Depression is a mental illness characterized by low mood. The clinical manifestations include low energy, slow thinking, reduced speech and movement, loss of interest or pleasure, and, in severe cases, suicidal tendencies [1
]. Depression is ranked second in the WHO ‘global burden of disease’ rankings [2
], just behind cancer. At present, depression is treated mainly by the use of drugs. The ‘monoamine hypothesis’, the decrease of monoamine neurotransmitter levels in the synaptic cleft, is still recognized as the major pathogenesis of depression in clinical settings [3
]. Most classical antidepressants, such as tricyclic antidepressants (TCAS), tetracyclic antidepressants (HCA), monoamine oxidase inhibitors (MAOI), and selective serotonin (5-HT) reuptake inhibitors (SSRIs), all presently on the market, are based on this theory. However, current monoamine neurotransmitter-based drugs are ineffective in about 30% of patients with depression, and long-term use can cause side effects such as cardiotoxicity, sleep disorders, and sexual dysfunction [4
]. Therefore, it is important to elucidate other possible mechanisms that may cause depression, and to develop drugs to inhibit these pathways with fewer adverse reactions and side effects.
Many studies have shown that the pathogenesis of depression is closely related to the inflammatory response [6
]. Clinical studies have found that levels of inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), are abnormally elevated in the peripheral blood and cerebrospinal fluid of patients with depression, and that inflammatory cytokines in patients with depression can return to normal levels after administration of antidepressants [9
]. There have also been similar indications in related animal studies: An abnormal increase in inflammatory cytokine levels has been observed in acute and chronic stress models of depression [12
]. Direct injection of inflammatory cytokines, such as TNF-α and IL-1β, into the brain of animals can produce depression-like behaviors, such as loss of pleasure, decreased activity, and decreased food intake [15
]. In addition, other studies have shown that depression in the inflammatory state is often accompanied by a disorder of the tryptophan–kynurenine (TRP–KYN) metabolic pathway, while indoleamine 2,3-dioxygenase (IDO), an enzyme involved in tryptophan metabolism, is a key factor connecting inflammation and depression [18
]. Clinical studies have found that patients with depression have a higher KYN/TRP ratio than non-depressed patients, suggesting that IDO activation is associated with depression [20
]. These studies all indicate that neuroinflammation is involved in the development of depression, contributing to a new understanding of the illness.
Honokiol is a bioactive polyphenolic substance and is one of the main active components extracted from the plant, Magnolia officinalis
, which is widely used in traditional Chinese medicine [21
]. Modern studies have shown that honokiol has few toxic side effects and many pharmacological activities, including anti-inflammatory, anti-oxidant, free radical scavenging, antibacterial, anti-cancer, and nerve cell protecting properties [22
]. Honokiol may exert its anti-inflammatory effects through multiple pathways, such as down-regulation of the MAPK and NF-κB signaling pathways, causing inhibition of lipopolysaccharide (LPS)-induced iNOS and COX-2 expression [29
]; inhibition of the phosphatidylinositol 3-hydroxykinase/protein kinase pathway (PI3K/AKT) [30
] and; inhibition of the activation of the NF-κB signaling pathway by the inhibition of IκB kinase activation [31
]. Additionally, honokiol is a small molecule that can easily pass through the blood–brain barrier, providing direct contact with the nerve tissue, thus, it has a direct effect on nerve cells [32
]. Studies have shown that honokiol can improve LPS-induced anxiety-like behavior by inhibiting the production of inflammatory cytokines [33
Our previous study [34
] found that honokiol improved depression-like behavior in acute and chronic stress mice. In the present study, we established a mouse depression model by peripheral administration of LPS, and measured the concentration of the pro-inflammatory cytokines, TNF-α, IL-1β, and interferon γ (IFN-γ), in peripheral blood, the concentration of IFN-γ in the brain, the expression of NF-κB P65 and IDO in the hippocampus, and the concentration of calcium in the brain. Measurement of TRP–KYN-related metabolites further confirmed the regulatory and neuroprotective effects of honokiol on depression-induced behavior in this model.
Relevant studies have shown that intraperitoneal injection of LPS can lead to an acute inflammatory response, causing depression-like behavior and cognitive changes in mice [35
]. LPS activates the innate immune response, resulting in the secretion of various pro-inflammatory cytokines, including TNF-α, IFN-γ, and IL-1β, and the induction of neuroendocrine and neurochemical changes, leading to depression [36
Neuroinflammation is considered to be an important pathological cause of depression, and this corresponds to the cytokine hypothesis of the pathogenesis of depression [39
]. Cytokines, activated and secreted by immune cells, are signaling molecules that have immunomodulatory biological activities. They may either be pro-inflammatory or anti-inflammatory. Pro-inflammatory cytokines include TNF-α, IFN-γ, and IL-1β [7
]. Normal levels of inflammatory cytokines are essential for cell signaling and a healthy protective immune response to pathogens, but long-term chronic inflammation caused by excessive stress and excessive production of inflammatory factors can cause tissue and cell damage [40
]. Studies have shown that IL-1β can stimulate the production of some neurotoxic proteins, inhibit the secretion of neuroprotective factors, have direct toxic effects on nerve cells, and inhibit the regeneration of neurons. Additionally, excessive IL-1β overexpression can over-activate the hypothalamus-pituitary-adrenal (HPA) axis, leading to raised glucocorticoid levels [42
]. Elevated levels of TNF-α can activate 5-HT transporters and promote pre-synaptic reuptake of 5-HT, and this can lead to depression caused by a decrease in 5-HT levels in the synapse [45
]. A high concentration of IFN-γ can aggravate the oxidative stress injury of local neurons, have a toxic effect on neural stem cells, and change the growth and differentiation state of neural stem cells [46
In addition, studies have shown that pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ are effective activators of NF-κB and IDO [48
The NF-κB signaling pathway plays an important role in depression-like behavior induced by acute and chronic stress and LPS [51
]. NF-κB is closely related to neuroinflammation: On one hand, NF-κB, as a multi-effect regulator, is involved in the regulation of inflammatory mediators and the transcription and expression of inflammatory cytokines [53
]; on the other hand, inflammatory factors such as TNF-α, IL-1β, and IFN-γ can activate NF-κB. This cycle aggravates the inflammatory response. Our results show that honokiol can effectively reverse the increase in NF-κB mRNA and protein expression in the hippocampi of LPS-treated depression model mice, suggesting that the improvement of LPS-induced depression-like behavior by honokiol may be related to the inhibition of NF-κB activation.
The ‘5-HT depression hypothesis’ suggests that immune-mediated activation of pro-inflammatory cells induces IDO activation, leading to decreased plasma tryptophan levels and increased synthesis of harmful tryptophan catabolic metabolites, both of which synergize to aggravate the development of depression [54
]. IFN-γ is a major inducer of IDO activation, while other pro-inflammatory cytokines such as TNF-α and IL-1β synergistically induce the activation of IDO [55
]. IDO, a rate-limiting enzyme involved in tryptophan metabolism, is widely distributed in various tissues and participates in TRP–KYN metabolism. Therefore, the activity of IDO plays a crucial role in depression. The KYN/TRP ratio is often used as an activity index to evaluate IDO activity: The higher the ratio, the greater the activity of IDO. Disordered activation of IDO causes excessive activation of the TRP–KYN pathway, depleting tryptophan, and degrading more TRP to KYN, resulting in insufficient synthesis of 5-HT [58
]. Studies have shown that the development of depressive symptoms was significantly correlated with the ratio of the kynurenine/kynurenic acid because kynurenine is neurotoxic, whereas kynurenic acid is neuroprotective. Thus, the KYN/KYNA ratio is often used as a measure of neurotoxicity and neuroprotection: The greater the ratio, the greater the neurotoxicity, which reflects an increase in the neurotoxic potential [60
]. In this study, the IDO
gene and protein expression levels and the KYN/TRP and KYN/KYNA ratios were significantly increased in the model group compared with the control group. However, in the drug-administered group, the IDO
gene and protein expression levels and the KYN/TRP and KYN/KYNA ratios were significantly decreased compared with those in the model group, indicating that the antidepressant effect of honokiol may also be related to inhibition of IDO activation and an increase in neuroprotective metabolites (Figure 7
Calcium can be called a ‘life and death signal’ and plays an important role in a variety of biological processes. Calcium homeostasis is essential for the function of the central nervous system [63
]. As a second messenger, free calcium is an important signal transduction factor, participating in many activities such as enzyme system activation, hormone secretion, nucleotide metabolism, cell proliferation, and synaptic plasticity [64
]. Under normal circumstances, calcium ions are maintained at a steady level, ensuring a dynamic balance. Disturbing this balance leads to apoptosis, neuronal degeneration, and necrosis [66
]. It is well known that NMDA receptors transmit important information through the conduction of calcium ions. NMDAR (N
-aspartate receptor) is a specific receptor for the excitatory amino acid glutamate, which is widely distributed in neurons of the hippocampus and hypothalamus, and is involved in synaptic plasticity regulation and advanced neural activity [67
]. NMDAR dysfunction is associated with schizophrenia, neurodegenerative diseases, etc. [68
]. Disordered activation of IDO also leads to an increase in the synthesis of harmful TRP catabolites such as QUIN, resulting in an imbalance of the QUIN/KYNA ratio. Quinolinic acid is an agonist of the NMDAR. Excessive accumulation of quinolinic acid over-activates NMDA receptors and excess NMDA receptor activity leads to cytotoxic calcium overload and neuronal damage further aggravates the occurrence of depression [62
]. In this study, the Ca2+
concentration in the honokiol group was significantly reduced compared with that in the model group, indicating that the anti-depressant effect of honokiol may, in part, be related to its ability to lower free calcium levels in brain tissue, thus inhibiting calcium overload.
Studies have shown that honokiol can cross the blood-brain barrier and induce neuroblastoma cell apoptosis [32
]. However, honokiol does not induce insults to non-neoplastic cells. In vitro and in vivo studies have also shown that honokiol has no obvious toxicity to normal brain cells [70
]. Considering previous studies on the toxicity of magnolia bark extract, its safety seems to be guaranteed [21
]. However, it has recently been reported that honokiol interacts with aristolochic acid in vitro to enhance the toxicity of aristolochic acid, suggesting that honokiol may also interact with other compounds derived from plants [72
]. Based on this, we should pay more attention to the side effects of honokiol in future research.
4. Materials and Methods
4.1. Animals and Ethical Review
Specific pathogen free (SPF) grade, adult male ICR mice, weighing 22–25 g, were provided by Beijing HuaFukang Biotechnology Co., Ltd., animal license number: SCXK (Beijing) 2014-0004. All animals were housed under the following standard conditions: A 12 h light/dark cycle; a humidity range of 40 ± 10%; and an ambient temperature of 22 ± 1 °C. The mice had free access to standard food and water. All experimental procedures involving the animals were approved by the Experimental Animal Ethics Committee of the Academic Committee of Beijing University of Chinese Medicine (project identification code: BUCM-4-2019011501-1006).
4.2. Animal Study Design
Thirty mice were randomly divided into a control group, an LPS group, and an LPS + Honokiol group (n = 10/group). Honokiol was suspended in 0.5% sodium carboxymethylcellulose (CMC-Na) at certain concentrations before use.
Studies have shown that pretreatment with honokiol (10 mg/kg) significantly improved depression-like behavior induced by chronic restraint stress [73
]. In addition, in our previous study [35
], we studied the antidepressant-like effects of honokiol on acute and chronic stress mice by setting up three dose groups (2.5, 5, and 10 mg/kg). Our results also showed that pretreatment with honokiol (10 mg/kg) significantly improved depression-like behavior.
Based on our previous experiments, honokiol was orally administered once daily for 11 days at a dose of 10 mg/kg body weight (bw), and the control and LPS groups were given an equal volume of 0.5% CMC-Na solution. Autonomic activity tests were performed 30 min after administration on Day 10, and on Day 11, the animals in the model and honokiol groups were injected intraperitoneally with 1 mg/kg bw LPS. Behavioral tests were performed 4 h later. After the behavioral tests, the eyeballs were removed for blood collection, the animals were killed by decapitation, the brain of each animal was removed and stored on ice for analysis.
4.3. Drugs and Reagents
Honokiol (batch number: P1095220) was obtained from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). The structure of honokiol was shown in Figure 8
. LPS (batch number: RH51487) was purchased from BioRuler (Connecticut, USA). IFN-γ (batch number: 228080442) ELISA kits were purchased from Multisciences (Lianke) Biotech, Co., Ltd. (Hangzhou, China). IL-1β (batch number: 20171012), TNF-α (batch number: 20171020), IFN-γ (batch number: 20171018) radioimmunoassay kits were purchased from Beijing Sino-UK Institute of Biological Technology. (Beijing, China). The Hipure Total RNA mini kit (No. R4111-02) was obtained from Magen Bio (Guangzhou, China). The Reveraid First Strand cDNA Synthesis kit (No. K1622) was from Thermo Scientific (San Diego, CA, USA); and the SYBR PCR master mix was from Invitrogen (San Diego, CA, USA).
The IDO1 antibody (No: 66528-1-Ig), NF-κB P65 antibody (No: 14220-1-AP), and the ECL chemiluminescence detection kit (No: B500022) were obtained from the Proteintech Group, Inc. (Chicago, IL. USA). The GAPDH antibody (No: cw0100) was obtained from Beijing Kangwei Century Biotechnology Co., Ltd. (Beijing, China). The calcium ion fluorescent probe Fluo-3-Am (batch number: KGAF023), PBS (No: KGB5001), RIPA buffer (No: KGP703-100), and the BCA protein quantitation kit were purchased from KeyGen BioTech Corp., Ltd. (Nanjing, China). The Minute™ Total Protein Extraction kit (animal cells/tissues) (No: SD-001/SN-002) was purchased from Invent Biotechnologies, Inc. (Eden Prairie, MN, USA). Foetal calf serum (No: hz001) was purchased from the Sijiqing Company Ltd. (Hangzhou, China), and collagenase type II (No: HA0890-100 mg) was purchased from Sigma-Aldrich (Saint Louis, MO, USA).
4.4. Behaviour Testing
4.4.1. Autonomic Activity Tests
Autonomic activity tests are used to measure the general behavior of animals and to assess behavioral changes in rodents exposed to new environments. The tests were performed on the 10th day of dosing, 30 min after drug administration. The mice were placed in a self-made opaque 60 cm by 60 cm by 40 cm activity box with a defined central square area of 30 cm by 30 cm. A small animal behavior analysis system (Etho-Vision XT9, Noldus, The Netherlands), was used to record the distance, velocity, and frequency of movement of the mice across both the central and marginal regions over 5 min.
4.4.2. Forced Swimming Test
This test is used to evaluate desperation in animals and is one of the classic models used to assess the efficacy of antidepressants [74
]. The experimental animals were subjected to an FST 4 h after the LPS injection (see Supplementary Materials
). The mice were placed individually in transparent cylindrical glass cylinders (25 cm high and 10 cm in diameter) with a water depth and temperature of 10 cm and 23–25 °C, respectively. Opaque baffles were placed between the cylinders to prevent the mice from seeing each other. The activity of the mice was observed a period of over 6 min using a small animal behavior analysis system (Etho-Vision XT9), and the duration of immobility over the last 4 min of the 6 min was recorded. Mice were judged to be immobile when they stopped struggling and floated on the water or made only small movements necessary to keep its head above water.
4.4.3. Tail Suspension Test
The tail suspension test is also one of the classic models used to assess the efficacy of antidepressants [75
]. The experimental animals were subjected to a TST 4 h after the LPS injection. The end of the tail of each mouse (2 cm from the tip) was fixed with medical tape to a tension transducer (JZ100, Shanghai Kang Wei Medical Technology Development Co., Ltd., China) so that the mouse was hanging upside down with its head approximately 15 cm off the ground. The activity of the animals was observed over 6 min using a MedLab BioInformatics acquisition system (MedLab-U/4C501H, Nanjing Medease Science and technology Co., Ltd., China), and the immobility time over the last 4 min was recorded.
4.5. Analysis of Pro-Inflammatory Cytokine Levels
4.5.1. Detection of TNF-α, IL-1β, and IFN-γ in Serum
After behavioral tests, the eyeballs were taken for blood collection and 300 µL samples of the supernatant were removed for radioimmunoassay. The concentration of each pro-inflammatory cytokine in the serum samples was determined by radioimmunoassay kits according to the manufacturer’s instructions.
4.5.2. Detection of IFN-γ in Brain Tissue
After the steps detailed in Section 4.5.1
, mice were killed and their brains were rapidly removed, and the hippocampus and cerebral cortex were isolated. The cerebral cortex was placed in a centrifuge tube and 4 °C saline was added at four times the volume of tissue. The tissue was thoroughly homogenized by an electric homogenizer and placed on ice. The IFN-γ content in the supernatant was determined according to the ELISA kit instructions.
4.6. RT–PCR Detection of Hippocampal IDO and NF-κB P65 Gene Expression
The hippocampus was dissected from the brain tissue, and the total RNA was extracted according to the Hipure Total RNA mini kit instructions. The RNA concentration was measured using an ultraviolet spectrophotometer (UV-2000, Unico, Shanghai, China). Reverse transcription was performed on a T100 Thermal Cycler PCR machine (Bio-Rad, USA) using the Reveraid First Strand cDNA Synthesis kit and the SYBR PCR master mix according to the manufacturers’ instructions. Amplification and quantitative detection were performed in a Real-Time PCR machine (Bio-Rad, USA). The RT–PCR protocol was as follows: initial denaturation at 95 °C for 15 min, then 55 cycles at 95 °C for 10 s, and finally 58 °C for 30 s. Primer sequences for the genes of interest were as follows:
IDO1 F: 5′ GGATCCTTGAAGACCACCACAT 3′
IDO1 R: 5′ AAGGACCCAGGGGCTGTAT 3′
NF-κB P65 F: 5′ ATCATCGAACAGCCGAAGCA 3′
NF-κB P65 R: 5′ TGATGGTGGGGTGTGTCTTG 3′
β-actin F: 5′ CCTAGGCACCAGGGTGTG 3′
β-actin R: 5′ CGGTGAGCAGCACAGGGT 3′
The 2−ΔΔCt method was used for relative quantitative analysis of the results.
4.7. Western Blot Detection of Hippocampal IDO and NF-κB P65 Protein Expression
The hippocampus sample was obtained as in Section 4.5.2
. RIPA buffer was added for tissue lysis and extraction of total protein. Protein concentration determination was performed using a BCA kit, and the samples were separated by electrophoresis on a 5–10% polyacrylamide gel. After separation, the proteins were transferred to a 0.45 μm PVDF membrane by the wet transfer method using constant piezoelectricity at 100 V for 1.5 h. The membrane was blocked in 5% skimmed milk for 2 h, incubated overnight with the primary antibody, washed in a TBST solution (SolarBio, Beijing, China) for 3 by 10 min, incubated in the secondary antibody at room temperature for 1 h on the shaker, washed a further three times in TBST solution, and placed on plastic wrap. Developer from the ECL chemiluminescence detection kit was added evenly, and the membrane was allowed to stand. The filter was blotted dry for 1 min, and an Azure multifunctional molecular imaging system (C600, Azure, USA) was used for detection of the labelled proteins. GAPDH was used as the control for protein loading and transfer efficiency Gray value analysis was performed using Image J software (The National Institutes of Health, Bethesda, MD, USA).
4.8. HPLC–MS Detection of TRP–KYN Metabolites
The serum content of 5-HT, KYN, TRP, KYNA, and QUIN was determined by HPLC–MS. The IDO activity is represented by the KYN/TRP ratio, and the neuroprotective effect by the KYN/KYNA ratio. HPLC analysis was performed using a DIONEX Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA). The liquid phase conditions used were: Column: MSLab C18 (150 by 4.6 mm 5 µm); mobile phase, A aqueous phase: Water (1% formic acid); B organic phase: acetonitrile (1% formic acid); injection volume: 5 µL; column temperature: 50 °C; flow rate: 1 mL/min; and gradient elution: 0–1 min, (10% B); 1–12 min, (10%–70% B); 12–12.1 min, (70%–100% B); 12.1–15 min, (100% B); 15–15.1 min, (100%–10% B); and 15.1–20 min, (10% B). Mass spectrometry was performed using an API 3200 QTRAP system (AB SCIEX, USA). The mass spectrometry conditions used were: Scanning mode: Multiple reaction monitoring (MRM); ion source: Electron spray ionization (ESI); atomization temperature (TEM): 500 °C; injection voltage (EP): 10; spray voltage (IS): +5500 V; collision chamber injection voltage (CXP): 2.0; atomizing gas (GS1): 55 psi; collision gas (CAD): medium; assist gas (GS2): 60 psi; and air curtain gas (CUR): 20 psi.
4.9. Determination of Free Ca2+ Concentration
After the brain was removed from the animals, the surface of the tissue was washed with PBS and the tissue was placed in a clean culture dish. The membrane on the tissue surface was removed with a cotton ball, and the tissue was washed in PBS and diced. A volume of 1 mL of 0.4% collagenase solution prepared in PBS was added to the dish, and the contents were transferred to a 15 mL centrifuge tube and made up to 2 mL with 0.4% collagenase. The sample was incubated at 37 °C for 40 min, shaking once every 10 min, and the reaction was terminated by adding 3 mL of 10% fetal calf serum. The solution was mixed, filtered through a 200 mesh, and centrifuged at 670× g for 5 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 5 mL PBS. A volume of 1 mL cell suspension (approximately 107 cells) was centrifuged again at 670× g for 5 min at 4 °C, and the supernatant was discarded. The cells were resuspended in 1 mL of PBS, Fluo-3-Am was added to a final concentration of 5 μM, and the solution was incubated at 37 °C for 30 min then rinsed twice with PBS to remove excess dye. The cells were resuspended and 300 μL of the solution was filtered into a flow tube, and the free calcium ion concentration was measured using a flow cytometer (FACS Canto II, BD, USA).
4.10. Statistical Analysis
Data were expressed as mean ± SEM, and were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistical analysis was performed using SAS 8.2 (IBM, Armonk, NY, USA) and GraphPad Prism 6.01 (GraphPad Software Inc, San Diego, CA, USA). p < 0.05 was considered statistically significant.