摘要OBJECTIVE:To investiage the effect of electroacupuncture(EA)at a single acupoint of Shenmen(HT7),Baihui(GV20),Sanyinjiao(SP6)and at combined acupoints of Shenmen(HT7)and Baihui(GV20)and Sanyinjiao(SP6)on the PKA/CREB and BDNF/TrkB signaling,as well as neuroapoptosis and neurogenesis in hippocampus and elucidate the underlying mechanism of single and combined acupoints on ameliorating spatial learning and memory deficits in a rat model of primary insomnia.METHODS:Primary insomnia was modeled by intraperitoneal injection of para-chlorophenylalanine(PCPA)once daily for 2 d.EA was applied at Shenmen(HT7),Baihui(GV20),Sanyinjiao(SP6),or Shenmen(HT7)+Baihui(GV20)+Sanyinjiao(SP6)(combined)for 30 min daily for 4 d.Spatial learning and memory function was evaluated by the Morris water maze(MWM)test.Protein expressions of hippocampal cAMP-dependent protein kinase(PKA)-Cβ,phosphorylated cAMP-responsive element-binding protein(p-CREB),brain-derived neurotrophic factor(BDNF),and tyrosine kinase receptor B(TrkB)were evaluated by Western blotting.Neuronal apoptosis in the hippocampus was detected with the transferase-mediated dUTP-X nick end labeling assay.Endogenous neurogenesis was examined with bromodeoxyuridine staining.The MWM test and hippocampal p-CREB,BDNF,and TrkB protein levels in the combined acupoints group were evaluated after the administration of a PKA-selective inhibitor(H89).RESULTS:Spatial learning and memory were significantly impaired in rats with insomnia.The spatial learning deficits were ameliorated in the Shenmen(HT7),Baihui(GV20),Sanyinjiao(SP6),and combined groups;this improvement was significantly greater in the combined group than the single acupoint groups.The spatial memory impairment was improved in the combined,Baihui(GV20),and Shenmen(HT7)groups,but not the Sanyinjiao(SP6)group.The expressions of PKA-Cβ,p-CREB,BDNF,and TrkB were decreased in rats with insomnia.All these proteins were significantly upregulated in the combined group.PKA/p-CREB protein levels were elevated in the Baihui(GV20)and Shenmen(HT7)groups,whereas BDNF/TrkB expression was upregulated in the Sanyinjiao(SP6)group.The staining results showed significant attenuation of hippocampal cell apoptosis and increased numbers of proliferating cells in the combined group,whereas the single acupoint groups only showed decreased numbers of apoptotic cells.In the combined group,the PKA inhibitor reversed the improvement of spatial memory and upregulation of p-CREB expression caused by EA,but did not affect its activation of BDNF/TrkB signaling.CONCLUSIONS:EA at the single acupoints Baihui(GV20),Shenmen(HT7),or Sanyinjiao(SP6)had an ameliorating effect on the spatial learning and memory deficits induced by insomnia.EA at combined acupoints exerted a synergistic effect on the improvements in spatial learning and memory impairment in rats with insomnia by upregulating the hippocampal PKA/CREB and BDNF/TrkB signaling,facilitating neurogenesis,and inhibiting neuronal apoptosis.These findings indicate that EA at combined acupoints[(Baihui(GV20),Shenmen(HT7),and Sanyinjiao(SP6)]achieves a more pronounced regulation of hippocampal neuroplasticity than EA at single acupoints,which may partly explain the underlying mechanisms by which EA at combined acupoints exerts a better ameliorative effect on the cognitive dysfunction caused by insomnia.