Effect of catalpol on behavior and neurodevelopment in an ADHD rat model

Biomedicine & Pharmacotherapy 118 (2019) 109033

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Effect of catalpol on behavior and neurodevelopment in an ADHD rat model a

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Haixia Yuan , Xinqiang Ni , Min Zheng , Xinmin Han , Yuchen Song , Minfeng Yu a b

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Institute of Pediatrics of traditional Chinese Medicine, First Clinical Medical College, Nanjing University of Chinese Medicine, Nanjing, 210029, Jiangsu Province, China Pediatrics of Traditional Chinese Medicine, Shenzhen Traditional Chinese Medicine Hospital, Shenzhen, 518038, Guangdong Province, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Catalpol ADHD Open field test Morris water maze Neurodevelopment

Studies suggest that abnormal neurodevelopment of prefrontal striatal circuits is implicated in the pathogenesis of attention deficit hyperactivity disorder (ADHD). In the present study, we investigated the effect of catalpol, an active ingredient of Rehmanniae radix preparata, which is the most frequently used Chinese medicinal herb for the treatment of ADHD, on behavior and neurodevelopment in spontaneously hypertensive rats (SHR). SHR were divided into SHR group (vehicle, i.g.), methylphenidate (MPH) group (2 mg/kg/day, i.g.), and catalpol group (50 mg/kg/day i.g.), and Wistar-Kyoto (WKY) rats were used as control group (vehicle, i.g.). Open Field Test (OFT) and Morris water maze (MWM) test were performed to assess the effect of catalpol on behavior. Results revealed that both catalpol and MPH treatment decreased average speed, time spent in the central area, rearing times, and central area visits, increased the immobility time of SHR in OFT, and increased number of visits to the annulus, and time spent in target quadrant in the MWM test. Hematoxylin and eosin (H&E) staining showed that catalpol reduced irregular neuronal arrangement, ruptured nuclear membranes, and resulted in disappearance of the nucleolus in the prefrontal cortex (PFC) and striatum of SHR. Moreover, immuno-fluorescent staining of NeuN and myelin basic protein (MBP) indicated that catalpol ameliorated neuronal loss and contributed to myelination. Finally, western blot and immunostaining analysis suggested that several regulatory proteins involved in PFC development were up-regulated by catalpol treatment, such as brain-derived neurotrophic factor (BDNF), cyclin-dependent kinase 5 (Cdk5), p35, fibroblast growth factor (FGF) 21 and its receptor (FGFR)1. Taken together, catalpol can effectively ameliorate hyperactive and impulsive behavior, improve spatial learning and memory in SHR, likely through the neurodevelopmental pathways. Nonetheless, whether catalpol could attenuate inattention in SHR and the pathway by which catalpol reduces neuronal loss remain to be further studied.

1. Introduction Attention deficit hyperactivity disorder (ADHD), a common neurodevelopmental disorder in childhood with a prevalence of 1.4–3.0% [1], is characterized by developmentally inappropriate and impairing inattention, hyperactivity, and impulsivity, and often continues into adolescence and adulthood [2]. It is a heritable and multifactorial disorder, and displays heterogenous etiologies. Neuroanatomical findings have indicated that ADHD is associated with structural brain abnormalities involving anomalous neurodevelopment of brain networks, in particular the prefrontal striatal circuits mediating 'executive function' [3,4]. Delayed attainment of peak cortical thickness has been

observed in children with ADHD, and the greatest maturational delay was localized in prefrontal cortical (PFC) regions that control cognitive and executive processes, and working memory [5]. Additionally, growing number of studies report reduced volumes of PFC [6–8] and striatum [9–11] in ADHD, implicating neurodevelopmental mechanisms regulating prefrontostriatal circuits maturation in ADHD etiology. This maturation involves specific processes, such as proliferation and migration of neurons, growth of dendrites, synaptogenesis, and the finetuning of synaptic contacts. Studies have revealed various genes, such FGF2, Cdk5/p35, associated with these neurodevelopmental processes and specifically linked to ADHD, which may contribute to research on underlying molecular mechanisms of ADHD [12].

Abbreviations: ADHD, attention deficit hyperactivity disorder; SHR, spontaneously hypertensive rats; MPH, methylphenidate; WKY, Wistar-Kyoto; OFT, Open Field Test; MWM, Morris water maze; H&E, Hematoxylin and eosin; PFC, prefrontal cortex; MBP, myelin basic protein; BDNF, brain-derived neurotrophic factor; Cdk5, cyclin-dependent kinase 5; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor ⁎ Corresponding author. E-mail addresses: [email protected] (H. Yuan), [email protected] (X. Ni), [email protected] (M. Zheng), [email protected] (X. Han), [email protected] (Y. Song), [email protected] (M. Yu). https://doi.org/10.1016/j.biopha.2019.109033 Received 23 April 2019; Received in revised form 22 May 2019; Accepted 22 May 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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field, and observed for 5 min. Between trials the floor of the box was wiped with 50% ethanol to remove the scent marks. Average speed (cm/s), immobility time (s), time spent in the central area (s), number of rearing and central area visits were recorded and analyzed by EasyTracking System (SLY-ETS Version 1.66, Beijing Sunny Instruments Co. Ltd.). OFT was performed in a quiet room from 8:00 to 12:00. MWM The experimental apparatus consisted of a circular black tank (150 cm in diameter and 50 cm in depth) divided into four quadrants, and filled with water (24 ± 1 °C). A circular platform (12 cm in diameter and 30 cm in depth) was submerged about 1 cm below the water surface in the center of the south-east quadrant. Spatial acquisition trials were conducted during day 1–5 with 4 trials per day, and one trial each day was from each of the four positions selected randomly. The interval between trials was 15 min. The rats were given 90 s to locate the hidden platform and allowed to rest on the platform for 10 s. If a rat failed to find the platform within the allotted time, it was guided to the platform and allowed to rest for 10 s. Twenty-four hours following this training period, the probe trial was performed on day 6 without the platform, and each rat was allowed to swim freely for 90 s. Escape latencies (s) in spatial acquisition trials, and time spent on the first visit to annulus (location of the removed platform) (s), swimming speed (cm/ s), number of annulus visits (platform crossings), and time spent in the target (south-east) quadrant (s) in probe trial were automatically calculated by the Easy-Tracking System (SLY-ETS Version 1.66, Beijing Sunny Instruments Co. Ltd.).

Catalpol, an iridoid glucoside, is the active ingredient of Rehmanniae radix preparata, which is the most frequently used Chinese medicinal herb for the treatment of ADHD according to a data mining report [13]. In our previous research, Rehmanniae radix preparata (RRP) could reduce spontaneous and impulsive behavior in juvenile spontaneously hypertensive rats (SHR) [14]. Catalpol possesses a broad range of biological and pharmacological activity including neuroprotection, antiapoptosis, anti-inflammation, and anti-oxidative stress property [15,16]. A study has demonstrated that catalpol has satisfactory bioavailability and can penetrate the blood brain barrier (BBB) [17,18]. Therefore, catalpol might be a potential therapeutic for neurodevelopmental and neurodegenerative diseases. This is the first study to investigate the impact of catalpol on behavior in SHR model of ADHD and the underlying molecular mechanisms of neurodevelopmental processes. 2. Materials and methods 2.1. Animals Young (3-week-old) male Wistar-Kyoto (WKY) wild-type rats (n = 10) and SHR (n = 30) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China, Certificate No. SCXK(Jing)2016-0011) and housed in plastic cages with a 12:12-h light–dark cycle under a constant temperature of 22–24 °C and relative humidity of 50–60%. The average weight of WKY was 59.1 g (range: 51.7–64.9 g), and of SHR was 52.4 g (range: 45.4–60.6 g). All rats were fed standard chow ad libitum and allowed to acclimatize to the laboratory environment for 1 week. All the procedures were done in strict accordance with the guidelines of Animal Management Rules of the Ministry of Health of the People’s Republic of China (documentation Number 55, 2001, Ministry of Health of PR China) and the international guidelines on the ethical use of laboratory animals [19]. The experiments were carried out with the approval of the Animal Ethic Committee of Nanjing University of Chinese Medicine.

2.4. Tissue collection and brain slice preparation After behavioral tests, the rats (n = 6 in each group) were fasted overnight and were sacrificed by bloodletting from abdominal aorta under anesthesia. The PFC and striatum were removed and stored at −80 °C for the subsequent biochemical examinations. For histological studies, rats from each group (n = 4) were anaesthetized and perfused transcardially with 100 mM cold PBS (pH 7.4) till the liver became pale, followed by 4% ice-cold paraformaldehyde solution (PFA) until the fixation tics are observed in the limbs. Brains were immediately isolated and post-fixed in 4% PFA for 24 h at 4 °C. The sections were cut on a standard microtome (Leica, Wetzlar, Germany) at 5 μm thickness from paraffin embedded brains, and mounted onto polylysine-coated slides.

2.2. Drug administration Methylphenidate (MPH), a commonly prescribed psychostimulant drug for ADHD, was used to compare the effects of catalpol in ADHD. MPH (Janssen Cilag Manufacturing LLC, Gurabo, U.S.A.) and catalpol (purity ≥ 98%, Tauto Biotech Co., Ltd., Shanghai, China) were dissolved in 0.5% sodium carboxyl methyl cellulose (CMC-Na). After the 1 week acclimation period, SHR were divided into three groups (n = 10 in each group) based on average speed in the Open Field Test (OFT): SHR group, MPH group, and catalpol group. MPH was intragastrically (i.g.) administrated at a dose of 2 mg/kg/day and catalpol at a dose of 50 mg/kg/day in a volume of 2 mL/kg of body weight for the subsequent 4 weeks as a chronic treatment. The dose of MPH and catalpol was selected according to references previously reported [20,21]. At the same time, WKY and SHR group were administered by gavage with the body weight-matched volume of vehicle (0.5% CMC-Na). After behavioral tests, the rats were sacrificed, and brain tissues were immediately collected for experiments or stored at −80 °C for further analysis.

2.5. Hematoxylin and eosin (H&E) staining H&E staining was performed according to the general protocol. Paraffin sections were deparaffinized in xylene and rehydrated in a series of diluted ethanol and double-distilled water, and then stained with 1% hematoxylin and eosin. The slides were subsequently dehydrated with ethanol at gradient concentrations. The morphology of the PFC and striatum was observed under a light microscope (Olympus, Japan). 2.6. Immunohistochemistry and immunofluorescence After being deparaffinized and rehydrated, the brain section slides were incubated in 0.01 M citrate buffer (pH 6.0) and subjected to hightemperature antigen retrieval for 30 min. Then the sections were immersed in 3% H2O2-methanol solution for 10 min at 24 °C to abolish endogenous peroxidase activity. The sections were washed in PBS and incubated for 1 h with 5% normal goat serum, and subsequently incubated overnight at 4 °C with anti-Cdk5, p35, FGF21, and FGFR1 antibodies diluted at 1:100 (Table 1). Next, the sections were washed with PBS and incubated with goat anti-rabbit IgG secondary antibodies diluted at 1:1000 (Table 1) for 30 min at 37 °C. The slides were visualized with 3,3-diaminobenzidine (DAB) after being washed in PBS. Following counterstain with hematoxylin, dehydration with ethanol, the slides were sealed with neutral gums. For immuno-fluorescent labeling, sections were incubated for 12 h at 4 °C with anti-MBP, NeuN, and BDNF

2.3. Behavioral tests OFT was conducted for all rats at the beginning and end of the 4 weeks of drug administration, and the Morris Water Maze (MWM) test after 3 weeks of drug administration. OFT OFT has been proven to be a useful method to assess behavioral performance, such as spontaneous activity, exploratory, and anxietylike behavior [22]. The open field is a black box (100 × 100 × 40 cm) and for data analysis, the total area was equally divided into 16 equal squares virtually, a central area (50 × 50 cm) and a peripheral area (25 cm on each side). Each rat was gently placed in the center of the 2

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3. Results

Table 1 Antibodies used in western blot analysis and immunofluorescence staining. Antibody For western blot analysis Anti-BDNF Anti-Cdk5 Anti-p35 Anti-FGF2 Anti-FGF21 Anti-FGFR1 Anti-β-actin Goat anti-Rabbit IgG (HRP) Goat anti-Mouse IgG (HRP) For immunofluorescence staining Anti-MBP Anti-NeuN Anti-BDNF Alexa Fluor 488 Goat Anti- Rabbit IgG For immunohistochemistry staining Anti-Cdk5 Anti-p35 Anti-FGF21 Anti-FGFR1 Goat anti-Rabbit IgG (HRP)

Manufacturer

Catalog number

Abcam Abcam Abcam ImmunoWay Abcam CST Santa Cruz Abcam Santa Cruz

ab108319 ab40773 ab10570 YT5549 ab171941 #9740 sc-47778 ab6721 sc-2005

CST CST Abcam Jackson ImmunoResearch

#78896 #24307 ab108319 111-545-003

Abcam Abcam Abcam CST Abcam

ab40773 ab10570 ab171941 #9740 ab6721

3.1. Effect of catalpol on hyperactivity and impulsivity in SHR To evaluate the effect of catalpol on spontaneous locomotor activity and impulsivity in SHR, OFT was performed. The results revealed that there was no significant difference in average speed, immobility time, rearing number, time spent in the central area, and number of central area visits among SHR, MPH, and catalpol groups before drug administration, but the three groups showed less immobility time and higher average speed, more rearing number, time spent in the central area, and number of central area visits than WKY rats (all p < 0.001, Fig. 1A-E). Rats in MPH and catalpol groups, rather than WKY and SHR groups, showed remarkable changes in these parameters before and after drug administration for 4 weeks. After drug intervention, both MPH and catalpol groups displayed lower average speed (both p < 0.01, Fig. 1A) and increased immobility time (both p < 0.001, Fig. 1B) compared to SHR group. In addition, MPH and catalpol administration significantly decreased rearing number (both p < 0.001, Fig. 1C), time spent in the central area (both p < 0.001, Fig. 1D), and central area visits (both p < 0.001, Fig. 1E). The representative movement pathways of each group before and after drug administration are shown in Fig. 1F.

Abcam (Abcam, Massachusetts, USA); ImmunoWay (ImmunoWay Biotechnology, Texas, USA); CST (Cell Signaling Technology, Massachusetts, USA); Santa Cruz (Santa Cruz Biotechnology, Texas, USA); Jackson ImmunoResearch (Jackson ImmunoResearch Laboratories, Pennsylvania, USA).

3.2. Effect of catalpol on spatial learning and memory of SHR In order to assess whether catalpol has an effect on spatial learning and memory in SHR, MWM assay was performed. Escape latency was calculated as the time taken by the rat to reach the hidden platform. During spatial acquisition trials, the escape latencies were found to be decreased in all the groups; however, as a model control, SHR group required less time to find the escape platform compared to WKY rats at the 5th day (p < 0.001, Fig. 2A). MPH and catalpol treatment for 3 weeks resulted in shorter escape latencies in comparison with SHR group at the 5th day of spatial acquisition trials (both p < 0.001, Fig. 2A). As observed for the average speed in OFT, a statistically significant difference was observed between WKY rats and SHR concerning the swimming speed (p < 0.001) in the probe trial; meanwhile, the treatment groups displayed lower swimming speed, when compared to SHR group (both p < 0.01) (Fig. 2B). SHR spent less time on the first visit to annulus without the platform and showed more number of visits to the annulus than WKY rats (p < 0.001, p < 0.05); meanwhile, both MPH and catalpol markedly reduced the time spent on the first visit (both p < 0.001) and increased the visits to the annulus (p < 0.01 and p < 0.05, respectively) (Fig. 2C, D). Similarly, SHR spent more time in the target quadrant than WKY rats (p < 0.001), and rats from MPH and catalpol groups learned the platform location better than SHR, indicated by the time spent in the target quadrant (p < 0.001 and p < 0.01, respectively) (Fig. 2E). The representative swimming patterns of each group during spatial acquisition and probe trials are shown in Fig. 2F.

primary antibodies diluted at 1:100 (Table 1). After washing with PBS, the sections were incubated in dark for 1 h at 37 °C with Alexa Fluor 488-conjugated secondary antibodies diluted at 1: 200 (Table 1). Then, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Finally, the images from PFC and striatum were captured by a fluorescence microscope (Olympus BX43, Tokyo, Japan) at 100 and 400× magnification. The Image-Pro Plus 6.0 was used to analyze mean optical density (OD) and the number of positive stained cells in the sections.

2.7. Western blotting The isolated PFC was homogenized in RIPA buffer (Beyotime Biotechnology, China), and then centrifuged at 12,000 rpm for 15 min at 4 °C. Protein concentration in the supernatant was measured according to BCA protein assay kit (Thermo Scientific, USA). After being normalized, 26–28 μg of proteins were loaded in each lane, separated using 8–12% Tris-glycine SDS-PAGE gels, and transferred to PVDF membrane (0.2 or 0.45 μm). The membranes were blocked in 5% skim milk powder in TBS-T (TBS plus 0.1% Tween 20) at room temperature and blotted overnight at 4 °C with primary antibodies: anti-BDNF (1:2000), Cdk5 (1:2000), p35 (1:1000), FGF2 (1:1000), FGF21 (1:1000), FGFR1 (1:1000), and β-actin (1:10,000) (Table 1). Following incubation with corresponding secondary antibodies (Table 1) at room temperature for 2 h, the membranes were visualized using Clarity Max Western ECL Substrate (Bio-Rad, USA). Protein bands were captured and analyzed with ChemiDoc MP Imaging System (Bio-Rad, USA).

3.3. Histopathological alterations in PFC and striatum after drug administration H&E staining was used to observe the morphological differences in PFC and striatum between WKY rats and SHR, and the alterations after drug administration. The results revealed that the neurons in PFC of SHR showed an irregular arrangement and abnormal aggregation compared to WKY rats, and this condition was effectively ameliorated in MPH and catalpol groups (Fig. 3A). In addition, the PFC and striatum of SHR exhibited increased pyknosis, neuronal loss, ruptured nuclear membrane, and disappeared nucleolus, in comparison with WKY rats (Fig. 3A, B). After drug administration, MPH and catalpol markedly alleviated pyknosis and neuronal loss in the PFC and striatum; moreover, ruptured nuclear membrane and disappeared nucleolus were decreased in catalpol, but increased in MPH group (Fig. 3A, B). These

2.8. Statistical analysis Data were presented as mean ± SEM. Difference between the groups was evaluated using one-way analysis of variance (ANOVA) followed by the LSD post hoc test. A value of p < 0.05 was considered statistically significant. Statistical analysis and figures were obtained by IBM SPSS Statistics 19.0 (IBM SPSS Software, NY, USA) and GraphPad Prism Version 7.01 (GraphPad Software, CA, USA). 3

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Fig. 1. Behavioral performance in OFT (n = 10 in each group). (A) Average speed (cm/s), (B) Immobility time (s), (C) Rearing number, (D) Time spent in the central area (s), and (E) Number of central area visits before and after drug administration for 4 weeks. (F) Representative pathways of each group drug administration. All data are presented as mean ± SEM. **p < 0.01, ***p < 0.001.

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Fig. 2. Behavioral performance in MWM test (n = 10 in each group). (A) Escape latencies (s) in spatial acquisition trial. (B) Swimming speed (cm/s) in probe trial. (C) Time spent on the first visit to annulus (s) in probe trial. (D) Number of annulus visits (platform crossings) in probe trial. (E) Time spent in the target (south-east) quadrant (s) in probe trial. (F) Representative pathways of each group during spatial acquisition and probe trials. All data are presented as mean ± SEM. *p < 0.05, ***p < 0.001 SHR versus WKY group; #p < 0.05, ##p < 0.01, ###p < 0.001, compared to SHR group.

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Fig. 3. Hematoxylin and Eosin (H&E) stained sections from PFC and striatum (n = 3 in each group). (A) H&E staining of PFC and (B) striatum of each group. The red arrows show pyknotic neurons, which are characterized by hyperchromatic nucleus and cytoplasm; the black arrows show ruptured nuclear membrane and disappeared nucleolus. Scale bar = 100 μm (up), 20 μm (below) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

immunostaining images showed that compared to WKY rats, SHR showed significantly decreased fluorescence density of MBP (p < 0.001, Fig. 4A, C), and lower percentage of NeuN+ cells (p < 0.001, Fig. 4A, D) in PFC; meanwhile both these conditions were ameliorated by catalpol (both p < 0.05), but aggravated by MPH administration (both p < 0.001). The same expression trend of MBP and NeuN was also observed in the striatum of four groups (Fig. 4B, E, F). These results reveal that SHR might show abnormal neurodevelopment in specific brain regions, and catalpol possesses neuroprotective effect.

observations suggest that there were pathological alterations in PFC and striatum of SHR, and catalpol has protective effect on neuronal morphology.

3.4. Alterations in MBP and NeuN expression in PFC and striatum In order to validate further the protective effect of catalpol on neurons, we also determined the expressions of MBP and NeuN in PFC and striatum of each group using immunofluorescence staining. The 6

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Fig. 4. Reduced expression of myelin marker MBP and neuronal nuclei marker NeuN in PFC and striatum was reversed by catalpol, but aggravated by MPH treatment. MBP (up, red), NeuN (below, red) and DAPI (blue) immuno-fluorescent staining in PFC sections shown in (A) and in striatum sections shown in (B). Quantitative data of the mean density of MBP fluorescence in PFC (C) and striatum sections (E). Percentage of positive cells stained with the NeuN antibody in PFC (D) and striatum sections (F). Scale bar =50 μm. n = 3 for each group; All data are expressed as the mean ± SEM. **p < 0.01, ***p < 0.001 SHR versus WKY group; ##p < 0.01, ### p < 0.001, compared to SHR group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Western blot analysis showed lower expression of BDNF in PFC of SHR compared to WKY rats (p < 0.05). Catalpol treatment observably increased the levels of BDNF (p < 0.05), however, no statistically significant difference in BDNF expression was observed between SHR and

3.5. Catalpol increases the BDNF levels in PFC of SHR As BDNF is known to play a crucial role in neurodevelopment, we assessed the effect of catalpol on the BDNF levels in PFC of SHR. 7

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Fig. 5. The BDNF expression in PFC of SHR was increased after catalpol treatment. (A) Western blot analysis of the BDNF levels in PFC. (B) Relative levels of BDNF protein in PFC (fold change relative to β-actin protein levels). (C) Immuno-fluorescent staining in PFC sections. BDNF (red), DAPI (blue); scale bar =50 μm. n = 3 in each group; All data are expressed as the mean ± SEM. *p < 0.05, SHR versus WKY group; #p < 0.05, compared to SHR group (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

significantly elevated FGF21 and FGFR1 levels (p < 0.05 and p < 0.01, respectively), but no obvious change in FGF2 levels was observed compared to those in the SHR group. Downregulation of FGF2 expression (p < 0.05), upregulation of FGF21 (p < 0.01), and no significant alterations in FGFR1 levels were found in MPH group when compared with SHR. These results reveal that catalpol to some extent regulated the FGFs and related receptor.

MPH groups (Fig. 5A, B). Additionally, the results were also confirmed by immuno-fluorescent staining of PFC sections from each group (Fig. 5C). 3.6. Catalpol increases the protein levels of Cdk5 and p35 expression in PFC of SHR Considering the role of Cdk5/p35 during neurodevelopment, we next compared the expression of Cdk5 and p35 in PFC of each group using western blot and immunohistochemistry staining. Results showed that the expression of Cdk5 was much lower in SHR group compared to WKY group (p < 0.05), and these differences were effectively reversed by the catalpol treatment (p < 0.001), but not MPH (Fig. 6A, B, D). No significant alterations in p35 levels in PFC were observed between WKY rats and SHR. After drug exposure for 4 weeks, catalpol, not MPH, dramatically elevated p35 protein level in PFC of SHR (p < 0.05, Fig. 6A, C, D). Our findings suggest that catalpol regulates Cdk5/p35 activity in PFC of SHR.

4. Discussion Psychostimulants, such as MPH, are the first-line pharmacological treatments for ADHD, and they mainly act by increasing the availability of synaptic dopamine. However, psychostimulant interventions alone cannot be one-size-fits-all treatment for ADHD due to the heterogenous etiologies and complex pathomechanism. In addition, side-effects such as growth restriction, headache, and sleep disturbances, restrict the clinical application of psychostimulants [1]. Therefore, alternative or complementary drug discovery are highly required. Chinese herbal medicine (CHM) has been used widely to treat ADHD and proven safe and effective [24]. A data mining report suggests that RRP is the most frequently used CHM for the treatment of ADHD [13]. As a bioactive component of RRP, catalpol has been reported to have various neuroprotective properties both in vivo and in vitro models of neurodegenerative disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [151625,26]; however, it has not yet been used in the study of ADHD. Hence, based on the neurodevelopmental mechanisms involved in the pathogenesis of ADHD and the pharmacological function of catalpol, we previously hypothesized that catalpol might be a

3.7. Catalpol increases the levels of FGF21 and FGFR1 in PFC of SHR We chose to measure the levels of FGFs and their receptors in PFC of each group, because it has been shown that these have an influence on the behavior, by affecting brain development [23]. As showed in western blot and immunohistochemistry analysis (Fig. 7A-F), except the statistical difference in FGFR1 expression between WKY and SHR groups (p < 0.05), no significant difference in the levels of FGF2 and FGF21 was observed between the two groups. Catalpol group displayed 8

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Fig. 6. The expression of Cdk5 and p35 in PFC of SHR was increased after catalpol treatment. (A) Western blot analysis of the Cdk5 and p35 levels in PFC. (B) and (C) Relative levels of Cdk5 and p35 in PFC (fold change relative to β-actin level). (D) Immunohistochemistry analysis of expression of Cdk5 and p35 in PFC sections. Scale bar =50 μm. n = 3 in each group; All data are expressed as the mean ± SEM. *p < 0.05, SHR versus WKY group; #p < 0.05, ###p < 0.001, compared to SHR group.

closely related to ADHD behavior [4,32]. Moreover, studies also provide evidence that there is a delayed neuronal maturation, and abnormalities in myelination of axons in SHR striatum and PFC, which is consistent with the developmental delay seen in children [33,34]. So, we investigated the morphology of PFC and striatum by H&E staining and observed an irregular arrangement and abnormal aggregation of neurons in SHR PFC; meanwhile, increased pyknosis, neuronal loss, and incomplete cytoarchitecture were also observed in the PFC and striatum of SHR, implying that there are potential disturbances in migration and axon guidance, and neuronal apoptosis. These histomorphological changes were shown to be evidently ameliorated by catalpol administration. Interestingly, it seems that MPH treatment aggravated neuroapoptosis, resulting in more ruptured nuclear membranes and disappeared nucleolus, which is in line with previous reports [35,36]. Next, we used immunofluorescence staining to check further for neuronal loss and abnormalities in myelination in SHR PFC and striatum. It was found that PFC and striatum of SHR displayed a significant decrease in NeuN+ cells compared to corresponding brain regions of WKY rats, and this condition was attenuated by catalpol treatment, but exacerbated by MPH treatment. It further strengthened the evidence for the earlier histological observations. MBP is a protein proved to play

promising natural medicine for the treatment of ADHD [14,27]. In the present study, we applied behavioral tests, histomorphological, and molecular analysis to evaluate the effect of catalpol on behavior and neurodevelopment in SHR model of ADHD. The OFT showed that catalpol and MPH treatment remarkably decreased average speed, time spent in the central area, rearing times, central area visits, and increased immobility time of SHR, revealing that catalpol ameliorates the hyperactive and impulsive behavior in SHR compared to WKY rats. It was also observed that SHR performed better than WKY rats in our MWM test, with shorter escape latencies, more visits to the annulus (without platform), and time spent in the target quadrant, which is consistent with previous reports [28–30]. This condition is commented to be attributed to higher swim speed of SHR, resulting in shorter latencies to find the platform and, thus, better performance in MWM test [31]. Without regard for strain differences, our study revealed that both catalpol and MPH not only lowered the swim speed, but also increased the number of visits to the annulus and time spent in the target quadrant by SHR in the probe trial, clearly indicating the effect on improving spatial learning and memory of SHR. It is well established that the structural and functional abnormalities in frontostriatal circuitry observed in neurodevelopmental disorders are 9

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Fig. 7. The effect of catalpol on the expression of FGF2, FGF21, and FGFR1 in PFC of SHR. (A) Western blot analysis of FGF2, FGF21 and FGFR1 levels in PFC. (B–D) Relative levels of FGF2, FGF21, and FGFR1 protein in PFC (fold change relative to β-actin protein level). (E) Immunohistochemistry analysis of FGF21 and FGFR1 in PFC sections. Scale bar =50 μm. n = 3 in each group; All data are expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 SHR versus WKY group; # p < 0.05, ##p < 0.01, ###p < 0.001, compared to SHR group.

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immobility time. These results imply that regulatory network of neurodevelopment is closely associated with certain behavioral characteristics of ADHD. Interestingly, FGF21 was strongly and positively related with time spent in target quadrant in probe trial of MWM, which suggests that it might be a superior target for spatial learning and memory ability. It is noteworthy that these results have inevitable limitations due to sample shortage. More work should be carried out to carefully evaluate the role of specific regulatory protein in the pathogenesis of ADHD. It has been shown that both MPH and catalpol treatment exhibited positive impacts on behavior characteristics of SHR. However, unlike catalpol, MPH aggravated neuroapoptosis and reduced myelination of neurons in PFC and striatum of SHR. In addition, MPH showed incongruent effects on the expression of regulatory proteins, when compared with catalpol. The seemingly contradictory results aroused some thoughts. 1) It seems that, as a stimulant, MPH plays a therapeutic role in controlling ADHD symptoms mainly by increasing the availability of synaptic dopamine, rather than protecting neurons or regulating neurodevelopment. 2) These contradictions may be helpful to explain the differences in outcome of MPH clinical treatment. 3) The etiology of ADHD is highly heterogeneous. Involvement of neurotransmission cannot fully reveal the diversity of clinical symptoms and stimulants are not the only option accordingly. Therefore, alternative or complementary treatment should be taken into account. Noteworthy, additional work to evaluate of the effects of MPH and catalpol on the development of more brain regions and other regulatory proteins involved in neurodevelopment will be required.

pivotal role in the process of myelination of nerves. When compared to WKY rats, SHR showed reduced MBP expression in PFC and striatum, which has not yet been reported, suggesting potential abnormality in the myelination of axons, in accordance with the findings of Dimatelis et al. [33]. Contrary to MPH, the neuroprotective effects of catalpol are embodied strongly due to increase in MBP expression and, hence, in myelination of SHR brain neurons. Maturation of PFC includes several developmental processes, such as proliferation and migration of neurons, growth of dendrites, formation of neural micro- and macro-circuits, and the fine-tuning of synapses [12]. Failure of any of these processes can result in neurodevelopmental abnormalities within the PFC, which then contributes to cognitive deficits seen in patients with neurodevelopmental disorders, including ADHD. Several regulatory proteins are believed to be crucial to PFC development, defects in which are associated with behavioral impairments of ADHD. In order to explore the underlying mechanisms of how catalpol affects neurodevelopment, we assessed the effect of catalpol on the expression of specific regulatory proteins. As a member of the neurotrophin family of growth factors, BDNF is essential for neuronal survival, differentiation, apoptosis, neurite outgrowth, synaptic plasticity, and maintenance of neural circuits [37,38]. Considerable evidence supports that BDNF is implicated in the pathogenesis and as potential therapeutic of ADHD [39,40]. It has been reported that compared to WKY rats, SHR display decreased BDNF levels in hippocampus [41,42], and this study obtained consistent result in PFC of SHR. Catalpol, but not MPH, treatment up-regulated the BDNF levels, in consistent with the study indicating that MPH reduced BDNF expression and signaling in PFC [43]. Cdk5 is a neuronal serine/threonine protein kinase critical to neuronal migration, microtubule remodeling, neurite outgrowth, axonal growth, synapse formation, and corticogenesis, crucial for spatial learning and memory [44,45], gene variants of which have been associated with the etiology of ADHD [46]. p35 is one of the two homologous non-cyclin activators, and is required for Cdk5 activity. Mice lacking p35 show reversed cortical lamination, and abnormal neurite outgrowth in culture [47]. Furthermore, juvenile p35 knockout mice display spontaneous hyperactivity and paradoxical response to psychostimulants [48]. Lower levels of Cdk5, but not p35 were observed in SHR PFC; expression of both Cdk5 and p35 was increased by catalpol treatment, however, no response to MPH exposure was observed in this study. These results suggest that dysregulation of Cdk5/p35 activity in SHR can be reverted by catalpol. FGF-FGFR signaling has a prominent role in axon extension, neuronal migration, and cortical circuit formation, contributing to the development of the nervous system [49,50]. Though evidence is lacking that implicates the role of FGF or FGFR in pathology of ADHD, it has been shown that FGF2 is involved in protection against neuronal death [51], and FGF2 knockout mice exhibit hyperactivity ([52,53]). In this study, SHR, WKY and catalpol groups showed no significant difference in FGF2 levels in PFC, while MPH treatment downregulated FGF2 levels in SHR. FGF21 signaling is implicated in neuroapoptosis, synaptic plasticity, and dendritic spine density, probably via metabolic regulation in brain [54,55]. Results showed that administration of MPH and catalpol increased FGF21 levels in SHR, but no significant difference in FGF21 expression was observed in PFC of SHR and WKY rats. Studies suggest that FGFR1 deficiency is closely related to spontaneous and persistent locomotor hyperactivity in mice ([56,52,53]). Interestingly, we also found relative deficiency of FGFR1 in SHR compared to WKY rats, and were able to attenuate this condition by treatment with catalpol, but not MPH. Taken together, it appears that dysregulation of FGFR, rather than FGFs, is linked to ADHD. We further assessed the correlation between behaviors and these regulatory proteins as shown in Fig. S1. BDNF, FGFR1, Cdk5 and p35 were strongly and negatively correlated with one or more behavioral parameters reflecting hyperactivity and impulsiveness in OFT. In addition, BDNF and p35 were closely and positively correlated with

5. Conclusion Overall, our findings suggest that catalpol can effectively ameliorate hyperactive and impulsive behavior, improve spatial learning and memory in SHR, a widely recognized animal model of ADHD. More importantly, the study provides evidence that catalpol exerts neuroprotective role by inhibiting neuronal apoptosis, increasing myelination and BDNF expression, and affects neurodevelopment, likely through regulation of specific regulatory proteins with a crucial role in PFC maturation. Nevertheless, this is a preliminary study with certain limitations, for instance, catalpol was administrated at only one dose (50 mg/kg/day), and its effect on the mRNA expression of the regulatory proteins was not assessed in the present study. It is worth stating that further research is undergoing to estimate the pharmacological action of catalpol in treating ADHD; 1) whether catalpol exerts protective effect in a dose dependent manner, 2) assess whether catalpol affects the attention of SHR in the related behavior testing such as 5-choice serial reaction time task (5-CSRTT), 3) assess the behavior characteristics after cessation of the catalpol administration and 4) research on underlying mechanisms of catalpol in anti-neuroapoptosis. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements This work was financially supported by National Natural Science Foundation of China (NO.81503616, 81873341), Natural Science Foundation of Guangdong Province of China (NO. 2017A030313749), Science and Technology Project of Shenzhen (NO.JCYJ20160428105935612), Shenzhen Sanming medical project of China (NO. SZSM201812064), Postgraduate Research & Practice Innovation Program of Jiangsu Province (NO. KYCX18_1545). References [1] C.M. Thapar A, Attention-deficit/hyperactivity disorder, Lancet, Elsevier, 2016, pp.

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