Effect of Yokukansan and Yokukansankachimpihange on Aggressive Behavior, 5-HT Receptors and Arginine Vasopressin Expression in Social Isolation-Reared Mice

Abstract

The traditional herbal medicines yokukansan (YKS) and yokukansankachimpihange (YKSCH) are prescribed for neurosis, insomnia or night crying and irritability in children. YKSCH comprises YKS and two additional herbs, a chimpi and a hange, and is used to treat digestive function deficiencies. However, the differences between the effects of YKS and YKSCH on brain function are unclear. The present study examined the effects of YKS and YKSCH on aggressive behavior in mice reared under a social isolation (SI) condition. Mice were housed individually for 6 weeks. YKS and YKSCH were administered orally for 2 weeks before aggression tests. SI increased aggressive behavior against naïve mice, and YKS, but not YKSCH, significantly attenuated this aggressive behavior. Because serotonin (5-HT)_2A and 5-HT_3A receptor antagonists are reported to have anti-aggressive effects, the mRNA levels of these receptors were examined. YKS attenuated the SI-induced increase in 5-HT_2A and 5-HT_3A receptor mRNA in the amygdala. On the other hand, YKSCH attenuated the SI-induced increase in 5-HT_1A receptor mRNA. YKS and YKSCH did not affect 5-HT and its metabolite 5-hydroxyindoleacetic acid content in the amygdala. However, YKSCH increased the mRNA level of arginine vasopressin (AVP), which is a neuropeptide that has been implicated in aggression, in the amygdala. These results suggest that YKS ameliorates aggressive behavior by decreasing 5-HT_2A and 5-HT_3A receptor expression. The YKSCH-induced increase in AVP may disrupt the anti-aggressive effect of YKS. YKS may be more effective than YKSCH for treating irritability if digestive function deficiencies are not considered.

INTRODUCTION

Yokukansan (YKS: Yi-gan san in Chinese) is a traditional herbal medicine comprising seven types of medicinal herbs. It has been approved by the Ministry of Health, Labour and Welfare of Japan as a remedy for neurosis, insomnia or night crying and irritability in children. Yokukansankachimpihange (YKSCH), which was developed in Japan, includes YKS combined with two medicinal herbs, Citrus unshiu peel (chimpi) and Pinellia tuber (hange). Although YKSCH is similar to YKS, it is more commonly prescribed for patients whose symptoms include digestive function deficiencies.¹⁾ However, it is unclear whether YKS and YKSCH have different effects on brain functions.

Social isolation (SI)-reared animals demonstrate increased aggressive behavior compared with group-reared animals.^2,3) This aggressive behavior is suggested to be related to the aggressiveness associated with schizophrenia and Alzheimer’s disease.^4,5) YKS improves agitation/aggression scores in patients with Alzheimer’s disease.⁶⁾ YKS also ameliorates aggressive behavior in SI-reared mice.⁷⁾ Therefore, SI-induced aggressive behavior may be a useful model to compare the effect of YKSCH with that of YKS on psychological symptoms. SI-induced aggressive behavior is reduced by serotonin (5-HT)_1A receptor agonists, 5-HT_2A receptor antagonists and serotonin reuptake inhibitors.^4,8,9) These reports suggest that 5-HT receptors participate in SI-induced aggressive behavior.

The neuropeptide arginine vasopressin (AVP) has been implicated in aggressive behavior. Human studies show that intranasal administration of AVP promotes aggressive behavior in response to presentations of faces of unfamiliar men.¹⁰⁾ Hypothalamic injection of AVP stimulates aggression in male hamsters but not in female hamsters.¹¹⁾ Vasopressin V1a receptor antagonists inhibit inter-male aggression.¹²⁾ These results suggest that AVP is related to aggression in males but not in females.

To examine the different effects of YKS and YKSCH on brain functions, in this study, we evaluated the effects of YKS and YKSCH on SI-induced aggressive behavior. mRNA levels of 5-HT receptors and AVP in the amygdala were examined because the amygdala plays a key role in modulating aggressive behavior in humans and mice.^13–15) Furthermore, the content of 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the amygdala was analyzed.

MATERIALS AND METHODS

Animals

Male ddY mice (4 weeks old) were supplied by Japan SLC, Inc. (Shizuoka, Japan). After habituation for 1 week, mice were housed individually in cages (136 × 208 × 115 mm) for 6 weeks prior to testing. Group-reared controls were housed with 3–5 mice per cage (182 × 260 × 128 mm). Naïve male mice (5 weeks old) that were used for the aggression test were also supplied by Japan SLC, Inc. and were group-housed for one week prior to the test. All mice were housed at a temperature of 23 ± 2°C with a relative humidity of 60 ± 10% under a 12 h light–dark cycle (lights on 07 : 00–19 : 00). Food and water were available ad libitum. All procedures regarding animal care and use were carried out in keeping with the regulations dictated by the Experimental Animal Care and Use Committee of Fukuoka University (#1507852, #1704040).

Drugs

Dry powdered extracts of YKS (Lot. No. 331039200) and YKSCH (Lot. No. 331036600) were provided by Tsumura & Co. (Tokyo, Japan). YKS comprises seven dried extracts as follows: Poria Sclerotium (4.0 g, sclerotium of Wolfiporia cocos Ryvarden et Gilbertson), Atractylodes Lancea Rhizome (4.0 g, rhizome of Atractylodes lancea De Candolle), Uncaria Hook (3.0 g, hook of Uncaria rhynchophylla Miquel), Cnidium Rhizome (3.0 g, rhizome of Cnidium officinale Makino), Japanese Angelica Root (3.0 g, root of Angelica acutiloba Kitag awa), Bupleurum Root (2.0 g, root of Bupleurum falcatum Linné) and Glycyrrhiza (1.5 g, root and stolon of Glycyrrhiza uralensis Fisher). YKSCH consists of YKS and two additional herbs, Pinellia tuber (5.0 g, tuber of Pinellia ternata Breitenbach, hange) and Citrus unshiu peel (3.0 g, peel of Citrus unshiu Markovich, chimpi). Each plant sample was authenticated by identification of the external morphology and marker compounds of plant specimens according to the methods of the Japanese Pharmacopoeia and the standards of Tsumura & Co. The seven or nine medicinal herbs were extracted with purified water at 95°C for 1 h, and the extraction solution was separated from the insoluble waste and concentrated by removing water under reduced pressure. Spray drying was used to produce a dried extract powder. The yields of YKS and YKSCH were 15.9 and 15.8%, respectively. The quality was standardized in keeping with the Good Manufacturing Practices defined by the Japanese Ministry of Health, Labour and Welfare. The components of YKS and YKSCH extract were previously confirmed by the three-dimensional HPLC analysis.¹⁶⁾

YKS and YKSCH were dissolved in distilled water (vehicle). The dose of YKS (1000 mg/kg) was selected according to a previous study, in which YKS significantly ameliorated aggressive behavior.⁷⁾ The same doses of YKSCH and YKS were used.

Aggression Test in a Neutral Area

The aggression test was performed according to a modified version of Bibancos’s procedure.¹⁷⁾ Each isolated or group-housed mouse was placed individually in a neutral area (fresh, clean cage, 182 × 260 × 128 mm) for a 60-min acclimation period. Subsequently, a naïve male mouse was introduced into the area as a stimulus. The naïve mice were of the same strain and younger than the experimental mice. The number of attacks against the naïve mouse was recorded for 10 min.

Quantitative (q)RT-PCR

After the aggression test, mice were rapidly decapitated, and the amygdala was quickly dissected. The total RNA was extracted using the TRI Reagent^® (Molecular Research Center, Inc., Cincinnati, OH, U.S.A.). First-strand cDNA was reverse-transcribed from total RNA using a ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan). Real-time PCR was conducted on a LightCycler^® 96 System (Roche, Basel, Switzerland) using THUNDERBIRD SYBR qPCR Mix (TOYOBO Co., Ltd.) and primers as per the manufacturer’s protocol. The following PCR conditions were employed: 95°C for 15 s, 59°C (for β-actin and 5-HT_1B receptor), 63°C (for 5-HT_1A, 5-HT_2A and 5-HT_3A receptors), 65°C (for 5-HT_2C receptor) or 66°C (for AVP) for 30 s and 72°C for 45 s. The sequences of the primers were as follows: 5′-GGA TGT TTT CCT GTC CTG GT-3′ and 5′-CAC AAG GCC TTT CCA GAA CT-3′ for 5-HT_1A receptor (NM_008308); 5′-TCA CAT GGC CAT TTT TGA CT-3′ and 5′-CAG TTT GTG GAA CGC TTG TT-3′ for 5-HT_1B receptor (NM_010482); 5′-AGA ACC CCA TTC ACC ATA GC-3′ and 5′-ATC CTG TAG CCC GAA GAC TG-3′ for 5-HT_2A receptor (NM_172812); 5′-CTA ATT GGC CTA TTG GTT TGG CA-3′ and 5′-CGG GAA TTG AAA CAA GCG TCC-3′ for 5-HT_2C receptor (NM_008312.4); 5′-CTT CCC CTT TGA TGT GCA G-3′ and 5′-CCA CTC GCC CTG ATT TAT G-3′ for 5-HT_3A receptor (NM_013561); 5′-GCC AGG ATG CTC AAC ACT ACG-3′ and 5′-TCT CAG CTC CAT GTC AGA GAT G-3′ for AVP (NM_009732.2); and 5′-GGC TGT ATT CCC CTC CAT CG-3′ and 5′-CCA GTT GGT AAC AAT GCC ATG T-3′ for β-actin (BC138611.1). The primer sequences for the 5-HT_1A, 5-HT_1B, 5-HT_2A and 5-HT_3A receptors were based on a study by Bibancos et al.¹⁷⁾ Other primer sequences were obtained from the PrimerBank database (https://pga.mgh.harvard.edu/primerbank/). Relative quantitative analysis was performed using LightCycler^® 96 software (Roche) to compare the target gene levels to those of the control gene (β-actin).

5-HT and 5-HIAA Content

The content of 5-HT and 5-HIAA were measured with an HPLC-electrochemical detector (ECD) system (Eicom Co., Ltd., Kyoto, Japan). Mice were sacrificed immediately after the aggression test. Brain regions were quickly dissected, weighed and homogenized in 150 µL of ice-cold 0.2 M perchloric acid containing 0.1 mM ethylenediaminetetraacetic acid (EDTA) and 10 ng/mL isoproterenol (internal standard). Following centrifugation at 20000 g × 15 min, 50 µL of the supernatant was mixed with 20 µL of 1 M sodium acetate and filtered through a membrane filter (0.45 µm; Millex, Merck, Darmstadt, Germany). Then, 10 µL of the sample was injected into the HPLC-ECD system, which used an Eicompak SC-5ODS column (3.0 mm i.d. × 150 mm, Eicom Co., Ltd.) and was set at a potential of +750 mV against an Ag/AgCl reference electrode with a graphite carbon working electrode (WE-3G, Eicom Co., Ltd.). The mobile phase consisted of 0.1 M acetate-citrate buffer (pH 3.5), 200 mg/L sodium 1-octanesulfonate, 5 mg/L EDTA and 17% methanol. The flow rate was maintained at 0.5 mL/min. The monoamine levels were calculated on the basis of standard values using PowerChrom (version 2.2.4; Eicom Co., Ltd.).

Experimental Procedure

Mice were isolated for 6 weeks, and daily YKS and YKSCH administration (1000 mg/kg/d, for 2 weeks, per os (p.o.)) was started at 4 weeks after the initiation of SI (Fig. 1). The aggression test was performed at 1 h after the final drug administration. Immediately after the aggression test, mice were sacrificed to analyze monoamine content and mRNA expression.

Fig. 1. Experimental Schedule

Mice (5 weeks old) were housed individually for 6 weeks (social isolation). Group-reared mice were housed with 3-5 mice/cage. Social isolation-reared mice were treated daily with distilled water (vehicle), YKS or YKSCH for 14 d before the aggression test. Mice were decapitated immediately after the aggression test.

Statistical Analysis

Data were evaluated for statistical significance using one-way ANOVA followed by Tukey’s multiple comparisons test. An outlier was removed from each PCR data set (5-HT_2A, 5-HT_3A and AVP mRNA) using the ROUT method (Prism 6.04 software, GraphPad, La Jolla, CA, U.S.A.). The criterion for statistical significance was p < 0.05. Data are shown as the mean ± standard error of the mean (S.E.M.).

RESULTS

Effect of YKS and YKSCH on Aggressive Behavior in SI-Reared Mice

SI-reared mice showed an increased number of attacks compared with group-reared mice [F(3,236) = 3.201, p < 0.05 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test, Fig. 2]. YKS administration to SI-reared mice significantly attenuated this increase in the number of attacks [F(3,236) = 3.201, p < 0.05 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test, Fig. 2], while YKSCH administration did not produce a significant attenuation. However, YKSCH administration also did not increase the number of attacks compared with group-reared mice. These results suggest that the anti-aggressive effect of YKSCH is weaker than that of YKS.

Fig. 2. Effect of YKS and YKSCH on Aggressive Behavior in Social Isolation-Reared Mice

Each isolated or group-housed mouse was placed individually in a neutral area for 1 h. Subsequently, a naïve mouse was introduced into the area. The aggressive behavior of the trial mouse against the naïve mouse was evaluated for 10 min. A final administration of YKS or YKSCH was conducted 1 h before the test. Values indicate the means ± standard error of the mean (S.E.M.) (n = 52–69/group). * p < 0.05, significantly different.

Effect of YKS and YKSCH on 5-HT Receptor Expression in the Amygdala of SI-Reared Mice

5-HT_1A receptor agonists, 5-HT_2A receptor antagonists and serotonin reuptake inhibitors reduce aggressive behavior in SI-reared mice,^4,8,9) which suggests that aggressive behavior is mediated by 5-HT receptors. Chronic administration of YKS decreases 5-HT_2A receptor levels in the mouse frontal cortex.¹⁸⁾ Therefore, the effect of YKS and YKSCH on 5-HT receptor expression was examined. The mRNA levels of several 5-HT receptors (5-HT_{1A, 1B, 2A and 3A} receptors) were increased in SI-reared mice compared with group-reared mice [5-HT_1A receptor, F(3,53) = 4.060, p < 0.05 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test; 5-HT_1B receptor, F(3,53) = 3.322, p < 0.05 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test; 5-HT_2A receptor, F(3,52) = 5.354, p < 0.01 by one-way ANOVA, p < 0.01 by Tukey’s multiple comparisons test; 5-HT_3A receptor, F(3,52) = 4.206, p < 0.01 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test]; however, the 5-HT_2C receptor mRNA level was not increased (Fig. 3). YKSCH administration significantly decreased the enhanced 5-HT_1A mRNA level [F(3,53) = 4.060, p < 0.05 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test], whereas, YKS administration did not (Fig. 3A). Neither YKS administration nor YKSCH administration decreased the enhanced 5-HT_1B mRNA level (Fig. 3B). On the other hand, YKS administration significantly decreased the enhanced 5-HT_2A and 5-HT_3A receptor mRNA levels [5-HT_2A receptor, F(3,52) = 5.354, p < 0.01 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test; 5-HT_3A receptor, F(3,52) = 4.206, p < 0.01 by one-way ANOVA, p < 0.05 by Tukey’s multiple comparisons test], whereas, YKSCH administration did not (Figs. 3C, E). These results suggest that a decrease in 5-HT_2A and 5-HT_3A receptor levels contributes to the anti-aggressive effect of YKS.

Fig. 3. Effect of YKS and YKSCH on 5-HT Receptor Expression in the Amygdala of Social Isolation-Reared Mice

5-HT_1A (A), 5-HT_1B (B), 5-HT_2A (C), 5-HT_2C (D) and 5-HT_3A (E) mRNA levels in the amygdala were examined after the aggression test. Data are expressed as a percentage of control values (group-reared mice). Values indicate the means ± S.E.M. (n = 12–15/group). * p < 0.05 and ** p < 0.01, significantly different.

Effect of YKS and YKSCH on the Content of 5-HT and Its Metabolite 5-HIAA in the Amygdala of SI-Reared Mice

Since the mRNA levels of 5-HT receptors were altered in the amygdala of SI-reared mice treated with YKS, the content of 5-HT and its metabolite 5-HIAA in the amygdala were then examined. The contents of 5-HT and 5-HIAA were unchanged in SI-reared mice compared with group-reared mice. Likewise, YKS and YKSCH did not affect the 5-HT and 5-HIAA content (Figs. 4A, B). These data suggest that YKS and YKSCH are not responsible for the underlying mechanisms of 5-HT metabolism.

Fig. 4. Effect of YKS and YKSCH on the Content of 5-HT and Its Metabolite 5-HIAA in the Amygdala of Social Isolation-Reared Mice

5-HT (A) and 5-HIAA (B) levels in the amygdala were examined using HPLC after the aggression test. Values indicate the means ± S.E.M. (n = 16–19/group).

Effect of YKS and YKSCH on AVP Expression in the Amygdala

AVP plays a key role in aggressive behavior.¹¹⁾ Therefore, AVP mRNA levels were examined. Although SI-reared mice did not show increased AVP mRNA levels, YKSCH administration significantly increased the AVP mRNA level compared with group-reared mice and YKS-administered mice [F(3,52) = 4.972, p < 0.01 by one-way ANOVA, p < 0.01 by Tukey’s multiple comparisons test (Fig. 5)].

Fig. 5. Effect of YKS and YKSCH on AVP Expression in the Amygdala

AVP mRNA levels in the amygdala were examined after the aggression test. Data are expressed as a percentage of control values (group-reared mice). Values indicate the means ± S.E.M. (n = 12–15/group). * p < 0.05 and ** p < 0.01, significantly different.

DISCUSSION

YKS and YKSCH are prescribed for neurosis, insomnia or night crying and irritability in children. Prescription of these medicines is dependent on the digestive condition. However, the differences between the effects of YKS and YKSCH on brain function are unclear. Therefore, the present study examined the difference between the effects of YKS and YKSCH on aggressive behavior in SI-reared mice. YKS, but not YKSCH, significantly ameliorated SI-increased aggressive behavior. The RT-PCR results suggested that the anti-aggressive effect of YKS may be mediated by a decrease in the expression levels of 5-HT_2A and 5-HT_3A receptors. In addition, the YKSCH-induced increase in the AVP level may disrupt the anti-aggressive effect because AVP is implicated in aggression.^11,12) The present study suggests that YKSCH is less effective in ameliorating aggressive behavior than YKS because it increases AVP expression.

5-HT_2A receptor agonists enhance SI-induced aggressive behavior. In contrast, 5-HT_2A receptor antagonists inhibit SI-induced aggressive behavior.^8,9) In other models of aggression, 5-HT_2A receptor antagonism reduces aggressive behavior.^19,20) 5-HT₃ receptor antagonists also reduce cocaine-, apomorphine- and alcohol-induced aggressive behavior.^21–23) Thus, activation of 5-HT_2A and 5-HT₃ receptors may induce aggressive behavior. The increased 5-HT_2A and 5-HT_3A receptor mRNA levels in this study could be due to SI-induced aggressive behavior. YKS administration attenuated aggressive behavior and the increase in 5-HT_2A and 5-HT_3A receptor mRNA in the amygdala (Figs. 2, 3). Taken together, our results suggest that YKS inhibits aggressive behavior via mediation of 5-HT_2A and 5-HT_3A receptor expression. Repeated administration of YKS has been reported to reduce 5-HT_2A receptor expression levels in the prefrontal cortex.^18,24) However, the effect of YKS on the 5-HT_3A receptor expression level is unknown. Our study is the first report to demonstrate that YKS mediates 5-HT_3A receptor expression.

AVP-producing cells are found in the hypothalamus (paraventricular nucleus [PVN], supraoptic nucleus [SON] and suprachiasmatic nucleus [SCN]) and the extended amygdala (bed nucleus of the stria terminals [BNST] and medial amygdala).^25,26) AVP projections from the amygdala are suggested to facilitate aggressive behavior.²⁷⁾ However, SI did not significantly increase AVP mRNA in the amygdala (Fig. 5). Another study also demonstrated no alteration of plasma AVP levels in prairie voles subjected to chronic SI-rearing.²⁸⁾ However, SI increased the density of AVP immunoreactive cells in the hypothalamus of prairie voles.²⁹⁾ We also observed increased AVP mRNA levels in the hypothalamus of SI-reared mice (data not shown). SI may induce aggressive behavior via hypothalamic AVP expression. Previous studies have reported that AVP mRNA expression in the amygdala is decreased by adrenalectomy, abolished by a combination of adrenalectomy and gonadectomy, and restored by androgen replacement.³⁰⁾ In addition, adrenalectomy and gonadectomy have been found to cause a reduction in aggressiveness of SI-reared mice.^31,32) These reports imply that AVP expression in the amygdala is mediated by androgen and enhances aggressiveness. Given that citrus peel extract has been found to increase serum androgen levels in mice,³³⁾ chimpi might enhance aggressiveness via androgen. YKSCH administration increased AVP mRNA levels in the amygdala compared with YKS administration (Fig. 5), implying that chimpi disrupts the anti-aggressive effects of YKS via AVP expression. Taken together, SI and YKSCH may induce aggressive behavior via AVP expression in the hypothalamus and amygdala, respectively. However, the mechanism by which YKSCH increases AVP mRNA is unknown.

The 5-HT and 5-HIAA content in amygdala were unchanged by SI although SI affected the 5-HT receptors expression (Figs. 3, 4). Other study also demonstrates the unchanged 5-HT and 5-HIAA content in hippocampus of SI-reared mice.³⁴⁾ Thus, SI may not affect the synthesis and metabolism of 5-HT. The present study also showed YKS and YKSCH do not alter the 5-HT and 5-HIAA content (Fig. 4). YKS attenuates SI-induced aggressive behavior in zinc-deficient mice while YKS does not affect the 5-HT content in brain tissue.³⁵⁾ These results suggest that the 5-HT and 5-HIAA content are not implicated in anti-aggressive effect of YKS.

5-HT_1A and 5-HT_1B receptor agonists are reported to attenuate aggressive behavior in SI-reared mice,^36,37) implying that the activation of 5-HT_1A and 5-HT_1B receptor signaling prevents aggression. However, the present study showed an increase in 5-HT_1A and 5-HT_1B receptor mRNA levels in SI-reared mice (Figs. 3A, B). YKSCH attenuated the increase in 5-HT_1A receptors (Fig. 3A). Other studies have demonstrated that SI increases 5-HT_1A receptor binding in the cortical amygdala, hippocampal CA1 field, septum and different cortical regions.^38,39) Additionally, 5-HT_1A receptor mRNA is increased in the hippocampus of SI-reared rats.⁴⁰⁾ Behavioral experiments with 5-HT receptor agonists suggest that SI induces supersensitivity of 5-HT_1A and 5-HT₂ receptors.⁴¹⁾ Although 5-HT_1B receptor expression was not reported, our results are similar to other results regarding 5-HT_1A receptors. However, the association between aggressive behavior and increased 5-HT_1A and 5-HT_1B receptor mRNA levels is unknown.

In this study, the dose of YKSCH (1000 mg/kg) was identical to that of YKS (1000 mg/kg). The YKS content (719 mg/kg) in YKSCH is less than that of YKS alone (1000 mg/kg). Therefore, the decreased effect of YKSCH on aggression and 5-HT receptor levels may be, in part, due to the decreased YKS content in YKSCH. However, YKSCH produced a significant increase in AVP mRNA compared with YKS, while the vehicle did not induce a significant increase. These results suggest that increased AVP expression is not due to the decreased YKS content in YKSCH.

In conclusion, the present study indicates that YKS ameliorates SI-induced aggressive behavior, likely by inhibiting the increase in 5-HT_2A and 5-HT_3A receptors. In addition, the YKSCH-induced increase in the AVP level may contribute to the weaker anti-aggressive effect of YKSCH. YKS may be more effective for the treatment of irritability than YKSCH if digestive function deficiencies are not considered.

Acknowledgments

The authors thank the Department of Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University for providing the LightCycler^® 96 System and Tsumura & Co. for providing YKS and YKSCH. The authors also thank Shuhei Fujita and Sho Yoshida, Chika Aramaki for technical assistance. We thank Lisa Kreiner, Ph.D., for editing a draft of this manuscript.

Conflict of Interest

Katsunori Iwasaki received a research grant from Tsumura & Co. The other authors declare no conflict of interest.

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