Treatment with Glycyrrhiza glabra Extract Induces Anxiolytic Effects Associated with Reduced Salt Preference and Changes in Barrier Protein Gene Expression.

We have previously identified that low responsiveness to antidepressive therapy is associated with higher aldosterone/cortisol ratio, lower systolic blood pressure, and higher salt preference. Glycyrrhiza glabra (GG) contains glycyrrhizin, an inhibitor of 11ß-hydroxysteroid-dehydrogenase type-2 and antagonist of toll-like receptor 4. The primary hypothesis of this study is that food enrichment with GG extract results in decreased anxiety behavior and reduced salt preference under stress and non-stress conditions. The secondary hypothesis is that the mentioned changes are associated with altered gene expression of barrier proteins in the prefrontal cortex. Male Sprague-Dawley rats were exposed to chronic mild stress for five weeks. Both stressed and unstressed rats were fed a diet with or without an extract of GG roots for the last two weeks. GG induced anxiolytic effects in animals independent of stress exposure, as measured in elevated plus maze test. Salt preference and intake were significantly reduced by GG under control, but not stress conditions. The gene expression of the barrier protein claudin-11 in the prefrontal cortex was increased in control rats exposed to GG, whereas stress-induced rise was prevented. Exposure to GG-enriched diet resulted in reduced ZO-1 expression irrespective of stress conditions. In conclusion, the observed effects of GG are in line with a reduction in the activity of central mineralocorticoid receptors. The treatment with GG extract or its active components may, therefore, be a useful adjunct therapy for patients with subtypes of depression and anxiety disorders with heightened renin-angiotensin-aldosterone system and/or inflammatory activity.

Stress Hormones Cortisol and Aldosterone, and Selected Markers of Oxidative Stress in Response to Long-Term Supplementation with Omega-3 Fatty Acids in Adolescent Children with Depression.

Late childhood and adolescence are crucial periods of brain development with high vulnerability to environmental insults. The aim of this study was to test the hypotheses that in adolescents with depression (a) 12 weeks-supplementation with omega-3 fatty acids results in the attenuation of salivary stress hormone concentrations; (b) the mentioned supplementation improves potentially disrupted daily rhythm of stress hormones; (c) stress hormone concentrations correlate with values of selected markers of oxidative stress. The sample consisted of 60 patients suffering from depression aged 11-18 years. Hormone concentrations in saliva were measured in the morning and midday before (baseline) and after (6, 12 weeks) food supplementation with omega-3 or omega-6 (as comparator) fatty acids. Morning cortisol decreased in response to omega-3 but not omega-6 fatty acids at 12 weeks compared to baseline. No changes were observed in aldosterone concentrations. The obtained results show that adolescent children with depression preserved the daily rhythm of both stress hormones. Baseline morning cortisol concentrations correlated positively with depression severity and lipoperoxides, and negatively with docosahexaenoic acid. Aldosterone concentrations correlated positively with 8-isoprostane. Thus, both hormones showed positive correlation with the selected markers of oxidative stress suggesting that enhanced stress hormone secretion may be associated with increased oxidative tissue damage in adolescent children with depression. This study was registered with the ISRCTN registry (DEPOXIN study, ISRCTN81655012).

Trophic factors as potential therapies for treatment of major mental disorders.

Notwithstanding major advances in psychotherapeutics, their efficacy and specificity remain limited. The slow onset of beneficial outcomes and numerous adverse effects of widely used medications remain of chief concern, warranting in-depth studies. The majority of frontline therapies are thought to enhance the endogenous monoaminergic drive, to initiate a cascade of molecular events leading to lasting functional and structural plasticity. They also involve alterations in trophic factor signalling, including brain-derived neurotrophic factor (BDNF), VGF (non-acronymic), vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), glial cell-derived neurotrophic factor (GDNF), and others. In several major mental disorders, emerging data suggest protective and restorative effects of trophic factors in preclinical models, when applied on their own. Antidepressant outcomes of VGF and FGF2, for instance, were shown in experimental animals, while BDNF and GDNF prove useful in the treatment of addiction, schizophrenia, and autism spectrum disorders. The main challenge with the effective translation of these and other findings in the clinic is the knowledge gap in action mechanisms with potential risks, as well as the lack of effective platforms for validation under clinical settings. Herein, we review the state-of-the-art and advances in the therapeutic use of trophic factors in several major neuropsychiatric disorders.

Food Enrichment with Glycyrrhiza glabra Extract Suppresses ACE2 mRNA and Protein Expression in Rats-Possible Implications for COVID-19.

Angiotensin converting enzyme 2 (ACE2) is a key entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus known to induce Coronavirus disease 2019 (COVID-19). We have recently outlined a concept to reduce ACE2 expression by the administration of glycyrrhizin, a component of Glycyrrhiza glabra extract, via its inhibitory activity on 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2) and resulting activation of mineralocorticoid receptor (MR). We hypothesized that in organs such as the ileum, which co-express 11betaHSD2, MR and ACE2, the expression of ACE2 would be suppressed. We studied organ tissues from an experiment originally designed to address the effects of Glycyrrhiza glabra extract on stress response. Male Sprague Dawley rats were left undisturbed or exposed to chronic mild stress for five weeks. For the last two weeks, animals continued with a placebo diet or received a diet containing extract of Glycyrrhiza glabra root at a dose of 150 mg/kg of body weight/day. Quantitative PCR measurements showed a significant decrease in gene expression of ACE2 in the small intestine of rats fed with diet containing Glycyrrhiza glabra extract. This effect was independent of the stress condition and failed to be observed in non-target tissues, namely the heart and the brain cortex. In the small intestine we also confirmed the reduction of ACE2 at the protein level. Present findings provide evidence to support the hypothesis that Glycyrrhiza glabra extract may reduce an entry point of SARS-CoV-2. Whether this phenomenon, when confirmed in additional studies, is linked to the susceptibility of cells to the virus requires further studies.

Adjunct Therapy With Glycyrrhiza Glabra Rapidly Improves Outcome in Depression-A Pilot Study to Support 11-Beta-Hydroxysteroid Dehydrogenase Type 2 Inhibition as a New Target.

Mineralocorticoid-receptor (MR) dysfunction as expressed by low systolic blood pressure and a high salivary aldosterone/cortisol ratio predicts less favorable antidepressant treatment outcome. Inhibition of peripheral 11-beta-hydroxysteroid-dehydrogenase type 2 (11betaHSD2) reverses these markers. We therefore tested the hypothesis that the 11betaHSD2 inhibitor glycyrrhizin affects treatment outcome via this mechanism. We administered Glycyrrhiza glabra (GG) extract containing 7-8 % of glycyrrhizin at a dose of 2 × 700 mg daily adjunct to standard antidepressants in hospitalized patients with major depression. These subjects were compared in an open-label fashion with patients, who did not receive GG (treatment as usual, TAU). Assessments were done at baseline and approximately 2 weeks after. Twelve subjects were treated with GG and compared to 55 subjects with TAU. At week 2, the Hamilton Depression Rating Scale (HAMD-21) change from baseline as well as the CGI-S change showed a significant time × treatment interaction (p < 0.03), indicating a possible therapeutic benefit of GG. Clinical benefit seems to be more pronounced in subjects with lower systolic blood pressure and significantly correlated with reduced sleep duration in the GG group. Our preliminary data show that treatment with the 11betaHSD2 inhibitor glycyrrhizin may possess a beneficial effect on antidepressant response, which may be specific to a defined depression subtype.

Target-based biomarker selection - Mineralocorticoid receptor-related biomarkers and treatment outcome in major depression.

Aldosterone and mineralocorticoid receptor (MR)-function have been related to depression. We examined central and peripheral parameters of MR-function in order to characterize their relationship to clinical treatment outcome after six weeks in patients with acute depression. 30 patients with a diagnosis of major depression were examined 3 times over a 6 week period. Aldosterone and cortisol salvia samples were taken at 7.00 a.m. before patients got out of bed. Easy to use e-devices were used to measure markers of central MR function, i.e. slow wave sleep (SWS) and heart-rate variability (HRV). Salt-taste intensity (STI) and salt pleasantness (SP) of a 0.9% salt solution were determined by a newly developed scale. In addition, systolic blood pressure (SBP) and plasma electrolytes were determined as markers for peripheral MR activity. The relationship between the levels of these biomarkers at baseline and the change in clinical outcome parameters (Hamilton depression rating scale (HDRS)-21, anxiety, QIDS and BDI) after 6 weeks of treatment was investigated. A higher aldosterone/cortisol ratio (Aldo/Cort) (n = 17 due to missing values; p < 0.05) and lower SBP (n = 24; p < 0.05) at baseline predicted poor outcome, as measured with the HDRS, independent of gender. Only in male patients higher STI, lower SP, lower SWS (all n = 13) and higher HRV (n = 11) at baseline predicted good outcome p < 0.05). Likewise, in male patients low baseline sodium appears to be predictive for a poor outcome (n = 12; p = 0.05; based on HDRS-6). In conclusion, correlates of higher central MR-activation are associated with poorer clinical improvement, particularly in men. This contrasts with the finding of a peripheral MR-desensitization in more refractory patients. As one potential mechanism to consider, sodium loss on the basis of dysfunctional peripheral MR function and additional environmental factors may trigger increased aldosterone secretion and consequently worse outcome. These markers deserve further study as potential biological correlates for therapy refractory depression.

Comparison of stress-induced changes in adults and pups: is aldosterone the main adrenocortical stress hormone during the perinatal period in rats?

Positive developmental impact of low stress-induced glucocorticoid levels in early development has been recognized for a long time, while possible involvement of mineralocorticoids in the stress response during the perinatal period has been neglected. The present study aimed at verifying the hypothesis that balance between stress-induced glucocorticoid and mineralocorticoid levels is changing during postnatal development. Hormone responses to two different stressors (insulin-induced hypoglycaemia and immune challenge induced by bacterial lipopolysaccharid) measured in 10-day-old rats were compared to those in adults. In pups corticosterone responses to both stressors were significantly lower than in adults, which corresponded well with the stress hyporesponsive period. Importantly, stress-induced elevations in aldosterone concentration were significantly higher in pups compared both to corticosterone elevations and to those in adulthood with comparable adrenocorticotropin concentrations in the two age groups. Greater importance of mineralocorticoids compared to glucocorticoids in postnatal period is further supported by changes in gene expression and protein levels of gluco- (GR) and mineralocorticoid receptors (MR) and selected enzymes measured by quantitative PCR and immunohystochemistry in the hypothalamus, hippocampus, prefrontal cortex, liver and kidney. Gene expression of 11beta-hydroxysteroid dehydrogenase 2 (11ß-HSD2), an enzyme enabling preferential effects of aldosterone on mineralocorticoid receptors, was higher in 10-day-old pups compared to adult animals. On the contrary, the expression and protein levels of GR, MR and 11ß-HSD1 were decreased. Presented results clearly show higher stress-induced release of aldosterone in pups compared to adults and strongly suggest greater importance of mineralocorticoids compared to glucocorticoids in stress during the postnatal period.

Genetic aspects of vitamin D receptor and metabolism in relation to the risk of multiple sclerosis.

Recent findings suggest that polymorphisms in vitamin D pathway genes are candidates for association with multiple sclerosis susceptibility. It has been now well demonstrated that vitamin D has immunomodulatory functions that may be favorable for reduction of multiple sclerosis risk. Current research has been focused on identification of new variants of genes involved in vitamin D pathway, namely in vitamin D receptor and enzymes of vitamin D metabolism. These variants have been intensively studied as possible genetic predictors of both vitamin D levels and the risk of multiple sclerosis. Considering the findings available up-to-date, we may recognize two groups of genetic variants. The first group of genes was found to predict vitamin D levels but not the risk of multiple sclerosis. The second group of genetic variants is represented by promising genes predicting vitamin D levels as well as the risk of multiple sclerosis. A strong association with increased risk of the disease has been observed for a rare variant in the CYP27B1 gene encoding a vitamin D-activating enzyme. Observed interaction between genetic and epidemiological findings brings the rationale for supplementation trials of vitamin D. Although promising effects of vitamin D supplementation have emerged, the results obtained so far are inconclusive and the real therapeutic significance of vitamin D supplementation remains to be elucidated.

Subchronic treatment of rats with oxytocin results in improved adipocyte differentiation and increased gene expression of factors involved in adipogenesis.

BACKGROUND AND PURPOSE: Treatment with thiazolidinediones, insulin-sensitizing drugs, enhances adipogenesis, which may result in unwanted increase in adiposity. Based on the suggested metabolic effects of oxytocin, the aims of the present study were to: (i) determine whether chronic treatment with oxytocin exerts positive effects on white adipose tissue growth without increasing adiposity; (ii) investigate possible mechanisms of action of oxytocin by measuring the level of gene expression of adipogenic factors; and (iii) test the hypothesis that oxytocin's effect on adipose tissue involves specific activation of eukaryotic elongation factor 2 (eEF2). EXPERIMENTAL APPROACH: Adult rats were subcutaneously treated with oxytocin (3.6 µg·100 g⁻¹ body weight day⁻¹) via osmotic minipumps for 2 weeks. Adipocytes from epididymal adipose tissue were isolated and their size evaluated by light microscopy. Gene expression of adipogenic and angiogenic factors was determined by real-time PCR and dephosphorylation of eEF2 by immunoblotting. KEY RESULTS: Oxytocin treatment decreased the diameter of adipocytes and increased the epididymal adipose tissue protein content without changing the adipose tissue mass. Increases in fatty acid binding protein, peroxisome proliferator-activated receptor γ, insulin-sensitive glucose transporter 4, leptin and CD31 mRNA levels were noted in the epididymal and/or retroperitoneal fat tissue of oxytocin-treated rats. Oxytocin enhanced the dephosphorylation of eEF2 in the epididymal adipose tissue. CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that subchronic treatment with oxytocin induces adipogenic and angiogenic effects and that the eEF2 signalling pathway is involved in these effects of oxytocin on adipose tissue in vivo. These findings are likely to motivate further research and indicate new approaches for modulating adipose tissue morphology and metabolism.

Effect of single treatment with the antihypertensive drug eplerenone on hormone levels and anxiety-like behaviour in rats.

OBJECTIVE: The aim of the study was to investigate the effects of single peripheral administration of mineralocorticoid receptor (MR) antagonist eplerenone on: 1. the hypothalamic-pituitary-adrenocortical (HPA) axis; 2. the renin-angiotensin-aldosterone (RAA) system, 3. anxiety-like behaviour. METHODS: Male Wistar rats were injected subcutaneously with eplerenone (100 mg/kg body weight) or vehicle. Two hours following the injections, a half of the animals from each treatment group were decapitated to obtain blood for measurement of hormone levels. The other half of the animals was subjected to behavioural testing in the elevated plus-maze test. To provide comprehensive behavioural profile of eplerenone, standard spatiotemporal and ethologically derived measures of anxiety were assessed. RESULTS: Single treatment with eplerenone resulted in a significant increase in plasma aldosterone levels. Plasma concentrations of ACTH and corticosterone were not modified by eplerenone injection. Administration of eplerenone failed to alter classical spatiotemporal measures of anxiety (number of entries and time spent in the open arms). However, ethological parameters related to exploration (head dipping) and risk assessment behaviour (stretched attend postures) were significantly affected by eplerenone injection. CONCLUSIONS: Single injection of eplerenone is followed by a reduction of ethological indices of anxiety-like behaviour and by an elevation of plasma aldosterone levels. Acute administration of eplerenone appears to affect the RAA system but not hormones of the HPA axis.

Urinary catecholamines in children with attention deficit hyperactivity disorder (ADHD): modulation by a polyphenolic extract from pine bark (pycnogenol).

Our study tested the hypothesis that treatment with a potent polyphenol complex not only reduces hyperactivity of children, but also catecholamine excretion and oxidative stress. Urine catecholamine concentrations were measured in attention deficit hyperactivity disorder (ADHD) children and healthy controls. ADHD children received either placebo (PL) or Pycnogenol (Pyc), a bioflavonoid extract from the pine bark, for one month. The study was performed in a randomized, double-blind, PL controlled design. Concentrations of catecholamines were higher in urine of ADHD patients compared to those of healthy children. Moreover, noradrenaline (NA) concentrations positively correlated with degree of hyperactivity of ADHD children. In ADHD patients, adrenaline (A) and NA concentrations positively correlated with plasma levels of oxidized glutathione. The treatment of ADHD children with Pyc caused decrease of dopamine (D) and trend of A and NA decrase and increased GSH/GSSG ratio. In conclusion, the data provide further evidence for the overactivity of the noradrenergic system in ADHD and demonstrate that A release may be increased, as well. Treatment of ADHD children with Pyc normalized catecholamine concentrations, leading to less hyperactivity, and, consequently, to reduced oxidative stress.

Activation of FOS in hypocretin neurons of the rat by insulin-induced hypoglycemia.

OBJECTIVE: The aim of the present study was to investigate the extent of activation of hypocretin (HCRT) synthesizing neurons after a single intraperitoneal administration of insulin using Fos-HCRT dual immunohistochemistry. In addition, there was also an attempt to depict the spatial organization of activated HCRT perikarya within the whole portion of the medial and lateral hypothalamic (LHA) areas. METHODS: The animals (rats) were fixed 90 min after i.p. administration of insulin (2.5 IU/kg). The brains were removed, and sectioned through the hypothalamus into 40 microm thick alternate coronal sections. Fos-HCRT perikarya were double immunostained with avidin-biotin-peroxidase (ABC) technique using Nickel-DAB and single DAB as the two chromogens. For the mapping of Fos-HCRT double-labeled perikarya a light microscopy was employed. Counting of Fos-labeled HCRT perikarya was performed manually and blindly with no insight into the treatment of the animals. RESULTS: The data demonstrate that in the early phase of the acute hypoglycemia, the number of the dually labeled Fos-HCRTir perikarya in the entire LHA was only moderately increased from 9.54 to 15.64% in spite of the fact that within the same period the plasma glucose levels were declined by more than 70%. Moreover, within the LHA, the distribution of activated double-labeled Fos-HCRTir perikarya did not show any special spatial organization. CONCLUSION: The present data indicate that a large fall in plasma glucose in early phase of acute hypoglycemia does not represent an appropriate stimulus for massive activation of HCRT neurons in the LHA of rats.

Repeated citalopram treatment but not stress exposure attenuates hypothalamic-pituitary-adrenocortical axis response to acute citalopram injection.

Many experimental, clinical and epidemiological studies have shown a direct connection between exposure to stress or adverse life events and disease, but little is known about the effect of stress on the action of drugs. The aim of this study was to test the hypothesis that previous exposure to stress changes the action of the antidepressant drug citalopram (10 mg/kg, i.p.) on hypothalamic-pituitary-adrenocortical (HPA) axis function, gene expression of selected neuropeptides and serotonin reuptake. Three different stress models were used, which included immobilization, restraint and unpredictable stress stimuli. Samples of plasma for hormone measurement were taken from conscious cannulated animals. Changes in corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) gene expression in the paraventricular nucleus of the hypothalamus and the anterior pituitary, respectively, and the ability of citalopram to inhibit serotonin reuptake were investigated. The exposure to three different stress models did not influence citalopram action on individual parameters of HPA axis and on serotonin reuptake. On the other hand, repeated administration of the drug led to significant attenuation of ACTH and CRH mRNA responses. The present results allow to suggest that the stressors used did not influence serotonergic neurotransmission to the extent that would modify HPA axis response to citalopram challenge. Activation of HPA axis by acute citalopram treatment was found to be accompanied by increased CRH gene expression in the hypothalamus. Repeated administration of the drug led to the development of tolerance to activation of central and peripheral components of HPA axis, but not to serotonin reuptake inhibition.