NADPH oxidase 4 (NOX4) as a biomarker and therapeutic target in neurodegenerative diseases.

Diseases like Alzheimer's and Parkinson's diseases are defined by inflammation and the damage neurons undergo due to oxidative stress. A primary reactive oxygen species contributor in the central nervous system, NADPH oxidase 4, is viewed as a potential therapeutic touchstone and indicative marker for these ailments. This in-depth review brings to light distinct features of NADPH oxidase 4, responsible for generating superoxide and hydrogen peroxide, emphasizing its pivotal role in activating glial cells, inciting inflammation, and disturbing neuronal functions. Significantly, malfunctioning astrocytes, forming the majority in the central nervous system, play a part in advancing neurodegenerative diseases, due to their reactive oxygen species and inflammatory factor secretion. Our study reveals that aiming at NADPH oxidase 4 within astrocytes could be a viable treatment pathway to reduce oxidative damage and halt neurodegenerative processes. Adjusting NADPH oxidase 4 activity might influence the neuroinflammatory cytokine levels, including myeloperoxidase and osteopontin, offering better prospects for conditions like Alzheimer's disease and Parkinson's disease. This review sheds light on the role of NADPH oxidase 4 in neural degeneration, emphasizing its drug target potential, and paving the path for novel treatment approaches to combat these severe conditions.

Effects of Saffron Extract (Affron®) with 100 mg/kg and 200 mg/kg on Hypothalamic-Pituitary-Adrenal Axis and Stress Resilience in Chronic Mild Stress-Induced Depression in Wistar Rats.

Stress-related symptoms are a global concern, impacting millions of individuals, yet effective and safe treatments remain scarce. Although multiple studies have highlighted the stress- alleviating properties of saffron extract, the underlying mechanisms remain unclear. This study employs the unpredictable chronic mild stress (CMS) animal model to investigate the impact of a standardized saffron extract, Affron® (AFN), on hypothalamic-pituitary-adrenal (HPA) axis regulation and neuroplasticity in Wistar rats following repeated oral administration. The research evaluates AFN's effects on various stress-related parameters, including hypothalamic gene expression, stress hormone levels, and the sucrose preference test. In animals subjected to continuous unpredictable CMS, repetitive administration of AFN at doses of 100 mg/kg and 200 mg/kg effectively normalized HPA axis dysregulation and enhanced neuroplasticity. Increased concentrations of AFN demonstrated greater efficacy. Following AFN oral administration, adrenocorticotropic and corticosterone hormone levels exhibited significant or nearly significant reductions in comparison to subjects exposed to stress only. These changes align with the alleviation of stress and the normalization of the HPA axis. These findings elucidate AFN's role in stress mitigation, affirm its health benefits, validate its potential as a treatment for stress-related symptoms, confirm its physiological effectiveness, and emphasize its therapeutic promise.

The microRNA-mediated gene regulatory network in the hippocampus and hypothalamus of the aging mouse.

Aging leads to time-dependent functional decline of all major organs. In particular, the aging brain is prone to cognitive decline and several neurodegenerative diseases. Various studies have attempted to understand the aging process and underlying molecular mechanisms by monitoring changes in gene expression in the aging mouse brain using high-throughput sequencing techniques. However, the effect of microRNA (miRNA) on the post-transcriptional regulation of gene expression has not yet been comprehensively investigated. In this study, we performed global analysis of mRNA and miRNA expression simultaneously in the hypothalamus and hippocampus of young and aged mice. We identified aging-dependent differentially expressed genes, most of which were specific either to the hypothalamus or hippocampus. However, genes related to immune response-related pathways were enriched in upregulated differentially expressed genes, whereas genes related to metabolism-related pathways were enriched in downregulated differentially expressed genes in both regions of the aging brain. Furthermore, we identified many differentially expressed miRNAs, including three that were upregulated and three that were downregulated in both the hypothalamus and hippocampus. The two downregulated miRNAs, miR-322-3p, miR-542-3p, and the upregulated protein-encoding coding gene C4b form a regulatory network involved in complement and coagulation cascade pathways in the hypothalamus and hippocampus of the aging brain. These results advance our understanding of the miRNA-mediated gene regulatory network and its influence on signaling pathways in the hypothalamus and hippocampus of the aging mouse brain.

NOX4 as a critical effector mediating neuroinflammatory cytokines, myeloperoxidase and osteopontin, specifically in astrocytes in the hippocampus in Parkinson's disease.

Oxidative stress and mitochondrial dysfunction have been believed to play an important role in the pathogenesis of aging and neurodegenerative diseases, including Parkinson's disease (PD). The excess of reactive oxygen species (ROS) increases with age and causes a redox imbalance, which contributes to the neurotoxicity of PD. Accumulating evidence suggests that NADPH oxidase (NOX)-derived ROS, especially NOX4, belong to the NOX family and is one of the major isoforms expressed in the central nervous system (CNS), associated with the progression of PD. We have previously shown that NOX4 activation regulates ferroptosis via astrocytic mitochondrial dysfunction. We have previously shown that activation of NOX4 regulates ferroptosis through mitochondrial dysfunction in astrocytes. However, it remains unclear why an increase in NOX4 in neurodegenerative diseases leads to astrocyte cell death by certain mediators. Therefore, this study was designed to evaluate how NOX4 in the hippocampus is involved in PD by comparing an MPTP-induced PD mouse model compared to human PD patients. We could detect that the hippocampus was dominantly associated with elevated levels of NOX4 and α-synuclein during PD and the neuroinflammatory cytokines, myeloperoxidase (MPO) and osteopontin (OPN), were upregulated particularly in astrocytes. Intriguingly, NOX4 suggested a direct intercorrelation with MPO and OPN in the hippocampus. Upregulation of MPO and OPN induces mitochondrial dysfunction by suppressing five protein complexes in the mitochondrial electron transport system (ETC) and increases the level of 4-HNE leading to ferroptosis in human astrocytes. Overall, our findings indicate that the elevation of NOX4 cooperated with the MPO and OPN inflammatory cytokines through mitochondrial aberration in hippocampal astrocytes during PD.

Reduction of fetuin-A levels contributes to impairment of Purkinje cells in cerebella of patients with Parkinson's disease.

Phenotypic features such as ataxia and loss of motor function, which are characteristics of Parkinson's disease (PD), are expected to be very closely related to cerebellum function. However, few studies have reported the function of the cerebellum. Since the cerebellum, like the cerebrum, is known to undergo functional and morphological changes due to neuroinflammatory processes, elucidating key functional factors that regulate neuroinflammation in the cerebellum can be a beneficial therapeutic approach. Therefore, we employed PD patients and MPTP-induced PD mouse model to find cytokines involved in cerebellar neuroinflammation in PD and to examine changes in cell function by regulating related genes. Along with the establishment of a PD mouse model, abnormal shapes such as arrangement and number of Purkinje cells in the cerebellum were confirmed based on histological finding, consistent with those of cerebellums of PD patients. As a result of proteome profiling for neuroinflammation using PD mouse cerebellar tissues, fetuin-A, a type of cytokine, was found to be significantly reduced in Purkinje cells. To further elucidate the function of fetuin-A, neurons isolated from cerebellums of embryos (E18) were treated with fetuin-A siRNA. We uncovered that not only the population of neuronal cells, but also their morphological appearances were significantly different. In this study, we found a functional gene called fetuin-A in the PD model's cerebellum, which was closely related to the role of cerebellar Purkinje cells of mouse and human PD. In conclusion, morphological abnormalities of Purkinje cells in PD mice and patients have a close relationship with a decrease of fetuin-A, suggesting that diagnosis and treatment of cerebellar functions of PD patients might be possible through regulation of fetuin-A. [BMB Reports 2023; 56(5): 308-313].

The anorexic effect of DL-fenfluramine is dependent on animals' habituation to different food types.

Background: As rates of obesity and diabetes have increased dramatically over the past few decades, the use of anti-obesity drugs has now become a routine therapeutic measure. However, the pharmacological effects of chronic use of these drugs in humans frequently lead to reduced efficacy in reducing appetite and body weight through as-yet-unidentified mechanisms. An example of this can be found in animal studies where the appetite suppressant DL-fenfluramine (FEN) is chronically administered and its tolerance develops in animals and humans. The appetite effects of FEN are typically measured in several animal studies by the feeding changes in a balanced standard diet. To determine whether FEN differentially altered appetite suppression in animals with long-term expression with different macronutrient diet compositions, its anorexic effects were measured specifically in male rats that had previously been chronically maintained on normal chow (NC) or a high-fat and high-carbohydrate western diet (WD). Methods: Three experiments were conducted by feeding the animals either NC or WD for 1 month to habituate them with their diet. Animals maintained on either NC or WD were subsequently offered both diet options ad libitum for a 2- or 7-day adaptation period while receiving daily systemic FEN treatment. Results: The results suggested that long-term habituated food affected the food preference of animals and their appetite even after chronic systemic FEN administration. Therefore, the effectiveness and success or failure of repeated use of chronic anti-obesity drugs may depend on habituated food type. Conclusion: The appetite suppressant effect was found to be determined by the palatability of a specific macronutrient and the habituated food rather than by a change in the concentration of the administered FEN. This results in a critical analysis of the rationale for taking medication considering the patient's past dietary habits to achieve successful weight loss.

Disease predictability review using common biomarkers appearing in diabetic nephropathy and neurodegeneration of experimental animals.

It is recently known that the kidney and brain have a very rich distribution of blood vessels, and the histological structures of micro-vessels are very similar. Therefore, a number of studies have reported that renal diseases like chronic kidney disease (CKD) caused by various causes have a very close relationship with the occurrence of neurodegenerative diseases. On the other hand, since diabetic nephropathy, which is caused by chronic inflammation, such as diabetes, often shows very different prognoses even in patients at the same clinical stage, the judgment of their disease prognosis will have a critical meaning in clinical practice. Recently, many studies of cerebro-renal interaction have been reported using experimental animals. The discovery of common biomarkers found in both organs can predict the prognosis of renal disease and the possibility of neurodegenerative disease progression. More associations can be found with novel common biomarkers found in the brain and kidneys that seem entirely unrelated. In that case, it will ultimately be a research field that can expand predictive models of patients' complex diseases through these biomarkers in clinical practice. It is presented biomarkers such as α-klotho, Nephrin, and Synaptopodin. These markers are observed in both the brain and kidney, and it has been reported that both organs show a very significant change in function according to their expression. Even though the brain and kidneys perform very independent functions, it is thought that it has a crucial diagnostic significance that the genes commonly expressed in both organs are functionally effective. With the discovery of novel biomarkers that share cerebro-renal interactions at the early stage of diabetic nephropathy, physicians can predict post-clinical symptoms and prevent severe neurodegenerative and cerebrovascular diseases. Therefore, further study for the diseases of these two organs in laboratory animals means that the field of research on this relationship can be expanded in the future. In the future, more attention and research will be needed on the possibility of prediction for the prevention of neurological diseases caused by CKD in disease animal models.

Respiratory Safety Evaluation in Mice and Inhibition of Adenoviral Amplification in Human Bronchial Endothelial Cells Using a Novel Type of Chlorine Dioxide Gas Reactor.

Since the onset of the COVID-19 pandemic, there has been a growing demand for effective and safe disinfectants. A novel use of chlorine dioxide (ClO2) gas, which can satisfy such demand, has been reported. However, its efficacy and safety remain unclear. For the safe use of this gas, the stable release of specific concentrations is a must. A new type of ClO2 generator called Dr.CLOTM has recently been introduced. This study aimed to investigate: (1) the effects of Dr.CLOTM on inhibiting adenoviral amplification on human bronchial epithelial (HBE) cells; and (2) the acute inhalation safety of using Dr.CLOTM in animal models. After infecting HBE cells with a recombinant adenovirus, the inhibitory power of Dr.CLOTM on the virus was expressed as IFU/mL in comparison with the control group. The safety of ClO2 gas was indirectly predicted using mice by measuring single-dose inhalation toxicity in specially designed chambers. Dr.CLOTM was found to evaporate in a very constant concentration range at 0-0.011 ppm/m3 for 42 days. In addition, 36-100% of adenoviral amplification was suppressed by Dr.CLOTM, depending on the conditions. The LC50 of ClO2 gas to mice was approximately 68 ppm for males and 141 ppm for females. Histopathological evaluation showed that the lungs of female mice were more resistant to the toxicity from higher ClO2 gas concentrations than those of male mice. Taken together, these results indicate that Dr.CLOTM can be used to provide a safe indoor environment due to its technology that maintains the stable concentration and release of ClO2 gas, which could suppress viral amplification and may prevent viral infections.

Spatial and temporal changes in the PGE2 EP2 receptor in mice hippocampi during postnatal development and its relationship with cyclooxygenase-2.

OBJECTIVES: Prostaglandin E2 E-prostanoid 2 receptor (PGE2 EP2), downstream of cyclooxygenase-2 (COX-2), plays an important role in inflammatory responses, but there are some reports about synaptic functions of COX-2 and PGE2 EP2 in the hippocampus. MATERIALS AND METHODS: C57BL/6J mice were sacrificed at postnatal days (P) 1, 7, 14, 28, and 56 for immunohistochemical staining for EP2 and doublecortin as well as western blot for EP2. In addition, COX-2 knockout and its wild-type mice were euthanized for immunohistochemical staining for EP2. RESULTS: EP2 immunoreactivity was observed in the majority of the cells in the dentate gyrus at P1 and P7, while at P14, it was detected in the outer granule cell layer and was confined to its subgranular zone at P28 and P56. EP2 protein levels in the hippocampal homogenates were also highest at P7 and lowest at P56. EP2 immunoreactivity was partially colocalized, with doublecortin (DCX)-immunoreactive neuroblasts appearing in the mid-zone of the granule cell layer at P14 and in the subgranular zone of the dentate gyrus at P28. Co-localization of EP2 and DCX was significantly decreased in the dentate gyrus in the P28 group compared with that in the P14 group. In COX-2 knockout mice, EP2 immunoreactivity was significantly decreased in the hippocampal CA1 region (P=0.000165) and dentate gyrus (P=0.00898). CONCLUSION: EP2 decreases with age, which is expressed in DCX-immunoreactive neuroblasts in the dentate gyrus. This suggests that EP2 is closely linked to structural lamination and adult neurogenesis in the dentate gyrus.

Natural Products in the Prevention of Metabolic Diseases: Lessons Learned from the 20th KAST Frontier Scientists Workshop.

The incidence of metabolic and chronic diseases including cancer, obesity, inflammation-related diseases sharply increased in the 21st century. Major underlying causes for these diseases are inflammation and oxidative stress. Accordingly, natural products and their bioactive components are obvious therapeutic agents for these diseases, given their antioxidant and anti-inflammatory properties. Research in this area has been significantly expanded to include chemical identification of these compounds using advanced analytical techniques, determining their mechanism of action, food fortification and supplement development, and enhancing their bioavailability and bioactivity using nanotechnology. These timely topics were discussed at the 20th Frontier Scientists Workshop sponsored by the Korean Academy of Science and Technology, held at the University of Hawaii at Manoa on 23 November 2019. Scientists from South Korea and the U.S. shared their recent research under the overarching theme of Bioactive Compounds, Nanoparticles, and Disease Prevention. This review summarizes presentations at the workshop to provide current knowledge of the role of natural products in the prevention and treatment of metabolic diseases.

NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases.

Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondrial dysfunction is linked to oxidative stress and reactive oxygen species (ROS) in neurotoxicity during AD. Impaired mitochondrial metabolism has been associated with mitochondrial dysfunction in brain damage of AD. While the role of NADPH oxidase 4 (NOX4), a major source of ROS, has been identified in brain damage, the mechanism by which NOX4 regulates ferroptosis of astrocytes in AD remains unclear. Here, we show that the protein levels of NOX4 were significantly elevated in impaired astrocytes of cerebral cortex from patients with AD and APP/PS1 double-transgenic mouse model of AD. The levels of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), a marker of oxidative stress-induced lipid peroxidation, were significantly also elevated in impaired astrocytes of patients with AD and mouse AD. We demonstrate that the over-expression of NOX4 significantly increases the impairment of mitochondrial metabolism by inhibition of mitochondrial respiration and ATP production via the reduction of five protein complexes in the mitochondrial ETC in human astrocytes. Moreover, the elevation of NOX4 induces oxidative stress by mitochondrial ROS (mtROS) production, mitochondrial fragmentation, and inhibition of cellular antioxidant process in human astrocytes. Furthermore, the elevation of NOX4 increased ferroptosis-dependent cytotoxicity by the activation of oxidative stress-induced lipid peroxidation in human astrocytes. These results suggest that NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in AD.

Physical Stress Induced Reduction of Proliferating Cells and Differentiated Neuroblasts Is Ameliorated by Fermented Laminaria japonica Extract Treatment.

Laminaria japonica is widely cultivated in East Asia, including South Korea. Fucoidan, a main component of L. japonica, protects neurons from neurological disorders such as ischemia and traumatic brain injury. In the present study, we examined the effects of extract from fermented L. japonica on the reduction of proliferating cells and neuroblasts in mice that were physically (with electric food shock) or psychologically (with visual, auditory and olfactory sensation) stressed with the help of a communication box. Vehicle (distilled water) or fermented L. japonica extract (50 mg/kg) were orally administered to the mice once a day for 21 days. On the 19th day of the treatment, physical and psychological stress was induced by foot shock using a communication box and thereafter for three days. Plasma corticosterone levels were significantly increased after exposure to physical stress and decreased Ki67 positive proliferating cells and doublecortin immunoreactive neuroblasts. In addition, western blot analysis demonstrated that physical stress as well as psychological stress decreased the expression levels of brain-derived neurotrophic factor (BDNF) and the number of phosphorylated cAMP response element binding protein (pCREB) positive nuclei in the dentate gyrus. Fermentation of L. japonica extract significantly increased the contents of reduced sugar and phenolic compounds. Supplementation with fermented L. japonica extract significantly ameliorated the increases of plasma corticosterone revels and decline in the proliferating cells, neuroblasts, and expression of BDNF and pCREB in the physically stressed mice. These results indicate that fermented L. japonica extract has positive effects in ameliorating the physical stress induced reduction in neurogenesis by modulating BDNF and pCREB expression in the dentate gyrus.

Elevated CLOCK and BMAL1 Contribute to the Impairment of Aerobic Glycolysis from Astrocytes in Alzheimer's Disease.

Altered glucose metabolism has been implicated in the pathogenesis of Alzheimer's disease (AD). Aerobic glycolysis from astrocytes is a critical metabolic pathway for brain energy metabolism. Disturbances of circadian rhythm have been associated with AD. While the role of circadian locomotor output cycles kaput (CLOCK) and brain muscle ARNT-like1 (BMAL1), the major components in the regulation of circadian rhythm, has been identified in the brain, the mechanism by which CLOCK and BMAL1 regulates the dysfunction of astrocytes in AD remains unclear. Here, we show that the protein levels of CLOCK and BMAL1 are significantly elevated in impaired astrocytes of cerebral cortex from patients with AD. We demonstrate that the over-expression of CLOCK and BMAL1 significantly suppresses aerobic glycolysis and lactate production by the reduction in hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) protein levels in human astrocytes. Moreover, the elevation of CLOCK and BMAL1 induces functional impairment by the suppression of glial fibrillary acidic protein (GFAP)-positive filaments in human astrocytes. Furthermore, the elevation of CLOCK and BMAL1 promotes cytotoxicity by the activation of caspase-3-dependent apoptosis in human astrocytes. These results suggest that the elevation of CLOCK and BMAL1 contributes to the impairment of astrocytes by inhibition of aerobic glycolysis in AD.

NOX4 Deficiency Exacerbates the Impairment of Cystatin C-Dependent Hippocampal Neurogenesis by a Chronic High Fat Diet.

Hippocampal neurogenesis is linked with a cognitive process under a normal physiological condition including learning, memory, pattern separation, and cognitive flexibility. Hippocampal neurogenesis is altered by multiple factors such as the systemic metabolic changes. NADPH oxidase 4 (NOX4) has been implicated in the regulation of brain function. While the role of NOX4 plays in the brain, the mechanism by which NOX4 regulates hippocampal neurogenesis under metabolic stress is unclear. In this case, we show that NOX4 deficiency exacerbates the impairment of hippocampal neurogenesis by inhibiting neuronal maturation by a chronic high fat diet (HFD). NOX4 deficiency resulted in less hippocampal neurogenesis by decreasing doublecortin (DCX)-positive neuroblasts, a neuronal differentiation marker, and their branched-dendrites. Notably, NOX4 deficiency exacerbates the impairment of hippocampal neurogenesis by chronic HFD. Moreover, NOX4 deficiency had a significant reduction of Cystatin C levels, which is critical for hippocampal neurogenesis, under chronic HFD as well as normal chow (NC) diet. Furthermore, the reduction of Cystatin C levels was correlated with the impairment of hippocampal neurogenesis in NOX4 deficient and wild-type (WT) mice under chronic HFD. Our results suggest that NOX4 regulates the impairment of Cystatin C-dependent hippocampal neurogenesis under chronic HFD.

Intensive morphometric analysis of enormous alterations in skeletal bone system with micro-CT for AHNAK-/- mice.

AHNAK has been reported to be involved in actin cytoskeleton rearrangement of some cell types, calcium homeostasis, and activation of T cells. Although the functional role of AHNAK in muscle cells, epidermis, and brain has been determined, its association with apparent clinical impairment has not been found yet. During phenotypic analysis of AHNAK knock out (KO) mice for many years, we observed that AHNAK KO mice showed very slow growth. Snouts of these animals were very short, and their bones were easily broken compared to normal mice. It is known that AHNAK is closely related to calcium. However, intensive morphological studies on phenotypes of bone have yet been reported for AHNAK. Thus, the objective of the present study was to analyze the morphology of skull, mandibular, limbs, and caudal bones of AHNAK KO mice intensively using micro-CT with many factors for various ages of these mice (6 weeks, 18 weeks, and 40 weeks). As a result, it was found that the facial region of AHNAK KO mouse showed a large difference in mandible than skull. Their both femur and tibia were shortened, and bone strength was also significantly decreased compared to normal mice. Particularly, the tail bone of AHNAK KO mice exhibited morphological abnormality by age. Taken together, these results suggest that AHNAK plays an important role in bone shape, development, and metabolism. Although our results demonstrated that AHNAK has a distinct role in bone, further investigations are needed to determine various features of bone metabolism related to AHNAK in the future.

Lipid alterations in the skeletal muscle tissues of mice after weight regain by feeding a high-fat diet using nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.

This study investigated lipid alterations in muscle tissues [gastrocnemius (Gas) and soleus (Sol)] of mice under different diet programs (weight gain, weight maintenance, weight regain, and controls) by nanoflow ultrahigh pressure liquid chromatography-electrospray ionization-tandem mass spectrometry. Since overloaded lipids in the skeletal muscle tissues by excessive fat accumulation are related to insulin resistance leading to type II diabetes mellitus, analysis of lipid alteration in muscle tissues with respect to high-fat diet (HFD) is important to understand obesity related diseases. A total of 345 individual lipid species were identified with their molecular structures, and 184 lipids were quantified by selected reaction monitoring method. Most triacylglycerol (TG) and phosphatidylethanolamine (PE) species displayed a significant (>2-fold, p < 0.01) increase in both the Gas and Sol and to a larger degree in the Gas. However, lipid classes involved in insulin resistance and anti-inflammatory response, including lysophosphatidylcholine (18:0), diacylglycerol (16:0_18:1, 16:0_18:2, and 18:1_18:1), ceramide (d18:1/24:0 and d18:1/24:1), and phosphatidylinositol (18:0/20:4), showed a significant accumulation in the Sol exclusively after HFD treatment. In addition, the lipid profiles were not significantly altered in mice that were fed HFD only for the last 4 weeks (weight gain group), suggesting that consuming HFD in the younger age period can be more effective in the Gas. This study reveals that lipid classes related to insulin resistance accumulated more in the Sol than in the Gas following HFD treatment and the weight regain program perturbed lipid profiles of the Sol to a greater extent than that by the other diet programs, confirming that the Sol tissue is more influenced by HFD than Gas.

Sleep-inducing effect of Passiflora incarnata L. extract by single and repeated oral administration in rodent animals.

Social cost of insomnia in modern society is gradually increasing. Due to various social phenomena and lifestyles that take away the opportunity of good quality of sleep, problems of insomnia cannot be easily figured out. Prescription of sleeping pills for insomnia patients can cause other inconveniences due to their side effects beyond their intended purposes. On the other hand, Passiflora incarnata L. (PI) has been widely used in South America for several centuries, showing effectiveness for sleep, sedation, anxiety, and so on in the civilian population. However, reports on the treatment efficacy of this herbal medicinal plant for insomnia patients through standardization as a sleeping agent have been very rare. Therefore, we obtained leaves and fruits of PI (8:2 by weight) as powder to prepare an extract. It was then applied to C6 rat glioma cells to quantitate mRNA expression levels of GABA receptors. Its sleep-inducing effect was investigated using experimental animals. PI extract (6 µg/ml) significantly decreased GABA receptors at 6 hr after treatment. Immobility time and palpebral closing time were significantly increased after single (500 mg/kg) or repeated (250 mg/kg) oral administration. In addition, blood melatonin levels were significantly increased in PI extract-treated animals after both single and repeated administrations. These results were confirmed through several repeated experiments. Taken together, these results confirmed that PI extract had significant sleep-inducing effects in cells and animals, suggesting that PI extract might have potential for treating human insomnia.

Aging-related lipidomic changes in mouse serum, kidney, and heart by nanoflow ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Aging refers to the intracellular accumulation of reactive oxygen species that damages proteins, DNA, and lipids. As alterations in lipid metabolism may trigger metabolic disorders and the onset of metabolic diseases, changes in lipid profiles can be closely related to aging. In this study, a comprehensive lipidomic comparison between 4- and 25-month-old mice was performed to investigate age-induced changes in the lipid profiles of mouse serum, kidney, and heart using nanoflow ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Quantitative analysis of 279 of the 542 identified lipids revealed significant changes upon aging, mainly showing decreased levels in the three types of samples. Exceptionally, most triacylglycerols showed significant increases in heart tissue. The kidney was influenced more by aging than the serum and heart. The highly abundant lipids in each lipid class with significant decreases (> 2-fold, p < 0.01) were lysophosphatidic acid 18:1, lysophosphatidylinositol 20:4, and ceramide d:18:1/24:0 in serum; lysophosphatidylglycerol 16:0 in heart tissue; and eight phosphatidylethanolamines (20:4, 22:6, 36:2, 36:3, 38:4, 38:5, 38:6, 40:6, and 40:7), two cardiolipins (72:7 and 72:8), and lysophosphatidylcholine 18:0 in kidney tissue. The findings indicate the potential of lipidomic analysis to study characteristic age-related lipid changes.

Edgeworthia papyrifera Regulates Osteoblast and Osteoclast Differentiation In Vitro and Exhibits Anti-osteoporosis Activity in Animal Models of Osteoporosis.

Osteoporosis is a clinical condition characterized by low bone strength that leads to an increased risk of fracture. Strategies for the treatment of osteoporosis involve inhibition of bone resorption by osteoclasts and an increase of bone formation by osteoblasts. Here, we identified the extract derived from the stem part of Edgeworthia papyrifera that enhanced differentiation of MC3T3-E1 cells to osteoblast-like cells and inhibited osteoclast differentiation of RAW 264.7 cells in vitro. In support of our observation, rutin and daphnoretin, which were previously reported to inhibit osteoclast differentiation, were identified in E. papyrifera extract. In an animal model of osteoporosis, the ovariectomy-induced increases in bone resorption biomarkers such as pyridinoline and tartrate-resistant acid phosphatase were significantly reduced by E. papyrifera extract administration at 25.6 and 48.1%, respectively. Furthermore, the ovariectomy-induced bone loss in animal models of osteoporosis was significantly prevented by the administration of E. papyrifera in our study. Taking these observations into account, we suggest that E. papyrifera is an interesting candidate for further exploration as an anti-osteoporotic agent.

Improvement in neurogenesis and memory function by administration of Passiflora incarnata L. extract applied to sleep disorder in rodent models.

Recently, there have been reports that chronic insomnia acts as an insult in the brain, causing memory loss through the production of ROS, inflammation, and, Alzheimer's disease if persistent. Insomnia remains the leading cause of sleep disturbance and as such has serious implications for public health. Patients with Alzheimer's disease are also known to suffer from severe sleep disturbance. Meanwhile, vitexin is a key ingredient in Passiflora incarnata L (passion flower, PF) extract, which is known to help with sleep. This medicinal plant has been used as a folk remedy for sedation, anxiety and sleep since centuries ago, but the standardization work has not been done and the extent of the effect has not been clearly demonstrated. For this reason, we tried to test the possibility that repeated administration of PF could improve the memory by promoting hippocampal neurogenesis at the DBA/2 mice known have inherited sleep disorders, as well as preventive effects of Alzheimer's disease. Here, we found that vitexin, which is the main bioactive component of ethanol extracts from leaves and fruits (ratio; 8:2) of PF, confirmed the improvement of neurogenesis (DCX) of DBA/2 mice repeated PF oral administration by immunohistochemistry (IHC) and western blot analysis. PF-treated group showed increased the neurotrophic factor (BDNF) in the hippocampus compared with that of vehicle-treated group, but the inflammation markers Iba-1 (microglial marker) and COX-2 were inconsistent between the groups. However, we found COX-2 signal is essential for hippocampal neurogenesis according to the additional IHC experiments using COX-2 inhibitor and pIkappaB have shown. In addition, although prescription sleeping pills have been reported to show significant changes in appetite and metabolic rate from time to time, no changes in the feeding behavior, body weight, metabolic rate and body composition of the animals were observed by administration of PF. Interestingly, we found that short-term oral administration of PF displayed improved memory according to the water maze test. Quantitative analysis of Tau protein, which is a marker of Alzheimer's disease, was performed in the SD rats and DBA/2 mice by repeated PF oral administration and pTau/Tau values were significantly decreased in PF-treated group than vehicle-treated group. In conclusion, our results suggest that PF lead high hippocampal neurogenesis in the animals even in inherited sleep-disturbed animals. The increased hippocampal neurogenesis functionally enhanced memory and learning functions by repeated PF oral administration. These results identify PF as a potential therapy for enhancing memory functions and prevention of Alzheimer's disease through actions on the hippocampus.