Prokinetic meranzin hydrate from Chaihu-Shugan-San improves depression-like behaviors and hypomotility in rats via ghrelin and neurocircuitry

Depression and functional dyspepsia (FD) are characterized by comorbidity, overlapping depression, and nausea. The pathogenesis of depression and FD is mediated by α2-adrenoreceptor and/or ghrelin. Antidepressant (A) or prokinetic (P) agents are numerous, but few have been investigated in this context. Ancient Gan-zhu-shu-xie (GZSX), whose representative traditional Chinese medicine(TCM) is Chaihu-Shugan-San (CSS), may exert antidepressant effects with prokinetic meranzin hydr-ate (MH) via α2-adrenoreceptors in the acute forced swimming (FS) test in rats.Therefore, the main aim of the study is to investigate the acute antidepressant and prokinet-ic effects of CSS and MH after acutely FS on rats, and its possible mechanism. Rockford, IL, USA). Protein lysates were separated by 15% and 8% SDS-PAGE electrophoresis and were transferred onto polyvinylidene diuoride (PVDF)membranes. After blocking with 5% bovine serum albumin (BSA) for 1 hr, the membranes were incubated with anti-BDNF (SANTAN, 1:200), anti-PMTOR (CST, 1:1000)and anti-beta-actin (Proteintech, 1:4000) antibodies at normal atmospheric temperature for 6 hr followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 hr. Then, the blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientic Inc.). BDNF and PMTOR were normalized to beta-actin bands. Then, all ReHo maps were smoothed with an isotropic Gaussian kernel of 2mm3 FWHM. Then all the calculated 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 Other regions affected by MH, [D-Lys3]-GHRP-6, uoxetine, and mosapride lie in proximity to the HTB. The HTB differentially regulates depression and FD. The hippocampus is implicated in depression and FD. The thalamus plays a major role in FD and minor role indepression. The hippocampus is a well-known target for the effects of antidepressants. Gastrectasia, nausea, and delayed gastric emptying, which are related to FD, match foci of BOLD regions centered in the thalamus [72–73]. In the present study, mosapride as a prokinetic stimulated the thalamus but not hippocampus. The feeding inhibitor [D-Lys3]-GHRP-6 acts on the thalamus, consistent with ghrelin’s prominent role in feeding behavior. Ghrelin antagonist [D-Lys3]-GHRP-6inhibits BOLD-activated HTB foci by MH. These effects are different from the BOLDsignal of areas stimulated by monistic nutrition regulated by ghrelin, including the thalamus, limbic areas, parahippocampal cortex, insula, and caudate BOLD datum of improved BOLD L.JF. data

Here, we aimed to assess whether ghrelin could affect DB&H [6,48] following acute FS and rapid A&P by MH and CSS. Further, we aimed to elucidate the uni ed neurocircuitry of depression and FD based on brain activation indicated by BOLD signal.

Materials And Methods
Preparation of CSS extract CSS consisted of seven medicinal plants (Radix Bupleuri, Dried Tangerine Peel, Fructus Aurantii, Rhizoma Ligustici Chuanxiong, Rhizoma Cyperi, Radix Paeoniae Alba, Liquorice Root) purchased from Xiangya Hospital of Central South University and identi ed by Dr. Xi Huang of Nanjing University of Chinese Medicine. First, 750 g of CSS was weighed according to the ratio (4:3:4:3:3:4:4) of the aforementionedherbs. A volume of 7,500 mL distilled water was added, and contents were soaked for 30 minutes. After soaking, contents were brought to a boil and continuously heated for 40 minutes, after which they were ltered with gauze. Decoctions were collected twice and lyophilized to obtain the powdered form of CSS,stored at 4℃ until use. The yield of lyophilized powder of CSS was about 20.5%(w/w). In additionally, the lyophilized powder was evenly dispersed in distilled water at dosage of CSS (30 g/kg) before the experiment.

Animals
Adult male Sprague Dawley rats weighing 180-200 g were obtained from Jiangning Qinglongshan Animal Cultivation Farm (Nanjing, China). Wild-type and GHSR KO male mice were purchased from Shanghai Model Animal Center. Rats were group-housed two per cage, and mice were group-housed four per cage in standard laboratory conditions (temperature, 23 ± 1ºC; humidity, 50% ±10%), under a 12-h light-dark cycle (lights on from 7 a.m.) with ad libitum access to water and food. All studies on animals were approved and conducted according to the institutional guidelines of the Animal Care and Use Committee at Nanjing University of Chinese Medicine. All animals were acclimatized 7 days before experimentation.
Except the sham group, all animals were forced to swim 15 min to establish a comorbidity model for inducing depressive-like behaviors and hypomotility. Vehicle,Sham WT and GHSR group refer to 0.9% saline (10 mL/kg).

Behavioral test
Forced swimming test (FST) Animals were placed in a cylinder (20 cm in diameter and 46 cm in height for rats; 10 cm in diameter and 25 cm in height for mice) containing 30 cm of water (23-25℃). Twenty-four hours before testing, animals were individually placed into the cylinder for 15 min FS. Test sessions (5 min in duration) were recorded with a videocamera. Periods of passive oating in the water and exerting minimal activity except for respiration were scored as immobility time [52][53].

Open eld test (OFT)
The size of the open box was 50 × 50 × 40 cm, which was divided into 16 grids. Rats were placed in the center of the open box. Behavior was recorded for 10 minutes using a video camera 24 h following acute stress [53].

Tail suspension test (TST)
Tape was used to suspend mice in a hanging box (25 × 35 × 60 cm). The entire 6-min session was recorded using a video camera, and immobility was analyzed. The mobility bouts of front limbs with momentum-induced oscillations and pendulum swinging were not regarded as mobility [53].

Western blot analysis
Animals were placed in a cylinder (20 cm in diameter and 46 cm in height for rats; 10 cm in diameter and 25 cm in height for mice) containing 30 cm of water (23-25ºC) for 15 min FS. After 30 min, MH or MH + Dlys was administered. Animals were anesthetized 30 min later, and the hippocampus was removed.
The whole hippocampus was lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors. Protein concentration was determined colorimetrically by BCA assay (Pierce, Rockford, IL, USA). Protein lysates were separated by 15% and 8% SDS-PAGE electrophoresis and were transferred onto polyvinylidene di uoride (PVDF)membranes. After blocking with 5% bovine serum albumin (BSA) for 1 hr, the membranes were incubated with anti-BDNF (SANTAN, 1:200), anti-PMTOR (CST, 1:1000)and anti-beta-actin (Proteintech, 1:4000) antibodies at normal atmospheric temperature for 6 hr followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 hr. Then, the blots were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scienti c Inc.). BDNF and PMTOR were normalized to beta-actin bands.

MRI acquisition and analysis
Rats were positioned in the scanner in a prone position after stress. Rectal temperature was maintained at 37.5ºC with a temperature-controlled water blanket placed beneath the rats. Respiratory rate was monitored at 60-80 breaths/min continuously throughout the entire experiment using an MRIcompatible pulse oximeter.Head position was stabilized with a bite bar and two rods located on opposite sides of the temporal surface of the head. Rats were anesthetized using inhaled iso urane (3% for induction and 1.5-2% for maintenance). Intraperitoneal injection of dexmedetomidine was performed 3 minutes later. SpmratIHEP toolbox [56][57][58] of the statistical parametric mapping (SPM8) software (Welcome Department of Imaging Science; http://www. l.ion.ucl.ac.uk/spm) was employed to preprocessing and data analysis. The SPM8 software comprises an fMRI rat brain template and atlas in Paxinos & Watson space.
The main steps for preprocessing in spmrat IHEP are as follows. In order to compass magnetization equilibrium, rst ten volumes of each individual were discarded. Slice timing was used to revise the differences of slice acquisition times of each individual. The temporal processed volumes of each subject were realigned to the rst 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 x, y, or z axis and 1° of rotation in each axis. Normalize the realigned volumes through the corresponding mean image and the rat brain EPI template, and spatially standardize it to Paxinos & Watson space. All the normalized images were resliced in 1.0 × 1.5 × 1.0 mm3 voxels. A Gaussian kernel of 2 × 4 × 2 mm3 FWHM (Full Width at Half-maximum) was employed to smooth the normalized functional series. Afterwards, use Data Processing Assistant for Resting-State fMRI (DPARSF, http://rfmri.org/DPARSF) to detrend and lter (0.01-0.08 Hz) the smoothed images.
Individual regional homogeneity (ReHo) maps were generated by calculating Kendall's coe cient of concordance of the time series of a given voxel with those of its nearest neighbor (26 voxels). Then, all ReHo maps were smoothed with an isotropic Gaussian kernel of 2mm3 FWHM. Then all the calculated 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, the two-sample t-test was performed using SPM8. Brain regions with signi cant ReHo changes in patients were yielded based on a voxel-level height threshold of P < 0.005 (uncorrected) and a cluster-extent threshold of 10 voxels.

Statistical analysis
Data were analyzed using SPSS software, version 25.0 (IBM SPSS,China) and values were presented as mean ± standard deviation(S.D.). The results were considered statistically signi cant if P < 0.05. ANOVA was applied by the LSD testing as post hoc analysis for further examination of group differences. The T-test was applied to the comparison between the two groups.

Results
The contents (mg/g) of 12 (ABCs) in CSS According to the UPLC method used, the contents (mg/g) of 12 (ABCs) in CSS wereas follows: albi orin, 559. 9  Quanti cation of MH in SD rats' hippocampal and intestinal by UPLC MH was determinted in healthy SD rats' hippocampus and intestine after administratedthe CSS ( Fig. 1.E-F), explain that the MH could be absorbed by the healthy SD rats' hippocampus and intestine.

Behavioral tests results
To validated the effect of CSS and MH on behavior performance of depression-like rat, FST and OFT were conducted.
Thedualism of gut-brain disorder, P&A, and their regulation implicated the HTB circuit based on BOLD activation foci. These results are causalities within and between counterparts 1-3, different from depressive comorbidity with somatic disease whose dichotomy is equivocal [11][12][13][32][33] in homogeneous studies. To date, an in vivo localized 1H-MRS study at 4.7 T without BOLD signal only coupled acute FS and rapid antidepressant desipramine [62]. We observed BOLD activated hippocampal responses to traditional Chinese medicine using rst-line antidepressant uoxetine as a control [27] and a connection from activated foci of the insular lobe, cingulate gyrus, and left amygdala cortex using subacute FS-induced visceral hyperalgesia in ovariectomized rats [63]. As described, it simultaneously induced DB&H (IT of 55.6%↑, NC of 20.8%↓,GE 39.1%↓, and InT 40.3%↓) compared with sham. MH affected IT, NC, GE, and INT (28.6%↓, 53.2%↑, 53.9%↑ and 25.1%↑ vs vehicle). P&A actions by MH were attenuated in acute FS after pretreatment with [D-Lys3] -GHRP (ghrelin antagonist). The regulatory effects of ghrelin in dual gut-brain disorder in homogeneous studies differ from its orthodox role in regulation of feeding reported since 1999 and effects on depression-like behavior reported since 2008 [64][65]. Further, prokinetic MH increased mTOR phosphorylation in parallel with increased GluA1 expression after ketamine administration [66] and activated the AMPA-ERK1/2-BDNF pathway [49], suggesting rapid antidepressant effects. MH-inducedantidepressant effects were almost absent in GHSR KO mice, suggesting ghrelin as a shared mediator of gut-brain disorder [51]. BOLD activation foci of the HTB circuit respond to stimuli within or between counterpart(s). Inside each counterpart, FS, MH, and ghrelin are bisected as gut-brain disorder and P&A, with shared regulation and interaction within the HTB circuit. Counterparts 1-3 represent FS→ pathophysiology, MH→therapy, and ghrelin→shared mechanisms, as shown in Fig. 3. The inside/outside counterparts indicate causalities. Here, each HTB circuit comprising comorbid-like pro les is different from the 19 other circuits identi ed from BOLD activated areas related to pathogenesis, therapy, and pharmacology for single diseases [17][18][19][20][21][22][23][24][25][26][27][28][29]. FS is widely used as a behavioral paradigm to assess depressive behaviors [52], but hypomotility has been overlooked. Similarly, the widely used chronic social stress model has been overlooked in heart disorder apart from when arrhythmia co-occurs with anxiety behavior during social stress and is integrated by an agent with simultaneous anti-anxiety and cardioprotective effects [67]. To date, nosingle compound except ferulic acid and MH [6,52] have been designed as a simultaneous A&P. Further, ghrelin and α2-AR [49] have been reported to commonly mediate A&P using distinct regulatorymechanisms [49,[64][65]. In mapping activated regions, the roles played by the intricate neurovascular couplings of BOLD signal are invaluable [68] but may be nonspeci c or have poor sensitivity [69]. The latter indicates spatial non-speci city of BOLD contrast especially from gradient echo pulse sequences [69] and lower sensitivity by spin echo pulse sequences which has higher speci city [69]. 7.0 T fMRI BOLD foci can precisely discriminate activity of neural populations in the sensorimotor cortex at 1.5 mm scale with high spatial delity, similar to electrophysiological determination of activated foci [46]. We address which foci are activated based on antagonist studies, using stress and SSRIs as control [17][18][19][20][21][22][23][24][25][26][27][28][29]. FS, MH, and ghrelin antagonist [D-Lys3]-GHRP-6 were used for activation and inactivation. Pre-inhibition of HTB matched the BOLD response map in Fig. 3.
Thenature and signal amplitude (Ke, cluster size or number of voxels) of uoxetine and mosapride differed from that of MH in the HTB. Mosapride only activated the thalamus and basal ganglia. BOLD signal intensity for MH was > 4.0 and 1.4 times greater than that for uoxetine in the hippocampus and thalamus, respectively. Fluoxetine-induced c-fos expression in the thalamus contradicts its effects on behavior [70][71]. [D-Lys3]-GHRP-6 inhibited MHreduced signal intensity by 84.8% and 23.3% in the hippocampus and thalamus, respectively. According to the top three standard criteria following acute FS-stimulated BOLD foci ranking among 17 regions (Table.1), we selected the HTB circuit. Other regions affected by MH, [D-Lys3]-GHRP-6, uoxetine, and mosapride lie in proximity to the HTB. The HTB differentially regulates depression and FD. The hippocampus is implicated in depression and FD. The thalamus plays a major role in FD and minor role indepression. The hippocampus is a well-known target for the effects of antidepressants. Gastrectasia, nausea, and delayed gastric emptying, which are related to FD, match foci of BOLD regions centered in the thalamus [72][73]. In the present study, mosapride as a prokinetic stimulated the thalamus but not hippocampus. The feeding inhibitor [D-Lys3]-GHRP-6 acts on the thalamus, consistent with ghrelin's prominent role in feeding behavior. Ghrelin antagonist [D-Lys3]-GHRP-6inhibits BOLD-activated HTB foci by MH. These effects are different from the BOLDsignal of areas stimulated by monistic nutrition regulated by ghrelin, including the thalamus, limbic areas, parahippocampal cortex, insula, and caudate [74][75].

Conclusions
GZSX and CSS are implicated in CDF. Ancient wisdom provides clues on related entities and their dualistic properties, rst by docking (in) activated HTB and integrating with neurocircuitry, as indicated via BOLD signals. If prede ned by one-disease-one-target-one-drug dogma, half of the available comorbidity-like dualism properties would be overlooked. MH-stimulated BOLD changes were modulated by uoxetine, mosapride, and [D-Lys3]-GHRP-6 in terms of the nature and intensity of signal foci. To conclude, we report on acute FS-stimulated DB&H, MH-induced rapid A&P, and ghrelin-related regulation coupled to BOLD signals in brain areas before, providing insight into a uni ed theory of depression pathogenesis and pharmacotherapy. Availability of data and materials The datasets used in this study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate
The protocols were approved by the Animal Care and Use Committee at Nanjing University of Chinese Medicine. (No.201905A013).

Consent for publication
The manuscript is approved by all authors for publication.