| ObjectiveTo compare the effects of electro-acupuncture and manul acupuncture on Siguan acupoints by observing the behavioral changes of CUMS rats.To find out the antidepression biochemical mechanism of acupuncture by observing brain glutamate metabolism changes of CUMS rats.Part One:Comparative study of EA, MA and Riluzole on rats behavioral tests Methods1. Animals60 male SD rats weighted between 200 and 270g were randomly divided into 5 groups:control group, model group, electrol-acupunture group (EA group), manul acupuncture group (MA group), and riluzole group. Each group has 12 rats.2. Experimental procedureRats in control group housed 3 per cage with free access to food and water. Rats in model group, EA group, MA group and riluzole group housed separated and receive 7 different kinds of chronic unpredictable mild stress for 35 days, (one kind of stress per day). During treatment, all 5 groups receive the high flat fixation.After completed CUMS successfully:(1) Model group:free access to food and water.(2) EA group:receive electro-acupunture treatment on Siguan acupoints with a 2Hz continuous wave. The first week once per day, next two weeks once two day(3) MA group:receive manul acupuncture treatment on Siguan acupoints with a 10s soft rotation once per 10mins, needles retain for 30mins,(4) Riluzole group:once every 12 h, lasts for 3 weeks.3. Behavioral testsCarry out behavioral tests on 0th day,21th day,35th day,42th day, and 56th day. With weight test, surcrose preference test, open field test and Morris water maze test to evaluate the CUMS model and treatment effects of EA, MA and riluzole.4. Statistical analysisUsing SPSS20.0 statistical software, measurement data with mean+/-standard deviation;The behavioral science test scores on the basis of repetitive measure analysis of variance, normality test and f test, accord with normal distribution and f test is to choose the single factor analysis of variance, if do not accord with the criterion to choose Kruskal Wallis H-rank and inspection, two comparison using nonparametric multiple comparison (Behrens-Fisher test program), USES the P value after correction, inspection standard (P< 0.05. Laboratory measurement data are based on normal distribution and f test results, respectively, using the single factor analysis of variance and rank and inspection.Results:1. Weight testAfter 5 weeks CUM stress, compare with the control group, weight of model group decreased significantly (P<0.05). After 3 weeks treatment, compared with model group, weight of EA group, MA group and riluzole group increased significantly (P=0.022, P= 0.022, P<0.01).2. Sucrose preference testAfter 5 weeks CUM stress, compared with control group, sucrose preference rate of model group significantly decreased (P<0.05). After 3 weeks treatment, compared with model group, sucrose preference rate of EA group, MA group and riluzole group increased significantly (P<0.01).3. Open field test(1)Horizon activity:After 5 weeks CUM stress, compared with control group, activities of model group significantly reduced (P<0.05). After 3 weeks treatment, compared with model group, activities of EA group, MA group and riluzole group increased, increase of MA group and riluzole group was statistically(P=0.234, P=0.234, P=0.038).(2) Vertical activities:After 5 weeks CUM stress, compared with control group, activities of model group significantly reduced (P<0.05). After 3 weeks treatment, compared with model group, activities of EA group, MA group and riluzole group increased (P=0.58,P=0.58, P=0.177).4. Morris water maze test(1)Escape latency:After 5 weeks CUM stress, compared with control group, time of model group used to find the flat increased (P<0.05). After 3 weeks treatment, compared with model group, time of EA group, MA group and riluzuole group shortened (P=0.168, P=0.168, P=0.007).(2)Total distance:After 5 weeks CUM stress, compared with control group, total distance of model group increased sinificantly (P<0.05). After 3 weeks treatment, compared with model group, total distance of EA group, MA group and riluzuole group shortened (P=0.015, P=0.015, P= 0.123).Part Two:Comparative study of EA, MA and Riluzole on fundction of Astrocyte Methods1. Animals and experimental procedure:same as part one 2. Evaluation indexUsing Western blot and Q-PCR technology to detect relative expression quantity of GFAP and GFAP mRNA in hippocampus and the prefrontal cortex of rats.3. Statistical analysis:same as part oneResults:1. GFAP and GFAP mRNA relative expression quantity in hippocampus(1) After 5 weeks CUM stress, compared with control group, GFAP expression quantity of model decreased (P=0.002). After 3 weeks treatment, compared with model group, GFAP expression quantity of EA group, MA group and riluzole increased significantly (P=0.003, P=0.003, P=0.003).(2) After 5 weeks CUM stress, compared with control group, GFAP mRNA expression quantity of model decreased (P<0.01). After 3 weeks treatment, compared with model group, GFAP mRNA expression quantity of EA group, MA group and riluzole group increased significantly (P=0.003, P=0.003, P=0.001).2. GFAP and GFAP mRNA relative expression quantity in prefontal cortex(1) After 5 weeks CUM stress, compared with control group, GFAP expression quantity of model group were decreased (P=0.001). After 3 weeks treatment, compared with model group, GFAP expression quantity of EA group, MA group and riluzole group increased significantly (P=0.019, P=0.019,P=0.001).(2) After 5 weeks CUM stress, compared with control group, GFAP mRNA expression quantity of model group were decreased (P=0.006). After 3 weeks treatment, compared with model group, GFAP mRNA expression quantity of EA group, MA group and riluzole group increased (P=0.016, P=0.016,P=0.017).Part Three:Comparative study of EA, MA and Riluzole on fundction of GSMethods:1. Animals and experimental procedure:same as part one2. Evaluation indexUsing Western blot and Q-PCR technology to detect relative expression quantity of GS and GS mRNA in hippocampus and the prefrontal cortex of rats.3. Statistical analysis:same as part oneResults:1. GS and GS mRNA relative expression quantity in hippocampus(1) Compared with control group, GS expression quantity of model group were decreased (P<0.01). After 3 weeks treatment, compared with model group, GS expression quantity of EA group, MA group and riluzole group increased significantly(P<0.01)(2) Compared with control group, GS mRNA expression quantity of model group were decreased (P=0.018). After 3 weeks treatment, compared with model group, GS mRNA expression quantity of EA group, MA group and riluzole group increased significantly (P<0.019, P=0.019, P=0.007).2. GS and GS mRNA relative expression quantity in prefontal cortex(1) Compared with control group, GS expression quantity of model group were decreased (P=0.018). After 3 weeks treatment, compared with model group, GS expression quantity of EA group, MA group and riluzole group increased significantly (P=0.019, P=0.019, P=0.007).(2) Compared with control group, GS mRNA expression quantity of model group were decreased (P=0.018). After 3 weeks treatment, compared with model group, GS mRNA expression quantity of EA group, MA group and riluzole group increased significantly (P=0.018, P=0.018, P<0.01)Part Four:Comparative study of EA, MA and Riluzole on metabolism of Glutamate Methods:1. Animals and experimental procedure:same as part one2. Evaluation indexDetect the metabolism of glutamate transmited by astrocyte in hippocampus and prefrontal cortex of rats with 1H-[13C]-NMR, record the content of Glu-C4, Gln-C4, and GABA-C23. Statistical analysis:same as part oneResults:(1) The enrichment of Glu-C4, Cln-C4 and GABA-C2 in hippocampusGlu-C4:Compared with control group, the enrichment of model group was significantly decreased (P=0.034). After 3 weeks treatment, compared with model group, the enrichment of EA group, MA group and riluzole group has a rising trend without statistical difference (P>0.05).Cln-C4:Compared with control group, the enrichment of model group was significantly decreased (P=0.034). After 3 weeks treatment, compared with model group, the enrichment of EA group, MA group and riluzole group has a rising trend without statistical difference (P>0.05).GABA-C2:Compared with control group, the enrichment of model group was significantly decreased (P<0.001). After 3 weeks treatment, compared with model group, the enrichment of EA group, MA group and riluzole group has a rising trend without statistical difference (P=0.001, P<0.01)(2) The enrichment of Glu-C4, Cln-C4 and GABA-C2 in prefontal cortexGlu-C4:Compared with control group, the enrichment of model group was significantly decreased (P<0.01). After 3 weeks treatment, compared with model group, the enrichment of EA group, MA group and riluzole group has a rising trend without statistical difference (P=0.002, P= 0.002, P=0.002)Cln-C4:Compared with control group, the enrichment of model group was significantly decreased (P=0.001). After 3 weeks treatment, compared with model group, the enrichment of EA group, MA group and riluzole group has a rising trend without statistical difference (P=0.019,P=0.019, P=0.001).GABA-C2:Compared with control group, the enrichment of model group was significantly decreased (P=0.034). After 3 weeks treatment, compared with model group, the enrichment of EA group, MA group and riluzole group has a rising trend without statistical difference (P>0.05).Conclusion:Electro-acupuncture and manul acupuncture on Siguan acupoints can reverse the behavioral changes of CUMS rats. There is a clearly trend that manul acupuncture has a better effect on automatic and exploratory activities.Electro-acupuncture and manul acupuncture on Siguan acupoints can regulate the metabolism of glutamate in the brain of CUMS rats which may be the antidepression biochemical mechanism of acupuncture. There is a clearly trend that manul acupuncture has a better effect on regulating the metabolism of Glutamate. |
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