左归降糖解郁方调控小胶质细胞CD300f改善糖尿病并发抑郁症海马突触微环境损伤的机制研究

Mechanism of Zuogui Jiangtang Jieyu Formula on improving hippocampal synaptic microenvironment damage via regulating microglia CD300f in diabetes-related depression

目的探讨左归降糖解郁方调控小胶质细胞免疫受体分子样家族成员f(immune receptor molecule-like family member f,CD300f)改善糖尿病并发抑郁症(diabetes-related depression,DD)海马突触微环境损伤的保护作用及机制。方法原代分离、培养、纯化和鉴定小胶质细胞和海马神经元,复制模拟DD环境下的海马突触微环境损伤体外细胞模型,设置正常组、模型组、CD300f阻断剂CLM-1组、左归降糖解郁方含药血清组和左归降糖解郁方含药血清+CLM-1组。采用细胞成像分析观察小胶质细胞和海马神经元形态结构;采用ELISA法检测细胞上清液中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase,IDO)、5-羟色胺(5-hydroxyteyptamine,5-HT)、多巴胺(dopamine,DA)含量;采用CCK-8实验及活死细胞染色评估海马神经元活性;采用尼氏染色观察海马神经元突触损伤;采用免疫荧光检测小胶质细胞中CD300f、Toll样受体4(Toll-like receptor 4,TLR4)和海马神经元中突触素(synaptophysin,SYN)、突触后密度蛋白95(postsynaptic density protein 95,PSD-95)蛋白表达;采用Western blotting检测SYN、PSD-95蛋白表达。结果左归降糖解郁方能有效改善小胶质细胞和海马神经元形态结构损伤,抑制细胞上清中神经炎症因子TNF-α、IL-1β和IDO水平(P<0.05、0.01),促进神经递质5-HT、DA水平(P<0.01),进而缓解海马神经元细胞活性及突触损伤。进一步的机制研究发现,左归降糖解郁方能显著增加海马小胶质细胞中CD300f表达(P<0.01),降低TLR4表达(P<0.05),并上调海马神经元中突触前膜SYN和突触后膜PSD-95表达(P<0.01),最终抑制海马突触微环境损伤。结论左归降糖解郁方能有效改善体外DD状态下的海马突触微环境损伤,其机制可能与上调小胶质细胞CD300f有关。

Objective To explore the protective effect and mechanism of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方)on regulating the immune receptor molecule like family member f(CD300f)of microglia to improve the hippocampal synaptic microenvironment damage in diabetes-related depression(DD).Methods Microglia and hippocampal neurons were primitively isolated,cultured,purified and identified.The in vitro cell model of hippocampal synaptic microenvironment damage was replicated and simulated in DD environment.The cultured cells were randomly divided into normal group,model group,CD300f blocker CLM-1 group,drug-containing serum of Zuogui Jiangtang Jieyu Formula group,and drug-containing serum of Zuogui Jiangtang Jieyu Formula+CLM-1 group.Morphology and structure of microglia and hippocampal neurons were observed by cell imaging analysis;ELISA was used to measure the contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),indoleamine 2,3-dioxygenase(IDO),5-hydroxytryptamine(5-HT)and dopamine(DA)in the cell supernatant;The activity of hippocampal neurons was evaluated by CCK-8 assay and live/dead cell staining;Nissl’s staining was used to observe synaptic damage in hippocampal neurons;The expressions of CD300f and Toll-like receptor 4(TLR4)proteins in microglia as well as synaptophysin(SYN)and postsynaptic density protein 95(PSD-95)in hippocampal neurons were detected by immunofluorescence staining;Western blotting was used to detect the expressions of SYN and PSD-95 proteins.Results Zuogui Jiangtang Jieyu Formula significantly improved the morphological and structural damagee of microglia and hippocampal neurons,inhibited the levels of neuroinflammatory factor TNF-α,IL-1βand IDO in the cell supernatant(P<0.05,0.01),promoted the levels of neurotransmitter 5-HT and DA(P<0.01),thereby alleviating hippocampal neuronal activity and synaptic damage.Further mechanism research found that Zuogui Jiangtang Jieyu Formula significantly increased the expression of CD300f in hippocampal microglia(P<0.01),reduced the expression of TLR4(P<0.05),up-regulate the expressions of SYN in presynaptic membrane and PSD-95 in postsynaptic membrane of hippocampal neurons(P<0.01),ultimately inhibiting hippocampal synaptic microenvironment damage.Conclusion Zuogui Jiangtang Jieyu Formula can effectively improve the hippocampal synaptic microenvironment damage in the state of DD in vitro,and its mechanism may be related to the up-regulation of microglial CD300f.