Research Article | Open Access
The Kampo Medicine Yokukansan Decreases MicroRNA-18 Expression and Recovers Glucocorticoid Receptors Protein Expression in the Hypothalamus of Stressed Mice
It is well known that glucocorticoid receptor (GR) signaling regulates the hypothalamic-pituitary-adrenal (HPA) axis, and GR expression level is associated with HPA axis activity. Recent studies revealed that microRNA- (miR-) 18 and/or 124a are candidate negative regulators of GR in the brain. The Kampo medicine Yokukansan (YKS) can affect psychological symptoms such as depression and anxiety that are associated with stress responses. In this study, we evaluated the effect of YKS on miR-18 and 124a and GR levels in mice exposed to stress. We found that YKS pretreatment normalized elevated plasma corticosterone levels in stress-exposed mice. In addition, GR mRNA levels were downregulated in the brain following stress exposure. While miR-124a expression levels were not altered in the hypothalamus of stress-exposed mice, miR-18 levels decreased in the hypothalamus of YKS-pretreated mice after stress exposure. Finally, GR protein levels in the paraventricular nucleus (PVN) of the hypothalamus after stress exposure recovered in YKS-pretreated mice. Collectively, these data suggest that YKS normalizes GR protein levels by regulating miR-18 expression in the hypothalamus, thus normalizing HPA axis activity following stress exposure.
Hypothalamic-pituitary-adrenal (HPA) axis activation is one of the key responses following physiological stress [1–5]. Corticosteroids provide negative feedback to the HPA system at the level of the hippocampus, hypothalamus, and pituitary gland by activating glucocorticoid receptors (GRs) [6–9]. At rest, the mineralocorticoid receptor has a high affinity for corticosterone . However, GR has a low affinity for corticosterone and is rarely activated in resting conditions. Thus, GR expression is a crucial indicator of stress response indicator [10–14]. However, little is known about the molecular mechanisms regulating GR expression in the hypothalamus.
Yokukansan (YKS) (Tsumura, TJ-54) is a traditional Japanese medicine administered to patients who show symptoms such as nervousness, short-tempered behavior, irritability, sleeplessness, eyelid twitching, and limb shaking . Furthermore, YKS ameliorates behavioral and psychological symptoms such as aggression, anxiety, and depression in patients with Alzheimer’s disease and other forms of dementia. However, the mechanism underlying YKS-mediated attenuation of psychological symptoms and several stress-response behaviors is unknown.
MicroRNAs (miRs) are noncoding RNAs that inhibit the translation and/or decrease the stability of their target mRNAs, ultimately decreasing their protein expression levels [16, 17]. Recent studies indicate that miRs are involved in various functions such as embryonic development, differentiation, and neural plasticity [18–21]. Interestingly, miR-18 and/or miR-124a are candidate negative regulators of GRs in the brain . Given YKS’s stress-modifying capabilities, we hypothesized that it could exert its effect by impacting the HPA axis, and more specifically, by regulating GR expression in the hypothalamus. In the present study, we demonstrate that YKS affects hypothalamic miR-18 expression levels in stress-exposed mice and investigate GR protein expression level in the paraventricular nucleus (PVN) of the hypothalamus in YKS-pretreated and stress-exposed mice.
2. Materials and Methods
2.1. Ethics Statement
All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of Kinki University (no. KAME-24-021), and the Guiding Principles for the Care and Use of Laboratory Animals and the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals were followed.
Adult male C57/BL6 mice weighing 25–35 g were obtained at 11 weeks of age from Japan SLC, Inc. (Hamamatsu, Japan). Three mice per cage were housed in a temperature- (°C), humidity- (%), and light- (12-h light/dark schedule, lights on at 07:00 and off at 19:00 h) controlled environment and were fed laboratory food and water ad libitum. The animals were allowed to adjust to the environment for 1 week before the experiments were performed. A randomized design was used to assign the mice to one of three groups ( in each group): no-stressed control, stress-exposed, and YKS-pretreated stress-exposed.
2.3. Stress Exposure
Stress exposure was performed as previously described . Briefly, the mice were placed in a 50 mL conical polypropylene centrifuge tube and immersed vertically to the level of the xiphoid process in a water bath at 23°C for 2 h. In our preliminary experiments, chronic stress exposure did not induce gastric ulcer formation. Control mice were removed from their home cages and placed in new breeding cages for 2 h. Immediately after the end of testing, mice were anesthetized with sodium pentobarbital (30 mg/kg) and perfused transcardially with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer.
2.4. Drug Administration
YKS is composed of seven dried medicinal herbs: 19.5% Atractylodes lancea rhizome (ALR; rhizome of Atractylodes lancea De Candolle, Compositae), 19.5% Poria sclerotium (PS; sclerotium of Poria cocos Wolf, Polyporaceae), 14.6% Cnidium rhizome (CR; rhizome of Cnidium officinale Makino, Umbelliferae), 14.6% Japanese Angelica root (JAR; root of Angelica acutiloba Kitagawa, Umbelliferae), 9.8% Bupleurum root (BR; root of Bupleurum falcatum Linné, Umbelliferae), 7.3% Glycyrrhiza (GR; root and stolon of Glycyrrhiza uralensis Fisher, Leguminosae), and 14.6% Uncaria hook (UH; hook of Uncaria rhynchophilla Miquel, Rubiaceae) . The seven medical 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 performed to produce dried extract powder. The dry powdered YKS extracts used in the present study were supplied by Tsumura & Co. (Tokyo, Japan) and dissolved in distilled saline. The mice ( in each group) received oral YKS (1.0 g/kg of body weight) or saline 1 h before stress exposure.
2.5. Measurement of Plasma Corticosterone Levels
After mice were deeply anesthetized at the end of the stress experiment, and blood samples were collected into tubes containing heparin between 11:00 and 13:00 h by cardiac puncture. The tubes were immediately placed on ice and then centrifuged at 1,000 g for 15 min at 4°C. Plasma was stored at −80°C prior to the enzyme immunoassays. Plasma corticosterone levels were determined in duplicate using an AssayMax Corticosterone enzyme-linked immunosorbent assay kit (Assaypro, St. Charles, MO, USA).
2.6. Reverse Transcriptase Reaction and Real-Time Polymerase Chain Reaction (PCR)
Total RNA was prepared from the hippocampus and hypothalamus of mice using ISOGEN II (NipponGene, Toyama, Japan) according to the manufacturer’s instructions. The total RNA extract was reverse transcribed by using oligo(dT)12–18 primers and ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Life Science, Tokyo, Japan) according to the manufacturer’s instructions. Real-time PCR was performed using an ABI PRISM 7900HT Sequence Detection System with the THUNDERBIRD qPCR Mix (Toyobo Life Science). To quantify the expression levels of GR, the following primers were used: GR forward primer, 5′-GTACCTCTGGAGGACAGATGTA-3′ (complement of bases 1025–1044); GR reverse primer, 5′-GCACCTATTCCAGTTTTCAG-3′ (complement of bases 1133–1152). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward primer 5′-GTGTTCCTACCCCCAATGTG-3′ and GAPDH reverse primer 5′AGGAGACAACCTGGTCCTCA-3′ were used as the internal controls. SYBR Green I fluorescence from the double-stranded PCR products was measured according to the manufacturer’s instructions (Toyobo Life Science).
2.7. Quantification of miR-18 and 124a
Total microRNA was prepared from the hippocampus and hypothalamus of mice by using ISOGEN II (NipponGene) according to the provided instructions. The microRNA extract was reverse transcribed with the TaqMan MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays has-miR-18a and mmr-miR-124a (Life Technologies, Inc., Carlsbad, CA, USA) as described by the manufacturer. Real-time PCR was performed using an ABI PRISM 7900HT Sequence Detection System with TaqMan MicroRNA Assays has-miR-18a (Life Technologies, Inc.). The comparative CT method (ΔΔCt) was used to quantify the relative expression levels of miR-18 and 124a according to the manufacturer’s instructions.
Immunohistochemical analysis was performed as described previously [23, 25]. The brain sections (10 to 12 sections of the PVN region from each mouse) were immersed in guinea pig anticorticotropin-releasing factor (CRF) (1 : 500; Bachem Inc., Torrance, CA, USA) or rabbit anti-GR (1 : 100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at 4°C for 24 h. The sections were then rinsed with phosphate-buffered saline (PBS) for 60 min and incubated at room temperature for 1 h with Alexa Fluor 568-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-guinea pig IgG antibody (Life Technologies, Inc.) at a dilution of 1 : 500 in PBS. Finally, the sections were washed with PBS for 1 h and mounted on slides using PermaFluor. Confocal microscopy (LSM-510 META) was performed with 20, 40, and 60x objective lenses (Carl Zeiss, Oberkochen, Germany).
2.9. Statistical Analyses
The statistical significances of differences were evaluated by two-tailed Student’s t-tests unless otherwise mentioned, and differences were considered significant at . All data are presented as the mean ± SEM, and the number of experiments is indicated.
3.1. YKS Normalizes Stress-Upregulated Plasma Corticosterone Levels
The HPA axis is reliably activated by stress exposure, and it is well known that this increases plasma corticosterone levels for up to 6 h after stress exposure [14, 23, 26]. YKS was previously reported to significantly affect mouse behavior just 1 h after a single oral dose of YKS, and several components of YKS cross the blood-brain barrier and function for at least 12 h after oral administration [27–30]. Thus, our study conditions were appropriate for studying stress responses and the effects of YKS.
To evaluate HPA axis activation in our mouse model of acute stress, we first measured plasma corticosterone levels in control and stress-exposed mice. The stress-exposed mice exhibited upregulated plasma corticosterone levels (Figure 1, Stress-YKS). However, YKS pretreatment of stress-exposed mice normalized plasma corticosterone to levels comparable to those measured in control mice (Figure 1, Stress + YKS). We previously reported that Sgk1 expression in the corpus callosum was upregulated after acute stress . However, this stress response was not detected in YKS pretreatment mice.
3.2. Stress Exposure Decreases GR mRNA Expression in the Hippocampus and Hypothalamus
GR expression level is likely associated with HPA axis activity . Thus, we examined GR mRNA levels in the hippocampus and hypothalamus after stress exposure. GR mRNA expression was decreased in both regions, and YKS pretreatment did not affect levels in stress-exposed mice (Figure 2, Stress + YKS). We further examined GR mRNA levels in YKS-pretreated and unstressed mice. GR mRNA levels were not significantly different between the two groups.
3.3. YKS Reduces miR-18 Expression in the Hypothalamus after Stress Exposure
The above findings indicated that YKS does not directly regulate GR mRNA. Next, we determined if YKS is involved in the posttranscriptional regulation of GR protein levels by evaluating the effect of YKS on miR-18 and 124a expression in the hippocampus and the hypothalamus following stress exposure. YKS pretreatment only reduced miR-18 expression in the hypothalamus (Figures 3(a) and 3(b)). miR-124a expression levels were unchanged in both regions after stress exposure (Figures 3(a) and 3(b)). These findings indicate that YKS regulates miR-18 expression in the hypothalamus but has no effect on GR mRNA or miR-124a after stress exposure.
3.4. YKS Normalizes GR Protein Level in the PVN of the Hypothalamus after Stress Exposure
We found that YKS pretreatment reduced miR-18 expression in the hypothalamus (Figure 3(a)). To obtain definitive evidence that this downregulation of miR-18 reduced inhibition of GR protein translation, we analyzed GR protein expression levels in the PVN of the hypothalamus.
It is well known that GR protein and CRF are expressed in the parvocellular division of the PVN of the hypothalamus and that CRF is secreted in response to stress [31, 32]. We first located the parvocellular division of the PVN by examining CRF expression (Figure 4(a)). CRF immunoreactivity (IR) was slightly upregulated in response to stress exposure (Figure 4(a)). However, YKS pretreatment suppressed this increase in CRF-IR in the PVN after stress exposure (Figure 4(a)). Furthermore, the number of CRF-IR cells in the PVN was not changed by stress exposure with or without YKS pretreatment (Figure 4(a)).
We found that stress exposure decreased the number of GR-IR cells, but this effect was blocked by YKS pretreatment (Figures 4(a) and 4(b)). These results suggest that YKS reduces miR-18 expression and increases GR protein expression by downregulating the inhibitory effects of GR translation in the PVN of the hypothalamus after stress exposure.
YKS has been used to treat the behavioral and psychological symptoms of dementia (BPSD). It is also administered to patients with schizophrenia, certain pain diseases, delirium and dementias, and Parkinson’s disease [33, 34]. Since YKS was found to improve BPSD, especially psychological symptoms such as anxiety, depression, and apathy in patients with several types of dementias, we initially examined HPA axis activity by measuring plasma corticosterone levels. We found that YKS regulated HPA axis activity by decreasing corticosterone levels in stress-exposed mice.
In the present study, we provide the first evidence that YKS downregulates miR-18 expression in the hypothalamus after stress exposure and ultimately normalizes GR protein levels in the PVN, thus affecting HPA axis activity.
It is well known that GR protein regulation is involved in HPA axis activity and reflects circulating plasma corticosterone levels and that corticosterone activates GRs in the hypothalamus [6–9]. Furthermore, GR expression level is regulated by several transcriptional factors and splicing regulators [35–37]. Recently, other regulatory mechanisms controlling GR expression levels have been reported including mRNA degradation, specific transcription factors activation, and regulation of microRNA expression [22, 38, 39]. We found that oral YKS administration did not affect GR mRNA levels, and we then focused on the posttranscriptional regulation of GR expression levels by microRNAs. In the brain, miR-18 and/or miR-124a posttranscriptionally regulate GR protein expression [22, 40, 41]. In our stress model, miR-124a expression was unchanged in the hypothalamus and hippocampus, whereas miR-18 expression was only downregulated in the hypothalamus of stress-exposed mice with YKS pretreatment. The finding of miR-18 reduction in the hypothalamus is in good agreement with a previous report . Therefore, miR-18 expression may crucially control GR protein expression in the hypothalamus after stress exposure (Figures 3 and 4).
Previous studies reported that GR is expressed in CRF-positive neurons of the PVN region [31, 32]. While the number of GR-expressing cells decreased after stress exposure, we found that stress-exposed animals that received YKS pretreatment had similar numbers of cells compared to control animals (Figure 4). However, little is known about the molecular mechanisms by which YKS regulates GR expression in the hypothalamus. Previous studies reported that the HPA axis is regulated by several neurotransmitters including serotonin, dopamine, and norepinephrine [42, 43]. YKS has a partial agonistic effect on serotonin 1A receptor (5-HT1A) and suppresses 5-HT2A receptor activity [44–47]. Another report described GRs as an indirect target of antidepressants that affect serotonin neurotransmission . However, YKS administration was not found to alter 5-HT2A receptor expression in the hypothalamus . Based on our results and these earlier reports, posttranscriptional GR regulation in the hypothalamus by YKS is likely unrelated to serotonin neurotransmission.
YKS is a mixture with seven components, and it is important to determine which components affect stress responses in the HPA axis and posttranscriptional GR regulation in the hypothalamus. Several components of YKS (e.g., geissoschizine methyl ether and glycyrrhetinic acid) can cross the blood-brain barrier and function in the brain for at least 1–12 h after oral YKS administration [27–30]. Thus, it is important to identify effective and metabolic components in YKS that are capable of crossing the blood-brain barrier.
In conclusion, our findings suggest that YKS downregulates miR-18 expression and normalizes HPA axis activity by recovering GR protein expression in the hypothalamus of stress-exposed mice. Elucidating the functional roles of the YKS-miR-18-GR protein-regulating pathway in the PVN of the hypothalamus is a primary goal of future research.
Conflict of Interests
The authors declare no conflict of interests.
The authors thank Drs. J. Horiguchi, T. Miyaoka, M. Tabuchi, K. Sekiguchi, T. Katayama, S. Matsuzaki, S. Shimada, and Y. Shigeyoshi for their valuable comments. The authors thank A. Matsumura, A Kawakami, E. Hisamatsu, and the members of the Central Research Facilities of Faculty of Medicine, Kinki University, and Center for Medical Research and Education of Graduate School of Medicine for their technical assistance. This work was supported in part by the Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research on Innovative Areas (Grant 26117519), a Grant-in-Aid for Scientific Research (C) (Grant 25430079), and the Sakamoto Research Foundation of Psychiatric Diseases. The authors are grateful to Tsumura & Co. (Tokyo, Japan) for generously providing the Yokukansan (TJ-54).
- V. Bartanusz, D. Jezova, L. T. Bertini, F. J. H. Tilders, J.-M. Aubry, and J. Z. Kiss, “Stress-induced increase in vasopressin and corticotropin-releasing factor expression in hypophysiotrophic paraventricular neurons,” Endocrinology, vol. 132, no. 2, pp. 895–902, 1993.
- K. S. Kendler, L. M. Karkowski, and C. A. Prescott, “Causal relationship between stressful life events and the onset of major depression,” American Journal of Psychiatry, vol. 156, no. 6, pp. 837–841, 1999.
- G. Aguilera and C. Rabadan-Diehl, “Vasopressinergic regulation of the hypothalamic-pituitary-adrenal axis: implications for stress adaptation,” Regulatory Peptides, vol. 96, no. 1-2, pp. 23–29, 2000.
- P. W. Gold and D. S. Charney, “Diseases of the mind and brain: depression: a disease of the mind, brain, and body.,” American Journal of Psychiatry, vol. 159, no. 11, p. 1826, 2002.
- B. S. McEwen, “Protection and damage from acute and chronic stress: allostasis and allostatic overload and relevance to the pathophysiology of psychiatric disorders,” Annals of the New York Academy of Sciences, vol. 1032, pp. 1–7, 2004.
- J. P. Herman, P. D. Patel, H. Akil, and S. J. Watson, “Localization and regulation of glucocorticoid and mineralocorticoid receptor messenger RNAs in the hippocampal formation of the rat,” Molecular Endocrinology, vol. 3, no. 11, pp. 1886–1894, 1989.
- D. Zhe, H. Fang, and S. Yuxiu, “Expressions of hippocampal mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the single-prolonged stress-rats,” Acta Histochemica et Cytochemica, vol. 41, no. 4, pp. 89–95, 2008.
- F. Thomson and M. Craighead, “Innovative approaches for the treatment of depression: targeting the HPA axis,” Neurochemical Research, vol. 33, no. 4, pp. 691–707, 2008.
- C. M. Pariante and S. L. Lightman, “The HPA axis in major depression: classical theories and new developments,” Trends in Neurosciences, vol. 31, no. 9, pp. 464–468, 2008.
- E. R. De Kloet, M. Joëls, and F. Holsboer, “Stress and the brain: from adaptation to disease,” Nature Reviews Neuroscience, vol. 6, no. 6, pp. 463–475, 2005.
- M. P. Boyle, J. A. Brewer, M. Funatsu et al., “Acquired deficit of forebrain glucocorticoid receptor produces depression-like changes in adrenal axis regulation and behavior,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 2, pp. 473–478, 2005.
- E. R. De Kloet and J. M. H. M. Reul, “Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems,” Psychoneuroendocrinology, vol. 12, no. 2, pp. 83–105, 1987.
- J. M. H. M. Reul and E. R. De Kloet, “Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation,” Endocrinology, vol. 117, no. 6, pp. 2505–2511, 1985.
- B. Myers, J. M. McKlveen, and J. P. Herman, “Glucocorticoid actions on synapses, circuits, and behavior: implications for the energetics of stress,” Frontiers in Neuroendocrinology, vol. 35, no. 2, pp. 180–196, 2014.
- T. Hiratsuka, S. Matsuzaki, S. Miyata et al., “Yokukansan inhibits neuronal death during ER stress by regulating the unfolded protein response,” PLoS ONE, vol. 5, no. 10, Article ID e13280, 2010.
- R. W. Carthew, “Gene regulation by microRNAs,” Current Opinion in Genetics & Development, vol. 16, no. 2, pp. 203–208, 2006.
- Y. Wang, H. M. Stricker, D. Gou, and L. Liu, “MicroRNA: past and present,” Frontiers in Bioscience, vol. 12, no. 6, pp. 2316–2329, 2007.
- X. Cao, G. Yeo, A. R. Muotri, T. Kuwabara, and F. H. Gage, “Noncoding RNAs in the mammalian central nervous system,” Annual Review of Neuroscience, vol. 29, pp. 77–103, 2006.
- T. Dalmay, “MicroRNAs and cancer,” Journal of Internal Medicine, vol. 263, no. 4, pp. 366–375, 2008.
- G. Stefani and F. J. Slack, “Small non-coding RNAs in animal development,” Nature Reviews Molecular Cell Biology, vol. 9, no. 3, pp. 219–230, 2008.
- H. Chiu, A. Alqadah, and C. Chang, “The role of microRNAs in regulating neuronal connectivity,” Frontiers in Cellular Neuroscience, vol. 7, article 283, 2014.
- E. Vreugdenhil, C. S. L. Verissimo, R. Mariman et al., “MicroRNA 18 and 124a down-regulate the glucocorticoid receptor: implications for glucocorticoid responsiveness in the brain,” Endocrinology, vol. 150, no. 5, pp. 2220–2228, 2009.
- S. Miyata, Y. Koyama, K. Takemoto et al., “Plasma corticosterone activates SGK1 and induces morphological changes in oligodendrocytes in corpus callosum,” PLoS ONE, vol. 6, no. 5, Article ID e19859, 2011.
- H. Kanno, Z. Kawakami, K. Mizoguchi, Y. Ikarashi, Y. Kase, and H. Ariga, “Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-,” PLoS ONE, vol. 9, no. 12, Article ID e116275, 2014.
- T. Ishikawa, S. Miyata, Y. Koyama et al., “Transient expression of Xpn, an XLMR protein related to neurite extension, during brain development and participation in neurite outgrowth,” Neuroscience, vol. 214, pp. 181–191, 2012.
- M. N. Silverman and E. M. Sternberg, “Glucocorticoid regulation of inflammation and its functional correlates: from HPA axis to glucocorticoid receptor dysfunction,” Annals of the New York Academy of Sciences, vol. 1261, no. 1, pp. 55–63, 2012.
- S. Imamura, M. Tabuchi, H. Kushida et al., “The blood-brain barrier permeability of geissoschizine methyl ether in Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan,” Cellular and Molecular Neurobiology, vol. 31, no. 5, pp. 787–793, 2011.
- A. Nishi, T. Yamaguchi, K. Sekiguchi et al., “Geissoschizine methyl ether, an alkaloid in Uncaria hook, is a potent serotonin1A receptor agonist and candidate for amelioration of aggressiveness and sociality by yokukansan,” Neuroscience, vol. 207, pp. 124–136, 2012.
- M. Tabuchi, S. Imamura, Z. Kawakami, Y. Ikarashi, and Y. Kase, “The blood-brain barrier permeability of 18β-glycyrrhetinic acid, a major metabolite of glycyrrhizin in Glycyrrhiza root, a constituent of the traditional Japanese medicine yokukansan,” Cellular and Molecular Neurobiology, vol. 32, no. 7, pp. 1139–1146, 2012.
- H. Kushida, M. Fukutake, M. Tabuchi et al., “Simultaneous quantitative analyses of indole and oxindole alkaloids of Uncaria Hook in rat plasma and brain after oral administration of the traditional Japanese medicine Yokukansan using high-performance liquid chromatography with tandem mass spectrometry,” Biomedical Chromatography, vol. 27, no. 12, pp. 1647–1656, 2013.
- Z. Liposits, R. M. Uht, R. W. Harrison, F. P. Gibbs, W. K. Paull, and M. C. Bohn, “Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF),” Histochemistry, vol. 87, no. 5, pp. 407–412, 1987.
- N. Laflamme, N. Barden, and S. Rivest, “Corticotropin-releasing factor and glucocorticoid receptor (GR) gene expression in the paraventricular nucleus of immune-challenged transgenic mice expressing type II GR antisense ribonucleic acid,” Journal of Molecular Neuroscience, vol. 8, no. 3, pp. 165–179, 1997.
- Y. Matsuda, T. Kishi, H. Shibayama, and N. Iwata, “Yokukansan in the treatment of behavioral and psychological symptoms of dementia: a systematic review and meta-analysis of randomized controlled trials,” Human Psychopharmacology, vol. 28, no. 1, pp. 80–86, 2013.
- K. Matsumoto, Q. Zhao, Y. Niu et al., “Kampo formulations, chotosan, and yokukansan, for dementia therapy: existing clinical and preclinical evidence,” Journal of Pharmacological Sciences, vol. 122, no. 4, pp. 257–269, 2013.
- R. Kumar and E. B. Thompson, “Transactivation functions of the N-terminal domains of nuclear hormone receptors: protein folding and coactivator interactions,” Molecular Endocrinology, vol. 17, no. 1, pp. 1–10, 2003.
- R. H. DeRijk, M. J. Schaaf, F. J. Stam et al., “Very low levels of the glucocorticoid receptor β isoform in the human hippocampus as shown by Taqman RT-PCR and immunocytochemistry,” Molecular Brain Research, vol. 116, no. 1-2, pp. 17–26, 2003.
- M. J. M. Schaaf and J. A. Cidlowski, “Molecular mechanisms of glucocorticoid action and resistance,” Journal of Steroid Biochemistry and Molecular Biology, vol. 83, no. 1–5, pp. 37–48, 2002.
- R. H. Derijk, M. J. M. Schaaf, G. Turner et al., “A human glucocorticoid receptor gene variant that increases the stability of the glucocorticoid receptor β-isoform mRNA is associated with rheumatoid arthritis,” Journal of Rheumatology, vol. 28, no. 11, pp. 2383–2388, 2001.
- N. Z. Lu and J. A. Cidlowski, “Glucocorticoid receptor isoforms generate transcription specificity,” Trends in Cell Biology, vol. 16, no. 6, pp. 301–307, 2006.
- S. Uchida, A. Nishida, K. Hara et al., “Characterization of the vulnerability to repeated stress in Fischer 344 rats: possible involvement of microRNA-mediated down-regulation of the glucocorticoid receptor,” European Journal of Neuroscience, vol. 27, no. 9, pp. 2250–2261, 2008.
- R. V. Durairaj and E. R. Koilmani, “Environmental enrichment modulates glucocorticoid receptor expression and reduces anxiety in Indian field male mouse Mus booduga through up-regulation of microRNA-124a,” General and Comparative Endocrinology, vol. 199, pp. 26–32, 2014.
- A. J. Dunn, “Cytokine activation of the HPA axis,” Annals of the New York Academy of Sciences, vol. 917, pp. 608–617, 2000.
- M. Hamon and P. Blier, “Monoamine neurocircuitry in depression and strategies for new treatments,” Progress in Neuro-Psychopharmacology and Biological Psychiatry, vol. 45, pp. 54–63, 2013.
- K. Terawaki, Y. Ikarashi, K. Sekiguchi, Y. Nakai, and Y. Kase, “Partial agonistic effect of yokukansan on human recombinant serotonin 1A receptors expressed in the membranes of Chinese hamster ovary cells,” Journal of Ethnopharmacology, vol. 127, no. 2, pp. 306–312, 2010.
- N. Egashira, K. Iwasaki, A. Ishibashi et al., “Repeated administration of Yokukansan inhibits DOI-induced head-twitch response and decreases expression of 5-hydroxytryptamine (5-HT)2A receptors in the prefrontal cortex,” Progress in Neuro-Psychopharmacology & Biological Psychiatry, vol. 32, no. 6, pp. 1516–1520, 2008.
- H. Kanno, K. Sekiguchi, T. Yamaguchi et al., “Effect of yokukansan, a traditional Japanese medicine, on social and aggressive behaviour of para-chloroamphetamine-injected rats,” Journal of Pharmacy and Pharmacology, vol. 61, no. 9, pp. 1249–1256, 2009.
- T. Ueda, S. Ugawa, Y. Ishida, and S. Shimada, “Geissoschizine methyl ether has third-generation antipsychotic-like actions at the dopamine and serotonin receptors,” European Journal of Pharmacology, vol. 671, no. 1-3, pp. 79–86, 2011.
- N. P. Maric and M. Adzic, “Pharmacological modulation of HPA axis in depression—new avenues for potential therapeutic benefits,” Psychiatria Danubina, vol. 25, no. 3, pp. 299–305, 2013.
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