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

Objective

To 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<italic/> 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.

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 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.