Jieyu Anshen Granule, a Chinese Herbal Formulation, Exerts Effects on Poststroke Depression in Rats

Jieyu Anshen granule (JY) is a traditional Chinese medicine formula for treating depression and anxiety. The aim of the study was to observe the effects of JY on poststroke depression (PSD) and investigate the underlying mechanism. PSD rat model was developed by middle cerebral artery occlusion following chronic unpredictable mild stress in conjunction with isolation rearing. We performed behavioral tests, Western blot, ELISA, and BrdU/NeuN staining. Treatment with JY showed significant antidepressant effect in open-field and sucrose preference tests, as well as significant improvement in beam-walking, cylinder, grip strength, and water maze tests. In addition, treatment with JY could restore the levels of neurotransmitters and decrease the levels of hormone and inflammation cytokines in serum and brain. Treatment with JY also showed significant regulation in the expression of neurotransmitter receptors and NF-κB/IκB-α signaling in the prefrontal cortex and hippocampus. Moreover, the numbers of newborn neurons in the hippocampus were increased by treatment with JY. Our results suggest that JY could ameliorate PSD and improve the neurological and cognitive functions. The antidepressive effect may be associated with the modulation of JY on monoamine system, neuroendocrine, neuroinflammation, and neurogenesis.


Introduction
Poststroke depression (PSD), which is different from general depression, is an extremely frequently neuropsychiatric disorder following ischaemic stroke, and common mood symptoms after stroke include anxiety and feelings of despair as well as anhedonia [1]. One-third of stroke patients suffer from depression, and depression negatively affects patients' ability to engage in rehabilitation therapies [2]. e pathophysiology of PSD is associated with a complex network of interrelated regulatory factors including the hypothalamic-pituitary-adrenal (HPA) axis, monoamine neurotransmitters, neuroinflammation, and neurogenesis [3]. Selective serotonin reuptake inhibitors (SSRIs) are the first choice for the pharmacological prevention and therapy of PSD [2,4]. Patients taking antidepressant drugs often experience high relapse rates and a variety of side effects, such as nausea, headaches, somnolence, and dry mouth [5,6].
Traditional Chinese medicine (TCM) is one of the commonly used complementary and alternative medicine therapies for depression, while a formula contains several herbs (Chaihu, Gancao, Fuling, Suanzaoren, Yujin, Baizhu, Yuanzhi, Shichangpu, Banxia, etc.) with a specific proportion interacting with each other to improve therapeutic effects and to reduce toxicities [7,8]. TCM or its major constituents are often used in clinical practice in China for treating depression; for example, polyphenols may show antidepressant effects through normalizing HPA hyperactivity [9]. Jieyu Anshen granules (JY) are a classical TCM with antidepressant activity that have been recognized by the China Food and Drug Administration [10]. e use of JY alone or in combination with additional antidepressants has already been widely implemented in China as a means of treating anxiety and depression [11]. However, it is not clear whether JY can be effective for PSD. us, a rat model of PSD was established using chronic unpredictable mild stress (CUMS) following middle cerebral artery occlusion (MCAO); then effects of JY were observed and underlying mechanism was investigated.

Animals.
Male Sprague-Dawley rats (240-260 g, 7-9 weeks) provided by Pengyue Animal Co. Ltd (License No. SCXK 20140007) had ad libitum food/water access while housed at 22 ± 2°C with 55 ± 5% humidity, as well as a 12 hour light/dark cycle. All animals had 1 week to acclimatize prior to study initiation. All studies were consistent with the guide from the National Institute of Health and received approval from the Ethical Committee of Yantai University.
CUMS consisted of a total of 7 unique stressors: swimming in 4°C water (5 min), 45°cage tilt (17 h), water deprivation (18 h, after which empty water bottles were given for 1 h), deprivation of food and water (20 h, followed by a sucrose preference test), soiled cages (200 ml water mixed into 100 g sawdust bedding; 21 h), paired caging (2 h), and overnight illumination (no darkness for 36 h). Neurological scores were determined 24 h after MCAO, then stressors were administered to rats in a random order for the following 18 days [12]. e rats were isolation-reared (one per cage) except CON group.
From the second day after MCAO, rats in the JY groups were administered JY (1 or 3 g/kg) daily for 4 weeks, while rats in the CIT10 group were administered citalopram (10 mg/kg) on the same schedule. Both JY and citalopram were administered intragastrically a volume of 1 mL/kg. Study design is illustrated in Figure 1. is test was performed both at baseline and following treatment, and sucrose preference was calculated as follows: sucrose preference � sucrose intake (g)/sucrose intake (g) + water intake (g) [12].

Open-Field Test.
Despair of rats was evaluated by open-field test (OFT). For this examination, rats were put into an open-topped cylindrical box with black walls (height, 40 cm; diameter, 75 cm). e bottom of the box was marked in 25 equally sized block sections. Rat locomotion and rearing activity were then monitored, with locomotor activity being quantified based on the number of blocks crossed and rearing activity being assessed based on how many times rats stood on their hind legs. Rats were each assessed one time for 3 min (isolation) [13].

Procedure for Evaluation of Neurological and Cognitive
Function in Rats 2.5.1. Beam-Walking Test. Rat coordination and motor movement integration was evaluated by beam-walking test (BWT) [14]. Before MCAO, rats underwent two training sessions per day for 2 days. During all sessions, a wooden beam (2.5 cm × 120 cm) was used, with a 30 × 30 × 30 wooden box placed on the other side to encourage rats to cross the beam. For training, rats were positioned on the center or starting points on the beam during the 1 st and 2 nd trials, respectively. Cushions were present under the beam to prevent any fall-induced injury to animals. Crossing time was determined as the time between when a forepaw was first extended onto the beam and when a forepaw was first extended into the wooden box, with a maximum cut-off time of 60 s.

Cylinder
Test. Asymmetry in forelimb use was evaluated by cylinder test (CT). For this assay, a transparent glass cylinder (30 cm high, 20 cm in diameter) was used. Rats were placed into the cylinder, and 20 movements were recorded for each animal, after which we calculated forelimb asymmetry as follows: 100 × (ipsilateral forelimb use +1/2 both)/ total forelimb use [15].

Forelimb Grip Force
Test. Muscular strength was evaluated by forelimb grip force test (FGFT), using a grip strength meter [16]. To measure this strength, rats were held by the tail over a grid in a position where they were able to reach the strength meter using their forepaws. Maximal grip strength was that at which rats were no longer able to grip the meter when they were pulled backwards away from it.

Memory Water Maze
Test. Spatial memory was evaluated by memory water maze test (MWMT), which is a standard approach for evaluating cognitive function [17]. For this test, we filled a pool, 1.2 m in diameter, with water and placed the study animals into the pool for 120 s during which time they sought to locate a hidden platform which was positioned 1 cm beneath the water surface. Surrounding the walls of the pool were specific visual cues to guide rats, including a horizontal tube on the north wall, an "X" on the south wall, an entrance door on the east wall, and a vertical tube on the west wall. Upon finding the hidden platform, rats were given 15 s during which they could rest on it. If they did not locate this platform within 60 s, they were put onto it for 15 s. Over a period of 4 days, rats received 4 total attempts to locate this platform. On the 5 th day this platform was removed and rats were given a 60 s performance assessment. e time spent in the quadrant where the platform had been located was used as a means of evaluating the degree to which rats were able to remember and navigate to the platform location.
2.6. Western Blotting. Prefrontal cortex and hippocampal tissue samples were washed with PBS and then lysed via the use of lysis buffer containing phenylmethylsulfonyl fluoride. Lysates were thoroughly homogenized and then spun for 10 minutes (12,000 ×g at 4°C), after which a Bradford assay was utilized to measure protein concentrations in supernatants. ese samples were then boiled in 5x loading buffer, electrophoretically separated using 12 % SDS-PAGE gels, and transferred to PVDF membranes that were blocked for 4 h with 5% nonfat dried milk. Membranes were then probed overnight using 1 :1000 dilutions of appropriate primary antibodies against 5-HT 1A R, ADRα2, NF-κB, IκB-α, GR, and β-actin at 4°C. Blots were then probed using appropriate secondary antibodies and were developed with ECL reagents and a gel imager (GE, LAS4000). Densitometric quantification was utilized as a means of assessing protein levels, and samples were normalized to levels of β-actin contained therein [18].   Evidence-Based Complementary and Alternative Medicine samples were measured by HPLC using an ultraviolet detector, with an approach adapted from previous work [19]. Briefly, we suspended brain tissue using ice-cold 0.6 mol/L perchloric acid supplemented with 0.5 mmol/L EDTA as well as 0.8 mmol/L L-cysteine. ese tissue homogenates then underwent 4°C centrifugation at 16,000 rpm for 20 minutes, and supernatants were filtered via 0.25 filtration into the chromatographic system. A Quaternary pump HPLC system (SHIMADZU: LC-20A, Shimadzu, Kyoto, Japan), 25 cm long by 4.6 mm in diameter, and a Cosmosil C 18 Column (5 mm particles) (Inertsil ODS-2) were used for all HPLC. e mobile phase contained 90% (v/v) 0.1 mol/L of a sodium acetate solution (0.1 mmol/L EDTA-2Na, pH 5.1), 10% (v/v) methanol. A 20 μL injection volume was used, with a 1 mL/min flow rate and a 275 nm wavelength. DA, 5-HT, and NE retention times were, respectively, 5.02, 10.08, and 3.37 minutes.

Statistical
Analysis. SPSS v.16.0 was used for all analyses, and data are expressed as means ± SEM. Two-way ANOVAs were used to compare SPT results, with treatment and day as the two factors. Other behavioral tests, Western blotting, and biochemical assays were performed by using one-way ANOVA via Tukey's post hoc comparison when comparing more than two groups. P < 0.05 was considered significant.

JY Improved Neurological and Cognitive Functions in PSD
Rats.
e MCAO + CUMS rats significantly decreased the time [F (4,35) � 2.71, P < 0.05] and distance [F (4,35) � 3.50, P < 0.05] in the target quadrant compared to CON rats. Treatment of rats with JY or CIT10 significantly increased the distance and time in the target quadrant compared to MCAO + CUMS rats (all P < 0.05) (Figure 4). e results suggested that JY could significantly improve neurological and cognitive functions in PSD rats.

JY Increased the Levels of Neurotransmitters in Prefrontal
Cortex, Hippocampus, Hypothalamus, and Striatum. As shown in Table 2, there was a significant increase in the levels of DA and 5-HT in prefrontal cortex of MCAO + CUMS rats compared to CON rats (all P < 0.01). Compared to MCAO + CUMS rats, the levels of DA and 5-HT were significantly increased in prefrontal cortex of CIT10 and JY1 groups (P < 0.05, P < 0.01). ere was also significant differences in the levels of NE, DA, and 5-HT in hippocampus between MCAO + CUMS and CON rats (P < 0.05, P < 0.01). JY1 treatment significantly increased the levels of these neurotransmitters in the hippocampus compared to MCAO + CUMS rats (P < 0.05, P < 0.01), whereas CIT10 significantly increased the level of 5-HT (P < 0.05). Levels of these neurotransmitters in the hypothalamus of MCAO + CUMS rats were significantly decreased compared to CON rats (all P < 0.01). JY1 treatment significantly increased the levels of these neurotransmitters in the hypothalamus compared to the MCAO + CUMS rats (P < 0.05, P < 0.01), whereas CIT10 increased the levels of DA and 5-HT (P < 0.05, P < 0.01). e levels of these neurotransmitters in the striatum of MCAO + CUMS rats were significantly decreased compared to CON rats (all P < 0.05). Compared to MCAO + CUMS rats, the level of NE in the striatum of CIT10 and JY1 treated rats was significantly increased (P < 0.05, P < 0.01). e results suggested that the levels of neurotransmitters in brain could be increased by administration of JY on PSD rat model.

JY Regulated the Expression of 5-HT 1A R and ADRα2 in Prefrontal Cortex and Hippocampus.
e expression of 5-HT 1A R was significantly reduced in prefrontal cortex in MCAO + CUMS rats compared to CON rats (P < 0.05). CIT10 and JY1 treatment significantly increased the expression of 5-HT 1A R (P < 0.05, P < 0.01) (Figure 5(a)). e expression of ADRα2 was significantly reduced in prefrontal cortex in MCAO + CUMS rats compared to CON rats (P < 0.01). JY1 and Evidence-Based Complementary and Alternative Medicine JY3 treatment significantly increased the expression of 5-HT 1A R (P < 0.01) ( Figure 5(b)). e expression of hippocampal 5-HT 1A R was significantly reduced in MCAO + CUMS rats compared to CON rats (P < 0.01). CIT10, JY1, and JY3 treatment significantly increased the expression of 5-HT 1A R (P < 0.01) ( Figure 5(c)). e expression of hippocampal ADRα2 was significantly increased in MCAO + CUMS rats compared to CON rats (P < 0.01), and CIT10, JY1, and JY3 significantly increased the expression of ADRα2 (P < 0.01) ( Figure 5(d)). e results suggested that the levels of neurotransmitters in brain could be increased by administration of JY on PSD rat model.

JY Regulated the HPA Axis Activity and GR Expression in Hippocampus.
e level of serum ACTH was significantly increased in MCAO + CUMS rats compared to CON rats (P < 0.01). CIT10, JY1, and JY3 treatment significantly decreased the level of serum ACTH (P < 0.05, P < 0.01) (Figure 6(a)). e level of serum CORT was significantly increased in MCAO + CUMS rats compared to CON rats (P < 0.01). JY1 and JY3 treatment significantly decreased the level of serum CORT (P < 0.05, P < 0.01) (Figure 6(b)). e expression of hippocampal GR was significantly decreased in MCAO + CUMS rats compared to CON rats (P < 0.01). CIT10, JY1, and JY3 treatment significantly increased the expression of GR (P < 0.01) (Figure 6(c)). e results suggested that the dysfunction of HPA axis could be reversed by administration of JY on PSD rat model.
e results suggested that the excess level of cytokines could be inhibited by administration of JY on PSD rat model.

Quantifying the BrdU+/NeuN+ Cells in Dentate Gyrus of Hippocampus.
To determine the effect of JY on the newly formed neurons in the dentate gyrus of hippocampus, the newly formed neurons were assessed by analysis of BrdU with the neuronal marker NeuN (Figure 9(a)). e number of BrdU+/NeuN+ cells was significantly decreased in the dentate gyrus of hippocampus in MCAO + CUMS rats compared to CON rats (P < 0.05). CIT10 and JY1 treatment Data are expressed as mean ± SEM. n � 7-10. # P < 0.05, ## P < 0.01 compared to CON group. * P < 0.05, * * P < 0.01 compared to MCAO + CUMS group. 6 Evidence-Based Complementary and Alternative Medicine significantly increased the number of BrdU+/NeuN+ cells in the dentate gyrus of hippocampus (P < 0.05) (Figure 9(b)). e results suggested that JY could significantly improve the neurogenesis of hippocampus on PSD rat model.

Discussion
PSD is the most frequent neuropsychiatric consequence, and experimental models may also pave the way for the discovery of novel therapeutic strategies [20]. e PSD model, which is developed by MCAO plus CUMS in our study, showed significant depressive behaviors, including decreased sucrose preference and motivation. Decreased sucrose preference in SPT is consistent with anhedonia, the major symptom of depression [21,22], whereas the OFT measures the horizontal and vertical movement of rats for the evaluation of activity and curiosity [23]. e SPTand OFTare widely accepted behavioral paradigms for assessing pharmacological antidepressant activity [24]. Treatment with JY reversed the decreased sucrose preference and motivation in PSD rats the same as treatment with citalopram, indicating that JY could exert an antidepressant-like activity in PSD rats. PSD is strongly associated with further worsening of physical and cognitive recovery, functional outcome, and quality of life [20]. e impairment of physical and cognitive function is thought to be the factor most closely associated with PSD development and severity [25]. It has been shown that the physical and cognitive impairments could be reversed by treatment with TCM via targeting multiple pathways [26]. In our study, we found that PSD rats exhibited both neurological impairments such as reduced asymmetry, strength, and decreased walking time, as well as impaired memory with MWMT test. BWT is a behavior test on assessing motor coordination after a stroke, and the CT and FGFT tests are behavior tests on assessing subjects for motor impairments [4,27,28]. e impairment of neurological and cognitive functions was observed in our PSD rat model, and treatment with JY significantly decreased the impairments, including improving grip force, decreasing asymmetry and beam crossing time. Lesions in the hippocampus are known to disrupt memory and spatial awareness [17]. It was shown that treatment with JY could improve the disrupted spatial memory in PSD rats.
Pathophysiology of PSD is complex and multifactorial, resulting from the combination of ischaemia-induced neurobiological dysfunctions and psychosocial distress [29]. An integrated view of the etiopathology of PSD posits the interlinking between monoamines, neuroinflammation, HPA axis, and neurogenesis as the common denominator [30,31]. e central monoamine hypothesis proposes that imbalances in 5-HT, NE, and DA could result in depression [32]. Axons between the brainstem and cerebral cortex which contain these neurotransmitters can be disrupted during PSD, thus leading to imbalances in 5-HT, NE, and Evidence-Based Complementary and Alternative Medicine 9 DA synthesis throughout the brain [33,34]. In this study, neurotransmitters such as 5-HT, NE, and DA were assessed in prefrontal cortex, hippocampus, hypothalamus, and striatum of rats, which showed that the level of neurotransmitters was suppressed by CUMS exposure, which is consistent with previous studies. However, treatment with JY upregulated the levels of neurotransmitters. Certain 5-HT 1A R ligands are currently used as a means of treating depression, such vilazodone and vortioxetine (SSRIs and partial 5-HT 1A R agonists), or generalized anxiety disorder, such as buspirone (a partial 5-HT 1A R agonist) [26]. Regulating ADRA2, associated with stress-dependent depression, may also improve treatment of a range of neuropsychiatric disorders [35]. We observed abnormal expression of 5-HT 1A R and ADRA2 in the prefrontal cortex and hippocampal in PSD rats compared to CON rats, which could be also reversed by treatment with JY. Depression is known to be linked with hyperfunctionality of the HPA axis [36], which primarily regulates physiological and psychological reactions to environmental changes [37]. Elevated stress hormone levels can be released as a result of chronic stress [38]. Impaired negative feedback in the HPA axis can cause levels of these stress hormones to continuously climb in a manner correlated with 5-HTsystem activity [39]. In this study, the level of serum CORT and ACTH was significantly increased in PSD rats. Hippocampal structure, acquisition of memory, and regulation of emotion could be affected by glucocorticoids [38]. We found that treatment with JY decreased the level of CORT and ACTH in the serum of PSD rats, consistent with reduced HPA axis hyperfunctionality. Glucocorticoids also have the potential to influence the structure of the hippocampus, as well as memory acquisition and emotional regulation [40]. us, JY could well regulate the functions of memory and emotion through HPA axis. GR is also most highly expressed in the hippocampus and can therein negatively regulate the HPA axis [41]. Altered HPA axis functionality is associated with both increased CORT level and decreased GR expression [42]. Chronic stress in animals is also known to reduce the plasticity and long-term potentiation of CA1 hippocampal neurons in a GR-dependent fashion, resulting in impairments to both learning and adaptation [43]. We found that CUMS treatment significantly decreased GR expression in the hippocampus of PSD rats, which could be reversed by JY treatment.
Increased levels of cytokines are also known to be linked to the pathophysiology of PSD [43]. Cerebral ischaemia leads to the production of increased proinflammatory IL-1β and TNF-α, which could further result in decreased 5-HT production, additionally promoting depression [44,45]. e cytokines could modulate 5-HT metabolism and HPA axis functionality, further modulating the pathophysiology of depression [46]. Reducing CORT level and inflammation can enhance the synthesis of 5-HT [47]. We observed increased levels of TNF-α and IL-1β in the serum, prefrontal cortex, and hippocampus of PSD rats, in addition to elevated CORT levels. at is to say, JY treatment could significantly inhibit neuroinflammation in PSD rat model. Moreover, we detected the expression of NF-κB and IκB-α, which regulates inflammatory cytokine production, and found that JY was able to inhibit neuroinflammation in the prefrontal cortex and hippocampus in PSD rats through the inhibition of NF-κB/IκB signaling.
It has been shown that newly formed neurons in the dentate gyrus were detected by the increasing of BrdU+/ NeuN+ cells [48]. Moreover, this study showed the decreased BrdU+/NeuN+ cells, while treatment with JY was associated with significant increase of BrdU+/NeuN+ cells in the dentate gyrus of hippocampus in PSD rats.

Conclusion
Our results indicate that JY could markedly exert antidepressant effects, also including improving neurological and cognitive functions in PSD rat model. ese beneficial effects of JY may be involved in the modulation of levels of neurotransmitters and their receptors, the restoration of dysfunction in HPA axis, the inhibition of neuroinflammation, and the improvement of neurogenesis in hippocampus.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All authors declare that they have no conflicts of interest.

Authors' Contributions
Yuan Du contributed to data curation, formal analysis, and methodology and wrote the original draft. Jian Ruan contributed to data curation and methodology. Leiming Zhang wrote, reviewed, and edited the manuscript. Fenghua Fu was responsible for conceptualization, funding acquisition, and supervision, in addition to manuscript writing, reviewing, and editing.