健脑解郁方对脑小血管病并发抑郁症大鼠海马突触可塑性的影响

Effect of Jiannao Jieyu Formula on hippocampal synaptic plasticity in rats with cerebral small vessel disease related with depression

目的探讨健脑解郁方对脑小血管病并发抑郁症(cerebral small vessel disease related with depression,CSVDD)模型大鼠海马突触可塑性的干预作用及机制。方法将40只SHR大鼠随机分为模型组、尼莫地平(21.6 mg/kg)+丁苯酞(54mg/kg)+氟西汀(5.4 mg/kg)组和健脑解郁方高、中、低剂量(28.08、14.04、7.02 g/kg)组,每组8只,另取8只WKY大鼠作为对照组,大鼠背部sc D-半乳糖(400 mg/kg)8周联合双侧颈总动脉狭窄(bilateral common carotid artery stenosis,BCAS)4周及慢性不可预见性温和刺激(chronic unpredicted mild stress,CUMS)4周制备CSVDD模型。采用糖水偏好率实验、旷场实验、强迫游泳实验和水迷宫实验评价大鼠抑郁样行为;ELISA法检测大鼠血清中一氧化氮(nitric oxide,NO)、内皮素-1(endothelin-1,ET-1)、5-羟色胺(5-hydroxytryptamine,5-HT)、谷氨酸(glutamate,Glu)、γ-氨基丁酸(γ-aminobutyric acid,GABA)、白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;激光微循环血流成像观察脑血流量;苏木素-伊红(HE)染色观察大鼠海马CA3区血管周围间隙情况;普鲁士蓝(LFB)染色观察大鼠脑白质胼胝体病理变化;TUNEL法检测大鼠海马CA3区神经元凋亡;免疫荧光法检测大鼠海马CA3区脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、突触素(synaptophysin,Syn)及突触后致密蛋白-95(post synaptic density-95,PSD-95)的表达。结果与模型组比较,健脑解郁方组大鼠糖水偏好率显著升高(P<0.05),自主活动显著增加(P<0.05),游泳不动时间显著减少(P<0.05),逃避潜伏期和目标象限潜伏期显著缩短(P<0.05、0.01);脑血流量显著上升(P<0.05、0.01);血清中5-HT和GABA水平显著升高(P<0.05),NO、ET-1、Glu、IL-6和TNF-α水平显著降低(P<0.05、0.01);海马CA3区扩大的血管周围间隙数量减少,病理损伤明显减轻,胼胝体髓鞘损伤减轻;海马CA3区凋亡神经元数量显著减少(P<0.01),BDNF、Syn和PSD-95表达显著升高(P<0.05、0.01)。结论健脑解郁方可能通过促进海马突触可塑性,提升神经细胞营养供给,降低海马神经元凋亡,以此改善脑组织海马及白质区域病理损伤,调节神经内分泌指标的表达,改善CSVDD模型大鼠认知及情绪障碍。

Objective To explore the intervention effect and mechanism of Jiannao Jieyu Formula(健脑解郁方)on hippocampal synaptic plasticity in rats with cerebral small vessel disease related with depression(CSVDD).Methods Forty SHR rats were divided into model group,nimodipine(21.6 mg/kg)+butylphthalide(54 mg/kg)+flouxetine(5.4 mg/kg)group,Jiannao Jieyu Formula high-,medium-,low-dose(28.08,14.04,7.02 g/kg)groups,with eight rats in each group,and eight WKY rats were taken as control group.The rats were sc D-galactose(400 mg/kg)on backs for eight weeks,combined with bilateral common carotid stenosis(BCAS)for four weeks and chronic unpredicted mild stress(CUMS)for four weeks to establish CSVDD model.The depression-like behavior of rats was evaluated by sugar water preference rate test,open field test,forced swimming test and water maze test;ELISA was used to detect nitric oxide(NO),endothelin-1(ET-1),5-hydroxytryptamine(5-HT),glutamate(Glu),γ-aminobutyric acid(GABA),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)levels in serum of rats;Cerebral blood flow was observed by laser microcirculation blood flow imaging instrument;Hematoxylin-eosin(HE)staining was used to observe the perivascular space in CA3 area of hippocampus;The pathological changes of corpus callosum in white matter of rats were observed by Prussian blue(LFB)staining;TUNEL method was used to detect neuronal apoptosis in CA3 region of hippocampus;Immunofluorescence method was used to detect the expressions of brain-derived neurotrophic factor(BDNF),synaptophysin(Syn)and post synaptic density-95(PSD-95)in CA3 region of hippocampus.Results Compared with model group,preference rate of sugar water in Jiannao Jieyu Formula group was significantly increased(P<0.05),autonomous activity was significantly increased(P<0.05),swimming immobility time was significantly reduced(P<0.05),and the escape latency and target quadrant latency were significantly shortened(P<0.05,0.01).Cerebral blood flow was significantly increased(P<0.05,0.01).The levels of 5-HT and GABA in serum were significantly increased(P<0.05),while the levels of NO,ET-1,Glu,IL-6 and TNF-αwere significantly decreased(P<0.05,0.01).The number of enlarged perivascular spaces in CA3 area of hippocampus was decreased,pathological damage and myelin sheath damage of corpus callosum were decreased.The number of apoptotic neurons in hippocampal CA3 area was significantly decreased(P<0.01),and the expressions of BDNF,Syn and PSD-95 were significantly increased(P<0.05,0.01).Conclusion Jiannao Jieyu Formula may improve the pathological damage in hippocampus and white matter area of brain tissue,regulate the expression of neuroendocrine indicators,and improve the cognitive and emotional disorders of CSVDD model rats by promoting the synaptic plasticity of the hippocampus,enhancing the nutrition supply of nerve cells,and reducing the apoptosis of hippocampal neurons.