数字中医药（英文）2022，Vol.5Issue(2)：210-221,12.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
Abstract
Objective To explore the protective effects and mechanism of Zuogui Jiangtang Jieyu For-mula (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-Daw-ley (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 streptozo-tocin (STZ) and four weeks of chronic unpredictable mild stress (CUMS). All drug administra-tion 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 evalu-ate 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-dioxy-genase (IDO) in the hippocampus were detected using enzyme-linked immunosorbent assay (ELISA); the protein expression levels of synaptophysin (SYN) and postsynaptic density ma-terial-95 (PSD-95) were detected via immunohistochemistry (IHC); and the protein expres-sion 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 gluc-ose + 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-trypto-phan (1-MT, IDO inhibitor) (G&P + 1-MT) groups. After 18 h of intervention by correspond-ing 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 signific-antly decreased in the hippocampus (P < 0.01) of rats after ZGJTJYF-M treatment. The pro-tein 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 res-ults 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. Conclusion ZGJTJYF 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 favor-able protective effect on hippocampal neuron injury caused by DD. Therefore, ZGJTJYF is an effective potential therapeutic drug for the prevention and treatment of DD.关键词
左归降糖解郁方/糖尿病/抑郁症/糖尿病并发抑郁症/TRP/KYN代谢通路/海马神经元/神经保护/5-羟色胺Key words
Zuogui Jiangtang Jieyu Formula (左归降糖解郁方, ZGJTJYF)/Diabetes/Depression/Diabetes complicated with depression/TRP/KYN metabolic pathway/Hippocampal neurons/Neuroprotection/5-Hydroxytryptamine (5-HT)引用本文复制引用
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].数字中医药（英文）,2022,5(2):210-221,12.基金项目
National Natural Science Foundation of China(81874464 and 82104793),and the Scientific Research Project of Education Department of Hunan Province(19K066). （81874464 and 82104793）
