[关键词]
[摘要]
目的 研究左归降糖解郁方通过调节吲哚胺-2,3-双加氧酶(indoleamine 2,3-dioxygenasel,IDO)信号抑制糖尿病并发抑郁症大鼠海马N-甲基D-天冬氨酸受体(N-methyl D-aspartate receptor,NR)过激致海马突触损伤的作用机制。方法 建立糖尿病并发抑郁症大鼠模型,并随机分为空白组、模型组、IDO抑制剂(3 mg/kg)组、左归降糖解郁方(20.52 g/kg)组和阳性对照(二甲双胍0.18 g/kg+氟西汀1.8 mg/kg)组。各组大鼠ig给药28 d后,采用旷场实验、强迫游泳实验和Morris水迷宫检测大鼠抑郁样行为和记忆认知功能;采用Western blotting和qRT-PCR检测海马IDO蛋白和基因表达;采用ELISA试剂盒检测血清和海马中γ-干扰素(interferon-γ,IFN-γ)、CC族趋化因子配体2(CC chemokine ligand 2,CCL2)、色氨酸、5-羟色胺(5-hydroxytryptamine,5-HT)、犬尿氨酸、犬尿喹啉酸(kynurenine acid,KA)、喹啉酸、白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和IL-6水平;采用免疫荧光法检测海马中离子钙接头蛋白-1(ionized calcium binding adaptor molecule-1,Iba-1)、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、突触素(synaptophysin,Syn)、突触后致密蛋白-95(post synaptic density protein-95,PSD-95)的表达;采用Western blotting检测海马中腺苷A1R受体(adenosine A1 receptor,A1R)、腺苷A2AR受体(adenosine A2A receptor,A2AR)、腺苷A3R受体(adenosine A3 receptor,A3R)、N-甲基D-天冬氨酸受体2A(N-methyl D-aspartate receptor 2A,NR2A)、N-甲基D-天冬氨酸受体2B(N-methyl D-aspartate receptor 2B,NR2B)和脑源性神经营养因子(brain derived neurotrophic factor,BDNF)蛋白表达;采用苏木素-伊红染色、Nissl染色和TUNEL法检测海马病理损伤及凋亡情况。结果 左归降糖解郁方能显著降低模型大鼠血糖水平(P<0.01),并改善抑郁样行为,表现为旷场实验总距离和中心时间显著增加(P<0.01)、强迫游泳不动时间显著缩短(P<0.01)、Morris水迷宫实验定位巡航时间显著降低(P<0.05)和空间探索时间显著增加(P<0.01)。左归降糖解郁方组和IDO抑制剂均能通过显著下调IDO蛋白和基因表达(P<0.001),并不同程度降低IFN-γ、CCL2、犬尿氨酸、喹啉酸水平(P<0.05、0.01),逆转色氨酸-犬尿氨酸代谢紊乱;同时显著抑制Iba-1、GFAP表达(P<0.05、0.01),并降低IL-1β、TNF-α、IL-6水平(P<0.05、0.01),抑制体内炎症水平;进而降低A2AR、NR2A、NR2B蛋白表达(P<0.05、0.01),增加A1R、A3R蛋白表达(P<0.05),改善NR过度激活;最终提高Syn、PSD-95、BDNF表达(P<0.05),显著改善海马病理损伤及降低神经元凋亡(P<0.05、0.01),对海马突触损伤和神经元产生保护作用。结论 左归降糖解郁方通过抑制IDO水平,调节糖尿病并发抑郁症大鼠海马色氨酸-犬尿氨酸代谢通路,从而改善NR过激致海马突触损伤,发挥抗抑郁作用。
[Key word]
[Abstract]
Objective To study the mechanism of Zuogui Jiangtang Jieyu Formula (左归降糖解郁方) on inhibiting hippocampal synaptic damage caused by excessive N-methyl D-aspartate receptor (NR) in diabetes with depression rats by regulating indoleamine 2,3-dioxygenasel (IDO) signal. Method Diabetic rats with depression were established and randomly divided into blank group, model group, IDO inhibitor (3 mg/kg) group, Zuogui Jiangtang Jieyu Formula (20.52 g/kg) group and positive control (metformin 0.18 g/kg + fluoxetine 1.8 mg/kg) group. After 28 d of administration, depression-like behavior and memory and cognitive function were detected by open field test, forced swimming test and Morris water maze. Western blotting and qRT-PCR were used to detect the expression levels of IDO protein and mRNA in hippocampus. The contents of interferon-γ (IFN-γ), CC chemokine ligand 2 (CCL2), tryptophan, 5-hydroxytryptamine (5-HT), kynurenine, kynurenine acid (KA), quinolinic acid, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-6 in serum and hippocampus were determined by ELISA. The expressions of ionized calcium binding adaptor molecule-1 (Iba-1), glial fibrillary acidic protein (GFAP), synaptophysin (Syn) and post synaptic density protein-95 (PSD-95) in hippocampus were detected by immunofluorescence assay. Protein expressions of adenosine A1 receptor (A1R), adenosine A2AR receptor (A2AR), adenosine A3 receptor (A3R), N-methyl D-aspartate receptor 2A (NR2A), N-methyl D-aspartate receptor 2B (NR2B) and brain derived neurotrophic factor (BDNF) in hippocampus were detected by Western blotting. Hematoxylin-eosin (HE), Nissl and TUNEL were used to detect the pathological injury and apoptosis of hippocampus. Results Zuogui Jiangtang Jieyu Formula significantly reduced blood sugar level and improved depression-like behavior in model rats (P < 0.01). The results showed that the total distance and center time of open field experiment were significantly increased (P < 0.01), the immobile time of forced swimming was significantly shortened (P < 0.01), the positioning cruise time of Morris water maze experiment was significantly decreased (P < 0.05), and the space exploration time was significantly increased (P < 0.01). Both Zuogui Jiangtong Jieyu Formula group and IDO inhibitor group could significantly down-regulate IDO protein and transcription levels (P < 0.001), and reduce IFN-γ, CCL2, kynurenine, quinolinic acid levels to varying degrees (P < 0.05, 0.01), and reverse tryptophan-kynurenine metabolic disorders. At the same time, the expressions of Iba-1 and GFAP were significantly inhibited (P < 0.05, 0.01), and the levels of IL-1β, TNF-α and IL-6 were decreased (P < 0.05, 0.01), and the levels of inflammation in vivo were inhibited. Furthermore, the protein expression levels of A2AR, NR2A and NR2B were decreased (P < 0.05, 0.01), and the protein expression levels of A1R and A3R were increased (P < 0.05) to improve the over-activation of NR. Finally, the expression levels of Syn, PSD-95 and BDNF were increased (P < 0.05), and the pathological injury of hippocampus was significantly improved and the apoptosis of neurons was decreased (P < 0.05, 0.01), which had a protective effect on the damage of hippocampus synapses and neurons. Conclusion Zuogui Jiangtong Jieyu Formula can effectively regulate the hippocampal tryptophan-kynurenine metabolic pathway in diabetic rats complicated with depression by inhibiting IDO level, so as to improve the hippocampal synaptic damage caused by NR overreaction and exert an antidepressant effect.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81874464);国家自然科学基金资助项目(82174357);湖南省科药联合基金资助项目(2023JJ60476);湖南省科药联合基金资助项目(2023JJ60482);湖南省中医药管理局科研课题(B2023141)