Methyl Donor Supplementation Prevents a Folate Deficiency-induced Depression-like State and Neuronal Immaturity of the Dentate Gyrus in Mice.

We have recently shown that folate deficiency induces depression-like behavior and neuronal immaturity in the dentate gyrus (DG) in mice. We also revealed that folate deficiency inhibits neuronal maturation, hypomethylates the promoter of certain neuronal genes and decreases intracellular levels of S-adenosylmethionine (SAM), a methyl donor, in cultured neural stem and progenitor cells. Based on these findings, we hypothesized that SAM reduction may be involved in a folate deficiency-induced depressive state and neural immaturity. In this study, we examined whether SAM supplementation prevents depression-like behavior and neural immaturity in low folate diet-fed mice. Intraperitoneal administration of SAM (50 mg/kg/day) for 14 days from 7 weeks old prevented increased immobility in low folate diet-fed mice. SAM supplementation also prevented an increase in the number of doublecortin (an immature neuron marker)-positive cells and a decrease in the number of NeuN (a mature neuron marker)/5-bromo-2'-deoxyuridine (a proliferation marker)-double positive cells in the DG of these mice. Furthermore, neurofunctional and neuromorphological abnormalities in the DG of low folate diet-fed mice, such as decreases in stress-induced expression of c-Fos (a neuronal activity marker), dendritic complexity and the number of mature spines, were improved by SAM supplementation. The disrupted expression of transcription factors involved in neuronal differentiation and maturation was also normalized by SAM supplementation. These results suggest that SAM reduction may be involved in a folate deficiency-induced depressive state.

Kihito prevents corticosterone-induced brain dysfunctions in mice.

Kihito (KIT; Gui Pi Tang) is a traditional herbal medicine that is used for treatment of neuropsychiatric disorders such as depression, anxiety, neurosis and insomnia in China and Japan. Recently, it has also been shown that KIT improves cognitive dysfunction in patients with Alzheimer's disease. In this study, to investigate the mechanisms underlying the effects of KIT on stress-induced brain dysfunctions such as a depressed state and memory impairment, we examined whether KIT prevents behavioral and neurophysiological abnormalities in mice treated chronically with corticosterone (CORT). CORT (40 mg/kg/day, s.c.) and KIT (1000 mg/kg/day, p.o.) were given to 7-week-old male ddY mice for 14 days. Twenty-four hours after the last treatment, depression-like behavior in the forced swim test, spatial memory in the Barnes maze test, cell survival and the number of new-born immature neurons, dendritic spine density and expression levels of mRNA for neurotrophic factors were analyzed. Depression-like behavior and spatial memory impairment were observed in CORT-treated mice without KIT treatment. Hippocampal cell survival, the number of hippocampal new-born immature neurons, hippocampal and accumbal dendritic spine density and mRNA levels for neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) were decreased in CORT-treated mice without KIT treatment. KIT prevented CORT-induced depression-like behavior, spatial memory impairment, and decreases in hippocampal cell survival, the number of hippocampal new-born immature neurons, accumbal dendritic spine density and GDNF mRNA. KIT may ameliorate stress-induced brain dysfunctions via prevention of adverse effects of CORT on cell survival, new-born immature neurons, spine density and neurotrophic factors.

Kami-shoyo-san ameliorates sociability deficits in ovariectomized mice, a putative female model of autism spectrum disorder, via facilitating dopamine D1 and GABAA receptor functions.

ETHNOPHARMACOLOGICAL RELEVANCE: Kami-shoyo-san (KSS) is a Kampo formula used clinically for menopause-related symptoms in Japan. However, the effect of KSS on autism spectrum disorder (ASD), a developmental disorder with a higher prevalence in males than in females, has not been reported yet. AIM OF THE STUDY: It is accepted generally that dysfunction in the GABAergic system is associated with pathogenesis of ASD. In our previous study, a decrease in brain allopregnanolone (ALLO), a positive allosteric GABAA receptor modulator, induced ASD-like symptoms such as impaired sociability-related performance and increased repetitive self-grooming behavior in male mice, and that KSS ameliorated these behavioral abnormalities via GABAA receptor- and dopamine D1 receptor-mediated mechanisms. In this study, to better understand a gender difference in the prevalence of ASD, we examined whether dissection of ovary (OVX), a major organ secreting progesterone in females, causes ASD-like behaviors in a manner dependent on brain ALLO levels, and if so, how KSS affects the behaviors. MATERIALS AND METHODS: Six-week-old ICR female mice received ovariectomy, and KSS (74 mg/kg and 222 mg/kg, p.o.) were treated before 1 h starting each behavioral test. The sociability, social anxiety-like behavior, and self-grooming behavior were analyzed by the resident-intruder test, mirror chamber test, and open field test, respectively. After finishing the behavioral experiment, the ALLO content in the brain was measured by ELISA. Furthermore, we examined the effects of OVX on the neuro-signaling pathways in the prefrontal cortex and striatum by Western blotting. RESULTS: The results revealed that OVX induced sociability deficits and social anxiety-related behaviors, but not repetitive self-grooming behavior, and that these behavioral changes were accompanied not only by a decrease of brain ALLO levels, but also by impairment of CREB- and CaMKIIα-mediated neuro-signaling in the prefrontal cortex. Moreover, the administration of KSS had no effect on the brain ALLO level, but significantly ameliorated the OVX-induced behavioral and neurochemical changes via facilitation of GABAA receptor and dopamine D1 receptor-mediated neurotransmission. CONCLUSIONS: These findings suggest that a decrease in gonadal hormone-derived ALLO plays a major role in ASD-like behaviors in female mice and that KSS is beneficial for the treatment of ASD in females.

Kami-shoyo-san improves ASD-like behaviors caused by decreasing allopregnanolone biosynthesis in an SKF mouse model of autism.

Dysfunctions in the GABAergic system are associated with the pathogenesis of autism spectrum disorder (ASD). However, the mechanisms by which GABAergic system dysfunctions induce the pathophysiology of ASD remain unclear. We previously demonstrated that a selective type I 5α-reductase inhibitor SKF105111 (SKF) induced ASD-like behaviors, such as impaired sociability-related performance and repetitive grooming behaviors, in male mice. Moreover, the effects of SKF were caused by a decrease in the endogenous levels of allopregnanolone (ALLO), a positive allosteric modulator of the GABAA receptor. In this study, we used SKF-treated male mice as a putative animal model of ASD and examined the effects of Kami-shoyo-san (KSS) as an experimental therapeutic strategy for ASD. KSS is a traditional Kampo formula consisting of 10 different crude drugs and has been used for the treatment of neuropsychiatric symptoms. KSS dose-dependently attenuated sociability deficits and suppressed an increase in grooming behaviors in SKF-treated mice without affecting ALLO content in the prefrontal cortex. The systemic administration of the dopamine D1 receptor antagonist SCH23390 reversed the ameliorative effects of KSS. On the other hand, the dopamine D2 receptor antagonist sulpiride and GABAA receptor antagonist bicuculline only attenuated the ameliorative effect of KSS on repetitive self-grooming behaviors. The present results indicate that KSS improves SKF-induced ASD-like behaviors by facilitating dopamine receptor-mediated mechanisms and partly by neurosteroid-independent GABAA receptor-mediated neurotransmission. Therefore, KSS is a potential candidate for the treatment of ASD.

Kamiuntanto increases prefrontal extracellular serotonin levels and ameliorates depression-like behaviors in mice.

Kamiuntanto (KUT; Jia Wei Wen Dan Tang in Pinyin) is a traditional Japanese Kampo medicine that is used to treat psychological dysfunction. However, the mechanisms of action of KUT are not understood. To investigate the mechanisms underlying the ameliorative properties of KUT, the effects of KUT on abnormal behaviors of isolation-reared mice and on the prefrontal monoaminergic system were examined. KUT (1000 mg/kg) reversed encounter-induced hyperactivity and increased immobility in the forced swim test in isolation-reared mice, as also found for an antidepressant, fluoxetine (30 mg/kg). In vivo microdialysis showed that KUT (1000 mg/kg) transiently increased the level of extracellular serotonin (5-HT) in the prefrontal cortex. In contrast, an incomplete KUT formula excluding Bambusae Caulis (BC), a component herb of KUT, did not reverse abnormal behaviors of isolation-reared mice or increase prefrontal extracellular 5-HT. Furthermore, the antidepressant-like effect of KUT in the forced swim test was prevented by pretreatment with GR127935, a 5-HT1B antagonist. These findings suggest that KUT may ameliorate depressive symptoms via 5-HTergic systems, and that BC plays an important role in the antidepressant-like effects of KUT.

Sansoninto, a traditional herbal medicine, ameliorates behavioral abnormalities and down-regulation of early growth response-1 expression in mice exposed to social isolation stress.

Social isolation (SI) mice exhibit behavioral abnormalities such as impairments of sociability- and attention-like behaviors, offering an animal model of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD). This study aimed to identify the effects of Sansoninto (SST; suan zǎo rén tang) on the psychiatric symptoms related to ADHD using SI mice. Four-week-old mice were socially isolated during the experimental period, and SST administration (800 or 2400 mg/kg, p.o.) was started at 2 weeks after starting SI. SST ameliorated SI-induced impairments of sociability- and attention-like behaviors in a dose-dependent manner, and tended to ameliorate contextual- and auditory-dependent fear memory deficit. Moreover, the expression level of Egr-1 was down-regulated by SI stress, and was restored by a high dose of SST. These findings suggest that SST is useful for improvement of psychiatric disorders such as ADHD.

Daily administration of yokukansan and keishito prevents social isolation-induced behavioral abnormalities and down-regulation of phosphorylation of neuroplasticity-related signaling molecules in mice.

BACKGROUND: Our previous studies demonstrated that post-weaning social isolation (ISO) in mice induces behavior abnormalities such as deficits of sociability- and attention-like behaviors. These deficits can be attenuated by methylphenidate (MPH), a drug used for attention deficit hyperactivity disorder (ADHD), suggesting that ISO mice offer a potential animal model of comorbid developmental disorder with ADHD and autism spectrum disorder symptoms. This study investigated the effects of Kampo formulae, yokukansan (YKS) and keishito (KST), on the neuropsychiatric symptoms of ISO mice to clarify the therapeutic or preventive/delaying potential of these formulae for the treatment of neurodevelopmental disorders. METHODS: Three-to-4-week old male ICR mice were socially isolated during an experimental period and YKS and KST (1523.6 and 2031.8 mg/kg, p.o.) was administered starting from week 2 and week 0 after starting ISO for the analysis of their therapeutic and preventive/delaying potentials, respectively. Sociability, attention-related behavior and fear memory were elucidated by a 3 chamber test, a water-finding test and fear conditioning test, respectively. Moreover, the phosphorylation of neuroplasticity-related signaling molecules in mice hippocampus was analyzed using western blotting. RESULTS: In a therapeutic procedure, YKS ameliorated ISO-induced impairments of attention-like behavior and context-dependent fear memory, but not of sociability, whereas KST had no beneficial effects in ISO mice. In experiments to analyze the preventive/delaying potentials of these treatments, both YKS and KST improved sociability, attention, and context-dependent fear memory deficits. The improvement of sociability in mice by YKS and KST was not inhibited by a dopamine D1 receptor antagonist, suggesting that YKS and KST improved the ISO-induced sociability deficit by other mechanisms besides activation of the dopaminergic system. On the other hand, the beneficial effects of YKS and KST on attention-like behavior were inhibited by a muscarinic antagonist, suggesting that YKS and KST ameliorated ISO-induced attention-like behavior through a cholinergic mechanism. Moreover, the phosphorylated forms of CaMKII and CREB were down-regulated by ISO stress and restored by YKS and KST administration. CONCLUSIONS: These findings suggest that YKS and KST may be useful for the improvement of neurodevelopmental disorders.

Gomisin N ameliorates lipopolysaccharide-induced depressive-like behaviors by attenuating inflammation in the hypothalamic paraventricular nucleus and central nucleus of the amygdala in mice.

Emotional impairments such as depressive symptoms often develop in patients with sustained and systemic immune activation. The objective of this study is to investigate the effect of gomisin N, a dibenzocyclooctadiene lignan isolated from the dried fruits of Schisandra chinensis (Turcz.) Baill., which exhibited inhibitory effects of the bacterial endotoxin lipopolysaccharide (LPS)-induced NO production in a screening assay, on inflammation-induced depressive symptoms. We examined the effects of gomisin N on inflammation induced by LPS in murine microglial BV-2 cells and on LPS-induced behavioral changes in mice. Gomisin N inhibited LPS-induced expression of mRNAs for inflammation-related genes (inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α) in BV-2 cells. Administration of gomisin N attenuated LPS-induced expression of mRNAs for inflammation-related genes, increases in the number of c-Fos immunopositive cells in the hypothalamus and amygdala, depressive-like behavior in the forced swim test and exploratory behavior deficits 24 h after LPS administration in mice. These results suggest that gomisin N might ameliorate LPS-induced depressive-like behaviors through inhibition of inflammatory responses and neural activation in the hypothalamus and amygdala.

Kamikihito Ameliorates Lipopolysaccharide-Induced Sickness Behavior via Attenuating Neural Activation, but Not Inflammation, in the Hypothalamic Paraventricular Nucleus and Central Nucleus of the Amygdala in Mice.

Sickness behavior is a series of behavioral and psychological changes that develop in those stricken with cancers and inflammatory diseases. The etiological mechanism of sickness behavior is not known in detail, and consequently there are no established standard therapies. Kamikihito (KKT), a Kampo (traditional Japanese herbal) medicine composed of 14 herbs, has been used clinically to treat psychiatric dysfunction. Previously, we found that KKT ameliorated sickness behavior in mice inoculated with murine colon 26 adenocarcinoma cells. In this study, we examined the effects of KKT on bacterial endotoxin lipopolysaccharide (LPS)-induced sickness behavior in mice. The administration of LPS caused the emotional aspects of sickness behavior, such as loss of object exploration, social interaction deficit, and depressive-like behavior. LPS also induced mRNA expression for cyclooxygenase (COX)-2, interleukin (IL)-1ß and IL-6, and increased the number of c-Fos immunopositive cells in the hypothalamus and amygdala. KKT ameliorated the behavioral changes and reversed the increases in c-Fos immunopositive cells in the two brain regions, but did not influence the mRNA expression. These results suggest that KKT ameliorates sickness behavior via the suppression of neural activation without anti-inflammatory effects, and that KKT has the potential to treat sickness behavior.

Modulation of chemokine expression on intestinal epithelial cells by Kampo (traditional Japanese herbal) medicine, Hochuekkito, and its active ingredients.

The intestinal epithelial cells sit at the interface between a lumen and a lamina propria or lymph nodes such as Peyer's patches, where they play important roles in maintaining intestinal homeostasis through chemokine secretion. This study investigated the effect of Hochuekkito (TJ-41)-a traditional Japanese herbal (Kampo) formula used as a tonic for weakness-on chemokine expression in intestinal epithelial cells in order to explore the mechanism of its modulating effect against mucosal immunity. When cells from the rat normal small intestinal epithelial cell-line IEC-6 were stimulated with TJ-41, mRNA expression of CC chemokine ligand (CCL) 11 (eotaxin), CCL20 (MIP-3α) and CCL25 (TECK) was enhanced. Oral administration of TJ-41 to methotrexate-treated mice enhanced mRNA expression of CCL25 and keratinocyte growth factor in the jejunum with, decreasing mRNA expression of the inflammatory marker tumor necrosis factor (TNF)-α. Although oral administration of TJ-41 did not affect CCL20 mRNA expression in villus epithelium of methotrexate-treated mice, enhancement of CCL20 mRNA expression was observed in Peyer's patches. Immunohistochemical analysis detected dense staining with anti-CCL20 antibody in the follicle-associated epithelium region of Peyer's patches in mice administered TJ-41. Analysis of active ingredients indicates that polysaccharide-containing macromolecules in TJ-41 contribute to the enhancement of CCL20 mRNA expression through an intracellular signal cascade via nuclear factor kappa B (NF-κB) activation.

Involvement of neuropeptide Y signaling in the antidepressant-like effect and hippocampal cell proliferation induced by kososan, a Kampo medicine, in the stress-induced depression-like model mice.

Neuropeptide Y (NPY) and Orexin-A (OX-A), well-known neuropeptides associated with feeding and arousal, show antidepressant-like properties via hippocampal cell proliferation. Previous studies have revealed that kososan, a Kampo (Japanese herbal) medicine, has an antidepressant-like effect in behavioral animal models of depression; the mechanisms underlying this effect may involve the orexinergic system and subsequent upregulation of hippocampal cell proliferation. However, the roles of NPY in kososan's antidepressant-like effect remain unclear. Here we investigated whether the regulation of the NPY system could play crucial roles in this effect in the stress-induced depression-like model mice. The antidepressant-like effect of kososan administered orally (1.0 g/kg) for 28 d was abolished by a continuous intracerebroventricular injection of BIBO3304, a neuropeptide Y1 receptor antagonist, for 7 d. Likewise, BIBO3304 injection blocked the kososan-induced increases in hippocampal cell proliferation and cluster formation of neural progenitor cells. On the other hand, BIBO3304 injection did not affect kososan-induced increases in hypothalamic OX-A-producing cells or in serum OX-A levels. These results suggest that the control of the NPY system in the brain plays an essential role in kososan's antidepressant-like effect and facilitates hippocampal cell proliferation, both of which could be attributed, at least in part, to the control of the NPY system subsequent to the control of the OX-A system.

A possible mechanism underlying an antidepressive-like effect of Kososan, a Kampo medicine, via the hypothalamic orexinergic system in the stress-induced depression-like model mice.

Kososan, a Kampo (Japanese herbal) medicine, has an antidepressive-like effect in behavioral animal models of depression and has been used clinically for the improvement of depressive mood. However, mechanism(s) underlying the antidepressive-like effect of kososan remain unknown. Previous studies showed that orexin-A (OX-A), a neuropeptide that is involved in feeding and arousal, exhibits an antidepressive-like property via hippocampal cell proliferation. Here, we used immunohistochemical analysis with bromodeoxyuridine (BrdU), a marker of proliferating cells, to investigate the effect of long-term treatment with kososan on the orexinergic system and on hippocampal cell proliferation. Oral administration of kososan (1.0 g/kg) or milnacipran (60 mg/kg), a serotonin and noradrenaline reuptake inhibitor, for 28 d led to an antidepressive-like effect in the stress-induced depression-like model mice and reversed the stress-induced decrease in the number of OX-A-positive cells in the lateral hypothalamic area. In addition, both kososan and milnacipran alleviated the stress-induced decrease in the number of BrdU-positive cells in the hippocampal dentate gyrus. Moreover, the antidepressive-like effect and the increase in cell proliferation and in the number of neuropeptide Y (NPY, which is closely associated with orexinergic system)-positive cells in the dentate gyrus induced by kososan were blocked by treatment with SB-334867, an orexin receptor 1 antagonist. These results suggest that kososan exerts an antidepressive-like effect via the improvement of the stress-induced decrease in hippocampal cell proliferation and that the mechanism underlying the antidepressive-like effect of kososan, but not of milnacipran, may be associated with the regulation of orexinergic and/or NPYergic transmission.

Anti-allergic activity of a Kampo (Japanese herbal) medicine "Sho-seiryu-to (Xiao-Qing-Long-Tang)" on airway inflammation in a mouse model.

Effects of a Kampo (Japanese herbal) medicine "Sho-seiryu-to (SST, Xiao-Qing-Long-Tang in Chinese)", which has been used for the treatment of allergic bronchial asthma clinically, were examined on ovalbumin (OVA)-sensitized allergic airway inflammation model (i.e., bronchial asthma) in a mouse. When SST was orally administered at 0.5 g/kg/day from day 1 to 6 days after OVA inhalation, SST reduced the OVA-specific IgE antibody titer in bronchoalveolar lavage (BAL) fluids at 7 days after the OVA inhalation. CD4(+) T cells obtained from the mouse lung produced more interleukin (IL)-4 and IL-5 but less interferon (IFN)-gamma than T cells from nonsensitized control animals. However, oral administration of SST reduced the production of IL-4 and IL-5 and the production of IFN-gamma returned to the control level. In addition, the IL-4 level was increased in the BAL fluid of the OVA-sensitized animals compared to the nonsensitized control, while the IFN-gamma levels decreased. SST reduced the IL-4 levels in the BAL fluids and returned the IFN-gamma level to control levels. Nerve growth factor (NGF) was increased in the BAL fluids of the OVA-sensitized mice over that of nonsensitized mice, but oral administration of SST augmented the NGF levels to approximately 2 times higher than in the sensitized mice. Although lung cells obtained from sensitized mice produced higher levels of NGF than nonsensitized mice, oral administration of SST augmented the production of NGF by the lung cells even higher ( approximately 2 times more than cells from sensitized mice). Administration of anti-NGF antibody to the airway blocked the effects of SST. These results suggest that SST modulates Th1/Th2 balance in the lungs and augmentation of NGF in the lungs may be related to the effects of SST. Pinellic acid (9S, 12S, 13S-trihydroxy-10E-octadecenoic acid), one component of the herbs of SST [Int. Immunopharmacol. 2 (2002) 1183], was purified from the tuber of Pinellia ternata Breitenbach. Oral administration of pinellic acid (50 microg/kg/day) also reduced the OVA-specific IgE antibody titer in BAL fluids from the sensitized mouse. This result suggests that pinellic acid is one of active ingredient(s) in SST.