基于嘌呤能信号转导途径探讨电针治疗抑郁大鼠的中枢机制

Discussion of the Central Mechanism of Electro-targeted Treatment in Depressed Rats Based on the Purinegic Signal Transduction Pathway

目的基于嘌呤能信号转导途径理论,观察电针治疗抑郁大鼠的中枢机制。方法选取SPF级SD大鼠150只,雌雄各半,查随机数字表将大鼠分为空白对照组、模型组、丙戊酸钠组、氟西汀组和针刺组,每组30只。除对照组外,其余4组采用慢性温和不可预见性应激刺激(chronic unpredictable mild stress,CUMS)建立大鼠抑郁模型,针刺组、丙戊酸钠组和氟西汀组在造模的同时进行治疗,实验全程共28 d。针刺组选取四神聪、内关、三阴交穴;丙戊酸钠组灌胃给予丙戊酸钠(300 mg·kg-1)药液,1次/d;氟西汀组灌胃给予氟西汀(1.8 mg·kg-1)药液,1次/d。采用糖水消耗实验、旷场实验、强迫游泳实验对大鼠进行行为学观察。之后选取10只大鼠采用免疫组化法结合图像分析技术检测海马脑区P2X7、A1、A2A阳性神经元的表达;另外10只采用Western blot法观察三磷酸二磷酸水解酶(E-NTPD/CD39)、核苷酸焦磷酸酶/磷酸二酯酶(NPPs)、5'-核苷酸酶(e5NT/CD73)、腺苷脱氨酶(ADA)和黄嘌呤氧化酶(XO)蛋白表达水平;其余10只采用脑部微透析技术收集脑组织液样品,然后用高效液相色谱法(HPLC)检测大鼠脑组织样品中腺苷、三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、单磷酸腺苷(AMP)以及尿酸的含量。结果与对照组相比,模型组大鼠体质量、糖水消耗、旷场实验的移动距离与平均速度明显减少,静止时间明显增加,强迫游泳实验中模型组的攀爬时间和游泳时间明显减少(P<0.05);而电针组、氟西汀组、丙戊酸钠组均有所好转。与对照组相比,模型组大鼠脑组织中P2X7、CD73、CD39、A2A受体细胞层数变多且较厚,排列紧密,结构清晰;ADA、腺苷激酶(ADK)、A1细胞较少,层数变薄,神经元排列紊乱、疏松,甚至有神经元缺失(P<0.05);电针组、氟西汀组与丙戊酸钠组细胞变化程度相对较轻。与对照组相比,模型组大鼠海马组织中P2X7明显升高,ATP相对减少(P<0.05);而电针组、氟西汀组、丙戊酸钠组二者均有所缓解。模型组大鼠海马组织中A1、A2A、CD39、CD73、ENPP1、ADA、ENT1、ENT2相较于对照组均有所增高,而ADK则有所降低(P<0.05)。模型组大鼠海马中AMP、腺苷(Adenosine)、肌苷(Inosine)、γ-氨基丁酸(GABA)相较于对照组均有所降低,而谷氨酸(Glu)则有所升高。结论电针可明显改善抑郁症(major depressive disorder,MDD)模型大鼠的抑郁行为情况,其机制可能是下调P2X7的表达来减少神经内炎症因子的释放,同时上调腺苷(ADO)的浓度来增强A1受体的表达,从而抑制Ca2+内流,降低神经兴奋性中毒,进而减少细胞损伤。

Objective Based on the purinegic signal transduction pathway theory,the central mechanism of electrotherapy in the treatment of depressed rats was observed.Methods 150 SPF-grade SD rats were selected,half male and female,the rats were divided into blank control group,model group,sodium valproate group,fluoxetine group and acupuncture group,with 30 rats in each group.In addition to the control group,the remaining four groups used chronic unpredictable mild stress(CUMS)to establish a rat depression model,and the acupuncture group,sodium valproate group and fluoxetine group were treated at the same time as modeling,and the whole experiment was completed for a total of 28 days.The acupuncture group selected“Sishencong(EX-HN1)”“Neiguan(PC6)”and“Sanyinjiao(SP6)”;Sodium valproate group was given sodium valproate(300 mg·kg-1)for gavage once a day,Fluoxetine was given gastric gavage(1.8 mg·kg-1)once a day.Behavioral observations were performed on rats by sugar water consumption experiment,open field experiment and forced swimming experiment.Then,10 rats were selected to detect the expression of P2X7,A1 and A2A positive neurons in the hippocampal brain region by immunohistochemistry combined with image analysis technology.In addition,Western-blotting technology was used to observe the expression levels of triphosphate diphosphate hydrolase(E-NTPD/CD39),nucleotide pyrophosphatase/phosphodiesterase(NPPs),5'-nucleotidase(e5NT/CD73),adenosine deoxygenase(ADA)and xanthine oxidase(XO)proteins.The remaining 10 were collected by brain microdialysis technology,and then the content of adenosine,ATP,ADP,AMP and uric acid in rat brain tissue samples were detected by high performance liquid chromatography(HPLC).Results Compared with the control group,the travel distance and average speed of the rats in the model group were significantly lower than those in the control group,the resting time was significantly more than that of the control group,and the climbing time and swimming time of the experimental group in the forced swimming experiment were significantly smaller than those in the control group(P<0.05).The electroacupuncture group,fluoxetine group and sodium valproate group all improved.Compared with the control group,the P2X7,CD73,CD39 and A2A receptor cells in the brain tissue of rats in the model group had more layers,thicker,tightly arranged and clear structure.ADA,ADK,A1 cells are few,the number of layers was thinned,the neuronal arrangement is disordered,loose,and even neuronal deletion(P<0.05).The degree of cell change in the electroacupuncture group,fluoxetine group and sodium valproate group was relatively mild.Compared with the control group,P2X7 was significantly elevated in rat hippocampal tissues in the model group and ATP was relatively reduced(P<0.05).The electroacupuncture group,fluoxetine group and sodium valproate group were all relieved.A1,A2A,CD39,CD73,ENPP1,ADA,ENT1 and ENT2 were increased in the hippocampus of rats in the model group compared with the control group,while ADK was lower(P<0.05).AMP,Adenosine,Inosine,and GABA were lower in the hippocampus in the model group than in the control group,while Glu was increased.Conclusion Electr oacupuncture can significantly improve the depressive behavior of MDD model rats,and its mechanism may be related to down-regulating the expression of P2X7 to reduce the release of inflammatory factors in the nerve,thereby reducing cell damage,and upregulating the concentration of ADO to enhance the expression of A1 receptors,thereby inhibiting Ca2+influx,reducing neuroexcitatory poisoning,and thereby reducing cell damage.