Cytoprotective Potential of Fucoxanthin in Oxidative Stress-Induced Age-Related Macular Degeneration and Retinal Pigment Epithelial Cell Senescence In Vivo and In Vitro.

Oxidative stress is identified as a major inducer of retinal pigment epithelium (RPE) cell dysregulation and is associated with age-related macular degeneration (AMD). The protection of RPE disorders plays an essential role in the pathological progress of retinal degeneration diseases. The pharmacological functions of fucoxanthin, a characteristic carotenoid, including anti-inflammatory and antioxidant properties, may ameliorate an outstanding bioactivity against premature senescence and cellular dysfunction. This study demonstrates that fucoxanthin protects RPE cells from oxidative stress-induced premature senescence and decreased photoreceptor cell loss in a sodium iodate-induced AMD animal model. Similarly, oxidative stress induced by hydrogen peroxide, nuclear phosphorylated histone (γH2AX) deposition and premature senescence-associated ß-galactosidase staining were inhibited by fucoxanthin pretreatment in a human RPE cell line, ARPE-19 cells. Results reveal that fucoxanthin treatment significantly inhibited reactive oxygen species (ROS) generation, reduced malondialdehyde (MDA) concentrations and increased the mitochondrial metabolic rate in oxidative stress-induced RPE cell damage. Moreover, atrophy of apical microvilli was inhibited in cells treated with fucoxanthin after oxidative stress. During aging, the RPE undergoes well-characterized pathological changes, including amyloid beta (Aß) deposition, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) expression and tight junction disruption, which were also reduced in fucoxanthin-treated groups by immunofluorescence. Altogether, pretreatment with fucoxanthin may protect against premature senescence and cellular dysfunction in retinal cells by oxidative stress in experimental AMD animal and human RPE cell models.

Cu2O/PEDOT:PSS/ZnO Nanocomposite Material Biosensor for Esophageal Cancer Detection.

A highly sensitive photoelectrochemical (PEC) biosensor without external bias was developed in this study. The biosensor was configured with a p-Cu2O and n-ZnO heterostructure. Hexamethylenetetramine (HMTA) and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) was used to improve the crystal structure of Cu2O and ZnO and reduce the defects in the Cu2O/ZnO interface. This fabrication method provided the highly crystallized Cu2O/ZnO structure with excellent electrical property and photoresponse in visible light. The structure was applied to a biosensor for detecting two different cancerous levels of esophageal cells, namely, OE21 and OE21-1, with a high gain in photocurrent (5.8 and 6.2 times, respectively) and a low detection limit (3000 cells in 50 µL). We believe that such a p-n heterojunction PEC biosensor could advance biosensor development and provide a promising candidate for biomedical applications.

Protective Effects of Fucoxanthin on Ultraviolet B-Induced Corneal Denervation and Inflammatory Pain in a Rat Model.

Fucoxanthin is a carotenoid with many pharmaceutical properties that is found in brown seaweed. However, the effects of fucoxanthin on corneal innervation and intense eye pain have not been extensively examined. To clarify the protective roles and underlying mechanisms of fucoxanthin on ocular lesions, we investigated the beneficial effects and mechanisms by which fucoxanthin ameliorates ultraviolet B (UVB)-induced corneal denervation and trigeminal pain. Treatment with fucoxanthin enhanced the expression of nuclear factor erythroid 2-related factor 2 in the cornea. Inhibition of typical denervation and epithelial exfoliation in the cornea were observed in rats treated with fucoxanthin following UVB-induced nerve disorders. Moreover, the active phosphorylated form of p38 MAP kinase (pp38) and the number of glial fibrillary acidic protein (GFAP)-positive neural cells were significantly reduced. Decreased expression of neuron-selective transient receptor potential vanilloid type 1 (TRPV1) in the trigeminal ganglia neurons was also demonstrated in rats treated with fucoxanthin after UVB-induced keratitis. Symptoms of inflammatory pain, including difficulty in opening the eyes and eye wipe behaviour, were also reduced in fucoxanthin-treated groups. Pre-treatment with fucoxanthin may protect the eyes from denervation and inhibit trigeminal pain in UVB-induced photokeratitis models.

Inhibition of Ultraviolet B-Induced Expression of the Proinflammatory Cytokines TNF-α and VEGF in the Cornea by Fucoxanthin Treatment in a Rat Model.

Ultraviolet B (UVB) irradiation is the most common cause of radiation damage to the eyeball and is a risk factor for human corneal damage. We determined the protective effect of fucoxanthin, which is a carotenoid found in common edible seaweed, on ocular tissues against oxidative UVB-induced corneal injury. The experimental rats were intravenously injected with fucoxanthin at doses of 0.5, 5 mg/kg body weight/day or with a vehicle before UVB irradiation. Lissamine green for corneal surface staining showed that UVB irradiation caused serious damage on the corneal surface, including severe epithelial exfoliation and deteriorated epithelial smoothness. Histopathological lesion examination revealed that levels of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF), significantly increased. However, pretreatment with fucoxanthin inhibited UVB radiation-induced corneal disorders including evident preservation of corneal surface smoothness, downregulation of proinflammatory cytokine expression, and decrease of infiltrated polymorphonuclear leukocytes from UVB-induced damage. Moreover, significant preservation of the epithelial integrity and inhibition of stromal swelling were also observed after UVB irradiation in fucoxanthin-treated groups. Pretreatment with fucoxanthin may protect against UVB radiation-induced corneal disorders by inhibiting expression of proinflammatory factors, TNF-α, and VEGF and by blocking polymorphonuclear leukocyte infiltration.

Human Umbilical Mesenchymal Stem Cell Xenografts Repair UV-Induced Photokeratitis in a Rat Model.

Most patients with a corneal injury are administered anti-inflammatory medications and antibiotics, but no other treatments are currently available. Thus, the corneal injury healing is unsatisfactory, affects the vision, and has a risk of blindness in severe cases. Human umbilical mesenchymal stem cells exhibit pluripotent and anti-inflammatory properties and do not cause immunological rejection in the host. Rats were irradiated with type B ultraviolet (UVB) light to generate a stable animal model of photokeratitis. After irradiation-induced photokeratitis, human umbilical mesenchymal stem cells were implanted into the subconjunctival space of the lateral sclera, and the changes in the corneal pathology were evaluated. Three weeks after implantation, many mesenchymal stem cells were visible in the subconjunctival space. These mesenchymal stem cells effectively reduced the extent of injury to the adjacent corneal tissue. They accelerated the epithelial layer repair, reduced the inflammatory response and neovascularization, and improved the disorganization of collagen and fibronectin in the corneal stroma caused by the injury. In conclusion, xenografted human umbilical mesenchymal stem cells can survive in rat eye tissues for a long time, effectively support the structural integrity of injured corneal tissues, restore corneal permeability, and reduce abnormal neovascularization. This study provides a new approach to the treatment of photokeratitis.

7,8-Dihydroxyflavone accelerates recovery of Brown-Sequard syndrome in adult female rats with spinal cord lateral hemisection.

BACKGROUND: 7,8-Dihydroxyflavone (DHF) mimicks the physiological action of brain-derived neurotrophic factor (BDNF). Since local BDNF delivery to the injured spinal cord enhanced diaphragmatic respiratory function, we aimed to ascertain whether DHF might have similar beneficial effects after Brown-Sequard Syndrome in a rat model of spinal cord lateral hemisection (HX) at the 9th thoracic (T9) vertebral level. METHODS: Three sets of adult female rats were included: sham+vehicle group, T9HX+vehicle group and T9HX+DHF group. On the day of surgery, HX+DHF group received DHF (5 mg/kg) while HX+vehicle group received vehicle. Neurobehavioral function, morphology of motor neurons innervating the tibialis anterior muscle and the transmission in descending motor pathways were evaluated. RESULTS: Adult female rats received T9 HX had paralysis and loss of proprioception on the same side as the injury and loss of pain and temperature on the opposite side. We found that, in this model of Brown-Sequard syndrome, reduced cord dendritic arbor complexity, reduced cord motoneuron numbers, enlarged cord lesion volumes, reduced motor evoked potentials, and cord astrogliosis and microgliosis were noted after T9HX. All of the above-mentioned disorders showed recovery by Day 28 after surgery. Therapy with DHF significantly accelerated the electrophysiological, histological and functional recovery in these T9HX animals. CONCLUSIONS: Our data provide a biological basis for DHF as a neurotherapeutic agent to improve recovery after a Brown-Sequard syndrome. Such an effect may be mediated by synaptic plasticity and glia-mediated inflammation in the spared lumbar motoneuron pools to a T9HX.

Neuronal degeneration in autonomic nervous system of Dystonia musculorum mice.

BACKGROUND: Dystonia musculorum (dt) is an autosomal recessive hereditary neuropathy with a characteristic uncoordinated movement and is caused by a defect in the bullous pemphigoid antigen 1 (BPAG1) gene. The neural isoform of BPAG1 is expressed in various neurons, including those in the central and peripheral nerve systems of mice. However, most previous studies on neuronal degeneration in BPAG1-deficient mice focused on peripheral sensory neurons and only limited investigation of the autonomic system has been conducted. METHODS: In this study, patterns of nerve innervation in cutaneous and iridial tissues were examined using general neuronal marker protein gene product 9.5 via immunohistochemistry. To perform quantitative analysis of the autonomic neuronal number, neurons within the lumbar sympathetic and parasympathetic ciliary ganglia were calculated. In addition, autonomic neurons were cultured from embryonic dt/dt mutants to elucidate degenerative patterns in vitro. Distribution patterns of neuronal intermediate filaments in cultured autonomic neurons were thoroughly studied under immunocytochemistry and conventional electron microscopy. RESULTS: Our immunohistochemistry results indicate that peripheral sensory nerves and autonomic innervation of sweat glands and irises dominated degeneration in dt/dt mice. Quantitative results confirmed that the number of neurons was significantly decreased in the lumbar sympathetic ganglia as well as in the parasympathetic ciliary ganglia of dt/dt mice compared with those of wild-type mice. We also observed that the neuronal intermediate filaments were aggregated abnormally in cultured autonomic neurons from dt/dt embryos. CONCLUSIONS: These results suggest that a deficiency in the cytoskeletal linker BPAG1 is responsible for dominant sensory nerve degeneration and severe autonomic degeneration in dt/dt mice. Additionally, abnormally aggregated neuronal intermediate filaments may participate in neuronal death of cultured autonomic neurons from dt/dt mutants.

Aircraft noise, like heat stress, causes cognitive impairments via similar mechanisms in male mice.

To our knowledge, little evidence is available about effects of aircraft noise (AN), a non-chemical stressor, on cognitive function. Again, it is unknown whether or not the heat stress (HS)-induced cognitive deficits can be exacerbated by AN. The adult male mice were assigned to four groups: group 1 mice exposed to non-HS (24-26 °C 2 h daily for 4 consecutive days) and white noise (WN) (2 h daily for 4 consecutive days), group 2 mice exposed to WN and HS (32-34 °C 2 h daily for 4 consecutive days), group 3 mice exposed to AN and non-HS (2 h daily for 4 consecutive days) and group 4 mice exposed to AN and HS (2 h daily for consecutive 4 days). Cognitive function were determined by passive avoidance, Y-maze, Morris water maze, and novel object recognition tests. Gut barrier and blood-brain-barrier (BBB) permeability, upload of lipopolysaccharide (LPS) translocation, systemic and central inflammation, and stress reactions were examined. Heat stressed mice displayed both increased stress reactions and learning and memory loss. Heat stress also caused gut barrier hyperpermeability, increased upload of LPS translocation, systemic inflammation, BBB disruption and hippocampal neuroinflammation. Aircraft noise stressed mice did not display systemic inflammation but caused gut barrier hyperpermeability, increased upload of LPS translocation, increased stress reactions, BBB disruption, hippocampal neuroinflammation and cognitive deficits. Aircraft noise exposure further exacerbated the heat stress-induced cognitive deficits and its complications. Our data suggest that AN, like HS, causes cognitive impairments via similar mechanisms in male mice.

Protective Effects of Fucoxanthin Dampen Pathogen-Associated Molecular Pattern (PAMP) Lipopolysaccharide-Induced Inflammatory Action and Elevated Intraocular Pressure by Activating Nrf2 Signaling and Generating Reactive Oxygen Species.

Inflammation and oxidative stress are closely related processes in the pathogenesis of various ocular diseases. Uveitis is a disorder of the uvea and ocular tissues that causes extreme pain, decreases visual acuity, and can eventually lead to blindness. The pharmacological functions of fucoxanthin, isolated from brown algae, induce a variety of therapeutic effects such as oxidative stress reduction and repression of inflammation reactions. However, the specific anti-inflammatory effects of fucoxanthin on pathogen-associated molecular pattern (PAMP) lipopolysaccharide-induced uveitis have yet to be extensively described. Therefore, the aim of present study was to investigate the anti-inflammatory effects of fucoxanthin on uveitis in rats. The results showed that fucoxanthin effectively enhanced the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in ocular tissues. Furthermore, fucoxanthin significantly increased the ocular activities of superoxide dismutase and decreased the levels of malondialdehyde stimulated by PAMP-induced uveitis. Ocular hypertension and the levels of inflammatory cells and proinflammatory cytokine tumor necrosis factor-alpha in the aqueous humor were alleviated with fucoxanthin treatment. Consequently, compared to the observed effects in lipopolysaccharide groups, fucoxanthin treatment significantly preserved iris sphincter innervation and pupillary function. Additionally, PAMP-induced corneal endothelial disruption was significantly inhibited by fucoxanthin treatment. Overall, these findings suggest that fucoxanthin may protect against inflammation from PAMP-induced uveitis by promoting the Nrf2 pathway and inhibiting oxidative stress.

Blocking the Spinal Fbxo3/CARM1/K+ Channel Epigenetic Silencing Pathway as a Strategy for Neuropathic Pain Relief.

Many epigenetic regulators are involved in pain-associated spinal plasticity. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic regulator of histone arginine methylation, is a highly interesting target in neuroplasticity. However, its potential contribution to spinal plasticity-associated neuropathic pain development remains poorly explored. Here, we report that nerve injury decreased the expression of spinal CARM1 and induced allodynia. Moreover, decreasing spinal CARM1 expression by Fbxo3-mediated CARM1 ubiquitination promoted H3R17me2 decrement at the K+ channel promoter, thereby causing K+ channel epigenetic silencing and the development of neuropathic pain. Remarkably, in naïve rats, decreasing spinal CARM1 using CARM1 siRNA or a CARM1 inhibitor resulted in similar epigenetic signaling and allodynia. Furthermore, intrathecal administration of BC-1215 (a novel Fbxo3 inhibitor) prevented CARM1 ubiquitination to block K+ channel gene silencing and ameliorate allodynia after nerve injury. Collectively, the results reveal that this newly identified spinal Fbxo3-CARM1-K+ channel gene functional axis promotes neuropathic pain. These findings provide essential insights that will aid in the development of more efficient and specific therapies against neuropathic pain.

Hyperspectral Ophthalmoscope Images for the Diagnosis of Diabetic Retinopathy Stage.

A methodology that applies hyperspectral imaging (HSI) on ophthalmoscope images to identify diabetic retinopathy (DR) stage is demonstrated. First, an algorithm for HSI image analysis is applied to the average reflectance spectra of simulated arteries and veins in ophthalmoscope images. Second, the average simulated spectra are categorized by using a principal component analysis (PCA) score plot. Third, Beer-Lambert law is applied to calculate vessel oxygen saturation in the ophthalmoscope images, and oxygenation maps are obtained. The average reflectance spectra and PCA results indicate that average reflectance changes with the deterioration of DR. The G-channel gradually decreases because of vascular disease, whereas the R-channel gradually increases with oxygen saturation in the vessels. As DR deteriorates, the oxygen utilization of retinal tissues gradually decreases, and thus oxygen saturation in the veins gradually increases. The sensitivity of diagnosis is based on the severity of retinopathy due to diabetes. Normal, background DR (BDR), pre-proliferative DR (PPDR), and proliferative DR (PDR) are arranged in order of 90.00%, 81.13%, 87.75%, and 93.75%, respectively; the accuracy is 90%, 86%, 86%, 90%, respectively. The F1-scores are 90% (Normal), 83.49% (BDR), 86.86% (PPDR), and 91.83% (PDR), and the accuracy rates are 95%, 91.5%, 93.5%, and 96%, respectively.

A possible cellular mechanism of neuronal loss in the dorsal root ganglia of Dystonia musculorum (dt) mice.

Dystonia musculorum (dt) is a mutant mouse with hereditary sensory neuropathy. A defective bullous pemphigoid antigen 1 (BPAG1) gene is responsible for this mutation. In the present study, we examined the distribution of neuronal intermediate filament proteins in the central and peripheral processes of the dorsal root ganglia (DRG) in adult dt mice using different approaches. We found that not only BPAG1, but also alpha-internexin was absent in the DRG neurons in adult dt mice. To study the relationship between the absence of alpha-internexin and the progressive neuronal loss in the DRG of dt mice, we further cultured DRG neurons from embryonic dt mutants. Immunocytochemical assay of cultured DRG neurons from dt embryos revealed that alpha-internexin was aggregated in the proximal region of axons and juxtanuclear region of the cytoplasma, yet the other intermediate filament proteins were widely distributed in all processes. The active caspase-3 activity was observed in the dt neuron with massive accumulation of alpha-internexin. From our observations, we suggest that the interaction between BPAG1 and alpha-internexin may be one of the key factors involved in neuronal degeneration, and abnormal accumulation of alpha-internexin may impair the axonal transport and subsequently turns on the cascade of neuronal apoptosis in dt mice.

The Therapeutic Potential of Human Umbilical Mesenchymal Stem Cells From Wharton's Jelly in the Treatment of Rat Peritoneal Dialysis-Induced Fibrosis.

A major complication in continuous, ambulatory peritoneal dialysis in patients with end-stage renal disease who are undergoing long-term peritoneal dialysis (PD) is peritoneal fibrosis, which can result in peritoneal structural changes and functional ultrafiltration failure. Human umbilical mesenchymal stem cells (HUMSCs) in Wharton's jelly possess stem cell properties and are easily obtained and processed. This study focuses on the effects of HUMSCs on peritoneal fibrosis in in vitro and in vivo experiments. After 24-hour treatment with mixture of Dulbecco's modified Eagle's medium and PD solution at a 1:3 ratio, primary human peritoneal mesothelial cells became susceptible to PD-induced cell death. Such cytotoxic effects were prevented by coculturing with primary HUMSCs. In a rat model, intraperitoneal injections of 20 mM methylglyoxal (MGO) in PD solution for 3 weeks (the PD/MGO 3W group) markedly induced abdominal cocoon formation, peritoneal thickening, and collagen accumulation. Immunohistochemical analyses indicated neoangiogenesis and significant increase in the numbers of ED-1- and α-smooth muscle actin (α-SMA)-positive cells in the thickened peritoneum in the PD/MGO 3W group, suggesting that PD/MGO induced an inflammatory response. Furthermore, PD/MGO treatment for 3 weeks caused functional impairments in the peritoneal membrane. However, in comparison with the PD/MGO group, intraperitoneal administration of HUMSCs into the rats significantly ameliorated the PD/MGO-induced abdominal cocoon formation, peritoneal fibrosis, inflammation, neoangiogenesis, and ultrafiltration failure. After 3 weeks of transplantation, surviving HUMSCs were found in the peritoneum in the HUMSC-grafted rats. Thus, xenografts of HUMSCs might provide a potential therapeutic strategy in the prevention of peritoneal fibrosis. Significance: This study demonstrated that direct intraperitoneal transplantation of human umbilical mesenchymal stem cells into the rat effectively prevented peritoneal dialysis/methylglyoxal-induced abdominal cocoon formation, ultrafiltration failure, and peritoneal membrane alterations such as peritoneal thickening, fibrosis, and inflammation. These findings provide a basis for a novel approach for therapeutic benefits in the treatment of encapsulating peritoneal sclerosis.

Characterising visual deficits in children of an urban elementary school in Taiwan.

PURPOSE: The aim was to screen children from Grades 1 to 6 in an urban elementary school in Central Taiwan for visual deficits and associated parameters and, as an extension, to examine the acceptance of cycloplegic therapy as well as the lag in optimal vision correction. METHODS: Of 900 students in one school, 731 participated in the study, with parental consent. Data from 694 students, who had also completed a vision correction history were analysed. In addition to body height and weight, the screening included vision, non-cycloplegic autorefraction and distance retinoscopy, axial length and functional testing. RESULTS: There was a decrease in students with vision of 1.0 or better from 55.8 per cent in Grade 1 to 20.0 per cent in Grade 6. The decreases between Grades 2 and 3 and Grades 5 and 6 were significant. These trends were in general agreement with those based on refractive error and axial length. The students had abnormal functional findings including: stereoscopic vision, 9.2 per cent; cover tests, 14.1 per cent; pupillary responses, 13.8 per cent; and less commonly in extraocular muscular functions (3.0 per cent) and colour vision (5.2 per cent). A full 40 per cent of students received cycloplegic therapy with 25 per cent dropping out for various reasons. These cases were generally associated with lower vision and higher myopia. A lag between subnormal vision and optical correction was also observed with 55.1 per cent apparently not optimally corrected. Other parameters, including body height, weight and body mass index were not correlated with vision or refractive error. CONCLUSIONS: Age-dependent increase in the prevalence of myopia appears to continue despite the common practice of topical cycloplegic therapy in Taiwan. Timely correction of the refractive error is also lacking. While maintaining a visual acuity of 1.0 or better for all students at all times is not possible, this lag might be shortened by more frequent screening and/or direct provision of optical aids.

Abnormal cellular translocation of alpha-internexin in spinal motor neurons of Dystonia musculorum mice.

Dystonia musculorum (dt) is a mutant mouse with hereditary neuropathy where the dysfunction is mainly found in the dorsal root ganglia (DRG) neurons but not in the spinal motor neurons. However, the accumulation of intermediate filament (IF) proteins in the swelling axons of spinal motor neurons could be found in dt/dt mice. In order to understand the pathological role of neuronal IFs in the swelling axons of spinal motor neurons from dt/dt mice, we extensively examined the distribution of neuronal IF proteins. By immunofluorescence staining, our results indicated that alpha-internexin was a major component in the swelling axon and showed abnormal translocation in the nuclei of spinal motor neurons in dt/dt mice. This abnormal translocation of alpha-internexin in the nuclei of spinal motor neurons was also confirmed by Western blotting and immunoelectron microscopy. Instead of the 10-nm filamentous structure, a diffuse immunopositive pattern of alpha-internexin was observed in the nucleus of spinal motor neurons in dt/dt mutants. We further examined the cell death of spinal motor neurons by TUNEL assay, and no TUNEL-positive cells could be identified from spinal motor neurons in dt/dt mice. From these observations we suggest that abnormal accumulation of neuronal IFs in the swelling axons and abnormal translocation of alpha-internexin in the nuclei of the spinal motor neurons from dt/dt mice may not directly cause cell death of the spinal motor neurons.

Diosgenin induces hypoxia-inducible factor-1 activation and angiogenesis through estrogen receptor-related phosphatidylinositol 3-kinase/Akt and p38 mitogen-activated protein kinase pathways in osteoblasts.

Diosgenin, extracted from the root of wild yam (Dioscorea villosa), has been reported to demonstrate an opportunity for medical application. Vascular endothelial growth factor-A (VEGF-A) plays an important role in bone-related angiogenesis, a critical process occurring during bone formation and fracture healing. In this study, we examine whether diosgenin is able to induce VEGF-A expression and to promote angiogenesis in osteoblasts. For murine MC3T3-E1 preosteoblast-like cells, VEGF-A mRNA and protein expression seemed to be significantly elevated in response to diosgenin in a concentration-dependent fashion. Conditioned media prepared from cells treated with diosgenin induced strong angiogenic activity in either in vitro or ex vivo angiogenesis assay. Furthermore, diosgenin treatment increased the stability and activity of HIF-1alpha protein. Inhibition of HIF-1alpha activity by transfection with DN-HIF-1alpha significantly diminished diosgenin-mediated VEGF-A up-regulation. The use of pharmacological inhibitors or genetic inhibition revealed that both the phosphatidylinositol 3-kinase (PI3K)/Akt and p38 signaling pathways were potentially required for diosgenin-induced HIF-1 activation and subsequent VEGF-A up-regulation. It is noteworthy that an estrogen receptor binding assay revealed that diosgenin has the strong ability to replace [(3)H]estradiol bound to estrogen receptor (IC(50), 10 nM). In addition, the specific estrogen receptor antagonists ICI 182,780 (faslodex) and tamoxifen were noted to be able to strongly inhibit diosgenin-induced, src kinase-dependent Akt and p38 MAPK activation. Taken together, such results provide evidence that diosgenin up-regulates VEGF-A and promotes angiogenesis in preosteoblast-like cells by a hypoxia-inducible factor-1alpha-dependent mechanism involving the activation of src kinase, p38 MAPK, and Akt signaling pathways via estrogen receptor.