OVX Mice Research Articles

Anti-Inflammatory and Osteogenic Effects of Vitamin K from Sargassum fulvellum Fermented by Lactococcus lactis KCCM12759P and Leuconostoc mesenteroides KCCM12756P

Vitamin K (VitK) is a vital nutrient that is newly recognized to support bone and cardiovascular health. As a nutraceutical, VitK is produced via plant extraction and bacterial fermentation. This study examined the potential anti-inflammatory and osteogenic benefits of VitK, i.e., VitK1 (phylloquinone; PK) and VitK2 (menaquinone; MKs), derived from Sargassum fulvellum fermented by Lactococcus lactis and Leuconostoc mesenteroides (SfLlLm) using lipopolysaccharide (LPS)-induced Raw264.7, MC3T3-E1 cells, and ovariectomized (OVX) mice. MK4, MK7, and MK9, as well as PK, were effectively acquired from SfLlLm and analyzed. SfLlLm_VitK reduced levels of proinflammatory cytokine in LPS-induced Raw264.7 cells and induced an osteogenesis regulating factor in MC3T3-E1 cells. In OVX mice, SfLlLm feeding reduced plasma levels of alkaline phosphatase, phosphate, and the pro-collagen type I alpha 2 gene (pro-Col1a2) while elevating cancellous bone volume and trabecular numbers. Accordingly, SfLlLm, comprising MKs, may be a candidate for preventing and treating immune and bone diseases.

NET-EN treatment leads to delayed HSV-2 infection, enhanced mucin and T cell functions in the female genital tract when compared to DMPA in a preclinical mouse model

Depot-medroxyprogesterone acetate (DMPA) and Norethisterone Enanthate (NET-EN) are progestin-only injectable contraceptives widely used by women in sub-Sharan Africa, where incidence of HIV-1 and HSV-2 infection remains high. Studies indicate that DMPA usage can increase the risk of HSV-2 infection, but limited data indicate no increased risk with use of NET-EN. We therefore investigated the effects of NET-EN and DMPA on susceptibility to vaginal HSV-2 infection in ovariectomized (OVX) mice and effects on immune responses, particularly in the vaginal tract (VT). OVX mice, when treated with NET-EN and infected intravaginally, had delayed genital pathology, decreased viral shedding, and extended survival compared to DMPA- or untreated OVX mice. CD4+ T cells isolated from VT showed no significant change in frequency with either contraceptive. However, DMPA significantly decreased the total number of VT CD4+ and CD8+ T cells and the number of IFN-γ producing CD4 and CD8 T cells and increased the percentage of CD4 and CD8 T cells producing TNF-α compared to untreated mice. In contrast, NET-EN significantly enhanced percentages of CD8+ T cells compared to DMPA treated mice, and frequencies of IFN-γ+ CD4 and CD8 T cells in the VT compared to untreated mice. Comparative analysis of splenic lymphocytes indicated that DMPA treatment resulted in reduction of CD4+ T cell frequency, but enhanced TNF-α+ CD4 T cells compared to untreated mice. NET-EN enhanced the frequency of CD8 T cells, as well as IFN-γ+ and TNF-α+ CD4, and IFN-γ+ CD8 T cells in the spleen compared to untreated mice. Importantly, we found DMPA treatment that significantly reduced mucin production, whereas NET-EN enhanced expression of cell-associated mucin in VT. High levels of mucin in NET-EN mice were associated with lower levels of HSV-2 virus detected in the vaginal tract. This study provides the first evidence that NET-EN treatment can delay HSV-2 infection compared to DMPA.

Teriparatide administration is osteoanabolic but does not impact atherosclerotic plaque calcification and progression in a mouse model of menopause

Menopause exacerbates osteoporosis and increases the risk of atherosclerotic plaque rupture, leading to cardiovascular mortality. Osteoporotic women are increasingly treated with teriparatide (TPTD, 1–34 parathyroid hormone), one of the few treatments that stimulate bone formation. Despite the fact that atherosclerotic plaque calcification is a hallmark of plaque development, the impact of TPTD administration on plaque calcification remain unclear. In this context, we sought to determine the effects of TPTD administration on atherosclerosis in ovariectomized (OVX) apolipoprotein E deficient mice (ApoE−/−), as a model of postmenopausal osteoporosis. OVX ApoE−/− mice, fed a high fat, high cholesterol diet to induce atherosclerosis, received either vehicle or TPTD daily injections (40 μg/kg/d) for 4 or 10 weeks, at which points plaques are respectively weakly and heavily calcified. After sacrifice, bone remodeling was evaluated by serum markers and bone histomorphometry. Bone architectural parameters were measured by μCT. Aortic plaques were analyzed histologically, and their calcification with von Kossa staining and the calcium tracer Osteosense. Plaque inflammation and calcification markers were measured by RT-qPCR. Intermittent TPTD increased bone volume in OVX mice, due to a higher stimulation of bone formation relatively to bone resorption. These effects were not accompanied by changes in serum levels of cholesterol, triglycerides, glucose or insulin. TPTD neither significantly affected aortic plaque size, inflammation, and calcification, even if it slightly increased vascular smooth muscle cell transdifferentiation into calcifying cells. In conclusion, TPTD exhibits osteoanabolic effects in OVX ApoE−/− mice, without significantly influencing atherosclerotic plaque progression or calcification in the short term.

Antiosteoporotic activity of nodakenetin, a coumarin compound from Angelica decursiva, by activation of the Wnt/β-catenin signaling pathway

Angelica decursiva (Umbelliferae) is a medicinal plant widely used to treat colds, coughs and fevers in Korea, Japan, and mainland China. The anti-inflammatory activity of nodakenetin, a furano-coumarin compound from A. decursiva, has been reported, although, the antiosteoporotic activity remains unknown. This study sought to investigate the antiosteoporotic activity and precise mechanism of action of nodakenetin in vitro cell culture and in vivo bone remodeling models. The transcriptional activity of nodakenetin on the Wnt signaling pathway was assessed using the TOPflash/FOPflash assay. The effect of nodakenetin on the osteoblast differentiation was measured using Alizarin red staining and alkaline phosphatase (ALP) activity. Western blotting and real-time RT-PCR were used to assess the effect of nodakenetin on the expression of markers related to Wnt/β-catenin pathway and osteoblast differentiation. The in vivo antiosteoporotic activity of nodakenetin was assessed using an ovariectomized (OVX)-induced bone loss mouse model. Nodakenetin activated the Wnt/β-catenin pathway through regulation of DKK1, β-catenin and other target proteins of the Wnt/β-catenin pathway in HEK293 and MC3T3-E1 cells. Nodakenetin induced the differentiation of MC3T3-E1 cells as shown by enhanced Alizarin red staining and ALP activity. Induction of osteoblast differentiation was related to upregulated expression of bone formation biomarkers such as bone morphogenic proteins and Runx2. Oral administration of nodakenetin in the OVX mouse model effectively protected the deterioration of bone microstructure in OVX mice. Nodakenetin exhibits antiosteoporotic activity in vitro and in vivo through the activation of the Wnt/β-catenin pathway and subsequent induction of osteoblast differentiation.

TGFβ2-Driven Ferritin Degradation and Subsequent Ferroptosis Underlie Salivary Gland Dysfunction in Postmenopausal Conditions.

Despite the high incidence of dry mouth in postmenopausal women, its underlying mechanisms and therapeutic interventions remain underexplored. Using ovariectomized (OVX) mouse models, here this study identifies ferroptosis, an iron-dependent regulated cell death, as a central mechanism driving postmenopausal salivary gland (SG) dysfunction. In the OVX-SGs, TGFβ signaling pathway is enhanced with the aberrant TGFβ2 expression in SG mesenchymal cells. Intriguingly, TGFβ2 treatment reduces iron-storing ferritin levels, leading to lipid peroxidation and ferroptotic death in SG epithelial organoids (SGOs). Mechanistically, TGFβ2 promotes the autophagy-mediated ferritin degradation, so-called ferritinophagy. A notable overexpression of the type III TGFβ receptor (TβRIII) is found in the OVX-SGs and TGFβ2-treated SGOs, while the silencing of TβRIII mitigates the ferroptosis-mediated deleterious effects of TGFβ2 on SGOs. Finally, administration of ferroptosis inhibitor, Liproxstatin-1 (Lip-1), improves saliva secretion in OVX mice. Present findings collectively suggest a link between TGFβ signaling, ferroptosis, and SG injury, offering new therapeutic avenues for postmenopausal xerostomia.

Water Extract of Pulsatilla koreana Nakai Inhibits Osteoclast Differentiation and Alleviates Ovariectomy-Induced Bone Loss.

Pulsatilla koreana Nakai (P. koreana) is a perennial herb traditionally used to treat malaria and fever. Although the pharmacological properties of P. koreana have been explored in various contexts, its effects on bone diseases, such as osteoporosis, remain poorly studied. In this study, we investigated the effects of water extracts of P. koreana (WEPK) on osteoclasts, which play a crucial role in bone remodeling, and an ovariectomized (OVX) mouse model, which mimics osteoporosis. Phytochemical profiling of WEPK revealed several compounds that regulate bone or fat metabolism. WEPK suppressed osteoclast differentiation by downregulating the expression of receptor activator of nuclear factor-κB ligand (RANKL), a cytokine that induces osteoclastogenesis. Additionally, WEPK directly inhibited RANKL-induced differentiation of osteoclast precursors by downregulating nuclear factor of activated T cells 1 (NFATc1), the master transcription factor for osteoclastogenesis, by modulating its upstream regulators. In vivo, oral administration of WEPK suppressed bone loss, reduced weight gain, and mitigated fat accumulation in the liver and gonadal tissues of OVX mice. Given its positive impact on bone and fat accumulation under estrogen deficiency, WEPK may serve as a promising alternative therapy for postmenopausal osteoporosis, especially when accompanied by other metabolic disorders, such as obesity and fatty liver.

Bone-targeted ultrasound-responsive nanobubbles for siRNA delivery to treat osteoporosis in mice

This project aimed to study the efficacy of a bone-targeted ultrasound-responsive nanobubble (NB) platform to deliver gene-silencing cathepsin K (CTSK) siRNA into the bone for osteoporosis treatment using in vitro and in vivo studies. To this end, characterization of CTSK siRNA loaded NB functionalized with alendronate (NB-CTSK siRNA-AL) was performed using transmission electron microscopy (TEM) imaging, and a release profile was obtained through fluorescent spectroscopy. In vitro studies were conducted by culturing NB-CTSK siRNA-AL with osteoclasts to evaluate siRNA uptake, CTSK expression, and the expression of tartrate-resistant acid phosphatase (TRAP). A control group and an NB-CTSK siRNA-AL treated group of ovariectomized (OVX) mice (n = 4) were tested. The OVX group that received treatment underwent weekly sessions for 4 weeks, during which they were exposed to low-intensity pulsed ultrasound (LIPUS) stimulation following administration of NB-CTSK siRNA-AL, prior to being sacrificed. Both groups underwent a series of tests to evaluate the bone targeting, safety, and efficacy of the nanoplatform. These tests included biodistribution studies conducted at 4 h and 24 h post-injection, a 3-point bending test of the femurs, nano-computed tomography analysis, as well as Hematoxylin & Eosin histological staining, Masson's Trichrome staining, and CTSK staining. The biodistribution showed the accumulation of NB-CTSK siRNA-AL in the bone and liver. Results showed that the OVX mice treated with NB-CTSK siRNA-AL had increased distal cortical bone thickness (174.4 ± 5.28 μm vs. 144.3 ± 10.66 μm, p > 0.05)) and bone volume fraction (16.5 ± 3.96 % vs. 6.55 ± 0.13 % (p > 0.05)). A reduced collagen degradation and downregulated CTSK expression were evident in the staining procedures. No adverse effects were recorded within histological assessments on the liver, kidney, and heart post-treatment. Morphology was shown to be normal and healthy within muscle cells post-LIPUS stimulation of NB-CTSK siRNA-AL. From these results, it can be concluded that an ultrasound-mediated NB-CTSK siRNA-AL can serve as a reliable, safe CTSK siRNA carrier to bone-specific targets for in vivo osteoporosis treatment.

Myopic shift in female mice after ovariectomy

Myopia is a global public health concern, with a higher prevalence in women than that in male. As the relationship between sex hormone and myopia remains unclear, we aimed to reveal the relationship between sex hormone and myopia by removing the ovaries of mice and measuring changes in ocular parameters related to myopia. Lens-induced myopia (LIM) surgeries were performed on 3-week-old male mice and age-matched female mice to evaluate the effects of sex on myopia development, which indicated a high degree of myopia and rapid progression of axial elongation in male mice. Bilateral ovariectomy (OVX) performed on 4-week-old female mice induced myopic refraction status in ovariectomized mice. Although axial length elongation was larger in ovariectomized mice than that in sham control mice, the result was insignificant. To further reveal the relationship between female sex hormones and myopia, LIM combined with OVX was performed, which revealed a magnified myopic refraction status in ovariectomized mice. Nevertheless, elongation of the anterior chamber depth in the − 30 D lens-treated eyes significantly increased in LIM + OVX mice compared with the frame-treated eyes in LIM + OVX mice and − 30 D lens-treated eyes in LIM + Sham surgery mice. Sex hormones play a role in regulating myopia development in female mice.

Dihydromyricetin ameliorate postmenopausal osteoporosis in ovariectomized mice: Integrative microbiomic and metabolomic analysis.

The gut microbiota may help mitigate bone loss linked to postmenopausal osteoporosis by affecting the immune and inflammatory responses and the gut-bone axis. Dihydromyricetin (DMY), a natural flavonoid, has some anti-inflammatory and antioxidant properties. This study aimed to investigate the mechanisms underlying the amelioration of bone loss in ovariectomized (OVX) mice treated with various doses of DMY. Eight-week-old C57/BL6 mice underwent ovariectomy and received varying DMY doses over 8 weeks. Thereafter, femoral bone microarchitecture, serum biomarker levels, and colon samples were analyzed to assess bone metabolism and inflammatory and hormonal responses. Fecal samples were subjected to 16S rDNA sequencing, and short-chain fatty acids were quantified. An untargeted metabolomics approach was applied to both serum and fecal samples to investigate alterations in the intestinal microbiota and metabolic profiles following DMY treatment in the OVX mice. The results show high-dose DMY has anti-osteoporotic effects. Compared to the OVX group, the DMY-treated group showed enhanced bone mineral density and reduced inflammation and colonic damage levels. The DMY treatment altered the gut microbiota composition, including the relative abundances at both the phylum and genus levels. In addition, DMY treatment increased the production of acetate and propionate. Metabolomic analysis revealed differential regulation of 37 and 70 metabolites in the serum and feces samples, respectively, in the DMY-treated group compared to those in the OVX group, affecting the serotonergic signaling, arachidonic acid metabolism, and unsaturated fatty acid biosynthesis pathways. In conclusion, these findings indicate that DMY can ameliorate bone loss in OVX mice via the gut-bone axis.

Effects of E2 on the IDO1-mediated metabolic KYN pathway in OVX female mice.

The aim of this study was to investigate the role of 17β-estradiol (E2)-mediated oestrogen receptor (ER) in modulating the depressive-like behaviours of ovariectomy (OVX) mice and the associated mechanisms. E2 was administrated in OVX mice. The behaviour and physiological changes of OVX mice including immobility time in tail suspension test (TST) and forced swimming test (FST), levels of serum E2, inflammatory mediators, oxidative stress factors, indoleamine2,3-dioxygenase 1 (IDO1) and the neurotransmitters mediated by IDO1 activation were then recorded. Cell injury models established by lipopolysaccharide (LPS) or H2O2 stimulation in HT22 and BV2 cells were employed to further explore the mechanisms of E2's function. E2 treatment improved OVX-induced increase of immobility time in FST and TST. Meanwhile, E2 ameliorated the changes of inflammatory factors (NF-κB, TNF-α and IL-6), IDO1, IDO1-mediated TRP/KYN pathway and oxidative stress factors (iNOS, MDA, GSH and SOD) in the hippocampus of OVX mice. Interestingly, ERβ inhibitor abolished E2's inhibitory effects on the inflammation and IDO1-mediated TRP/KYN pathway; ERβ inhibitor also abolished E2's anti-oxidative stress effect. In cell experiments, ERβ small interfering RNA (siRNA) pretreatment reversed E2's anti-inflammatory effect on LPS-treated HT22 and BV2 cells and E2's inhibitory effect on IDO1 expression in LPS-treated BV2 cells. ERβ siRNA pretreatment also reversed E2's anti-oxidation effect on H2O2-treated HT22 cells. E2 exert the antidepressant function in OVX mice via ERβ-modulated suppression of NF-κB-mediated inflammatory pathway, oxidative stress factors and IDO1-mediated TRP/KYN pathway in the hippocampus.

Sex differences in the metabolic activation of and platelet response to vicagrel in mice: Androgen as a key player

As a biological variable, sex influences the metabolism of and/or response to certain drugs. Vicagrel is being developed as an investigational new drug in China; however, it is unknown whether sex could affect its metabolic activation and platelet responsiveness. This study aimed to determine whether such differences could exist, and to elucidate the mechanisms involved. Orchiectomized (ORX) or ovariectomized (OVX) mouse models were used to investigate the effects of androgens or estrogens on the metabolic activation of and platelet response to vicagrel. Plasma vicagrel active metabolite H4 concentrations, platelet inhibition of vicagrel, and protein levels of intestinal hydrolases Aadac and Ces2 were measured, respectively. Further, p38-MAPK signaling pathway was enriched, whose role was determined using SB202190. Results showed that female mice exhibited significantly elevated systemic exposure of H4 and enhanced platelet responses to vicagrel than males, and that protein expression levels of Aadac and Ces2 differed by sex. OVX mice exhibited less changes than sham mice. ORX mice exhibited increases in protein levels of intestinal hydrolases, systemic exposure of H4, and platelet inhibition of vicagrel, but dihydrotestosterone (DHT) reversed these changes in ORX mice and suppressed these changes in OVX mice. Phosphorylated p38 levels were reduced in female or ORX mice but increased in ORX mice by DHT. SB202190 reversed DHT-induced changes observed in ORX mice. We concluded that sex differences exist in metabolic activation of and platelet response to vicagrel in mice through elevation of p38 phosphorylation by androgens, suggesting sex-based vicagrel dosage adjustments for patient care.

Ovariectomy and High Fat-Sugar-Salt Diet Induced Alzheimer's Disease/Vascular Dementia Features in Mice.

While the vast majority of Alzheimer's disease (AD) is non-familial, the animal models of AD that are commonly used for studying disease pathogenesis and development of therapy are mostly of a familial form. We aimed to generate a model reminiscent of the etiologies related to the common late-onset Alzheimer's disease (LOAD) sporadic disease that will recapitulate AD/dementia features. Naïve female mice underwent ovariectomy (OVX) to accelerate aging/menopause and were fed a high fat-sugar-salt diet to expose them to factors associated with increased risk of development of dementia/AD. The OVX mice fed a high fat-sugar-salt diet responded by dysregulation of glucose/insulin, lipid, and liver function homeostasis and increased body weight with slightly increased blood pressure. These mice developed AD-brain pathology (amyloid and tangle pathologies), gliosis (increased burden of astrocytes and activated microglia), impaied blood vessel density and neoangiogenesis, with cognitive impairment. Thus, OVX mice fed on a high fat-sugar-salt diet imitate a non-familial sporadic/environmental form of AD/dementia with vascular damage. This model is reminiscent of the etiologies related to the LOAD sporadic disease that represents a high portion of AD patients, with an added value of presenting concomitantly AD and vascular pathology, which is a common condition in dementia. Our model can, thereby, provide a valuable tool for studying disease pathogenesis and for the development of therapeutic approaches.

Low-dose estrogen release from silastic capsule enhanced flap wound healing in an animal model.

Deep and extensive wounds usually cannot be closed directly by suturing or skin grafting. Flap transplantation is typically used to reconstruct large wounds clinically. The flap survival is based on a stable blood perfusion. It is established that estrogen promotes wound healing and angiogenesis, and regulates the inflammatory response, leading to enhanced flap survival after transplantation. However, estrogen concentrations administered in previous studies were significantly higher than physiological levels, potentially causing systemic side effects. Estrogen-sustained-release silastic capsules can maintain blood serum estrogen closer to physiological levels. This study aimed to investigate whether administering estrogen at a lower concentration, closer to physiological levels, could still enhance flap survival. This study was performed in a random skin flap model in ovariectomized (OVX) mice. Sustained-release estrogen silastic capsules were implanted into OVX mice to determine the functional role of estrogen in wound healing after flap transplantation. Flap blood perfusion was analysed using a colour laser Doppler scanner. Immunohistochemical staining of CD31, hypoxia-inducible factor 1 alpha (HIF-1α), alpha-smooth muscle actin (α-SMA), cleaved caspase 3 and apoptotic terminal dUTP nick end-labelling stain was used to investigate flap angiogenesis, tissue hypoxia, wound healing and cell death in the flap tissue, respectively. We observed that administering estrogen at a lower concentration enhanced superficial blood perfusion while reducing the flap's ischemic area and tissue necrosis. HIF-1α expression was significantly decreased in the dermis layer but not in the fascia, whereas cleaved caspase 3 levels decreased in the fascia but remained unchanged in the dermis. Additionally, there was no significant difference in CD31and α-SMA expression between the groups. In summary, the study showed that an estrogen silastic capsule maintained physiological estrogen levels and improved superficial perfusion, thereby reducing dermal hypoxia, and cell death in a mouse random pattern skin flap model. Although no significant promotion of angiogenesis was observed, the study suggests that appropriate estrogen supplements could enhance flap wound recovery.

Targeting the osteoclastogenic cytokine IL-9 as a novel immunotherapeutic strategy in mitigating inflammatory bone loss in post-menopausal osteoporosis.

Recent discoveries have established the pivotal role of IL-9-secreting immune cells in a wide spectrum of inflammatory and autoimmune diseases. However, little is known about how IL-9 contributes to the etiology of inflammatory bone loss in PMO. We observed that IL-9 has a pathological impact on inflammatory bone loss in ovariectomized (Ovx) mice. Our in vivo temporal kinetics analysis revealed that estrogen deprivation enhanced the production of IL-9 from Th cells (majorly Th9 and Th17). Both our ex vivo and in vivo studies corroborated these findings in Ovx mice, as estrogen diminishes the potential of Th9 cells to produce IL-9. Mechanistically, Th9 cells in an IL-9-dependent manner enhance osteoclastogenesis and thus could establish themselves as a novel osteoclastogenic Th cell subset. Therapeutically neutralizing/blocking IL-9 improves bone health by inhibiting the differentiation and function of osteoclasts, Th9, and Th17 cells along with maintaining gut integrity in Ovx mice. Post-menopausal osteoporotic patients have increased IL-9-secreting Th9 cells, which may suggest a potential role for IL-9 in the development of osteoporosis. Collectively, our study identifies IL-9-secreting Th9 cells as a driver of bone loss with attendant modulation of gut-immune-bone axis, which implies IL-9-targeted immunotherapies as a potential strategy for the management and treatment of inflammatory bone loss observed in PMO.

Endothelial estrogen - myocardial cGMP axis critically determines angiogenesis and cardiac performance during pressure-overload.

Estrogen exerts beneficial cardiovascular effects by binding to specific receptors on various cells to activate nuclear and non-nuclear actions. Estrogen receptor α (ERα) non-nuclear signaling confers protection against heart failure remodeling, involving myocardial cyclic guanosine monophosphate (cGMP) - cGMP-dependent protein kinase G (PKG) activation; however, its tissue-specific role remains elusive. Herein, we examined the cell type-specific role of ERα non-nuclear signaling in estrogen-conferred protection against heart failure. We first assessed the tissue-specific impacts of ERα in estrogen's cardiac benefits, utilizing endothelial ERα deletion (ERαf/f/Tie2Cre+) and myocyte ERα deletion (ERαf/f/αMHCCre+) female mice. Female mice were ovariectomized and the effect of estradiol (E2) was assessed in hearts exposed to 3week pressure-overload (TAC). E2 failed to improve cardiac function in ERαf/f/Tie2Cre+ TAC hearts but provided benefits in ERαf/f/αMHCCre+ TAC hearts, indicating that endothelial ERα is essential. We next assessed the role of non-nuclear signaling in endothelial cells, employing animals with endothelial-specific inactivation of ERα non-nuclear signaling (ERαKI/KI/Tie2Cre+). Female OVX mice were supplemented with E2 and subjected to 3-week TAC. ERαKI/KI/Tie2Cre+ TAC hearts revealed exacerbated cardiac dysfunction and reduced myocardial PKG activity as compared to littermate TAC hearts, which was associated with attenuated myocardial induction of vascular endothelial growth factor (VEGF) and angiogenesis as assessed with CD31-stained capillary density. This phenotype of ERαKI/KI/Tie2Cre+ was rescued by myocardial PKG activation from chronic treatment with soluble guanylate cyclase (sGC) stimulator. We performed co-culture experiments to determine endothelial-cardiomyocyte interactions. VEGF induction by E2 in cardiac myocytes required co-existence of intact endothelial ERα signaling in a NOS-dependent manner. On the other hand, VEGF was induced in myocytes directly with an sGC stimulator in the absence of endothelial cells. An endothelial estrogen - myocardial cGMP axis stimulates angiogenic response and improves cardiac performance during pressure-overload.

Effectiveness of Tri-Kaysorn-Mas Extract in Ameliorating Cognitive-like Behavior Deficits in Ovariectomized Mice via Activation of Multiple Mechanisms.

Postmenopausal women have a higher probability of experiencing cognitive alterations compared to men, suggesting that the decline in female hormones may contribute to cognitive deterioration. Thailand traditionally uses Tri-Kaysorn-Mas (TKM), a blend of three medicinal herbs, as a tonic to stimulate appetite and relieve dyspepsia. Due to its antioxidant and anti-acetylcholinesterase activities, we investigated the effects of TKM (50 and 100 mg/kg/day, p.o., for 8 weeks) on cognitive deficits and their underlying causes in an ovariectomized (OVX) mouse model of menopause. OVX mice showed cognitive impairment in the Y-maze, novel object recognition task (NORT), and Morris water maze (MWM) behavioral tests, along with atrophic changes to the uterus, altered levels of serum 17β-estradiol, and down-regulated expression of estrogen receptors (ERα and ERβ). These behavioral effects were reversed by TKM. TKM decreased malondialdehyde (MDA) levels and mitigated oxidative stress in the brain by enhancing the activity of superoxide dismutase (SOD) and catalase (CAT) and by up-regulating the antioxidant-related gene Nrf2 while down-regulating Keap1. TKM also counteracted OVX-induced neurodegeneration by enhancing the expression of the neurogenesis-related genes BDNF and CREB. The results indicate that TKM extract alleviates oxidative brain damage and neurodegeneration while enhancing cognitive behavior in OVX mice, significantly improving cognitive deficiencies related to menopause/ovariectomy through multiple targets.

Ameliorating Effect of Standardized Rice Bran Supplement on Depressive-Like Behaviors in Ovariectomized Mice.

Menopausal depression, often associated with hormonal fluctuations such as decreased estrogen levels, imposes significant mental health burdens. Despite the antidepressant biological properties of standardized rice bran supplement (RBS), its impact on menopausal depression and underlying mechanisms remains largely unexplored. In this study, we investigated the antidepressant effects of RBS in a mouse model of estrogen deficiency-induced depression. Ovariectomized (OVX) mice received oral doses of RBS (250 and 1000 mg/kg) and 17β estradiol over a 20-week period. RBS administration resulted in decreased immobility time in the tail suspension and forced swim tests, along with increased locomotor activity in the open field test. Furthermore, RBS enhanced nitric oxide production and neuronal nitric oxide synthase (nNOS) expression in the hippocampi of OVX mice. Additionally, RBS administration phosphorylated extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), and tropomyosin receptor kinase B and increased the protein expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. These findings suggest that RBS alleviated depressive behaviors in OVX mice by augmenting hippocampal nNOS expression and activating the ERK-CREB-BDNF signaling pathway. Therefore, based on these results, we propose that RBS is a promising agent to treat menopausal depression, a challenging condition.

Abstract P228: Kdm6a Regulates Extracellular Matrix Genes During Arterial Stiffening and High Blood Pressure in Female Mice

Arterial stiffness measured by pulse wave velocity (PWV) increases steeply during menopause due to estradiol decline. Longitudinal studies show higher PWV in postmenopausal women compared to age-matched men. Previously, we have demonstrated increased PWV in ovariectomized (OVX) and middle-aged female mice. However, the impact of sex chromosomes on PWV is unknown in female mice. We hypothesize that a decline in estradiol unleashes the female sex chromosomes effect that promotes arterial stiffening. This study used four core genotype mice (n=6-10/group) of XX or XY sex chromosomes left intact or OVX for 8 weeks. Arterial stiffening was assessed with pulse wave Doppler and 24-hour blood pressure by radiotelemetry. Carotid artery passive myography and vascular reactivity of 2 nd order mesenteric artery were assessed by pressure myography. To induce hypertension, XX and XY mice were treated with deoxycorticosterone acetate (DOCA; 50 mg/pellet) for 21 days and 1% saline. In vitro, isolated aortic vascular smooth muscle cells (VSMC) were stretched on collagen matrix or treated with Kdm6a inhibitor GSKJ-1. PWV was significantly increased in OVX than in gonadal intact XX and XY mice (P<0.001). However, an interaction effect was indicated with higher PWV in XX than XY OVX mice (4.9±0.1 vs. 4.2±0.2 m/s; P<0.001), indicating a sex chromosome effect. Carotid artery stress-strain curves showed a leftward shift of XX than XY mice, suggesting increased structural stiffness. Masson’s trichrome staining of the aorta showed increased collagen in the adventitia of XX than XY (30% vs. 26%; P=0.005) mice. Higher systolic blood pressure (SBP) was indicated in XX than in XY mice (Baseline; 123±3 vs. 116±4 mmHg; P=0.02). Aortic RNAseq showed increased X-linked genes, including Kdm6a, in XX compared to XY mice. Stretched aortic VSMC showed increased Col1a1 and decreased Itga4 and Lama2 genes reversed by GSKJ-1. DOCA-induced hypertension blunted the PWV difference between XX and XY mice; however, SBP was higher in XX than in XY mice (Week 1: 135±13 vs.127±3 mmHg; P=0.03), but not weeks 2 and 3. We observed impaired acetylcholine-mediated relaxation in hypertensive XX than XY mice (47% vs. 24%; P=0.03). Our data suggested that OVX mice with XX sex chromosomes promote arterial stiffening and high blood pressure, and Kdm6a regulates extracellular matrix genes in XX female VSMC. DOCA-induced hypertension promotes endothelial dysfunction and blunts PWV in ovariectomized XX and XY mice.

METTL14-mediated HOXA5 m6A modification alleviates osteoporosis via promoting WNK1 transcription to suppress NLRP3-dependent macrophage pyroptosis

BackgroundOsteoporosis is a commonly diagnosed metabolic bone disease. NLRP3 inflammasome activation and pyroptosis are observed during osteoporosis. However, the mechanism by which NLRP3-mediated pyroptosis contributes to osteoporosis remains largely undefined. MethodsOvariectomized (OVX) mice were employed as an in vivo model of osteoclastogenesis. H&E staining and micro-CT detected the histological changes and bone parameters in the femur tissues. RANKL-treated macrophages were used as the in vitro model of osteoclastogenesis, and LPS/ATP treatment was used as the macrophage pyroptosis model. The cytotoxicity, cytokine secretion and caspase-1 activity were assessed by LDH release assay, ELISA and flow cytometry, respectively. The osteoclast formation ability was detected by TRAP staining. qRT-PCR, IHC and Western blotting detected the expression and localization of METTL14, pyroptosis-related or osteoclast-specific molecules in femur tissues or macrophages. Mechanistically, MeRIP assessed the m6A modification of HOXA5. Luciferase and ChIP assays were employed to detect the direct association between HOXA5 and WNK1 promoter in macrophages. ResultsMETTL14, HOXA5 and WNK1 were decreased in OVX mice, which was associated with pyroptosis. METTL14 or HOXA5 overexpression suppressed macrophage-osteoclast differentiation and pyroptosis, along with the upregulation of WNK1. METTL14-mediated m6A modification stabilized HOXA5 mRNA and increased its expression, and HOXA5 regulated WNK1 expression via direct binding to its promoter. Functional studies showed that WNK1 knockdown counteracted METTL14- or HOXA5-suppressed pyroptosis and macrophage-osteoclast differentiation. In OVX mice, overexpression of METTL14 or HOXA5 alleviated osteoporosis via suppressing WNK1-dependent NLRP3 signaling. ConclusionMETTL14-mediated HOXA5 m6A modification increased its expression, thereby inducing WNK1 expression and suppressing NLRP3-dependent pyroptosis to alleviate osteoporosis. The combination of METTL14 or HOXA5 agonist with pyroptosis targeted therapy may be a promising therapeutic approach for osteoporosis. The Translational Potential of this Article·•METTL14 or HOXA5 overexpression suppressed macrophage-osteoclast differentiation and pyroptosis in macrophages.·•METTL14-mediated m6A modification stabilized HOXA5 mRNA and increased its expression.•HOXA5 regulated WNK1 expression via direct binding to its promoter.•Silencing of WNK1 reversed METTL14- or HOXA5-suppressed pyroptosis and macrophageosteoclast differentiation.·•METTL14 or HOXA5 overexpression alleviated osteoporosis via suppressing WNK1-dependent NLRP3 signaling in OVX mice.

Ovariectomy and Estradiol Supplementation Prevents Cyclophosphamide- and Doxorubicin-Induced Spatial Memory Impairment in Tumor-Bearing MMTV-PyVT Mice.

Chemotherapy-related cognitive impairments (CRCIs) encompass cognitive deficits in memory, attention, and executive function that arise during and following chemotherapy. CRCI symptoms are predominantly reported by female cancer patients but also occur in males. These impairments may involve reduced estradiol levels, which then increases vulnerability to the impact of tumors and chemotherapy on cognition. This study utilized the MMTV-PyVT mouse model of breast cancer to test the hypothesis that impaired ovarian function and associated estradiol levels play a critical role in CRCI susceptibility. Mice were either ovariectomized (OVX) or underwent sham surgery. The OVX group then received supplemental estradiol (E2) ad libitum in the drinking water to maintain physiological hormone levels. After tumor development, mice were trained in the Morris water maze to assess spatial memory, and subsequently, they received weekly injections of either saline or a combination of cyclophosphamide (CYP; 66.7 mg/kg, i.v.) and doxorubicin (DOX; 6.7 mg/kg, i.v.) for 4 weeks. Spatial memory was reassessed 10 d and then 35 d, after the final injections. Results demonstrated a significant disruption of normal ovarian cycling in sham-operated mice treated with CYP + DOX, as well as significant spatial memory impairments when compared with OVX mice supplemented with E2 This study suggests that chemotherapy-induced ovarian damage and the consequent drop in circulating estrogens significantly contribute to vulnerability to CRCIs, underscoring the importance of estradiol in mitigating CRCI risks.