋白激酶C表达无明显变化,与模型组、正常组、非经非穴组比较(均P>0.01)。谷胱甘肽S转移酶、醛脱氢酶的ELISA结果与二维凝胶电泳结果一致,蛋白激酶C的ELISA结果与二维凝胶电泳蛋白激酶C抑制蛋白1的结果不一致。结论1.针刺太冲穴可显著降低SHR高血压,但未能使SHR高血压恢复至正常水平。2.针刺非经非穴降低SHR高血压效果不明显,甚至可能异常升高SHR高血压。3.针刺太冲可下调SHR延髓内促进兴奋性神经递质释放、诱发炎症反应、氧化磷酸化及信号转导相关蛋白的表达,上调氨基酸氧化分子伴侣、抑制氧化应激及信号转导相关蛋白的变化,其作用机制是多途径、多环节、多靶点的综合效应。4.针刺非经非穴可下调SHR延髓内能量代谢相关蛋白的表达,上调氨基酸氧化分子伴侣、蛋白质分解代谢以及诱发炎症反应相关蛋白的表达,其作用机制是较少途径、较少环节、较少靶点的简单效应。5.经穴效应及其作用机制存在一定的特异性。更多还原

【Abstract】 ObjectiveIn order to research the effect of acupuncture at TaiChong point (LR3) and non-acupoint on the blood pressure in Spontaneously Hypertensive Rats (SHR), and investigate, in whole level of protein expression, the central nerve mechanism of acupuncturing TaiChong point (LR3) to treat SHR, which provide the experimental clue for searching the specific indicators of biological effects that connect to the meridian effect, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) technologies were used and the differentially expressed proteins in the medulla oblongata of SHR were also separated and identified.Methods309-week-old male SHR were randomly divided into model group, TaiChong group and non-acupoint group, each group involve 10 SHR. And another normal control group involves 10 SD rats of normal blood pressure with the same weight with SHR. In the TaiChong group, acupuncture bilateral Taichong points (located between the 1st and the 2nd metatarsal of dorsal hind); while in non-acupoint group acupuncture bilateral non-acupoint (located at the indentation between the 3rd and 4th metatarsal of dorsal hind). As rats awake, in Taichong group and non-acupoint group, daily acupuncture 5 minutes per times, twisting nedlle 80±5 times/min frequently, Twist nedlle 180±5 degrees in rang. In Taichong group, the needle towards Yongquang but not penetrate or reach the plantar skin, rather than in non-acupoint group, needle perpendicular to the skin and penetrate the skin.5 minutes after acupuncture, using BP-6 non-invasive blood pressure measuring system for tail arterial blood pressure measurement in rats. Acupuncture treatment continnues totally 7 weeks, all the rats brains were removed at the end, then separated in the ice for the medulla oblongata. And the medulla oblongata is cracked, to Sonication, desalted, seperated for proteins with the 2-DE. Geting the gel images with Typhoon9200 scanner, analyzing the differences between the various groups of protein with Imagesaster 6.0. With MALDI-TOF-TOF tandem MS analysis, protein mass image was obtained. And with the GPS software, to search and identify protein in the MASCOT database identification, then used enzyme-linked immunosorbent assay (ELISA) test to further verify the identification of the protein.Results1. The influence of acupuncture at Taichong point (LR3) or non-acupoints on the blood pressure SHR’blood pressure. (1) Comparison results among groups: The original, before acupuncture, systolic blood pressure of Taichong group, and the non-acupoint group and model group were not significantly different (all P> 0.05). The 1st day in acupuncture, in Taichong group, and the non-acupoint group and model group, systolic blood pressure between the rats were not significantly different (all P> 0.05). The next day in acupuncture, compared with the model group and non-acupoints group, systolic blood pressure of Taichong group significantly decreased (P<0.01), prompting that systolic blood pressure of Taichong group started to decrease in the next day in acupuncture. The 3rd day in acupuncture, compared with model group, systolic blood pressure of the non-acupoint group were significantly higher, suggesting that the acupuncture at non-acupoint may be a non-benign stimulus, resulting in a significantly increased of systolic blood pressure in rats. On the 4th in acupuncture, systolic blood pressure of Taichong group and the non-acupoint group and model group were not significantly different (all P> 0.05). On the 5th day of acupuncture, compared with non-acupoint, Taichong group’s blood pressure decreased significantly (P<0.01). In acupuncture 6th, 7th day, compared with the model group and non-acupoints group, Taichong group’ systolic blood pressure were significantly lower (P<0.01), suggested acupuncture at Taichong points begin to stabilize the blood pressure level in the 5th. In different time periods, in the model group, Taichong group and non-Meridian Group, systolic blood pressure were significantly higher than normal group (P<0.01), prompted acupuncture at Taichong or non-acupoint were not able to restore blood pressure to normal levels in this study.(2) Comparison results in each group: Compared with the 1st day, Taichong group systolic blood pressure decreased significantly in the next day (P<0.01); but on the 3rd and the 4th day, the systolic blood pressure were increased in small rate (P> 0.05), and then began to decrease at the 5th day (P<0.01), until the 6th day,7th day the reducing of systolic blood pressure remained stable (P<0.01), suggested acupuncture at Taichong points begin to stabilize the blood pressure level in the 5th. Non-acupoints group’systolic blood pressure were not significantly different in different time (P> 0.05). Compared with the 3rd day, the model rats’systolic blood pressure significantly increased on the 6th day (P<0.01); compared with the 4th day, model group’s systolic blood pressure were significantly increased in the 6th day and the 7th days (P<0.01), prompting that systolic blood pressure of model group was rising. In different time, in nomal group, systolic blood pressure between the rats were not significantly different (all P> 0.05).2. The two-dimensional gel electrophoresis (2-DE) image of rat’s medulla in each group, and its analysis. In this study,2-DE image were successfully obtained for normal SD rats, SHR and the SHR after acupuncturing Taichong or non-acupoint; and 621±30 proteins in normal SD rats’medulla,621±20 proteins in SHR’s medulla, 621±40 proteins in medulla of SHR after acupuncturing Taichong,621±20 proteins in medulla of SHR after acupuncturing non-acupoint were found also. Compared with SHR in model group,88 proteins were found differentially expressed in medulla from Taichong group (expression ratio of protein was beyond 2 folds); 91 proteins were found differentially expressed in medulla oblongata from non-acupoints group (expression ratio of protein was beyond 2 folds),47 proteins were found differentially expressed in medulla oblongata from normal group (expression ratio of protein was beyond 2 folds).3. Differences in protein expression in the medulla of rats that dentified. (1) In medulla of Taichong group, compared with model group,5 proteins were down-regulated (expression ratio of protein was beyond 2 folds):Synapsin-1, NAD dependent deacetylase, electron transfer flavin protein and myelin basic protein; 6 proteins were up-regulated (expression ratio of protein was beyond 2 folds): glutamate dehydrogenase, glutathione S transferase, aldehyde dehydrogenase 2, Rho GDP dissociation inhibitor, DJ-1 protein and superoxide dismutase. (2) In medulla of non-acupoint group, compared with model group,1 proteins were up-regulated (expression ratio of protein was beyond 2 folds):electron transfer flavoprotein; 3 proteins were down-regulated (expression ratio of protein was beyond 2 folds): Heat shock protein 90, the Ubiquitin hydrolase isozyme and DJ-1 protein. (3)In medulla of Taichong group, compared with non-acupoints group,7 proteins were down-regulated (expression ratio of protein was beyond 2 folds):Heat shock protein 90, Synapsin-1, pyruvate kinase, NAD dependent deacetylases enzyme, protein kinase C inhibitor protein 1, Ubiquitin hydrolase isozymeand myelin basic protein; 6 proteins were up-regulated (expression ratio of protein was beyond 2 folds):glutamate dehydrogenase, aldehyde dehydrogenase 2, glutathione S transferase, Rho GDP dissociation inhibitor, DJ-1 protein and superoxide dismutase. (4) In medulla of model group, compared with normal group,6 proteins were down-regulated (expression ratio of protein was beyond 2 folds):pyruvate kinase, glutamate dehydrogenase, glutathione S transferase, aldehyde dehydrogenase 2, Ubiquitin hydrolase isozyme and superoxide dismutase; 7 proteins were up-regulated (expression ratio of protein was beyond 2 folds):Heat shock protein 90, Synapsin-11, NAD dependent deacetylase, electron transfer flavin protein, protein kinase C inhibitor protein 1, and myelin basic protein. (5) In medulla of Taichong group, compared with normal group,3 proteins were down-regulated (expression ratio of protein was beyond 2 folds):superoxide dismutase and myelin basic protein;4 proteins were up-regulated (expression ratio of protein was beyond 2 folds): Heat shock protein 90, Rho GDP dissociation inhibitor, DJ-1 protein and superoxide dismutase. (6) In medulla of non-acupoint group, compared with the normal group, 4 proteins were down-regulated (expression ratio of protein was beyond 2 folds): glutamate dehydrogenase, aldehyde dehydrogenase 2, glutathione S transferase and over superoxide dismutase; 7 proteins were up-regulated (expression ratio of protein was beyond 2 folds):Heat shock protein 90, synapsin 1, NAD dependent deacetylase, protein kinase C inhibitor protein 1, Ubiquitin hydrolase isozyme, DJ-1 protein and myelin basic protein.5. results of ELISA(1) The expression changes of glutathione S transferase in medullary among each group. In the medulla model group, compared with normal group, the expression of glutathione S transferase was significantly down-regulated (P<0.01). After acupuncture treatment, the expression of glutathione S transferase in the medulla of Taichong group were significantly up-regulated, compared with the model group, and the non-acupoint group, (P<0.01); but the expression of glutathione S transferase in the medulla of non-acupoint group has no significant difference form the model group (P> 0.01), and lower than the normal group (P<0.01). (2) The expression changes of aldehyde dehydrogenase in the medulla among each group. In the medulla model group, compared with normal group, the expression of aldehyde dehydrogenase was significantly down-regulated (P<0.01). After acupuncture treatment, the expression of aldehyde dehydrogenase in the medulla of Taichong group were significantly up-regulated, compared with the model group, and the non-acupoint group, (P<0.01); but the expression of aldehyde dehydrogenase in the medulla of non-acupoint group has no significant difference form the model group (P> 0.01), and lower than the normal group (P<0.01) (3) The expression changes of protein kinase C in medullary among each group. The expression of protein kinase C in the medulla of model group has no significant difference form the nomal group (P> 0.01); After acupuncture treatment, expression of protein kinase C in medullary of Taichong group has no significant changes from the model group, normal group, and the non-acupoint group (all P> 0.01). ELISA results of Glutathione S transferase, aldehyde dehydrogenase are consistent with the two-dimensional gel electrophoresis; meanwhile the ELISA results of protein kinase C are inconsistent with the results of two-dimensional gel electrophoresis of protein kinase C inhibitor protein 1.Conclusion1. Acupuncture Taichong can reduce the hypertension of SHR, but were not able to restore blood pressure to normal levels in this study.2. acupuncture at non-acupoints didn’t have an obvious effect on decreasing the blood pressure in SHR, even cause increase in SHR hypertension.3. Acupuncture Taichong Point can down-regulated expressions of the proteins that promote excitatory neurotransmitter, induced inflammatory response, oxidative phosphorylation and induce oxidative stress protein, and up-regulate amino acid oxidation chaperones and energy metabolism and oxidative stress in SHR’ medulla. Its mechanism is a multi-channel, multi-link, the combined effect of multiple targets.4. Acupuncture non-acupoint can down-regulated expressions of the proteins that promote energy metabolism, and up-regulate amino acid oxidation, protein catabolism and expressions of protein that induced inflammatory response in medullary of SHR. The mechanism is less means, fewer links, fewer target Point of simple effect.5. Acupoints effect and its mechanism has some specificity.更多还原