电针与手针四关穴对CUMS大鼠脑内谷氨酸代谢的影响

发布时间：2018-04-28 19:47

本文选题：抑郁 + 谷氨酸代谢　；参考：《广州中医药大学》2016年博士论文

【摘要】：目的：通过观察电针及手针四关穴对慢性不可预见性温和刺激致郁大鼠行为学及脑内谷氨酸代谢功能的影响,对比两种针刺方法改善CUMS大鼠抑郁行为的效应差异,并探讨其内在机制。实验一电针与手针四关穴改善CUMS大鼠行为的效应比较方法：1.动物分组雄性SD大鼠60只,体重220-270g,随机分为5组：空白组、模型组、电针组、手针组、药物组,每组12只。2.干预措施空白组3只/笼,正常饲养。模型组、电针组、手针组与药物组采用慢性不可预见性温和刺激联合孤养方法建立抑郁大鼠模型(选择7种不同温和刺激,每日施加一种刺激,连续施加35日)。行为学结果提示造模成功后开始治疗,治疗期间各组大鼠进行相同程度的捉抓与束缚。(1)模型组：建模成功后不予其他干预。(2)电针组：建模成功后第二日予电针四关穴,连续波,频率2Hz,留针30min,强度以见大鼠肢体微微颤抖而未嘶叫为度；治疗第一周每日一次,治疗第二、第三周每两日一次。(3)手针组：建模成功后第二日予针刺四关穴,施捻转行针法,平补平泻,每10min行针1次,每次捻转10s,留针30min；疗程同电针组。(4)药物组：造模成功第二日予利鲁唑灌胃治疗(4mg/kg, i.p),每12h灌胃一次,连续治疗3周。3.行为学评价指标在造模前、造模3周结束时及造模5周结束时对各组大鼠进行体质量、糖水偏好率、旷场实验及Morris水迷宫实验检测,以评价造模成功与否：在治疗1周结束时及治疗3周结束时对各组大鼠进行上述行为学检测,评价电针、手针与西药的治疗效应。4.统计分析采用SPSS20.0统计软件整理数据,计量资料采用均数±标准差表示；各行为学检测评分组间比较在重复测量方差分析基础上,进行正态性检验及方差齐性检验,符合正态分布及方差齐性检验则选用单因素方差分析,检验标准P0.05。若不符合者则选用Kruskal-Wallis H秩和检验观察整体差异,进一步采用wilcoxon秩和检验两两比较,采用SPSS调整后显著性结果,检验标准P0.05。实验室计量资料则根据正态性检验及方差齐性检验结果选用单因素方差分析或秩和检验。结果：1.电针、手针与利鲁唑对CUMS大鼠体质量的影响CUMS造模5周后,与空白组比较,各造模组体重显著下降(P0.05)；治疗3周后,与模型组比较,电针组、手针组与西药组体重显著增加(P=0.022,P=0.004,P0.001)；西药组与手针组疗效相当,电针组与西药组差异有统计学意义(P=0.019)。2.电针、手针与利鲁唑对CUMS大鼠糖水偏好的影响CUMS造模5周后,与空白组比较,各造模组糖水偏好率显著降低(P0.05)；治疗3周后,与模型组比较,电针组、手针组与西药组糖水偏好率显著升高(均为P0.001)；各组疗效比较无统计学差异(P0.05)。3.电针、手针与利鲁唑对CUMS大鼠旷场实验评分的影响①水平活动：CUMS造模5周后,与空白组比较,各造模组水平活动显著减少(P0.001)；治疗3周后,与模型组比较,电针组、手针组与西药组水平活动均有增加,手针与西药组有统计学差异(P=0.234,P=0.038,P=0.038)；手针组与西药组疗效相当(P0.05)。②垂直活动：CUMS造模5周后,与空白组比较,各造模组垂直活动显著减少(P0.05)；治疗3周后,与模型组比较,电针组、手针组与西药组垂直活动均有增加,手针组有统计学差异(P=0.58,P=0.035,P=0.177)。4.电针、手针与利鲁唑对CUMS大鼠Morris水迷宫评分的影响①潜伏期：CUMS造模5周后,与空白组比较,各造模组潜伏期明显高于空白组(P0.05)；治疗3周后,与模型组比较,电针组、手针组与西药组潜伏期均有缩短,手针组、西药组有统计学差异(P=0.168,P=0.016,P=0.007)；手针组与西药组疗效比较无统计学差异(P0.05)。②总距离：CUMS造模5周后,与空白组比较,各造模组移动总距离明显大于空白组(P0.001)；治疗3周后,与模型组比较,电针组、手针组与西药组移动总距离均有缩短,电针组有统计学差异(P=0.015,P=0.079,P=0.123)。实验二电针与手针四关穴改善CUMS大鼠脑内As功能的效应比较方法：1.动物分组与处理同实验一2.评价指标采用Western blot技术与Q-PCR技术观察治疗结束时各组大鼠海马与前额叶皮层星形胶质细胞胶质纤维酸性蛋白(GFAP)及其mRNA表达量的变化。3.数据处理与统计同实验一结果：1.海马脑区GFAP与GFAP mRNA表达量①GFAP含量：与空白组比较,模型组GFAP含量降低(P=0.002)；与模型组比较,电针组、手针组与西药组GFAP含量明显升高(P=0.003,P=0.007,P=0.003)；电针组、手针组与西药组比较无统计学差异(P0.05)。②GFAP mRNA表达量：与空白组比较,模型组GFAP mRNA表达量降低(P0.001)；与模型组比较,电针组、手针组与西药组GFAP mRNA表达量明显升高(P=0.003,P=0.011,P=0.001)；电针组、手针组与西药组比较无统计学差异(P0.05)。2.前额叶皮层脑区GFAP与mRNA表达量①GFAP含量：与空白组比较,模型组GFAP含量降低(P=0.001)；与模型组比较,电针组、手针组与西药组GFAP含量明显升高(P=0.019,P=0.004,P=0.001)；电针组、手针组与西药组比较无统计学差异(P0.05)。②GFAP mRNA表达量：与空白组比较,模型组GFAP mRNA表达量降低(P=0.006)；与模型组比较,电针组、手针组与西药组GFAP mRNA表达量均有升高,电针组、西药组有统计学差异(P=0.016,P=0.061,P=0.017)；电针组与西药组比较无统计学差异(P0.05)。实验三电针与手针四关穴改善CUMS大鼠脑内GS功能的效应比较方法：1.动物分组与处理同实验一2.评价指标采用W estern blot技术与Q-PCR技术观察治疗后各组大鼠海马与前额叶皮层内谷氨酰胺酶(GS)及其mRNA表达量的变化。3.数据处理与统计同实验一结果：1.海马脑区GS及其mRNA表达量①GS含量：与空白组比较,模型组GS含量降低(P0.001)；与模型组比较,电针组、手针组与西药组GS含量明显升高(均为P0.001)；电针组、手针组与西药组比较无统计学差异(P0.05)。②GS mRNA表达量：与空白组比较,模型组GS mRNA表达量降低(P=0.018)；与模型组比较,电针组、手针组与西药组GS mRNA表达量均有升高,电针组与西药组有统计学差异(P=0.019,P=0.036,P=0.007)；电针组、与西药组比较无统计学差异(P0.05)。2.前额叶皮层脑区GS及其mRNA表达量①GS含量：与空白组比较,模型组GS含量降低(P=0.002);与模型组比较,电针组、手针组与西药组GS含量明显升高(P=0.032,P=0.015,P=0.001)；电针组、手针组与西药组比较无统计学差异(P0.05)。②GS mRNA表达量：与空白组比较,模型组GS mRNA表达量降低(P=0.006)；与模型组比较,电针组、手针组与西药组GS mRNA表达量均有升高,电针组与西药组有统计学差异(P=0.018,P=0.015,P0.001)；电针组与西药组比较无统计学差异(P0.05)。实验四基于1H-[13C]-NMR比较变电针与手针四关穴对CumS大鼠脑内谷氨酸代谢的影响方法：1.动物分组与处理同实验一2.评价指标采用1H-[13C]-NMR技术观察治疗后各组大鼠海马与前额叶皮层内经星形胶质细胞代谢产生的谷氨酸(Glu-C4)、谷氨酰胺(Gln-C4)及γ-氨基丁酸(GABA-C2)含量的变化。3.数据处理与统计同实验一结果(1)海马谷氨酸-C4、谷氨酰胺-C4及Y-氨基丁酸-C2的富集量谷氨酸-C4：CUMS造模5周后,与空白组比较,模型组富集量显著下降(P=0.034)；治疗3周后,与模型组比较,电针组、手针组与西药组富集量有上升趋势,但无统计学差异(P0.05)。谷氨酰胺-C4：CUMS造模5周后,与空白组比较,模型组富集量显著下降(P=0.034)；治疗3周后,与模型组比较,电针组、手针组与西药组富集量有上升趋势,但无统计学差异(P0.05)。Y-氨基丁酸-C2-CUMS造模5周后,与空白组比较,模型组富集量显著降低(P0.001);治疗3周后,与模型组比较,电针组、手针组与西药组富集量明显上调(P=0.001,P0.001)；手针组优于电针组,差异有统计学意义(P=0.015),与西药组疗效相当(P0.05)。(2)前额叶皮层谷氨酸-C4、谷氨酰胺-C4及Y-氨基丁酸-C2的富集量谷氨酸-C4:CUMS造模5周后,与空白组比较,模型组富集量显著下降(P0.001)；治疗3周后,与模型组比较,电针组、手针组与西药组富集量有显著回升(P=0.002,P=0.001, P=0.002),电针组、手针组与西药组比较无统计学差异(P0.05)。谷氨酰胺-C4-CUMS造模5周后,与空白组比较,模型组富集量显著降低(P=0.001)；治疗3周后,与模型组比较,电针组、手针组与西药组富集量显著升高(P=0.019,P=0.007, P=0.001)；电针组、手针组与西药组比较无统计学差异P0.05)。Y-氨基丁酸-C2:CUMS造模5周后,与空白组比较,模型组富集量显著降低(P=0.034);治疗3周后,与模型组比较,电针组、手针组与西药组差异无统计学意义(P0.05)。结论：电针与手针四关穴可改善CUMS大鼠体重降低、糖水偏好率下降、活动能力减退等抑郁样行为,与利鲁唑相当；其中手针四关穴在改善自主活动及探索行为方面有优于电针与利鲁唑的趋势。电针与手针四关穴可提高CUMS大鼠海马及前额叶皮层的GFAP、GS、谷氨酸-C4、谷氨酰胺-C4及Y-氨基丁酸-C2的表达量；因此调控脑内谷氨酸的分解与代谢可能是其发挥抗抑郁效应的内在机制；其中手针四关穴在提高星形胶质细胞对谷氨酸代谢量方面有优于电针趋势,与利鲁唑类同。
[Abstract]:Objective: To observe the effect of electroacupuncture and hand needle four point on the behavior of rats with chronic unforeseeable mild stimulation and the function of glutamic acid metabolism in the brain, and compare the difference of the effect of two kinds of acupuncture methods to improve the depressive behavior of CUMS rats, and explore the internal mechanism. The effect of the experimental acupuncture and the hand needle four point on the behavior of CUMS rats is improved. 1. male SD rats were divided into 5 groups randomly: blank group, model group, electroacupuncture group, hand needle group, drug group, 12.2. intervention group in each group 3 / cage, normal feeding, model group, electroacupuncture group, hand acupuncture group and drug group established by chronic unforeseeable mild stimulation combined soliton method. The rat model of depression was selected (7 different mild stimuli, one stimulus was exerted daily for 35 days). The behavioral results suggested that the model was successfully treated after the model was successful. (1) the model group: the model group did not interfere with his intervention. (2) the electroacupuncture group: Second days after the successful modeling, the electroacupuncture group was given. Four, continuous wave, continuous wave, frequency 2Hz, retention of needle 30min, strength to see the limbs of rats trembling and not hoarse; treatment for the first week, second, third weeks once every two days. (3) hand acupuncture group: the second days after the modeling successful acupuncture four point, twisting and transferring needle, flat supplement, 1 times each 10min needle, twist 10s, leave needle 30m each time In; course of treatment with electroacupuncture group. (4) drug group: 4mg/kg (i.p) was given to ralurazole (i.p) for second days, and every 12h was given to the stomach once, and the evaluation indexes of.3. behavior for 3 weeks were at the end of the model, at the end of the model and at the end of the model for 5 weeks, the body mass, the sugar water preference rate, the open field experiment and the Morris water maze test were tested. Test to evaluate the success of the model: at the end of 1 weeks of treatment and at the end of the 3 week of treatment, the behavioral tests were carried out at the end of the 1 weeks and at the end of the treatment. The statistical analysis of the therapeutic effect of electroacupuncture, hand needle and Western Medicine was made by the statistical analysis of SPSS20.0 software, and the measurement data were expressed by mean number of standard deviation, and the comparison between the scores of behavioral test scores was compared. On the basis of the repeated measurement of variance analysis, the normal test and variance homogeneity test were carried out. The single factor variance analysis was selected according to the normal distribution and variance homogeneity test. If the standard P0.05. was not conformed, the Kruskal-Wallis H rank sum test was selected to observe the whole difference, and the Wilcoxon rank sum test 22 was compared, and SPSS was adopted. After the adjustment, the standard P0.05. laboratory measurement data were selected by single factor variance analysis or rank sum test according to normal test and variance homogeneity test. Results 1. electroacupuncture, hand acupuncture and ralurazole effect on the body mass of CUMS rats. After 5 weeks, the body weight of each model group decreased significantly (P0.05) compared with the empty white group. After 3 weeks of treatment, compared with the model group, the weight of electroacupuncture group, hand acupuncture group and Western medicine group increased significantly (P=0.022, P=0.004, P0.001), the curative effect of the western medicine group and the hand needle group was equal, the difference between the electroacupuncture group and the western medicine group was statistically significant (P=0.019).2. electroacupuncture, the effect of hand acupuncture and ralurazole on the sugar water preference of CUMS rats CUMS after the establishment of CUMS model, compared with the blank group. The sugar water preference rate of each model group decreased significantly (P0.05). After 3 weeks of treatment, the sugar water preference rate of the electroacupuncture group, the hand needle group and the western medicine group increased significantly (P0.001) compared with the model group, and there was no statistical difference (P0.05).3. electroacupuncture, hand needle and ralurazole on the open field test score of CUMS rats: level activity: CUMS model 5 After 3 weeks, compared with the model group, the level activity of the acupuncture group and the western medicine group increased, the hand needle and the western medicine group had a statistical difference (P=0.234, P=0.038, P=0.038), and the hand acupuncture group and the western medicine group had the same effect (P0.05). (2) the vertical activity: the CUMS model was made for 5 weeks. After 3 weeks of treatment, the vertical activity of the electroacupuncture group, hand needle group and Western medicine group increased, and the hand acupuncture group had statistical difference (P=0.58, P=0.035, P=0.177).4. electroacupuncture, hand acupuncture and ralurazole on the CUMS rat Morris water maze score. The incubation period: CUMS After 5 weeks, the incubation period of each model group was significantly higher than that in the blank group (P0.05). After 3 weeks of treatment, the latency of electroacupuncture group, hand needle group and Western medicine group shortened, hand acupuncture group and Western medicine group had statistical difference (P=0.168, P=0.016, P= 0.007), and there was no statistical difference between the hand needle group and the western medicine group (P0.05). Total distance: after 5 weeks of CUMS modeling, the total distance of each model group was significantly greater than that of the blank group (P0.001). After 3 weeks of treatment, the total distance between the electroacupuncture group, the hand acupuncture group and the western medicine group shortened, and the electroacupuncture group had a statistical difference (P=0.015, P=0.079, P=0.123). The experiment two electroacupuncture and hand needle four point improved CUMS big. The comparison of the effects of As function in the rat brain: 1. group and treatment of animals with the same experiment one 2. evaluation index using Western blot technique and Q-PCR technology to observe the changes of astroglia glial fibrillary acidic protein (GFAP) and mRNA expression in the hippocampus and prefrontal cortex of rats at the end of the treatment and.3. data processing and statistics of the same experiment Results: 1. the content of GFAP and GFAP mRNA expression in the hippocampus of the hippocampus: the content of GFAP: compared with the blank group, the GFAP content of the model group decreased (P=0.002). Compared with the model group, the GFAP content in the electroacupuncture group, the hand acupuncture group and the western medicine group increased significantly (P=0.003, P=0.007, P=0.003), and there was no statistical difference between the hand acupuncture group and the western medicine group (P0.05). Compared with the blank group, the expression of GFAP mRNA in the model group decreased (P0.001). Compared with the model group, the expression of GFAP mRNA in the electroacupuncture group, hand needle group and Western medicine group increased significantly (P=0.003, P=0.011, P=0.001), and there was no significant difference between the hand acupuncture group and the western medicine group (P0.05) in the electroacupuncture group (P0.05).2. prefrontal cortex brain region GFAP and mRNA expression content (1) content Compared with the blank group, the content of GFAP in the model group decreased (P=0.001). Compared with the model group, the content of GFAP in the electroacupuncture group and the western medicine group increased significantly (P=0.019, P=0.004, P=0.001), and there was no statistical difference between the acupuncture group and the western medicine group (P0.05). (2) the mRNA expression of GFAP in the group was lower than that in the blank group, and the GFAP mRNA expression in the model group was reduced. P=0.006); compared with the model group, the expression of GFAP mRNA in electroacupuncture group, hand acupuncture group and Western medicine group increased, and there was statistical difference between the electroacupuncture group and the western medicine group (P=0.016, P=0.061, P=0.017); there was no statistical difference between the electroacupuncture group and the western medicine group (P0.05). Experimental three electroacupuncture and hand needle four points improved the effect of GS function in the brain of CUMS rats: 1. W estern blot and Q-PCR techniques were used to evaluate the changes of glutaminase (GS) and mRNA expression in the hippocampus and prefrontal cortex of rats by W estern blot technique and Q-PCR technique. The results were as follows: GS in the hippocampus and the mRNA expression of GS in the hippocampus: compared with the blank group The content of GS in the model group decreased (P0.001); compared with the model group, the GS content in the electroacupuncture group and the western medicine group increased significantly (all P0.001); the electroacupuncture group, the hand acupuncture group and the western medicine group had no statistical difference (P0.05). The GS mRNA expression was compared with the blank group, the GS mRNA expression in the model group decreased (P=0.018); compared with the model group, the electroacupuncture group was compared with the model group, and the electroacupuncture group, The expression of GS mRNA in the hand acupuncture group and the western medicine group increased, and there was a statistical difference between the electroacupuncture group and the western medicine group (P=0.019, P=0.036, P=0.007). There was no statistical difference between the electroacupuncture group and the western medicine group (P0.05) the GS of the prefrontal cortex and the mRNA expression of GS in the.2. prefrontal cortex (P0.05): the GS content of the model group was lower than that in the blank group (P=0.002); compared with the model group, it was compared with the model group. The content of GS in electroacupuncture group, hand acupuncture group and Western medicine group increased significantly (P=0.032, P=0.015, P=0.001), and there was no statistical difference between the acupuncture group and the western medicine group (P0.05). The expression of GS mRNA was compared with the blank group, and the GS mRNA expression in the model group decreased (P=0.006), and the expression of GS mRNA in the electroacupuncture group and the hand acupuncture group and the western medicine group were all compared with the model group. There was a statistical difference between the electroacupuncture group and the western medicine group (P=0.018, P=0.015, P0.001), and there was no statistical difference between the electroacupuncture group and the western medicine group (P0.05). Experiment four based on 1H-[13C]-NMR, the methods of comparing the effects of variable electroacupuncture and hand needle four point on the metabolism of glutamic acid in the brain of CumS rats: 1. animal groups and treatment with experimental one 2. evaluation index using 1H-[13 C]-NMR technique observed the changes of glutamic acid (Glu-C4), glutamine (Gln-C4) and gamma aminobutyric acid (GABA-C2) in the hippocampus and prefrontal cortex of rats after treatment with.3. data processing and statistical data processing and statistical results (1) hippocampus glutamic acid -C4, glutamine -C4 and Y- aminobutyric acid -C2 concentration of glutamic acid After 5 weeks of acid -C4:CUMS, the concentration of the model group decreased significantly compared with the blank group (P=0.034). After 3 weeks of treatment, the concentration of the electroacupuncture group, the hand needle group and the western medicine group increased, but there was no statistical difference (P0.05). After 5 weeks of the glutamine -C4:CUMS model, the concentration of the model group decreased significantly (P=0.034 After 3 weeks of treatment, compared with the model group, the concentration of the electroacupuncture group, the hand needle group and the western medicine group increased, but there was no statistical difference (P0.05).Y- aminobutyric acid -C2-CUMS for 5 weeks. Compared with the blank group, the concentration of the model group decreased significantly (P0.001). After 3 weeks of treatment, the concentration of the electroacupuncture group, the hand needle group and the western medicine group was obviously higher than the model group. P=0.001 (P0.001); hand acupuncture group was better than electroacupuncture group, the difference was statistically significant (P=0.015), compared with the western medicine group (P0.05). (2) after 5 weeks of glutamic acid -C4 in prefrontal cortex, glutamine -C4 and Y- aminobutyric acid -C2, the concentration of the model group decreased significantly (P0.001) compared with the empty white group (P0.001); after 3 weeks, and after treatment, Compared with the model group, the concentration of the electroacupuncture group and the western medicine group increased significantly (P=0.002, P=0.001, P=0.002). There was no statistical difference between the electroacupuncture group and the western medicine group (P0.05). After 5 weeks of the glutamine -C4-CUMS model, the concentration of the model group decreased significantly (P=0.001). After 3 weeks of treatment, the electroacupuncture group was compared with the model group, electroacupuncture group, The concentration of the hand acupuncture group and the western medicine group increased significantly (P=0.019, P=0.007, P=0.001), and in the electroacupuncture group, there was no statistical difference between the hand acupuncture group and the western medicine group (P0.05). After 5 weeks, the concentration of.Y- aminobutyric acid -C2:CUMS was significantly reduced (P=0.034) compared with the blank group. After 3 weeks of treatment, the difference between the electroacupuncture group, the hand acupuncture group and the western medicine group was compared with the model group. No statistical significance (P0.05). Conclusion: Electroacupuncture and hand needle four point can improve the weight loss of CUMS rats, decrease the sugar water preference rate and decrease the activity ability, which is equivalent to raluzole, and the four point of hand needle is superior to electroacupuncture and raluzole in improving autonomic activity and exploration behavior. Electroacupuncture and hand needle four point can be used. Improve the expression of GFAP, GS, glutamic acid -C4, glutamine -C4 and Y- aminobutyric acid -C2 in the hippocampus and prefrontal cortex of CUMS rats; therefore, the regulation of the decomposition and metabolism of glutamic acid in the brain may be an intrinsic mechanism for its antidepressant effect. The four point of hand needle is superior to electricity in improving the metabolism of glutamic acid in glutamic cells. The needle trend is the same as that of ralurazole.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

【相似文献】