Meranzin hydrate elicits antidepressant effects and restores reward circuitry

Behavioural Brain Research 398 (2021) 112898

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Meranzin hydrate elicits antidepressant effects and restores reward circuitry XiangFei Liu a, JiaLing Zhou a, Tian Zhang a, Ken Chen a, Min Xu a, Lei Wu a, Jin Liu b, YunKe Huang a, f, BinBin Nie c, Xu Shen d, Ping Ren e, Xi Huang a, * a

State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Institute of TCM-Related Comorbid Depression, Nanjing University of Chinese Medicine, Nanjing, China Department of Traditional Chinese Medicine, Xiamen University, China c Key Laboratory of Nuclear Analytical Techniques, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China d State Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, China e Department of Geriatrics, Jiangsu Province Hospital of TCM, Nanjing University of Chinese Medicine, Nanjing, China f Master Degree Candidate at Department of Gynaecology and Obstetrics, Fudan University Medical School, China b

A R T I C L E I N F O

A B S T R A C T

Keywords: MH fMRI-BOLD UCMS Reward system Comorbidity

The burden of depression is enormous, and numerous studies have found that major depressive disorder (MDD) induces cardiovascular disorders (CVD) and functional dyspepsia (FD). Excitingly, meranzin hydrate (MH), an absorbed bioactive compound of Aurantii Fructus Immaturus, reverses psychosocial stress-induced mood disor­ ders, gastrointestinal dysfunction and cardiac disease. Pharmacological methods have repeatedly failed in an­ tidepressant development over the past few decades, but repairing aberrant neural circuits might be a reasonable strategy. This article aimed to explore antidepressant-like effects and potential mechanisms of MH in a rat model of unpredictable chronic mild stress (UCMS). Utilizing blood oxygen level-dependent (BOLD) functional mag­ netic resonance imaging (fMRI), we sought to find reliable neurocircuits or a dominant brain region revealing the multiple effects of MH. The results show that compared with UCMS rats, MH (10 mg/kg/day for 1 week i.g.)treated rats exhibited decreased depression-like behaviour; increased expression of brain-derived neurotrophic factor (BDNF) in the hippocampal dentate gyrus; and normalized levels of adrenocorticotropic hormone (ACTH), corticosterone (CORT), and acylated ghrelin (AG). Additionally, the UCMS-induced rise in BOLD activation in the reward system was attenuated after MH treatment. A literature search shown that nucleus accumbens (NAc) and hypothalamus of the reward system might reveal multiple effects of MH on MDD-FD-CVD comorbidity. Further research will focus on the role of these two brain regions in treating depression associated with comorbidities.

1. Introduction Depression, which affects 350 million people worldwide, is the predominant cause of disability and suicide [1]. However, selective se­ rotonin reuptake inhibitors (SSRIs), the first-line antidepressants, have a slow onset of action [2], cause inhibition of gastrointestinal motor

activities [3] and cardiotoxic side effects [4,5], and even increase the risk of suicidal thoughts and behaviour [6]. The N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine elicits rapid antidepressant ef­ fects via blockade of NMDAR-dependent bursting activity of LHb neu­ rons [7]. However, it has many adverse effects, such as dissociation, addiction, neurotoxicity, palpitation and nausea, that may restrict its

Abbreviations: MDD, major depressive disorder; FD, functional dyspepsia; CVD, cardiovascular disorders; MH, Meranzin hydrate; UCMS, chronic unpredictable mild stress; BOLD, blood oxygen level-dependent; fMRI, functional magnetic resonance imaging; BDNF, brain-derived neurotrophic factor; ACTH, adrenocortico­ tropic hormone; CORT, corticosterone; AG, acylated ghrelin; NAc, nucleus accumbens; NMDAR, N-methyl-d-aspartate receptor; GZSX, Gan-zhu-shu-xie; CSS, Chaihushugan-San; VTA, ventral tegmental area; SPT, sucrose preference test; OFT, open-field test; FST, forced swimming test; SPM8, statistical parametric mapping; FWHM, Full Width at Half-maximum; HPA, hypothalamic-pituitary-adrenocortical; ROIs, regions of interest. * Corresponding author at: Street address: No.138 XianLin Avenue, QiXia District, NanJing City, Jiangsu Province, China. E-mail addresses: [email protected] (X. Liu), [email protected] (J. Zhou), [email protected] (T. Zhang), [email protected] (K. Chen), [email protected] (M. Xu), [email protected] (L. Wu), [email protected] (J. Liu), [email protected] (Y. Huang), [email protected] (B. Nie), xshen88@ 163.com (X. Shen), [email protected] (P. Ren), [email protected] (X. Huang). https://doi.org/10.1016/j.bbr.2020.112898 Received 23 February 2020; Received in revised form 28 August 2020; Accepted 31 August 2020 Available online 6 September 2020 0166-4328/© 2020 Published by Elsevier B.V.

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Behavioural Brain Research 398 (2021) 112898

clinical use [8–10]. A plethora of literature has demonstrated that MDD has a close and bidirectional association with cardiovascular disorders (CVD) [4,5] and functional dyspepsia (FD) [11], and current antide­ pressants are unable to solve these problems. Available pharmacological strateges have not, however, precisely targeted the pathophysiology of mental diseases, resulting in no improvement in the efficacy of new drugs developed in the past few decades [12]. Complex neural circuits, functional collections of brain cells, are easily destroyed in depressed patients with mood disorders [13]. Repairing aberrant neural circuits may give us a reasonable strategy forward in the study of mental illness, and of course, successful drug development requires a pharmacological approach [14]. According to the traditional Chinese medicine theory “Gan-zhushu-xie (GZSX)”, the “Gan” can improve psychosocial stress-induced mood disorders, gastrointestinal dysfunction, and cardiac disease by regulating the ascent and descent of “Gan-Qi” [15]. Guided by GZSX, Chaihu-shugan- San (CSS), a famous traditional Chinese medicine (TCM) formula consisting of Chuanxiong Rhizoma, Citri Reticulatae Pericarpium Viride, Bupleuri Radix, Cyperi Rhizoma, Aurantii Fructus Immaturus, Paeoniae Radix Alba and Glycyrrhizae Radix, is utilized to remedy depression [16,17] and functional gastrointestinal disorders for long time [18–20]. Our previous study found that MH, an absorbed bioactive compound of Aurantii Fructus Immaturus [21,22], elicits fast antidepressant action via the AMPA–ERK1/2–BDNF pathway [23] and reverses gastrointestinal hypomotility of rats through increasing ghrelin levels [24]. Surprisingly, CSS and MH reverse atherosclerosis and depressive-like behaviours of mice via anti-inflammatory and BDNF-TrkB pathways [25]. MH may be a potential agent for treatment-resistant MDD-FD-CVD comorbidity. Functional magnetic resonance neuroimaging (fMRI) is one of the most powerful methods for studying neural circuits [26]. Furthermore, few studies have utilized BOLD–fMRI to detect disturbances in the mood regulating circuit, fronto-limbic circuit or limbic structures of rats or mice after exposure to UCMS or maternal separation [27–31]. BOLD–fMRI signals of brain activity predominantly depend on functional hyperaemia induced by neurotransmitters, particularly glutamate, indicating that fMRI primarily images neurovascular signalling as a consequence of synaptic activity [32]. This neuroimaging technique has greatly facili­ tated the investigation of brain activity in basic and clinical research in recent decades and is crucial for developing treatments for neurological disorders [33]. In addition, environmental stimuli lead to glucocorticoid secretion by the hypothalamic-pituitary-adrenocortical (HPA) axis, and glucocorticoids have important effects on neurocircuit regulation and behaviour [34]. Brain- derived neurotrophic factor (BDNF) has been implicated in mood disorders, and antidepressants reverse stress-induced decreases in hippocampal BDNF levels [35]. We strongly want to know the effect of MH on the brain regions of depressed rats, and try to find a reliable neurocircuit or a dominant brain region to explain the multiple effects of MH. In the current study, we employed the UCMS as a experimental model of depression. Our results showed that MH improved UCMS-induced depression behaviours, restore the dysfunction of the HPA axis, improve the expression of hippocampal BDNF, and repair the reward system function changes in depressed rats measured by BOLD.

approved by the Animal Care and Use Committees of Nanjing University of Chinese Medicine.

2. Materials and methods

The open-field test was carried out according to previously described protocols [38]. Before the experiment, rats were placed in the test room for 30 min. The experiment was conducted from 9:00 a.m. to 11:00 a.m. Rats were placed into the centre of the open-field apparatus (52 cm long ×48 cm wide ×32 cm high) with a total of 4 open boxes, and one rat was placed in each open box. The 60 W light tube above the central square was the only lighting source for the behaviour test room, and a quiet environment was kept. The spontaneous activity of rats was measured for 5 min by the Topscan intelligent analysis system, and the total dis­ tance of rat activity was recorded and analysed as the evaluation parameter.

2.2. Experimental design All rats were randomly divided into 4 groups (n = 6 rats/group), which include: (1) control group, (2) UCMS group, (3) UCMS + MH (10 mg/kg) treatment group, (4) UCMS + fluoxetine (10 mg/kg) treatment group. They were allowed to acclimatize for 7 days before use. Rats in the control group received normal conditions, and rats in the other groups received UCMS. On the third week of UCMS, the Control and UCMS groups received physiological saline (intragastrically). All treat­ ment groups were intragastrically administered once a day only in the last week of the UCMS procedure, and UCMS training was performed half an hour after the administration. All animals were subjected to the behavioural tests on consecutive days in the following order: sucrose preference test, open field test, and forced swimming test. 2.3. UCMS procedure The UCMS procedure was performed as described with a minor modification [36]. Briefly, the UCMS procedure involved seven different stress events, which were randomly arranged throughout 21 consecutive days. The stressors were: (1) 24 h of food and water deprivation, (2) 24 h of cage tilting (45◦ ), (3) 5 min of cold swim in 4℃ water, (4) 5 min of tail pinch, (5) overnight illumination, (6) 24 h of wet pad material, (7) 4 h of restricted movement. 2.4. Drugs and reagents MH (DIAO, Chengdu, China) and fluoxetine (mb1555) were dis­ solved in 0.9 % saline at a concentration of 10 mg/kg with a purity > 98 %. Enzyme-linked immunosorbent kits (ELISA) for CORT (CSB-E07014 r), ACTH (CSB-E06875 r), AG (CSB-E13167 r) were purchased from Wuhan Huamei (China). The anti-BDNF (ab108319) antibody was purchased from Proteintech Group (USA). 2.5. Sucrose preference test (SPT) The SPT was performed following a previous study [37]. In the beginning of the test, animals were habituated to two bottles of 2 % su­ crose solution (w/v) for 48 h to avoid neophobia. After adaptation, rats were deprived of water and food for 16 h. The sucrose preference test was conducted at 9:00 a.m. Rats were housed in individual cages and were free to access to two bottles of solution, one containing 2 % sucrose so­ lution and the other containing tap water, were weighed and presented to each rat. The positions of the two bottles (right/left) were varied randomly across animals and were reversed after 2 h. After 4 h, the consumption volume of each solution was recorded and analysed with the formula: sucrose preference (%) = sucrose consumption/(sucrose con­ sumption + water consumption) × 100 %. 2.6. Open-field test (OFT)

2.1. Animals Adult male Wister rats weighing 180− 220 g were purchased from Shanghai Xipuer-Bikai Laboratory Animal Co., Ltd. (Shanghai, China). All Animals were maintained on a 12-h light/dark cycle (lights on 06:00 – 18:00) under controlled temperature (22 ± 2℃) and humidity (55 % ± 5 %), with free access to water and food ad libitum. All procedures for animal care and use were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were 2

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2.7. Forced swimming test (FST)

(uncorrected) and a cluster-extent threshold of 10 voxels. Moreover, according to the voxel-wise analysis results, bilateral of four important regions of interest (ROIs) were selected, comprising Amygdaloid body, hippocampus, hypothalamus, visual cortex. The mean signal intensity of all voxels in these ROIs was further calculated.

For the forced swimming test, the method was performed as described previously [37] with slight modifications. Briefly, rats were forced to swim individually in plastic cylinders (height: 50 cm, diameter: 10 cm) that contained 38 cm of tap water at 22 ± 1℃. Animals were exposed pre-swimming for 15 min. After 24 h, rats were subjected to 5 min. of forced swimming. If the rat did not move or only did necessary movements to keep its head on the water, it was determined as immobile time.

2.10. Sample preparation Blood samples were collected and centrifuged (15 min, 3500 r/min, 4℃), and supernatant was stored at –80℃ until analysis. After blood collection, rats were transcardially perfused with saline solution until the blood was clear followed by 4 % paraformaldehyde (dissolved in 0.1 M phosphate buffer saline (PBS); pH = 7.4). Brains were removed and post-fixed with the same fixative overnight. Brain samples were paraffinembedded and cut into serial 2 μm-thick coronal frozen sections used for subsequent immunofluorescence staining analysis.

2.8. fMRI data acquisition After performing behavioural tests, rats were anaesthetized with isoflurane (3–5 % for induction and 1–2% for maintenance). Each rat was positioned in the scanner in a prone position. The head was stabi­ lized with a bite bar and two rods located on opposite sides of the temporal surface of the head. A pressure-sensor (SAII Instruments, Model 1030 Monitoring & Gating System, USA) was used to measure body temperature and respiration rates. A 9.4 T/21 cm horizontal bore magnet (Magnex, UK) equipped with an elliptical surface radiofrequency coil (24 × 18 mm2) and Biospec console (Bruker, Germany) was used to acquire functional brain images. An echo-planar imaging (EPI) sequence (with parameters of matrix size = 128 × 128, flip angle = 80◦ , resolution = 0.3 mm × 0.3 mm, slice thickness = 1.5 mm, slice gap = 1.5 mm, repetition time (TR) =2000 ms, echo time (TE) =18 ms, volume = 24) was used. In addition to the functional data, coplanar T2-weighted scanning was also performed.

2.11. Enzyme-linked immunosorbent assays (ELISA) Serum concentrations of CORT, ACTH and AG were measured using a commercial ELISA kit (Wuhan Huamei Bioengineering Institute, China) according to the manufacturer’s instructions. 2.12. Immunofluorescence (IF) staining Sections of 2 μm thickness were baked at 60℃ for 12 h. Paraffinembedded tissue was deparaffinized and hydrated through a series of graded alcohols. The sections were immersed in 0.01 M citrate buffer (pH 6.0) for heat repair antigen retrieval, and sections were washed three times (3 × 3 min.) in 0.1 M PBS solution (pH 7.4). Then, the slice was placed in a sodium borohydride solution at room temperature for 30 min., rinsed with water for 5 min., placed in Sudan black dye solution for 5 min., and finally rinsed with water for 3 min. After blocking in 5% BSA for 60 min, the section was incubated with primary rabbit polyclonal anti-BDNF antibody (1:100, Abcam) overnight at 4℃. Sections were rinsed and incubated with secondary antibodies (goat anti-rabbit IgG, Proteintech) at 37℃ for 90 min. Sections were then washed three times (3 × 5 min.) with PBS and incubated with 4′ , 6-diamidino-2-phenylin­ dole dihydrochloride (DAPI) (Thermo Fisher Scientific, USA) at room temperature for 10 min. Images were acquired with a fluorescence mi­ croscope (BA410E, Motic, China).

2.9. MRI data analysis All the functional image post-processing was performed by a single experienced observer who was unaware to whom the scans belonged. The pretreatment and data analysis were performed using the toolbox named spmratIHEP [39–43] in statistical parametric mapping (SPM8) software (Welcome Department of Imaging Science; http://www.fil.ion. ucl.ac.uk/spm), which comprised a fMRI rat brain template and atlas in Paxinos & Watson space. The functional data sets of all individuals were pre-processed in spmratIHEP by the following steps: (1) The first ten volumes of each individual were discarded to allow for magnetization equilibrium; (2) Slice timing: the differences in slice acquisition times of each individual were corrected using slice timing; (3) Realign: the temporal processed volumes of each subject were realigned to the first volume to remove the head motion, and a mean image was created over the 310 realigned volumes. All participants had less than 1 mm of translation in the x, y, or z-axis and 1◦ of rotation in each axis; (4) Spatial normalization: the realigned volumes were spatially standardized into the Paxinos & Watson space by normalizing with the EPI template of the rat brain via their corresponding mean image. Then, all normalized images were resliced by 1.0 × 1.5 × 1.0 mm3 voxels; (5) Smooth: normalized func­ tional series were smoothed with a Gaussian kernel of 2 × 4×2 mm3 Full Width at Half-maximum (FWHM). Afterwards, detrending and filtering (0.01− 0.08 Hz) were performed on the smoothed images using Data Processing Assistant for Resting-State fMRI (DPARSF, http://rfmri. org/DPARSF). Individual regional homogeneity (ReHo) maps were generated by calculating Kendall’s coefficient of concordance of the time series of a given voxel with those of its nearest neighbour (26 voxels). Then, all ReHo maps were smoothed with an isotropic Gaussian kernel of 2 mm3 FWHM. Then all the smoothed ReHo maps were analyzed within SPM8 based on the framework of the general linear model. To identify the difference in ReHo between the patients and the healthy controls, two-sample t-test was performed. Brain regions with significant ReHo changes in patients were yielded based on a voxel-level height threshold of P < 0.005

2.13. Statistics All data were analysed using GraphPad Prism 8 (La Jolla, CA, USA) and are presented as the means ± SEM. After verifying the normal dis­ tribution of the data, they were analysed using one-way ANOVA with independent measurement, followed by the Dunnett post hoc multiplerange analysis for individual means. P values of 0.05 or less were considered significant. 3. Result 3.1. UCMS-induced effects on depressive-like and anxiety-like behaviours are reversed by MH or fluoxetine treatment As shown in Fig. 1, the UCMS group exhibited a significantly longer immobility time in the FST than the control group [Fig. 1B, ANOVA, F(3,20) = 27.64, P < 0.0001]. MH and fluoxetine significantly decreased the duration of immobility time compared to the UCMS group (P < 0.001 or P < 0.01). The results of SPT are shown in Fig. 1C. The sucrose preference index was significantly reduced by UCMS compared to the control group [Fig. 1C, ANOVA, F(3,20) = 9.849, P = 0.0003] at the end of the experiment. MH and fluoxetine showed significant cures after oneweek treatment compared with the UCMS group (P < 0.01 or P < 0.05). Fig. 1D exhibited characteristic distances of rat movement in the OPT. 3

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Fig. 1. Schedule of the UCMS procedure for rats (A). Effect of UCMS and MH treatment (10 mg/kg) on forced swimming (B), sucrose preference (C), open field test (D). Values were expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus Control. ##P < 0.01, ###P < 0.001 versus UCMS model.

Compared to the control group, the UCMS group exhibited a significant reduction in total distance travelled [Fig. 1D, ANOVA, F(3,20) = 6.972, P = 0.0021], which was significantly reversed after one week of MH or fluoxetine administration (each P < 0.01).

3.4. UCMS-induced BOLD signal changes are reversed by MH treatment The voxel-level height threshold was P < 0.005 (uncorrected), and the cluster-extent threshold was 10 voxels. UCMS, unpredictable chronic mild stress; L, left; R, right; ReHo, regional homogeneity. According to regional homogeneity (ReHo) analysis, UCMS-induced increased BOLD activation in rat brain regions was primarily in the midbrain and reward system (including the hippocampus, hippocampus: dentate gyrus, amygdaloid body, hypothalamus, cortex, NAc, etc.). On the other hand, the decreased BOLD activation of rat brain regions induced by UCMS was evident in the sensory cortex, hippocampus, corpus callosum, visual cortex, auditory cortex, striatum, parietal cortex posterior area, parietal association cortex, septal area, olfactory cortex, third ventricle, and hippocampus: dentate gyrus (Table 1, Fig. 4). The voxel-level height threshold was P < 0.005 (uncorrected), and the cluster-extent threshold was 10 voxels. UCMS, unpredictable chronic mild stress; L, left; R, right; ReHo, regional homogeneity. The UCMS-induced increase in BOLD activation was reversed by MH treatment. Decreased BOLD activation was evident in the striatum, limbic system: septal area, thalamus: lateral nucleus group, bed nucleus of stria terminalis, hippocampus, midbrain superior colliculus, accum­ bens nucleus, and third ventricle. Several brain regions also demon­ strated increased BOLD activation in the auditory cortex, amygdaloid body, sensory cortex, third ventricle, and temporal association cortex (Table 2, Fig. 5). The BOLD-fMRI images of rats were based on significant ROIs. The data analysis results are shown in Fig. 6. Comparing with Control group, the BOLD signals of bilateral hippocampus was decreased by UCMS

3.2. UCMS-induced dysregulation of the HPA axis is reversed by MH or fluoxetine treatment As shown in Fig. 2, the UCMS procedure caused a significant increase in plasma ACTH [Fig. 2A, ANOVA, F(3,20) = 5.514, P = 0.0063], CORT [Fig. 2B, ANOVA, F(3,20) = 10.46, P = 0.0002] and AG [Fig. 2C, ANOVA, F(3,20) = 8.196, P = 0.0009] levels compared with the control group. Plasma levels of the three were strikingly reduced by MH after one week of treatment (P < 0.05 or P < 0.01). Meanwhile, fluoxetine group exhibited markedly reduced plasma levels of CORT and AG (each P < 0.001). 3.3. UCMS-induced BDNF expression reduction is reversed by MH treatment The effects of MH on BDNF levels in the dentate gyrus of the hip­ pocampus in UCMS-induced rats are shown in Fig. 3. The results revealed that UCMS induced a significant reduction in the expression of BDNF [Fig. 3D and B, ANOVA, F(2,6)=34.84, P = 0.0005] compared with the control group. One-week MH administration significantly increased the BDNF levels compared with the UCMS group (Fig. 3D, P < 0.01).

Fig. 2. Effect of UCMS and MH treatment (10 mg/kg) on ACTH (A), CORT(B), AG (C). Values were expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus Control. ##P < 0.01, ###P < 0.001 versus UCMS model.

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Fig. 3. Representative micrographs of IF staining for the BDNF proteins (scale bar =100 μm,agnification) in the hippocampal DG region (A-C). The quantitative analysis of the mean number of BDNF (D). Values were expressed as means ± SEM. Scale bar =100 μm. **P < 0.01, versus Control. ###P < 0.001 versus UCMS model.

procedure, however MH further decreased the BOLD signals [Fig. 6A, ANOVA, F (2, 15) = 0.5188, P = 0.6055], [Fig. 6B, ANOVA, F (2, 15) = 0.4873, P = 0.6237]. Comparing with Control group, the BOLD signals was decreased in left amygdaloid body and was increased right amyg­ daloid body by UCMS procedure, while MH respectively reversed the changes above. [Fig. 6C, ANOVA, F (2, 15) = 0.9544, P = 0.4072], [Fig. 6D, ANOVA, F (2, 15) = 4.028, P = 0.0398]. As shown in Fig. 6E, the BOLD signals changed slightly in left visual cortex [Fig. 6E, ANOVA, F (2, 15) = 0.08924, P = 0.9151]. Comparing with Control group, the UCMS procedure decreased BOLD signals in right visual cortex, while MH reversed the changes above [Fig. 6F, ANOVA, F (2, 15) = 1.421, P = 0.2723]. As shown in Fig. 6G-H, the UCMS procedure increased BOLD signals in left hypothalamus: preoptic region and slightly decreased BOLD signals in right hypothalamus: preoptic region, while MH decreased BOLD signals of bilateral hypothalamus: preoptic region [Fig. 6G, ANOVA, F (2, 15) = 2.441, P = 0.1209], [Fig. 6H, ANOVA, F (2, 15) = 0.1639, P = 0.8503].

circuits [46]. However, inappropriate supplies of environmental en­ ergetic demand and chronic activation of the abovementioned adaptive systems generate stress-related depression. Dysfunctions of the HPA axis first demonstrate release of ACTH secretagogues from neurose­ cretory neurons in the hypothalamus [47]. Then, ACTH induces the release of CORT in rats and cortisol and glucocorticoids in humans through the systemic circulation [48,49]. Nevertheless, glucocorti­ coids are not the only hormones involved in the energetic integration of stress, as emerging evidence has demonstrated that ghrelin plays an important role in coordinating context-appropriate energetic demand for organisms that undergo different types of stressors [50]. Jeffrey et al. found elevated ghrelin or AG in circulating levels when mice were exposed to chronic stress or 60 % calorie restriction, which helps animal cope with stress to achieve homeostatic adaptation through increased caloric intake [51]. In addition, endogenous or exogenous ghrelin ex­ erts antianxiety or antidepression-like responses and induces secretion of ACTH and CORT through HPA axis activation under environmental stimuli [45,50]. BOLD–fMRI signals discovered in the early 90 s have become a fundamental technique applied to investigate neural activity through harnessing complex neurovascular coupling [33]. Neurovascular coupling, a response termed functional hyperaemia, posits that the brain evolved to sustain neuronal function by increasing the flow of blood after disorders [32]. Traditional concepts suggest that a decline in glucose or O2 concentrations, or increasing carbon dioxide con­ centrations, trigger a rise in blood flow. However, this concept has shifted following the discovery that neurotransmitters, particularly glutamate rather than energy expenditure, are the main factors inducing blood flow by generating nitric oxide and arachidonic acid [32]. Reward circuit comprising the mPFC, hippocampus, and amyg­ dala mediates stress integrative functions. Numerous studies have found that stress and depression lead to decreased expression of BDNF in key limbic brain regions, regulating mood and cognition, including the hippocampus, prefrontal cortex, amygdala, and most notably the hippocampus [52]. The hippocampus plays an important role in mood disorders [52], and BDNF in the dentate gyrus of the hippocampus may be crucial for regulating the remedy of antidepressants [53]. Gluco­ corticoids secreted during acute or chronic stress in several limbic and cortical areas, including the abovementioned brain regions, rapidly increase glutamate release [45]. In the present study, UCMS-induced high levels of CORT, ACTH, AG and a significant reduction in the expression of BDNF were reversed by MH treatment. These data sug­ gest that MH can exert antianxiety or antidepression-like actions by restoring dysfunction of the HPA axis.

4. Discussion Our previous studies have found the antidepressant effects of MH on rats that undergo 15 min of forced swimming acute stress and im­ provements in stress-induced gastrointestinal disorders [24]. In this study, we first employed the well-validated UCMS model to investigate the brain function and pathophysiology of depression and ultimately to evaluate the antidepressant-like effect of MH. Anhedonia is a core symptom of depression, for which we use the sucrose preference index as parameter [37]. In the present study, rats subjected to UCMS showed a decreased consumption of sucrose solutions in SPT, which indicated declining sensitivity to rewards. FST, known as a “behavioural despair” test, is widely used to assess depression-like behaviour in animal models with mood disorders [37]. In accordance with acute stress employed in previous studies [24], UCMS induced increased immobility times in the FST. OFT is a very popular animal model used to assess anxiety-like behaviour that is highly comorbid with depression [38,44]. UCMS induced decreased locomotor activity in OPT. It is noteworthy that MH significantly ameliorated UCMS-induced behavioural despair, anhe­ donia responses and anxiety-like behaviours. Chronic stress inducing threats to homeostasis results in increased secretion of glucocorticoid by the HPA axis [45]. In response to stress, glucocorticoids promote energy reallocation for organismal adaptation presented with environmental stimuli and help to meet context-appropriate energetic demands through integration of multiple systems and activation of the forebrain, hypothalamic, and hindbrain 5

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Table 1 Results of ReHo in the BOLD–fMRI Analysis. Coordinates of the peak voxel Structural name voxels T-max X Y Z Increased BOLD activation in the UCMS group in comparison with the Control group L Midbrain: superior 166 5.4174 − 1.268 3.1649 − 6.1179 colliculus R Midbrain: superior 137 5.7766 1.5346 4.3535 − 6.1179 colliculus R Hippocampus 79 4.7818 1.5214 3.3833 − 5.1579 R Thalamus: lateral 79 6.2365 2.594 4.3303 − 5.1579 nucleus group L Amygdaloid body 66 5.1022 − 4.6399 8.8831 − 3.7179 L Hippocampus 66 4.7601 − 3.6529 5.3485 − 7.5579 L Midbrain: inferior 57 4.6659 − 2.4419 4.091 − 7.5579 colliculus L Midbrain: periaqueductal 42 6.8682 − 0.1954 4.6783 − 6.1179 grey matter R Pretectal area 37 6.5396 2.1961 4.3073 − 5.1579 R Hippocampus: dentate 34 4.474 1.5016 3.698 − 3.7179 gyrus R Midbrain: inferior 33 3.853 1.1697 3.0744 − 8.5179 colliculus L Third ventricle 33 4.5548 − 0.2086 4.009 − 5.1579 R Midbrain: 31 7.7643 0.2025 4.7013 − 6.1179 periaqueductal grey matter L Hippocampus: dentate 28 4.6728 − 4.4685 6.927 − 6.1179 gyrus R Nucleus accumbens 25 3.957 2.495 7.0366 2.0421 L Thalamus: lateral nucleus 25 4.4531 − 3.3761 5.0636 − 7.5579 group L Pretectal area 24 5.0653 − 0.0702 4.4595 − 5.1579 L Hypothalamus: tuberal 23 5.5104 − 0.2614 9.0844 − 1.3179 region L Olfactory cortex 22 5.0045 − 4.1892 6.7689 − 7.5579 R Retrosplenial cortex 21 4.7837 1.0156 2.8544 − 6.1179 L Thalamus: medial 17 8.0725 − 0.057 4.6863 − 6.1179 nucleus group L Striatum 16 3.6486 − 4.5147 6.3279 − 2.7579 R Corpus callosum 15 3.9706 0.2617 3.1892 − 0.3579 R Midbrain: tegmentum of 15 5.4574 1.9366 4.5755 − 5.1579 midbrain L Pontine tegmentum of 14 4.4006 − 3.3629 5.1488 − 8.5179 pons R Hypothalamus: tuberal 12 4.7062 − 0.1032 9.0609 − 2.7579 region L Midbrain: tegmentum of 11 4.6715 − 0.1756 5.107 − 7.5579 midbrain R Striatum 11 3.6852 2.6334 7.2039 2.0421 L Bed nucleus of stria 10 5.1671 − 4.1036 8.0468 − 3.7179 terminalis Decreased BOLD activation in the UCMS group in comparison with the Control group L Sensory cortex 334 5.6847 − 5.7587 3.8501 − 0.3579 L Hippocampus 95 6.7421 − 5.8138 3.2602 − 5.1579 L Corpus callosum 74 6.6487 − 6.0906 3.2442 − 5.1579 L Visual cortex 70 5.157 − 4.6201 0.6919 − 5.1579 L Auditory cortex 58 5.4908 − 6.1104 3.1164 − 3.7179 R Auditory cortex 56 4.1385 6.2229 4.182 − 6.1179 L Striatum 35 4.8455 − 4.9126 3.526 − 2.7579 L Parietal cortex posterior 33 4.5398 − 5.018 0.8105 − 5.1579 area L Parietal association 29 5.6046 − 4.6399 0.7057 − 3.7179 cortex R Sensory cortex 25 4.3849 6.3283 3.3929 − 3.7179 R Visual cortex 18 3.5415 5.825 2.4067 − 6.1179 L Limbic System: septal 16 6.4085 − 4.6399 3.6262 − 3.7179 area R Olfactory cortex 15 3.6938 5.9659 4.7363 − 7.5579 L Third ventricle 15 6.9715 − 4.8994 3.6112 − 3.7179 L Hippocampus: dentate 12 5.8404 − 5.018 3.731 − 5.1579 gyrus R Parietal cortex posterior 12 3.8231 5.535 2.3055 − 5.1579 area R Corpus callosum 10 4.0246 5.9461 4.166 − 6.1179

Fig. 4. Statistical results of BOLD in the UCMS group in comparison with the Control group. The voxel-level height threshold was P < 0.005 (uncorrected) and the cluster-extent threshold were 10 voxels. UCMS, unpredictable chronic mild stress.

Table 2 Results of ReHo in the BOLD–fMRI Analysis. Coordinates of the peak voxel Structural name voxels T-max X Y Z Increased BOLD activation in the MH group in comparison with the UCMS group R Auditory cortex 45 4.6645 6.7592 4.6555 − 6.1179 R Amygdaloid body 23 4.7018 4.7194 8.2559 − 3.7179 L Sensory cortex 23 4.2539 − 5.3476 0.8962 − 1.3179 R Third ventricle 16 4.1493 4.581 8.2479 − 3.7179 L Temporal association 11 4.542 − 5.6597 3.0377 − 7.5579 cortex Decreased BOLD activation in the MH group in comparison with the UCMS group R Striatum 92 4.0511 2.1103 4.4792 1.0821 R Limbic System: septal 45 4.7963 0.5014 5.2535 1.0821 area L Thalamus:lateral nucleus 23 4.7643 − 3.3761 4.922 − 7.5579 group R Bed nucleus of stria 17 4.4304 1.4554 6.0194 − 0.3579 terminalis L Limbic System: septal 16 3.5649 − 1.0572 3.1974 − 1.3179 area L Hippocampus 13 4.5553 − 3.6529 5.0476 − 7.5579 L Midbrain: superior 13 3.4921 − 2.044 3.8308 − 7.5579 colliculus R Accumbens nucleus 12 4.1748 0.9166 5.72 1.0821 R Hippocampus 11 3.5179 1.0905 2.7048 − 2.7579 R Third ventricle 10 3.6306 1.4554 4.568 − 0.3579

Earlier psychiatric studies mostly focused on the hippocampus and frontal cortex regions, but these regions cannot dock all symptoms of mood disorders [54]. Emerging studies in humans and animals suggest that disruption of neural circuits of reward and aversion induce mood disorders [55]. The brain’s reward system primarily comprises ventral VTA, substantia nigra pars compacta (SNc), dorsal raphe nucleus (DRN), median raphe nucleus (MRN), NAc, lateral habenula, lateral hypothal­ amus, hippocampus, amygdala, and cortex [26]. In the present study, the regions in which UCMS induced a rise or decline in BOLD activity compared with the control group include the midbrain (including VTA, SNc, DRN, and MRN), cortex, hippocampus, thalamus, amygdala, NAc 6

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Taken together, all of the above results suggest that MH produces antianxiety and antidepressant-like responses in the UCMS group rats. According to distinctive changes in cyclic adenosine monophosphate, reduced expression of endothelial cell tight junction protein claudin-5, and abnormal blood vessel morphology in NAc of stress-susceptible mice, NAc might be a crucial brain region docking comorbid CVD and MDD [56,57]. The brain-gut axis seems to be bidirectional in that they interact via the vagus nerve and immune system, and a series of elegant brain-lesion studies has demonstrated that the hypothalamus plays an important role in the control of energy homeostasis and integration of metabolic status [58,59]. In summary, the data presented in this study show that MH signifi­ cantly reversed UCMS-induced broad and complex neuropsychiatric phenotypes, and NAc and the hypothalamus of reward circuit might reveal multiple effects of MH. Epidemiological studies have shown that the incidence of depression is higher among women, and the symptoms of women are different from men [60,61]. The responses of HPA axis and autonomic nervous system to psychosocial stress show significant and consistent differences depend on sex, with men may experience alcohol-use disorders, while women show greater depression and anxi­ ety [62,63]. We neglect gender differences in depression in current study, but we will focus on gender differences in depression in future research.

Fig. 5. Statistical results of BOLD in the UCMS group in comparison with the MH group. The voxel-level height threshold was P < 0.005 (uncorrected) and the cluster-extent threshold were 10 voxels. UCMS, unpredictable chronic mild stress.

CRediT authorship contribution statement XiangFei Liu: Conceptualization, Investigation, Methodology, Writing - original draft. JiaLing Zhou: Investigation. Tian Zhang: Validation. Ken Chen: Data curation. Min Xu: Formal analysis. Lei Wu: Software. Jin Liu: Visualization. YunKe Huang: Writing - review & editing. BinBin Nie: Data curation, Software. Xu Shen: Supervision. Ping Ren: Resources, Supervision. Xi Huang: Resources, Supervision, Funding acquisition.

and striatum. Increased glutamate release that induced a rise in BOLD activity and a decline in BOLD activity might reflect UCMS-induced reductions of excitatory synapses or decreases in neurotransmitter output [32]. The respondent regions seem to be consistent with the reward system. Compared with the UCMS, MH may attenuate BOLD activity on the reward system and limbic system by regulating dysfunction of the HPA axis, and the increased BOLD signals in the cortex regions, amygdala and third ventricle of the MH group may be a result of organism adaptation.

Declaration of Competing Interest There are no conflicts of interest.

Fig. 6. ROI-based quantitative analysis results. According to the voxel-wise analysis results, four significant ROIs, hippocampus, amygdaloid body, visual cortex and hypothalamus: preoptic region are further analyzed quantitatively. The BOLD-fMRI signals of bilateral ROIs were analyzed between Control, UCMS and MH rats respectively. Values were expressed as means ± SEM. #P < 0.001 versus UCMS model.

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Acknowledgements [21]

This work was supported mainly by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Integration of Chinese and Western Medicine) (PI, H.X.), Foundation for High-Level Talents from Nanjing University of Chinese Medicine (PI, H.X.) and grants No. 81573797 (PI, H.X.) and No: 81072967 (PI, R.P.) from the National Natural Science Foundation of China. We would like to thank LinRan Han for providing analysis help.

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