凌佳, 刘检, 金狮, 邹蔓姝, 蒋雅杰, 王宇红. 基于TRP/KYN代谢通路探讨左归降糖解郁方对糖尿病并发抑郁症大鼠海马神经元保护作用[J]. Digital Chinese Medicine, 2022, 5(2): 210-221. DOI: 10.1016/j.dcmed.2022.06.010
引用本文: 凌佳, 刘检, 金狮, 邹蔓姝, 蒋雅杰, 王宇红. 基于TRP/KYN代谢通路探讨左归降糖解郁方对糖尿病并发抑郁症大鼠海马神经元保护作用[J]. Digital Chinese Medicine, 2022, 5(2): 210-221. DOI: 10.1016/j.dcmed.2022.06.010
LING Jia, LIU Jian, JIN Shi, ZOU Manshu, JIANG Yajie, WANG Yuhong. Protective effects of Zuogui Jiangtang Jieyu Formula on hippocampal neurons in rats of diabetes complicated with depression via the TRP/KYN metabolic pathway[J]. Digital Chinese Medicine, 2022, 5(2): 210-221. DOI: 10.1016/j.dcmed.2022.06.010
Citation: LING Jia, LIU Jian, JIN Shi, ZOU Manshu, JIANG Yajie, WANG Yuhong. Protective effects of Zuogui Jiangtang Jieyu Formula on hippocampal neurons in rats of diabetes complicated with depression via the TRP/KYN metabolic pathway[J]. Digital Chinese Medicine, 2022, 5(2): 210-221. DOI: 10.1016/j.dcmed.2022.06.010

基于TRP/KYN代谢通路探讨左归降糖解郁方对糖尿病并发抑郁症大鼠海马神经元保护作用

Protective effects of Zuogui Jiangtang Jieyu Formula on hippocampal neurons in rats of diabetes complicated with depression via the TRP/KYN metabolic pathway

  • 摘要:
    目的探讨左归降糖解郁方(ZGJTJYF)对糖尿病并发抑郁症(DD)大鼠海马神经元的保护作用及其机制。
    方法(i)体内实验中,取60只SPF级雄性SD大鼠,随机分为6组,每组10只:空白组、模型组、阳性药组(1.8 mg/kg 氟西汀+ 0.18 g/kg二甲双胍)、高剂量ZGJTJYF组(ZGJTJYF-H,40.500 g/kg ZGJTJYF)、中剂量ZGJTJYF组(ZGJTJYF-M,20.250 g/kg ZGJTJYF)和低剂量ZGJTJYF组(ZGJTJYF-L,10.125 g/kg ZGJTJYF)。除空白组外,其余各组均予以高脂乳剂灌胃联合单次尾静脉注射链脲佐菌素(STZ)及4周慢性不可预见性温和应激(CUMS)建立DD模型。所有给药组在CUMS造模期间分别经灌胃给予对应药物处理,空白组和模型组给予等量蒸馏水灌胃。处理4周后,通过检测大鼠的血糖、血清中糖化血红蛋白含量判断ZGJTJYF的降糖效果;通过开野实验和Morris水迷宫测试评价ZGJTJYF的抗抑郁作用;采用高效液相色谱联合电化学检测器(HPLC-ECD)检测大鼠海马中5-羟色胺(5-HT)水平;采用酶联免疫吸附实验(ELISA)检测大鼠海马中色氨酸(TRP)、犬尿氨酸(KYN)及吲哚胺2,3-双加氧酶(IDO)的含量变化;采用免疫组织化学染色法(IHC)检测大鼠海马中突触素(SYN)、突触后致密物质95 (PSD-95)蛋白的表达水平;采用蛋白印迹法检测N-甲基-D-天冬氨酸受体(NR)2A 和NR2B蛋白的表达水平。(ii)体外实验中,分别取5只SPF级SD孕鼠(E16 – 18),6只SPF级SD新生鼠用于原代海马神经元(Ne)、星形胶质细胞(As)和小胶质细胞(MG)提取,构建Ne-As-MG共培养体系,并将其分为6组:空白组(PBS)、模型组150 mmol/L葡萄糖+ 200 μmol/L皮质酮(G&P)+ PBS、空白血清组(G&P + 10%空白血清)、阳性药组(G&P + 10%阳性药含药血清)、ZGJTJYF组(G&P + 10% ZGJTJYF含药血清)和1-甲基-D-色氨酸组(1-MT,IDO阻断剂)(G&P + 1-MT),给予对应处理18 h后,采用免疫荧光法检测神经元突触相关蛋白SYN、PSD-95、NR2A和NR2B表达水平;采用ELISA法检测炎症因子白介素(IL)-1β、IL-6、肿瘤坏死因子(TNF)-α 及TRP/KYN代谢通路中相关因子TRP、KYN、犬尿喹啉酸(KYNA)和喹啉酸(QUIN)水平。
    结果(i)体内研究结果表明,ZGJTJYF-M和ZGJTJYF-L可显著改善DD大鼠的血糖升高状态(P < 0.01,P < 0.05),ZGJTJYF-H、ZGJTJYF-M和ZGJTJYF-L均显著增加其自主活动能力和学习记忆能力(P < 0.01,P < 0.01,P < 0.05);同时,ZGJTJYF-M可有效升高大鼠海马中5-HT和TRP水平(P < 0.01),降低KYN和IDO水平(P < 0.01),并能显著提高海马神经元中SYN和PSD-95的表达水平(P < 0.01),抑制海马内NR2A和NR2B异常活化(P < 0.01)。(ii)体外研究结果表明,ZGJTJYF含药血清可显著提高海马神经元中SYN和PSD-95的表达水平(P < 0.01);有效降低IL-1βP < 0.01)、IL-6 (P < 0.05)、TNF-αP < 0.01)、IDO (P < 0.05)、KYN (P < 0.05)和QUIN的水平(P < 0.01),升高TRP和KYNA的水平(P < 0.01);并对DD环境下神经元中的NR2A和NR2B异常活化具有显著的抑制作用(P < 0.05)。
    结论 中药复方ZGJTJYF可通过抑制大鼠海马中IDO表达,调节TRP/KYN代谢途径,有效改善海马中的5-HT不足,对DD导致的海马神经元损伤具有良好的保护作用,是防治DD的有效潜在治疗药物。

     

    Abstract:
    ObjectiveTo explore the protective effects and mechanism of Zuogui Jiangtang Jieyu Formula (左归降糖解郁方, ZGJTJYF) on hippocampal neurons in rats of diabetes complicated with depression (DD) via the TRP/KYN metabolic pathway.
    Methods(i) In vivo experiments: 60 specified pathogen free (SPF) grade male Sprague-Dawley (SD) rats were randomly divided into six groups with 10 rats in each groups: control, DD model, positive (1.8 mg/kg fluoxetine + 0.18 g/kg metformin), high-dose ZGJTJYF (ZGJTJYF-H, 40.500 g/kg ZGJTJYF), middle-dose ZGJTJYF (ZGJTJYF-M, 20.250 g/kg ZGJTJYF), and low-dose ZGJTJYF (ZGJTJYF-L, 10.125 g/kg ZGJTJYF) groups. Except for the control group, other groups were established DD model by high-fat emulsion intake with single tail vein streptozotocin (STZ) and four weeks of chronic unpredictable mild stress (CUMS). All drug administration groups were treated by gavage during CUMS modeling, and the control and model groups were given equal amount of distilled water. After four weeks, the serum levels of blood glucose and glycosylated hemoglobin were measured to determine the hypoglycemic effect of ZGJTJYF. Moreover, the open field test and Morris water maze test were performed to evaluate the antidepressant effect of ZGJTJYF. Changes in 5-hydroxytryptamine (5-HT) level were detected via high-performance liquid chromatography with electrochemical detection (HPLC-ECD); the levels of tryptophan (TRP), kynurenine (KYN), and indoleamine 2,3-dioxygenase (IDO) in the hippocampus were detected using enzyme-linked immunosorbent assay (ELISA); the protein expression levels of synaptophysin (SYN) and postsynaptic density material-95 (PSD-95) were detected via immunohistochemistry (IHC); and the protein expression levels of N-methyl-D-aspartate receptor (NR) 2A and NR2B were detected using Western blot. (ii) In vitro experiments: five SPF grade SD pregnant rats (E16 – 18) were used to obtain primary hippocampal neurons (Ne), six SD new-born rats were used to collected primary astrocytes (As) and microglia (MG), and to establish a Ne-As-MG co-culture system. All co-culture systems were divided into six groups: control (PBS), model 150 mmol/L glucose + 200 μmol/L corticosterone (G&P) + PBS, blank (G&P + blank serum), positive (G&P + positive drug-containing serum), ZGJTJYF (G&P + ZGJTJYF serum), and 1-methyl-D-tryptophan (1-MT, IDO inhibitor) (G&P + 1-MT) groups. After 18 h of intervention by corresponding treatment, immunofluorescence was used to analyze the protein expression levels of SYN, PSD-95, NR2A, and NR2B; ELISA was performed to measure the levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α , and TRP/KYN metabolic pathway-related factors TRP, KYN, kynurenine acid (KYNA), quinolinic acid (QUIN).
    Results(i) In vivo experimental results showed that ZGJTJYF-M and ZGJTJYF-L significantly improved the elevated blood glucose state of DD rats (P < 0.01 and P < 0.05, respectively); ZGJTJYF-H, ZGJTJYF-M, and ZGJTJYF-L increased their autonomous activity, learning, and memory ability (P < 0.01, P < 0.01, and P < 0.05, respectively). Moreover, the levels of 5-HT and TRP were significantly increased (P < 0.01), and the levels of KYN and IDO were significantly decreased in the hippocampus (P < 0.01) of rats after ZGJTJYF-M treatment. The protein expression levels of SYN and PSD-95 were significantly upregulated in hippocampal neurons (P < 0.01), while the abnormal activation of NR2A and NR2B was markedly inhibited in hippocampus (P < 0.05) of rats after ZGJTJYF-M treatment. (ii) In vitro experimental results showed that ZGJTJYF-containing serum significantly increased the protein expression levels of SYN and PSD-95 in hippocampal neurons (P < 0.01), decreased the levels of IL-1β (P < 0.01), IL-6 (P < 0.05), TNF-α (P < 0.01), IDO (P < 0.05), KYN (P < 0.05), and QUIN (P < 0.01), and increased the levels of TRP and KYNA (P < 0.01) in the simulated DD state. ZGJTJYF also had an significantly inhibitory effect on the abnormal activation of NR2A and NR2B in neurons (P < 0.05) in a stimulated DD state.
    ConclusionZGJTJYF can effectively improve 5-HT deficiency in the hippocampus of rats by inhibiting IDO expression and regulating the TRP/KYN metabolic pathway, and it has a favorable protective effect on hippocampal neuron injury caused by DD. Therefore, ZGJTJYF is an effective potential therapeutic drug for the prevention and treatment of DD.

     

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