Rutin ameliorated lipid metabolism dysfunction of diabetic NAFLD via AMPK/SREBP1 pathway.

BACKGROUND: In diabetic liver injury, nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease. Rutin is a bioflavonoid produced by the hydrolysis of glucosidases to quercetin. Its biological activities include lowering blood glucose, regulating insulin secretion, regulating dyslipidemia, and exerting anti-inflammatory effects have been demonstrated. However, its effect on diabetic NAFLD is rarely reported. PURPOSE: Our study aimed to investigate the protective effects of Rutin on diabetic NAFLD and potential pharmacological mechanism. METHODS: We used db/db mice as the animal model to investigate diabetic NAFLD. Oleic acid-treated (OA) HeLa cells were examined whether Rutin had the ability to ameliorate lipid accumulation. HepG2 cells treated with 30 mM/l d-glucose and palmitic acid (PA) were used as diabetic NAFLD in vitro models. Total cholesterol (TC) and Triglycerides (TG) levels were determined. Oil red O staining and BODIPY 493/503 were used to detect lipid deposition within cells. The indicators of inflammation and oxidative stress were detected. The mechanism of Rutin in diabetic liver injury with NAFLD was analyzed using RNA-sequence and 16S rRNA, and the expression of fat-synthesizing proteins in the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway was investigated. Compound C inhibitors were used to further verify the relationship between AMPK and Rutin in diabetic NAFLD. RESULTS: Rutin ameliorated lipid accumulation in OA-treated HeLa. In in vitro and in vivo models of diabetic NAFLD, Rutin alleviated lipid accumulation, inflammation, and oxidative stress. 16S analysis showed that Rutin could reduce gut microbiota dysregulation, such as the ratio of Firmicutes to Bacteroidetes. RNA-seq showed that the significantly differentially genes were mainly related to liver lipid metabolism. And the ameliorating effect of Rutin on diabetic NAFLD was through AMPK/SREBP1 pathway and the related lipid synthesis proteins was involved in this process. CONCLUSION: Rutin ameliorated diabetic NAFLD by activating the AMPK pathway and Rutin might be a potential new drug ingredient for diabetic NAFLD.

Clinical Research Progress of the Post-Stroke Upper Limb Motor Function Improvement via Transcutaneous Auricular Vagus Nerve Stimulation.

Stroke is a disease with high morbidity and disability, and motor impairment is a common sequela of stroke. Transcutaneous auricular vagus nerve stimulation (taVNS) is a type of non-invasive stimulation, which can effectively improve post-stroke motor dysfunction. This review discusses stimulation parameters, intervention timing, and the development of innovative devices for taVNS. We further summarize the application of taVNS in improving post-stroke upper limb motor function to further promote the clinical research and application of taVNS in the rehabilitation of post-stroke upper limb motor dysfunction.

[Relationship between acupoint sensitization and changes in dorsal root ganglion neuron excitability in myocardial ischemia mice].

OBJECTIVE: To investigate the relationship between the sensitization state of acupoints on the surface of the myocardial ischemia (MI) model mice and the changes in the electrophysiological properties of the dorsal root ganglion (DRG) neurons in the corresponding spinal cord segment, and its underlying mechanism. METHODS: Sixty-eight male C57BL/6J mice were randomly divided into control and model groups (34 mice in each group). The model group received an intraperitoneal injection of 160 mg/kg isoproterenol (ISO) to establish the MI model, and the control group received an injection of the same dose of normal saline as the model group. After modeling for about 6 days, MI proportion was measured by HE staining to verify the pathological changes in the heart tissue. Evans blue (EB) dye was injected into the tail vein of mice to reflect the size, location, distribution, and number of exudates on the body surface. Then, whole-cell membrane currents, intrinsic excitability and membrane properties of different types of DRG neurons were evaluated by electrophysiological experiment in vitro. RESULTS: Compared with the control group, the heart size was larger, with pathological outcomes showing enlarged myocardial hypertrophy, destroyed structure of cardiomyocytes, with mononuclear cell infiltration among the cardiomyocytes in the model group. Compared with the control group, the number of EB exudation points was significantly increased (P<0.01), which were mainly concentrated in the epidermis near the T1-T5 segment of the spinal cord, "Feishu" (BL13), "Jueyinshu" (BL14) and "Xinshu" (BL15) in the model group. Compared with the control group, the rheobase and action potential amplitude (APA) of DRG medium-sized neurons were obviously decreased (P<0.01, P<0.05), while the whole-cell membrane currents, the spike numbers, the average instantaneous frequency, and the average discharge frequency were markedly increased (P<0.01). There were no significant alterations in the membrane properties and intrinsic excitability induced by depolarized currents of small-sized neurons between groups. Compared with the control group, the whole-cell membrane currents, spike numbers, and the average instantaneous frequency were significantly increased in the model group(P<0.05, P<0.01) while rheobase was significantly decreased (P<0.05) in DRG medium-sized neurons labeled with biotin and CGRP. CONCLUSION: After the mice were modeled by ISO, the DRG medium-size neurons in the T1-T5 segment of the spinal cord may mediate the sensitization of acupoints on the body surface through their different neuronal membrane properties and intrinsic excitabilities.

Enhanced encapsulation of lutein using soy protein isolate nanoparticles prepared by pulsed electric field and pH shifting treatment.

In this study, soy protein isolate (SPI) was modified by a pulsed electric field (PEF) combined with pH shifting treatment (10 kV/cm, pH 11) to prepare SPI nanoparticles (PSPI11) for efficient loading of lutein. The results showed that when the mass ratio of SPI to lutein was 25:1, the encapsulation efficiency of lutein in PSPI11 increased from 54% to 77%, and the loading capacity increased by 41% compared to the original SPI. The formed SPI-lutein composite nanoparticles (PSPI11-LUTNPs) had smaller, more homogeneous sizes and larger negative charges than SPI7-LUTNPs. The combined treatment favored the unfolding of the SPI structure and could expose its interior hydrophobic groups to bind with lutein. Nanocomplexation with SPIs significantly improved the solubility and stability of lutein, with PSPI11 showing the greatest improvement. As a result, PEF combined with pH shifting pretreatment is an effective method for developing SPI nanoparticles loaded and protected with lutein.

Effects of integrated action and sensory observation therapy based on mirror neuron and embodied cognition theory on upper limb sensorimotor function in chronic stroke: a study protocol for a randomised controlled trial.

INTRODUCTION: This study protocol aims to explore the effectiveness and neural mechanism of the integration of action observation therapy (AOT) and sensory observation therapy (SOT) for post-stroke patients on upper limb sensorimotor function. METHODS AND ANALYSIS: This is a single-centre, single-blind, randomised controlled trial. A total of 69 patients with upper extremity hemiparesis after stroke will be recruited and randomly divided into an AOT group, a combined action observation and somatosensory stimulation therapy (AOT+SST) group, and a combined AOT and SOT (AOT+SOT) group in a 1:1:1 ratio. Each group will receive 30 min of daily treatment, five times weekly for 4 weeks. The primary clinical outcome will be the Fugl-Meyer Assessment for Upper Extremity. Secondary clinical outcomes will include the Box and Blocks Test, modified Barthel Index and sensory assessment. All clinical assessments and resting-state functional MRI and diffusion tensor imaging data will be obtained at pre-intervention (T1), post-intervention (T2) and 8 weeks of follow-up (T3). ETHICS AND DISSEMINATION: The trial was approved by the Ethics Committee of Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Chinese Traditional Medicine (Grant No. 2020-178). The results will be submitted to a peer-review journal or at a conference. TRIAL REGISTRATION NUMBER: ChiCTR2000040568.

[Research on mechanisms of acupoint sensitization in gastric ulcer mice from perspective of dorsal root ganglion neuronal excitability].

OBJECTIVE: To investigate the relationship between acupoint sensitization on the body surface and neuronal intrinsic excitability of the medium- and small-size dorsal root ganglion (DRG) neurons from the perspective of ion channel kinetics in mice with gastric ulcer. METHODS: Male C57BL/6J mice were randomly divided into control (n=32) and model groups (n=34). The gastric ulcer model was established by injection of 60% glacial acetic acid (0.2 mL/100 g) into the gastric wall muscle layer and submucosa near the pylorus in the minor curvature of the stomach. In contrast, the same dose of normal saline was injected in the same way in the control group. Six days after modeling, Evans blue (EB) solution was injected into the mouse's tail vein for observing the number and distribution of the exudation blue spots on the body surface. Histopathological changes of the gastric tissue were observed by H.E. staining. Then, whole-cell membrane currents and intrinsic excitability of medium- and small-size neurons in the spinal T9-T11 DRGs were measured by in vitro electrophysiology combining with biocytin-ABC method. RESULTS: In the control group, EB exudation blue spots were not obvious, while in the model group, the blue spots on the body surface were densely distributed in the area of spinal T9-T11 segments, the epigastric region, and the skin around "Zhongwan" (CV12) and "Huaroumen" (ST24) regions, and near the surgical incision region. Compared with the control group, the model group had a high level of eosinophilic infiltrates in the submucosa of gastric tissues, severe gastric fossa structure damage, gastric fundus gland dilation and other pathological manifestations. The number of exudation blue spots was proportional to the degree of inflammatory reaction in the stomach. In comparison with the control group, the spike discharges of type II of medium-size DRG neurons in T9-T11 segments were decreased, and the current of whole-cell membrane was increased, basic intensity was decreased (P<0.05), discharge frequency and discharge number were increased (P<0.01,P<0.000 1); while the discharges of type I small-size DRG neurons were decreased, those of type II neurons increased, the whole-cell membrane current was decreased, and discharge frequency and discharge number were decreased (P<0.01, P<0.000 1). CONCLUSION: Both the medium- and small-size DRG neurons from the spinal T9-T11 segments involve in gastric ulcer-induced acupoint sensitization via their different spike discharge activities. And intrinsic excitability of these DRG neurons can not only dynamically encode the plasticity of acupoint sensitization, but also can help us understand the neural mechanism of acupoint sensitization induced by visceral injury.

Genome-centered metagenomics illuminates adaptations of core members to a partial Nitritation-Anammox bioreactor under periodic microaeration.

The partial nitritation-anaerobic ammonium oxidation (anammox; PN-A) process has been considered a sustainable method for wastewater ammonium removal, with recent attempts to treat low-strength wastewater. However, how microbes adapt to the alternate microaerobic-anoxic operation of the process when treating low ammonium concentrations remains poorly understood. In this study, we applied a metagenomic approach to determine the genomic contents of core members in a PN-A reactor treating inorganic ammonium wastewater at loading as low as 0.0192 kg-N/m3/day. The metabolic traits of metagenome-assembled genomes from 18 core species were analyzed. Taxonomically diverse ammonia oxidizers, including two Nitrosomonas species, a comammox Nitrospira species, a novel Chloroflexota-related species, and two anammox bacteria, Ca. Brocadia and Ca. Jettenia, accounted for the PN-A reactions. The characteristics of a series of genes encoding class II ribonucleotide reductase, high-affinity bd-type terminal oxidase, and diverse antioxidant enzymes revealed that comammox Nitrospira has a superior adaptation ability over the competitors, which may confer the privileged partnership with anammox bacteria in the PN-A reactor. This finding is supported by the long-term monitoring experiment, showing the predominance of the comammox Nitrospira in the ammonia-oxidizing community. Metagenomic analysis of seven heterotrophs suggested that nitrate reduction is a common capability in potentially using endogenous carbohydrates and peptides to enhance nitrogen removals. The prevalence of class II ribonucleotide reductase and antioxidant enzymes genes may grant the adaptation to cyclically microaerobic/anoxic environments. The predominant heterotroph is affiliated with Chloroflexota; its genome encodes complete pathways for synthesizing vitamin B6 and methionine. By contrast, other than the two growth factors, Nitrospira and anammox bacteria are complementary to produce various vitamins and amino acids. Besides, the novel Chloroflexota-related ammonia oxidizer lacks corresponding genes for detoxifying the reactive oxygen species and thus requires the aid of co-existing members to alleviate oxidative stress. The analysis results forecast the exchanges of substrates and nutrients as well as the collective alleviation of oxidative stress among the core populations. The new findings of the genomic features and predicted microbial interplay shed light on microbial adaptation to intermittent microaeration specific to the PN-A reactor, which may aid in improving its application to low-strength ammonium wastewater.

Gamma oscillations and application of 40-Hz audiovisual stimulation to improve brain function.

BACKGROUND: Audiovisual stimulation, such as auditory stimulation, light stimulation, and audiovisual combined stimulation, as a non-invasive stimulation, which can induce gamma oscillation, has received increased attention in recent years, and it has been preliminarily applied in the clinical rehabilitation of brain dysfunctions, such as cognitive, language, motor, mood, and sleep dysfunctions. However, the exact mechanism underlying the therapeutic effect of 40-Hz audiovisual stimulation remains unclear; the clinical applications of 40-Hz audiovisual stimulation in brain dysfunctions rehabilitation still need further research. OBJECTIVE: In order to provide new insights into brain dysfunction rehabilitation, this review begins with a discussion of the mechanism underlying 40-Hz audiovisual stimulation, followed by a brief evaluation of its clinical application in the rehabilitation of brain dysfunctions. RESULTS: Currently, 40-Hz audiovisual stimulation was demonstrated to affect synaptic plasticity and modify the connection status of related brain networks in animal experiments and clinical trials. Although its promising efficacy has been shown in the treatment of cognitive, mood, and sleep impairment, research studies into its application in language and motor dysfunctions are still ongoing. CONCLUSIONS: Although 40-Hz audiovisual stimulation seems to be effective in treating cognitive, mood, and sleep disorders, its role in language and motor dysfunctions has yet to be determined.

Evaluating nitric oxide produced by rat inflamed microglial cell Lline, CHME-5, under the effect of IMOD.

Nitric oxide (NO), as a free radical, is produced by inflamed microglia cells and is one of the destructive factors of the immune system and a factor in myelin degradation. Therefore, inhibition of microglia activity is a chief strategy in reducing neurotoxic damage to the central nervous system. In this study, an herbal Immunomodulatory Drug (IMOD) was used to evaluate the effects of this drug in controlling the amount of nitric oxide. Nitric oxide induction was performed by bacterial lipopolysaccharide (LPS) in rat inflamed microglial cell line, CHME-5. ELISA test was used to measure the produced nitric oxide at 24, 48, and 72 hours. The results showed that the high concentrations of IMOD (1.2, and 4% V/V) had anti-inflammatory effects on microglial cells and were able to reduce the amount of nitric oxide in these cells but the effective dose of IMOD was in the range of 1.2% V/V. Therefore, the safest dose and the best time for the effect of IMOD on inflammatory cell groups are 1.2% V/V and 72h, respectively. Hence, with further studies, IMOD can be considered as an herbal anti-inflammatory drug that is effective in controlling neurodegenerative diseases.

Characterization of TMAO productivity from carnitine challenge facilitates personalized nutrition and microbiome signatures discovery.

The capability of gut microbiota in degrading foods and drugs administered orally can result in diversified efficacies and toxicity interpersonally and cause significant impact on human health. Production of atherogenic trimethylamine N-oxide (TMAO) from carnitine is a gut microbiota-directed pathway and varies widely among individuals. Here, we demonstrated a personalized TMAO formation and carnitine bioavailability from carnitine supplements by differentiating individual TMAO productivities with a recently developed oral carnitine challenge test (OCCT). By exploring gut microbiome in subjects characterized by TMAO producer phenotypes, we identified 39 operational taxonomy units that were highly correlated to TMAO productivity, including Emergencia timonensis, which has been recently discovered to convert γ-butyrobetaine to TMA in vitro. A microbiome-based random forest classifier was therefore constructed to predict the TMAO producer phenotype (AUROC = 0.81) which was then validated with an external cohort (AUROC = 0.80). A novel bacterium called Ihubacter massiliensis was also discovered to be a key microbe for TMA/TMAO production by using an OCCT-based humanized gnotobiotic mice model. Simply combining the presence of E. timonensis and I. massiliensis could account for 43% of high TMAO producers with 97% specificity. Collectively, this human gut microbiota phenotype-directed approach offers potential for developing precision medicine and provides insights into translational research. Video Abstract.

Pollen-Specific Protein PSP231 Activates Callose Synthesis to Govern Male Gametogenesis and Pollen Germination.

Spatiotemporally regulated callose deposition is an essential, genetically programmed phenomenon that promotes pollen development and functionality. Severe male infertility is associated with deficient callose biosynthesis, highlighting the significance of intact callose deposition in male gametogenesis. The molecular mechanism that regulates the crucial role of callose in production of functional male gametophytes remains completely unexplored. Here, we provide evidence that the gradual upregulation of a previously uncharacterized cotton (Gossypium hirsutum) pollen-specific SKS-like protein (PSP231), specifically at the post pollen-mitosis stage, activates callose biosynthesis to promote pollen maturation. Aberrant PSP231 expression levels caused by either silencing or overexpression resulted in late pollen developmental abnormalities and male infertility phenotypes in a dose-dependent manner, highlighting the importance of fine-tuned PSP231 expression. Mechanistic analyses revealed that PSP231 plays a central role in triggering and fine-tuning the callose synthesis and deposition required for pollen development. Specifically, PSP231 protein sequesters the cellular pool of RNA-binding protein GhRBPL1 to destabilize GhWRKY15 mRNAs, turning off GhWRKY15-mediated transcriptional repression of GhCalS4/GhCalS8 and thus activating callose biosynthesis in pollen. This study showed that PSP231 is a key molecular switch that activates the molecular circuit controlling callose deposition toward pollen maturation and functionality and thereby safeguards agricultural crops against male infertility.

Establishment and Preclinical Therapy of Patient-derived Hepatocellular Carcinoma Xenograft Model.

Hepatocellular carcinoma (HCC) is a world-wide health problem. Poor and delayed diagnoses as well as high recurrence rate resulting in high mortality rate. In this study, we established a patient-derived xenograft (PDX) model from HCC patient, and continuously maintained with subcutaneous passage more than 20 times. This HCC PDX tumor exhibited the same histological characteristics with the HCC patient and could be used to verify therapeutic effect of liver cancer. We further evaluated this PDX model by experimental chemotherapy, demonstrating that this HCC PDX model was sensitive to sorafenib treatment. Further, the potential of natural killer cell-based immunotherapy for HCC was tested using this model. We found that NK92 cells effectively suppressed the tumor growth in vivo and prolonged the survival time of HCC-bearing PDX mice. This study indicates that HCC PDX model is a good platform to testify the efficacy of preclinical chemotherapy and immunotherapy.

Artificial intelligence-assisted prediction of preeclampsia: Development and external validation of a nationwide health insurance dataset of the BPJS Kesehatan in Indonesia.

BACKGROUND: We developed and validated an artificial intelligence (AI)-assisted prediction of preeclampsia applied to a nationwide health insurance dataset in Indonesia. METHODS: The BPJS Kesehatan dataset have been preprocessed using a nested case-control design into preeclampsia/eclampsia (n = 3318) and normotensive pregnant women (n = 19,883) from all women with one pregnancy. The dataset provided 95 features consisting of demographic variables and medical histories started from 24 months to event and ended by delivery as the event. Six algorithms were compared by area under the receiver operating characteristics curve (AUROC) with a subgroup analysis by time to the event. We compared our model to similar prediction models from systematically reviewed studies. In addition, we conducted a text mining analysis based on natural language processing techniques to interpret our modeling results. FINDINGS: The best model consisted of 17 predictors extracted by a random forest algorithm. Nine∼12 months to the event was the period that had the best AUROC in external validation by either geographical (0.88, 95% confidence interval (CI) 0.88-0.89) or temporal split (0.86, 95% CI 0.85-0.86). We compared this model to prediction models in seven studies from 869 records in PUBMED, EMBASE, and SCOPUS. This model outperformed the previous models in terms of the precision, sensitivity, and specificity in all validation sets. INTERPRETATION: Our low-cost model improved preliminary prediction to decide pregnant women that will be predicted by the models with high specificity and advanced predictors. FUNDING: This work was supported by grant no. MOST108-2221-E-038-018 from the Ministry of Science and Technology of Taiwan.

Central adiponectin induces trabecular bone mass partly through epigenetic downregulation of cannabinoid receptor CB1.

Central adiponectin (APN) in either the globular (gAPN) or full-length forms decreases sympathetic tone and increases trabecular bone mass in mice through the hypothalamus. It is known that cannabinoid type-1 (CB1) receptors are expressed in the hypothalamic ventromedial nucleus and participate in energy metabolism by controlling sympathetic activity. However, whether central APN could influence endocannabinoid signaling through CB1 receptor to regulate bone metabolism has not been characterized. Here we demonstrate that gAPN downregulated CB1 expression in embryonic mouse hypothalamus N1 cells in vitro. gAPN intracerebroventricular (icv) infusions also decreased hypothalamic CB1 expression and bone formation parameters in APN-knockout (APN-KO) and wild-type mice. Most importantly, mice pretreated with icv infusions with the CB1 receptor agonist arachidonyl-2'-chloroethylamine or antagonist rimonabant attenuated or enhanced respectively central APN induction of bone formation. We then investigated whether epigenetic signaling mechanisms were involved in the downregulation of hypothalamic CB1 expression by gAPN. We found gAPN enhanced expression levels of various histone deacetylases (HDACs), especially HDAC5. Furthermore, chromatin immunoprecipitation assays revealed HDAC5 bound to the transcriptional start site transcription start site 2 region of the CB1 promoter. In summary, our study identified a possible novel central APN-HDAC5-CB1 signaling mechanism that promotes peripheral bone formation through epigenetic regulation of hypothalamic CB1 expression.

Cell-type-specific inhibition of the dendritic plateau potential in striatal spiny projection neurons.

Striatal spiny projection neurons (SPNs) receive convergent excitatory synaptic inputs from the cortex and thalamus. Activation of spatially clustered and temporally synchronized excitatory inputs at the distal dendrites could trigger plateau potentials in SPNs. Such supralinear synaptic integration is crucial for dendritic computation. However, how plateau potentials interact with subsequent excitatory and inhibitory synaptic inputs remains unknown. By combining computational simulation, two-photon imaging, optogenetics, and dual-color uncaging of glutamate and GABA, we demonstrate that plateau potentials can broaden the spatiotemporal window for integrating excitatory inputs and promote spiking. The temporal window of spiking can be delicately controlled by GABAergic inhibition in a cell-type-specific manner. This subtle inhibitory control of plateau potential depends on the location and kinetics of the GABAergic inputs and is achieved by the balance between relief and reestablishment of NMDA receptor Mg2+ block. These findings represent a mechanism for controlling spatiotemporal synaptic integration in SPNs.

Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis.

Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

Input- and cell-type-specific endocannabinoid-dependent LTD in the striatum.

Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD) is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs; however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN) subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1) receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.

Central adiponectin administration reveals new regulatory mechanisms of bone metabolism in mice.

Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.

Effects of 6-Hydroxyflavone on Osteoblast Differentiation in MC3T3-E1 Cells.

Osteoblast differentiation plays an essential role in bone integrity. Isoflavones and some flavonoids are reported to have osteogenic activity and potentially possess the ability to treat osteoporosis. However, limited information concerning the osteogenic characteristics of hydroxyflavones is available. This study investigates the effects of various hydroxyflavones on osteoblast differentiation in MC3T3-E1 cells. The results showed that 6-hydroxyflavone (6-OH-F) and 7-hydroxyflavone (7-OH-F) stimulated ALP activity. However, baicalein and luteolin inhibited ALP activity and flavone showed no effect. Up to 50 µ M of each compound was used for cytotoxic effects study; flavone, 6-OH-F, and 7-OH-F had no cytotoxicity on MC3T3-E1 cells. Moreover, 6-OH-F activated AKT and serine/threonine kinases (also known as protein kinase B or PKB), extracellular signal-regulated kinases (ERK 1/2), and the c-Jun N-terminal kinase (JNK) signaling pathways. On the other hand, 7-OH-F promoted osteoblast differentiation mainly by activating ERK 1/ 2 signaling pathways. Finally, after 5 weeks of 6-OH-F induction, MC3T3-E1 cells showed a significant increase in the calcein staining intensity relative to merely visible mineralization observed in cells cultured in the osteogenic medium only. These results suggested that 6-OH-F could activate AKT, ERK 1/2, and JNK signaling pathways to effectively promote osteoblastic differentiation.

Screening of flavonoids for effective osteoclastogenesis suppression.

Flavonoids are natural compounds derived from plants and some of them have been shown to inhibit osteoclast formation, implicating their potential use for the treatment of osteoporosis. Conventionally, the screening of antiosteoclastic agents is a tedious process that requires visual counting of the number of osteoclasts produced. The purpose of this study was to establish an easier and faster method for screening the antiosteoclastogenic flavonoids by using an enzyme assay. Tartrate-resistant acid phosphatase (TRAP) is a marker enzyme of the osteoclast. Results obtained demonstrated that cellular TRAP activity tended to correlate with the number of osteoclasts formed. However, the secreted TRAP activity was actually responsible for the resorption activities of the functional osteoclasts. Consequently, the effectiveness of antiosteoclastogenic agents was screened for by assessing their inhibition on receptor activator of NF-κB ligand (RANKL)-induced TRAP secretion. The half-inhibitory concentrations of flavonoids on TRAP secretion were employed as indices to compare the effectiveness of various flavonoids. The effective flavonoids also exhibited similar inhibitory potencies in the pit-formation analysis. This protocol provides a rapid analysis to screen for effective antiosteoclastogenic agents.