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Journal of Traditional Chinese Medicine logoLink to Journal of Traditional Chinese Medicine
. 2022 Sep 2;42(6):956–964. doi: 10.19852/j.cnki.jtcm.20220902.001

Compound Gaoziban tablet (复方高滋斑片) alleviates depression via toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-kappa B pathway

Xinshuang ZOU 1, Lei SHI 1, Hailong YIN 2, Haiping LI 1, Mengheng WANG 1, Wanci SONG 1, Laichun LUO 1, Hezhen WU 1, Yanfang YANG 1, Junfeng ZAN 1, Yanwen LIU 1, Hanxiong DAN 1, Qiang YIN 2,✉, Pengtao YOU 1,✉
PMCID: PMC9924672  PMID: 36378054

Abstract

OBJECTIVE:

To evaluate the effect of compound Gaoziban tablet (复方高滋斑片, CGZBT) on depression, and to investigate the underlying mechanism.

METHODS:

The components of CGZBT were analysed by high-performance liquid chromatography. Then, we assessed the effects of varying doses of CGZBT on an established chronic unpredictable mild stress (CUMS) model in rats. Whether animals were depressed was evaluated by sucrose preference test, open field test and forced swimming test. Neurotransmitters of hippocampus were detected by liquid chromatography-mass spec-trometry. Serum levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-4, and IL-10 were measured by enzyme-linked immunosorbent assay. Expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phospho-nuclear factor-kappa B (p-NF-κB), cyclooxygenase-2 (COX-2), ionized calcium binding adapter molecule-1 (IBA-1) were assessed by immunohistochemical staining and western blotting.

RESULTS:

Eight compounds were identified from CGZBT, moreover, our results showed that CGZBT effectively reversed the CUMS-induced decrease in sucrose preference, shortened the movement distance and prolonged immobility time. CGZBT significantly increased levels of 5-hydroxytryptamine, dopamine, norepinephrine, 5-hydroxyindoleacetic acid levels, and reduced the expression of TNF-α, IL-1β, IL-6, yet increased IL-4 and IL-10. Furthermore, the expressions of TLR4, MyD88, COX-2, p-NF-κB and IBA-1 in hippocampus were effectively reversed after treatment with CGZBT.

CONCLUSIONS:

These results indicated that CGZBT could, at least in part, alleviate depression induced by CUMS via the TLR4/MyD88/NF-κB pathway, suggesting its potential as an antidepressant drug.

Keywords: depression, toll-like receptor 4, myeloid differentiation factor 88, NF-kappa B, chronic unpredictable mild stress, compound Gaoziban tablet

1. INTRODUCTION

Depression is a serious mental illness that is mainly manifested as emotional, cognitive, behavioral, sleep, and diet symptoms. Depression ultimately damages the overall social functioning of patients and is one of the main causes of suicide.1 At present, mainstream anti-depressants effectively treat only 30%-35% of patients, and so serious economic and mental burdens to patients and their families remain. All available antidepressants are almost exclusively based on the serendipitous discovery of mood-elevating substances in the 1950s, which were later named monoamine neurotransmitters.2 Decreased monoamine neuro-transmitters, such as 5-hydroxytryptamine (5-HT) and norepinephrine (NE) are often regarded as the remarkable signs of depression. Moreover, accumulating evidence suggests that inflammatory cytokines also play an important role in the occurrence and development of depression.3 Animal studies have found that pro-inflammatory cytokine levels in the hippocampus and serum of depressed mice are significantly higher than those in healthy individuals.4 Based on this, more and more researchers made an effort to identify the key mediators of stress inflammation associated with depre-ssion in recent years. toll-like receptor 4 (TLR4) is a pathogen recognition receptor that recognizes various pathogen-associated molecular patterns to regulate innate and acquired immune responses,5 which is highly expressed in microglia cells in the brain and neurons.6 A growing number of studies have shown that TLR4 activation and its mediating inflammatory cytokines are closely related to some depressive symptoms and may be related to their occurrence and development, although the relationship between TLR4 and depression remains inconclusive.7,8

There is a long history and rich experience in the study of traditional Chinese medicine (TCM) anti-depression. Chinese Medicine Classics also have many discourses on the symptoms, pathogenesis, and treatment of depression. Although the name of depression is not recorded directly, it is classified into the category of emotion and called "Yu Disease".9 At present, only four Chinese herbal formulas are used for depression according to their functions and indications in instructions, namely Shugan Jieyu capsule (舒肝解郁胶囊), Jinxiang Shugan tablet (金香疏肝片), Shumian capsule (舒眠丸) and Anle capsule (安乐丸). At the stage of drug discovery, there are Yueju pill (越鞠丸), Ganmai Dazao decoction (甘麦大枣汤), Chaihu Shugan San (柴胡疏肝散), Sini San, Kaixin San (四逆散), and compound Chaigui formula (复方柴归汤).10 Few Chinese herbal formulas prescriptions that point out for depression, but more TCM symptom pattern of depression has been added to these indications, showing unique effects of sedation and sleep promotion.

Compound Gaoziban tablet (复方高滋斑片, CGZBT) is a traditional Chinese herbal formula developed by Xinjiang Uygur Pharmaceutical Co. Ltd. CGZBT is composed of 11 kinds of TCM, including 10 kinds of medicinal plants and one kind of insect drug: Chiguoyangti (Radix Rumicis Dentati), Oushichejugen (Centaurea behen L.), Tanxiang (Lignum Santali Albi), Daye-buxuecao (Herba Limonii Gmelinii), Xiangqinglan (Herba Dracocephali Moldavicae), Tinglizi (Semen Lepidii Apetali), Zisuzi (Fructus Perillae Argutae), Niushecaohua (Echium vulgare L.), Xunyicao (Lavandula angustifolia Mill.), Yuansuizi (Fructus Coriandri) and Canjian (Silkworm cocoon). CGZBT, as a Uygur Medicine, has long been used to treat neurasthenia and hypertension in China.11 Our pilot study showed that CGZBT reduced the levels of inflammatory factors in rats and improved depression-like behaviours (unpublished data). However, the antidepressant effect of CGZBT has not been reported. Based on this, the current study aimed to evaluate the effect of CGZBT on depression, as well as to explore the underlying mechanism.

2. METHODS

2.1. Chemicals and reagents

CGZBT was obtain from Xinjiang Uygur Pharm-aceutical Co. Ltd. (Xinjiang, China) (181023). Fluo-xetine hydrochloride was purchased from Changzhou Siyao Pharmaceuticals Co. Ltd. (Suzhou, China) (7686A). The drug dose in rats was obtained by the conversion of the human dose to rat equivalent dose based on body surface areas. After grinding, all agents were dissolved in distilled water separately and administered at a volume of 10 mL/kg.

2.2. Identification of the CGZBT compounds

A proper amount of methanol was used as the solvent of CGZBT and the mixed standards, then fully dissolved by ultrasonic for 1 h. The mobile phase was composed of methanol (A) and 0.05% phosphoric acid (B) with gradient elution system (0-5 min, 5%A; 5-17 min, 5% ⟶ 10%A; 17-20 min, 10% ⟶ 18%A; 20-25 min, 18% ⟶ 25%A; 25-35 min, 25% ⟶ 35%A; 35-40 min, 35% ⟶ 70%A; 40-45 min, 70% ⟶ 5%A) with a flow rate of 1.0 mL/min (the detection conditions follow a modified version of the protocol described previously).12 The ZORBAX SB-C18 chromatographic column (4.6 mm × 250 mm, 5 μm) was used for high-performance liquid chromatography and the column temperature was 30°C. The injection volume was 10 μL and detected under 330 nm ultraviolet light.

2.3. Animals and treatment

Adult male Sprague-Dawley rats weighting 160-180 g were purchased from Hubei Provincial Center for Disease Control and Prevention. Rats were housed in individual ventilated cages at a temperature of (22 ± 2) ℃, humidity of 50% ± 10%, and 12 h light/dark cycle. They were acclimatized in these conditions for one week before induction of depression via established chronic unpredictable mild stress, as previously described.13 Briefly, chronic unpredictable mild stress (CUMS) consists of the following steps: food deprivation/water deprivation, cold swimming, cage shaking, electric foot shocks, tail clamp, light/dark reversal, and cage housing tilting.

After first SPT, 72 rats were randomly divided into seven groups (n = 12 per group): Control group, Model group, 0.4 g/kg CGZBT group, 0.8 g/kg CGZBT group, 1.6 g/kg CGZBT group and Fluoxetine group (3.69 mg/kg). The Control group was only treated with normal saline. The Model group was treated with normal saline after CUMS. The drug-treated groups were given the corresponding dose of drugs after CUMS for two weeks. All animals were sacrificed 6 weeks later, then the whole brain and blood were carefully collected immediately for subsequent experiments. The whole experimental procedure is shown in Figure 1A. The animal study was reviewed and approved by Animal Ethics Committee of Hubei University of Chinese Medicine (Approval Number: 2017-0067).

Figure 1. CGZBT reduced depressive-like behaviours in rats.

Figure 1

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Schematic representation of the experimental procedure for CUMS in rat (A). Effects of CGZBT treatment on the sucrose consumption coefficient of rats at 0, 2, 4, and 6 weeks (B). Effects of CGZBT treatment on the movement traces of rats (C); Control group, Model group, 0.4 g/kg CGZBT group, 0.8 g/kg CGZBT group, 1.6 g/kg CGZBT group and Fluoxetine group (C1-C6, respectively). Effects of CGZBT treatment on distance of movement (D), distance through the center (E), and number of grid crossings (F) in the open field test. Effects of CGZBT treatment on immobility time in the forced swimming test after CUMS (G). The Control group was only treated with normal saline. The Model group was treated with normal saline after CUMS.CUMS consists of the following steps: food deprivation/water deprivation, cold swimming, cage shaking, electric foot shocks, tail clamp, light/dark reversal, and cage housing tilting. 0.4 g/kg CGZBT group, 0.8 g/kg CGZBT group, 1.6 g/kg CGZBT group and Fluoxetine group (3.69 mg/kg) were given the corresponding dose of drugs after CUMS for two weeks. CGZBT: compound Gaoziban tablet; CUMS: chronic unpredictable mild stress; FST: forced swimming test; OFT: open field test; SPT: sucrose preference test. Data are presented as mean ± standard deviation. aP < 0.01 vs Control group; bP < 0.01 vs Model group.

2.4. Behavioural assessment

The animal’s depression was assessed by the sucrose preference test (SPT), open field test (OFT), and the forced swimming test (FST). (a) SPT:14 We measured the sucrose consumption coefficient of each group at week 0, 2, 4 and 6. Sucrose consumption was calculated using the following formula: sucrose consumption coefficient = sucrose consumption/(water consumption + sucrose consumption) × 100%. (b) OFT:15 Locomotor activity was measured by open field test. The open field apparatus was composed of a light box (100 cm × 100 cm × 50 cm) and video analysis system. (c) FST:16 The rats were placed in a round transparent glass tank (40 cm in height × 20 cm in diameter) with a water temperature of 23-25 ℃ and a depth of 20 cm. Rats are considered immobile only when they float in the water without struggling.

2.5. Liquid chromatography-mass spectrometry (LC-MS) analysis for 5-HT, DA, NE and 5-hydroxyindoleacetic acid (5-HIAA)

The hippocampus was treated with methanol before LC-MS analysis. LC-MS conditions were in accordance with our previous study: 17 An Agilent ZORBAX SB-C18 chromatographic column (2.1 mm × 100 mm, 1.8 μm) was used, the mobile phase was 0.1% formic acid solution (A) and acetonitrile (B), the flow rate was 0.4 mL/min, and the column temperature was 40 ℃. The source injection voltage was 5 500 V (–4 500 V), the heating temperature was 550 ℃, Gas1 was 60 psi (1 psi ≈ 6.895 kPa), and Gas2 was 65 psi. The instrument we used was an ultraperformance liquid chromatograph (Shimadu, Japan), tandem 4500 AB Sciex triple quadrupole mass spectrometer (AB Sciex, Boston, MA, USA).

2.6. Enzyme-linked immunosorbent assay

The whole blood of rats was centrifuged at 12 000 rpm to obtain serum. Tumor necrosis factor-alpha (TNF-α), interleukin (IL) 1β, IL6, IL4, and IL10 kits were purchased from Shanghai Fusheng Industrial Co. Ltd. (Shanghai, China). The levels of TNF-α, IL-1β, IL-4, IL-6, IL-10 in serum of rats were determined. Concentrations were calculated by referring to the standard curve according to the manufacturer's instructions.

2.7. Immunohistochemical staining

The whole brain was fixed with paraformaldehyde and embedded in paraffin. Sections were dewaxed in water in a 65 °C oven and placed in ethylene diamine tetraacetic acid buffer for microwave repair. Next, sections were treated with 3% hydrogen peroxide solution and sealed at 5% Albumin from bovine serum after shaking dry. The diluted primary antibodies of TLR4 (1:100, NB100-56580SS, Novus Biologicals, Denver, CO, USA), myeloid differentiation factor 88 (MyD88) (1:150, ab131071, abcam, Beijing, China), phospho-nuclear factor-kappa B (NF-κB)-p65 (p-p65) (1 : 200, ab86299, abcam, Beijing, China), cyclooxygenase-2 (COX-2) (1:150, AF7003, Affinity Biosciences, Jiangsu, China), ionized calcium binding adapter molecule-1 (IBA-1) (1:300, GB11105, Servicebio, Wuhan, China) were added to cover each slice and kept at 4 °C overnight. Sections were subsequently incubated with biotinylated goat anti-rabbit secondary antibody (1:200, abcam, Beijing, China). Each section was mixed with 50-100 μL 3, 3-N-Diaminobenzidine Tertrahydrochloride (DAB) solution. Three high-power (400×) fields of view were randomly selected from each section. The images were observed under an IX51 microscope and analysed by the MicroPublisher imaging system.

2.8. Western blot analysis

The hippocampal tissue was homogenized with radio immunoprecipitation assay (RIPA) buffer containing 1% protease inhibitor PMSF. Protein concentrations in the supernatant were measured using the bicinchoninic acid protein assay kit (TaKaRa, Shiga, Japan). Equal amounts of proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) and trans-ferred onto nitrocellulose membranes (Millipore, Shang-hai, China). The membranes were blocked with 5% skimmed milk in Tris Buffered Saline with Tween (TBST) buffer for 2 h and then incubated overnight with antibodies for rabbit anti-TLR4 [1:1000; Cell Signaling Technology (CST), Shanghai, China], rabbit anti-MyD88 (1:1000; CST, Shanghai, China), rabbit anti-p-NF-κB (1:1000; CST, Shanghai, China), horseradish peroxidase (HRP)-conjugated glyceraldehyde-3-phos-phate dehydrogenase (1:10000, CST, Shanghai, China). TBST was used to wash the membranes three times and they were then incubated with HRP-goat anti-rabbit immunoglobulin G (1:2000; CST, Shanghai, China) for 1 h. Membranes were developed using enhanced chemi-luminescence chemiluminescent kits (Thermo Scientific, Shanghai, China) and exposed with x-ray film. The gray values were measured using Image J software (v1.51, National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated three times.

2.9. Statistical analyses

Data were obtained from three independent experiments. All results are presented as mean ± standard deviation ($\bar{x} \pm s$). SPSS version 11.0 (IBM Corp., Armonk, NY, USA) software was used for statistical analysis. Student’s t-test was used to detect difference between groups. P < 0.05 was considered statistically significant.

3. RESULTS

3.1. CGZBT reduced depressive-like behaviours in rats

From the second week, the sucrose consumption coefficient of the Model group was lower than that of the Control group (P < 0.01) (Figure 1B). It was signify-cantly increased with a dose of 0.4 g/kg CGZBT or greater in CGZBT-treated rats compared to the Model group (P < 0.01). Interestingly, the sucrose consumption coefficient of the 1.6 g/kg CGZBT group was higher than Fluoxetine group from the fourth week, indicating the superior effect of 1.6 g/kg CGZBT in reversing anhedonia.

The results of OFT showed that the distance of movement, distance through the center, and number of grid crossings in the Model group were significantly decreased compared with the Control group (P < 0.01) (Figure 1D-1F). Notably, these indicators in 1.6 g/kg CGZBT group were significantly increased compared with Model group, illustrating that higher dose of CGZBT reduced fear of empty space, altered spontaneous activity, exploring ability, and reduced anxiety levels.

In FST, the immobility times of the 0.8 g/kg CGZBT group and 1.6 g/kg CGZBT group were significantly decreased compared with the Model group (P < 0.01) (Figure1G). Together, these results indicated that CGZBT could significantly reverse CUMS-induced behavioural depression in rats.

3.2. CGZBT reversed changes in hippocampus neuro-transmitter levels

The detection results of 5-HT, DA, NE and 5-HIAA by LC-MS are shown as follows (Figure 2A-2D). Compared with the Control group, 5-HT, DA, NE and 5-HIAA concentrations in the hippocampus of the Model group were significantly decreased (P < 0.01), whereas they were significantly reversed with a dose of 0.8 g/kg or higher in CGZBT-treated rats compared to the Model group (P < 0.01). The results indicated that CGZBT had a conspicuous reversal effect on the decreases of 5-HT, DA, NE and 5-HIAA.

Figure 2. CGZBT restores the levels of neurotransmitters in the hippocampus.

Figure 2

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A-E: levels of 5-HT (A), DA (B), NE (C) and 5-HIAA (D) in the hippocampus after CUMS measured by LC-MS. The Control group was only treated with normal saline. The Model group was treated with normal saline after CUMS. CUMS consists of the following steps: food deprivation/water deprivation, cold swimming, cage shaking, electric foot shocks, tail clamp, light/dark reversal, and cage housing tilting. 0.4 g/kg CGZBT group, 0.8 g/kg CGZBT group, 1.6 g/kg CGZBT group and Fluoxetine group (3.69 mg/kg) were given the corresponding dose of drugs after CUMS for two weeks. CGZBT: compound Gaoziban tablet; CUMS: chronic unpredictable mild stress; 5-HT: 5-hydroxytryptamine; DA: dopamine; NE: norepinephrine; 5-HIAA: 5-hydroxyindoleacetic acid. Data are presented as mean ± standard deviation. aP < 0.01 vs control group; bP < 0.01 vs Model group.

3.3. CGZBT reversed serum levels of inflammatory factors

As shown in Figure 3, the levels of TNF-α, IL-1β and IL-6 in the Model group were significantly increased compared to the Control group (P < 0.01; Figure 3A-3C), while the levels of IL-4 and IL-10 were decreased (Figure 3D, 3E). After treatment with CGZBT, the levels of TNF-α, IL-1β, and IL-6 in serum were significantly decreased compared with Model group (P < 0.01), while levels of IL-4 and IL-10 were significantly increased in a dose-dependent manner (P < 0.01). These findings indicate that the antidepressant effect of CGZBT is related to its anti-inflammatory properties.

Figure 3. CGZBT reversed serum levels of inflammatory factors.

Figure 3

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Levels of serum TNF-α (A); IL-1β (B); IL-6 (C); IL-4 (D); IL-10 (E) were quantified by ELISA. The Control group was only treated with normal saline. The Model group was treated with normal saline after CUMS. CUMS consists of the following steps: food deprivation/water deprivation, cold swimming, cage shaking, electric foot shocks, tail clamp, light/dark reversal, and cage housing tilting. 0.4 g/kg CGZBT group, 0.8 g/kg CGZBT group, 1.6 g/kg CGZBT group and Fluoxetine group (3.69 mg/kg) were given the corresponding dose of drugs after CUMS for two weeks. CGZBT: compound Gaoziban tablet; CUMS: chronic unpredictable mild stress; TNF-α: tumor necrosis factor-alpha; IL: interleukin; ELISA: enzyme-linked immunosorbent assay. Data are presented as mean ± standard deviation. aP < 0.01 vs Control group; bP < 0.01 vs Model group.

3.4. CGZBT regulated TLR4, MyD88, p-NF-κB, COX-2 and IBA-1 expression in the hippocampus

To confirm the relationship between the TLR4/MyD88/ NF-κB pathway and the antidepressant effect of CGZBT, we measured these indicators in CA1 region of the hippocampus (Figure 4A). As shown by immune-ohistochemical staining, the nuclei of hippocampal cells in Control group were blue, had small cell bodies, and were evenly distributed. By contrast, the nuclei of hippocampal cells in Model group showed a dark brown color and shrinkage, the cell bodies were generally larger, and the distribution was irregular. In addition, many positive cells were found (Figure 4B). Similar results were observed in the comparison of the Model group with the 0.4 g/kg CGZBT group and 0.8 g/kg CGZBT group. However, the color and morphology of hippocampal cells in 1.6 g/kg CGZBT group were the same as those in Control group, which were consistent with the western blot results. As shown in Figure 4C and 4D, TLR4, MyD88 and p-NF-κB protein expression in the CGZBT group was significantly up-regulated compared with Control group (P < 0.01). Treatment with CGZBT significantly alleviated their increase in a dose-dependent manner (P < 0.01). IBA-1 is a marker of microglia and its expression increases significantly when microglia are activated. The results showed that Control group had fewer hippocampal microglia cells, exhibited smaller cell bodies, and had elongated protuberances (Figure 6B). After CUMS, we observed larger cell bodies and shorter and thicker protuberances in microglia in Model group. Notably, CGZBT significantly reversed this activation: there were fewer positive cells and microglia were smaller with fewer and more elongated protuberances. The combined results indicate that the TLR4/MyD88/NF-κB pathway is involved in the antidepressant effect of CGZBT.

Figure 4. CGZBT alleviated depression via the TLR4/MyD88/NF-κB pathway.

Figure 4

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A: the CA1 region we observed is shown in the black box; B: effects of CGZBT treatment on TLR4 (b1), MyD88 (b2), p-NF-κB (b3), COX-2 (b4) and IBA-1 (b5) in the hippocampus of rats by immunohistochemical staining (x 200 magnification; x 400 in the red box); C: Western blot analysis of TLR4, MyD88, and p-NF-κB proteins in the hippocampus; D: relative density of proteins. The Control group was only treated with normal saline. The Model group was treated with normal saline after CUMS. CUMS consists of the following steps: food deprivation/water deprivation, cold swimming, cage shaking, electric foot shocks, tail clamp, light/dark reversal, and cage housing tilting.0.4 g/kg CGZBT group, 0.8 g/kg CGZBT group, 1.6 g/kg CGZBT group and Fluoxetine group (3.69 mg/kg) were given the corresponding dose of drugs after CUMS for two weeks. DG: dentate gyrus; CGZBT: compound Gaoziban tablet; CUMS: chronic unpredictable mild stress; TLR4: toll-like receptor 4; MyD88: myeloid differentiation factor 88; p-NF-κB: phospho-nuclear factor-kappa B; COX-2: cyclooxygenase-2; IBA-1: ionized calcium binding adapter molecule-1. Data are presented as mean ± standard deviation. aP < 0.01 vs Control group; bP < 0.01 vs Model group.

4. DISCUSSION

The pathophysiology of depression includes a variety of molecular mechanisms and signaling pathways. TLR4 signaling pathway may alter in the peripheral circulatory system or central nervous system of depressed animal or patients.8 In the present study, CGZBT effectively inhibited depressive-like behaviours, including decreased sucrose preference, decreased spontaneous activity, and longer immobility time. Furthermore, CGZBT reversed depression-induced decreases in neurotransmitters in the hippocampus. Our findings suggested that CGZBT alleviated depression via the TLR4/MyD88/NF-κB pathway.

Eight compounds were identified from CGZBT, including sinapic acid, rutin, rosmarinic acid, myricetin, quercetin, luteolin, kaempferol and apigenin. Among them, myricetin, quercetin, luteolin, kaempferol and apigenin belong to flavonoids. It is worth noting that flavonoids have previously been reported to have significant antidepressant effects by the mechanism of reversing the attenuation of 5-HT, NA, DA, and 5-HIAA in the brain.18,19 Therefore, we speculate that CGZBT alleviates depression-like behaviours just because it contains this kind of substance, but more evidence is needed to confirm.

The CUMS-induced behavioural changes are thought to mimic depression-like behaviours in depressed pa-tients.20 Depressive-like conditions in animals are mea-sured by behavioural phenotypes considered analogous to those seen in humans.2 CGZBT significantly improved depressive-like behaviour in our study, thus confirming that CGZBT has antidepressant effects in rats. 5-HT and 5-HIAA are important monoamine neurotransmitters related to human emotion, and decreased levels of these neurotransmitters are indicators of depression.21

Significant changes in 5-HT and 5-HIAA after treatment with CGZBT were detected, which is consistent with previous studies.21 Alterations in the innate immune system and inflammatory responses have been repeatedly observed in patients with mood disorders,22 including increased circulating cytokines such as TNF-α, IL-1β, and IL-6.23 Studies have also reported that inhibition of inflammation can improve depressive-like behaviour in rats.24,25 Our results clearly showed that the expression of TNF-α, IL-1β, and IL-6 was decreased while IL-4 and IL-10 was increased after treatment with CGZBT, indicating that the antidepressant effect of CGZBT might be ascribed to its anti-inflammatory properties.

In our study, CGZBT downregulated the expression of TLR4, MyD88 and p-NF-κB in a dose-dependent manner. As key members of the inflammation-related signaling pathway, TLR4, MyD88 and NF-κB have been reported to be closely associated with depression.26 Some medicines, such as N-acetylcysteine and melatonin, which reduce the expression of TLR4 mRNA or protein, have been shown to ameliorate CUMS-induced behavioural dysfunction in mice or depressive symptoms in human.27,28 NF-κB, a crucial transcription factor, is an important downstream regulator of the MyD88-dependent pathway, which involved in cellular inflammatory response and immune response. Consistent with the findings of Hajebrahimi et al 29 and Qu et al29, 30 our results indicated that CGZBT had strong inhibitory effect on TLR4, MyD88 and p-NF-κB. The effects of CGZBT were most significant at a higher dose.

However, there are still some limitations in this work. Because of the complexity of TCM compounds, only eight compounds have been identified. And it is difficult to identify one or more compounds that actual function. Furthermore, we have not demonstrated whether the TLR4 signalling pathway is necessary and the only pathway for antidepressant effect of this drug. A deeper study of the intrinsic relationship between them is necessary.

In conclusion, our results suggested that that CGZBT had a significant therapeutic effect on the model of dep-ression and the mechanism of this effect is closely related to the TLR4/MyD88/NF-κB pathway. More importantly, our findings provide a theoretical basis for the potential application of CGZBT in the treatment of depression.

Contributor Information

Qiang YIN, Email: yinqiang@renfu.com.cn.

Pengtao YOU, Email: tptyou@hbtcm.edu.cn.

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