目的 研究左归降糖解郁方调控模拟糖尿病并发抑郁症脑环境下的体外海马神经干细胞增殖与分化作用及分子机制。方法 纯化和培养SD大鼠海马神经干细胞并通过标记巢蛋白（Nestin）进行荧光鉴定。取第3代海马神经干细胞进行实验，分为正常组、模型组、空白血清组、左归降糖解郁方含药血清组、左归降糖解郁方含药血清＋海马无翅基因3a（recombinant wingless type MMTV integration site family member 3a，Wnt3a）信号抑制剂XAV-939组。在干细胞培养液中添加葡萄糖和皮质酮模拟糖尿病并发抑郁症脑环境进行造模，空白血清组和左归降糖解郁方含药血清组于造模同期分别给予不同血清。干预18 h后，采用WST-1检测干细胞活力，采用Brdu免疫荧光结合高内涵细胞成像技术评价干细胞的增殖能力，分别采用双皮质素（doublecortin，DCX）、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）、神经元核抗原（neuron specific nuclear protein，NeuN）免疫荧光评估干细胞的分化能力，采用Western blotting检测各组细胞Wnt3a、低密度脂蛋白受体相关蛋白5（low density lipoprotein receptor-related protein 5，LRP5）、LRP6、β-连环蛋白（β-catenin）表达，采用qRT-PCR法检测神经原素2（neurogenin 2，Ngn2）、细胞周期蛋白（cyclinD1）基因表达。结果 左归降糖解郁方能显著增加受损海马神经干细胞的活力（P＜0.01），增加Brdu、DCX、GFAP、NeuN的表达（P＜0.01），增强其增殖和分化的能力。机制研究发现，左归降糖解郁方能显著增加神经干细胞中的Wnt3a、LRP5、LRP6、总β-catenin、核β-catenin蛋白的表达（P＜0.05、0.01），激活Wnt3a信号并增加β-catenin的蓄积和入核，从而促进下游Ngn2、cyclinD1靶基因的转录（P＜0.05、0.01）。XAV-939可通过增加β-catenin的降解（P＜0.01），减少其进入细胞核，减少下游Ngn2、cyclinD1的mRNA转录（P＜0.05、0.01），从而降低干细胞的增殖与分化能力。结论 左归降糖解郁方能明显改善葡萄糖与皮质酮对体外海马神经干细胞增殖与分化的抑制作用，其改善作用与激活Wnt3a/β-catenin信号有关。

Objective To explore the effects of Zuogui Jiangtang Jieyu Decoction (左归降糖解郁方, ZJJ) on proliferation and differentiation of hippocampal neural stem cells in vitro under the environment of diabetes with depression. Method The neural stem cells acquired from newborn SD rat hippocampus were purified and cultured, and then were identified by Nestin using fluorescence. The third generation neural stem cells were taken for experiments and divided into normal group, model group, blank serum group, ZJJ-containing serum group, ZJJ-containing serum + recombinant wingless type MMTV integration site family member 3a (Wnt3a) signaling inhibitor XAV-939 group. Glucose and corticosterone were added to stem cell culture medium for establishing the environment of diabetes with depression. The blank serum group and ZJJ-containing serum group were given different serum at the same period of molding, respectively. After 18 h of the intervention, viability of stem cell was determined by WST-1. The proliferative capacity of stem cells was evaluated by immunofluorescence combined with high-connotation cell imaging technology. The differentiation capacity of stem cells was assessed by doublecortin (DCX), glial fibrillary acidic protein (GFAP) and neuron specific nuclear protein (NeuN) immunofluorescence. The expressions of Wnt3a, low density lipoprotein receptor-related protein 5 (LRP5), LRP6, and β-catenin were detected by Western blotting. The expressions of neurogenin 2 (Ngn2) and cyclinD1 were determined by qRT-PCR. Results ZJJ significantly increased the vitality of hippocampal neural stem cells (P ＜ 0.01), and increased the expressions of Brdu, DCX, GFAP, NeuN (P ＜ 0.05, 0.01), enhanced ability of proliferation and differentiation. The mechanism study found that ZJJ significantly increased Wnt3a, LRP5, LRP6, total β-catenin and nuclear β-catenin protein expressions in neural stem cells (P ＜ 0.05, 0.01), activated Wnt3a signaling and increased the accumulation and entry of β-catenin, then promoted the transcription of downstream Ngn2 and cyclinD1 target genes (P ＜ 0.05, 0.01). XAV-939 inhibited proliferation and differentiation capacity of neural stem by promoting the degradation of β-catenin (P ＜ 0.01), and reducing β-catenin entry into nucleus, decreasing the transcription of downstream Ngn2 and cyclinD1 genes (P ＜ 0.05, 0.01). ConclusionZJJ can significantly improve the proliferation and differentiation of hippocampal neural stem cells induced by glucose and corticosterone, which may be related to the activation of Wnt3a/β-catenin signaling.

R285.5

国家自然科学基金资助项目（82174357）；国家自然科学基金资助项目（82104793）；湖南省自然科学基金资助项目（2022JJ30451）；湖南省卫生健康委科研课题（202102041447）；湖南省中药粉体与创新药物研究省部共建国家重点实验室培育基地开放基金项目（21PTKF1006）