Exploration of beauvericin's toxic effects and mechanisms in human astrocytes and N-acetylcysteine's protective role.

Beauvericin (BEA) is a newly identified mycotoxin produced by various Fusarium species, and its contamination in food and animal feed is widespread globally. This mycotoxin demonstrates cytotoxic effects by inducing oxidative stress in multiple models. Furthermore, evidence indicates that BEA possesses diverse toxic activities, making it a promising candidate for toxicological research. Recent studies have highlighted the ability of BEA to traverse the blood-brain barrier, suggesting its potential neurotoxicity. However, limited information is available regarding the neurotoxic effects of BEA on human astrocytes. Therefore, this study aimed to assess the neurotoxic effects of BEA on the Gibco® Human Astrocyte (GHA) cell line and elucidate the underlying mechanisms. Additionally, the study aimed to investigate the protective effects of the antioxidant N-acetylcysteine (NAC) against BEA-induced toxicity. The data show that exposure to BEA within the 2.5-15 µM concentration range resulted in concentration-dependent cytotoxicity. BEA-treated cells exhibited significantly increased levels of reactive oxygen species (ROS), while intracellular glutathione (GSH) content was significantly reduced. Western blot analysis of cells treated with BEA revealed altered protein levels of Bax, cleaved caspase-9, and caspase-3, along with an increased Bax/Bcl-2 ratio, indicating the induction of apoptosis. Additionally, BEA exposure triggered antioxidant responses, as evidenced by increased protein expression of Nrf2, HO-1, and NQO1. Significantly, pretreatment with NAC partially attenuated the significant toxic effects of BEA. In conclusion, our findings suggest that BEA-induced cytotoxicity in GHA cells involves oxidative stress-associated apoptosis. Furthermore, NAC demonstrates potential as a protective agent against BEA-induced oxidative damage.

Dapagliflozin prevents ERK activation and SGLT2-dependent endoglin upregulation in a mechanically provoked cardiac injury model.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are rapidly gaining ground in the treatment of heart failure (HF) with reduced ejection fraction (HFrEF) and acute myocardial infarction (AMI) by an unknown mechanism. Upregulation of Na+/H+ exchanger 1 (NHE1), SGLT1, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the diseased hearts was found to be attenuated by prolonged SGLT2i treatment. Unfortunately, dapagliflozin is not well understood as to how Na+/Ca2+ homeostasis is affected in cardiomyocytes. In this study, we aimed to investigate whether mechanical stretch in cardiomyocytes upregulate SGLT2, resulted to loss of Na+/Ca2+ homeostasis via ERK and eNOS signaling. AMI (+) and AMI (-) serum levels were estimated using ELISA assays of TGFß-1 or endoglin (CD105). Human cardiomyocyte cell line AC16 was subjected to different stresses: 5% mild and 25% aggressive, at 1 Hz for 24 h. Immunofluorescence assays were used to estimate troponin I, CD105, SGLT1/2, eNOSS633, and ERK1/2T202/Y204 levels was performed for 5% (mild), and 25% elongation for 24 h. AMI (+) serum showed increased TGFß1 and CD105 compared to AMI (-) patients. In consistent, troponin I, CD105, SGLT1/2, eNOSS633 and ERK1/2T202/Y204 were upregulated after 25% of 24 h cyclic stretch. Dapagliflozin addition caused SGLT2 inhibition, which significantly decreased troponin I, CD105, SGLT1/2, eNOSS633, and ERK1/2T202/Y204 under 25% cyclic stretching. In summary, SGLT2 may have sensed mechanical stretch in a way similar to cardiac overloading as in vivo. By blocking SGLT2 in stretched cardiomyocytes, the AMI biomarkers (CD105, troponin I and P-ERK) were decreased, potentially to rescue eNOS production to maintain normal cellular function. This discovery of CD105 and SGLT2 increase in mechanically stretched cardiomyocytes suggests that SGLT2 may conceive a novel role in direct or indirect sensing of mechanical stretch, prompting the possibility of an in vitro cardiac overloaded cell model, an alternative to animal heart model.

Microglial activation and toll-like receptor 4-Dependent regulation of angiotensin II type I receptor-mu-opioid receptor 1 heterodimerization and hypertension in fructose-fed rats.

Our previous study reported that the heterodimer of Angiotensin II Type I Receptor (AT1R) and Mu-Opioid Receptor 1 (MOR1) involves Nitric Oxide (NO) reduction which leads to elevation of blood pressure. Secondly, we showed that Toll-like Receptor 4 (TLR4) may be involved in the heterodimerization of AT1R and MOR1 in the brainstem Nucleus Tractus Solitarii (NTS), which regulates systemic blood pressure and gastric nitric oxide through the insulin pathway. Here, we investigated the role of microglial activation and TLR4 in the heterodimerization of AT1R and MOR1. Hypertensive rats were established after four weeks of fructose consumption. SBP of rats was measured using non-invasive blood pressure method. PLA technique was utilized to determine protein-protein interaction in the nucleus tractus solitarii. Results showed that the level of MOR-1 and AT1R was induced significantly in the fructose group compared with control. PLA signal potentially showed that AT1R and MOR1 were formed in the nucleus tractus solitarii after fructose consumption. Meanwhile, the innate immune cell in the CNS microglia was observed in the nucleus tractus solitarii using biomarkers and was activated. TLR4 inhibitor CLI-095, was administered to animals to suppress the neuroinflammation and microglial activation. CLI-095 treatment reduced the heterodimer formation of AT1R and MOR1 and restored nitric oxide production in the nucleus tractus solitarii. These findings imply that TLR4-primed neuroinflammation involves formation of heterodimers AT1R and MOR1 in the nucleus tractus solitarii which leads to increase in systemic blood pressure.

Attenuation of epithelial-mesenchymal transition via SGLT2 inhibition and diabetic cataract suppression by dapagliflozin nanoparticles treatment.

AIMS: Chronic hyperglycemia triggers overproduction of AKR1B1 (aldo-keto reductase family 1 member B) and receptor for advanced glycation end product (RAGE), which causes epithelial-mesenchymal transition (EMT) in the lens epithelial cells (LECs) of diabetic mellitus (DM) cataracts. However, it is unclear whether EMT in LECs is related to abnormal increase of SGLT2. Sodium glucose cotransporter 2 (SGLT2) inhibitor, also known as dapagliflozin (Dapa) can be used to treat diabetes. Here, we examined how Dapa or nano eye-drops (DapaN) reduce EMT in LECs of DM cataracts. The nano eye-drop provides an ophthalmic treatment that suppressed diabetic cataract progression and improved potency with reduced side effects. MAIN METHODS: SD rats were injected with streptozocin (STZ) (65 mg/kg, ip), nano-Dapa drops (0.456 mg/10 ml/eye) or Dapa (1.2 mg/kg/day) treatment for 6-12 weeks. Immunofluorescence staining was used for protein quantification of RAGE, SGLT2, N-cadherin and E-cadherin in the LECs of rats. KEY FINDINGS: In this study, Dapa applies nanotechnology-based delivery system and it contains polyvinylpyrrolidone (PVP) and HPBCD. Dapa showed therapeutic effect on DM cataracts, wherein it targeted EMT biomarker, E-cadherin. The nano-Dapa drops or oral Dapa inhibited SGLT2, suppressed AKR1B1 expression, decreased AcSOD2- and RAGE-induced EMT in diabetic cataracts. Our findings suggest that nanotechnology-based Dapa eye drops (Dapa-PVP-HPBCD) can effectively improve solubility of Dapa in aqueous solution. SIGNIFICANCE: Taken together, results suggest that the SGLT2-mediated DM cataract therapy may involve the AKR1B1-RAGE-AcSOD2-EMT pathway. The nano eye drops and Dapa show potential beneficial effects for cataract prevention. This study conveys new insights into cataract treatment and supplementation of nano-Dapa drops shows promising result in preventing diabetic cataracts.

Hyperlactatemia is associated with increased risks of long-term mortality and major adverse cardiovascular events in sepsis survivors.

INTRODUCTION: Serum lactate is a potentially valuable biomarker for risk assessment for patients with sepsis, as hyperlactatemia is associated with elevated short-term mortality risks. However, the associations between hyperlactatemia and long-term clinical outcomes in sepsis survivors remain unknown. The objective of this study was to investigate whether hyperlactatemia at the time of hospitalisation for sepsis was associated with worse long-term clinical outcomes in sepsis survivors. METHODS: In total, of 4983 sepsis survivors aged ≥ 20 years were enrolled in this study between January 1, 2012, and December 31, 2018. They were divided into low (≤18 mg/dL; n = 2698) and high (>18 mg/dL; n = 2285) lactate groups. The high lactate group was then matched 1:1 by propensity-score method to the low lactate group. The outcomes of interest were all-cause mortality, major adverse cardiac events (MACEs), ischaemic stroke, myocardial infarction, hospitalisation for heart failure, and end-stage renal disease. RESULTS: After propensity score matching, the high lactate group had greater risks of all-cause mortality (hazard ratio [HR] 1.54, 95% confidence interval [CI] 1.41-1.67), MACEs (HR 1.53, 95% CI 1.29-1.81), ischaemic stroke (HR 1.47, 95% CI 1.19-1.81), myocardial infarction (HR 1.52, 95% CI 1.17-1.99), and end-stage renal disease (HR 1.42, 95% CI 1.16-1.72). Subgroup analyses stratified by baseline renal function revealed almost similarity across groups. CONCLUSION: We found that hyperlactatemia is associated with long-term risks of mortality and MACEs in sepsis survivors. Physicians may consider more aggressive and prompter management of sepsis in patients who present with hyperlactatemia to improve long-term prognoses.

Blocking of SGLT2 to Eliminate NADPH-Induced Oxidative Stress in Lenses of Animals with Fructose-Induced Diabetes Mellitus.

Chronic hyperglycemia triggers an abnormal rise in reactive oxygen species (ROS) that leads to blindness in patients with diabetes mellitus (DM) and cataracts. In this study, the effects of dapagliflozin, metformin and resveratrol on ROS production were investigated in lens epithelial cells (LECs) of animals with fructose-induced DM. LECs were isolated from patients without DM, or with DM devoid of diabetic retinopathy. Animals were treated with 10% fructose for 8 weeks to induce DM, which was verified by monitoring blood pressure and serum parameters. For drug treatments, 1.2 mg/day of dapagliflozin was given for 2 weeks, 500 mg/kg/day of metformin was given, and 10 mg/kg/day of resveratrol was given. Dihydroethidium was used to stain endogenous O2˙- production in vivo of the LECs. Superoxide production was expressed in the cataract of DM, or patients without DM. Sodium-glucose cotransporter 2 (SGLT2), glucose transporter 1 (GLUT1), GLUT5, the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p47/p67-phox, NOX4 and RAGE were significantly increased in LECs with DM. In addition, the dapagliflozin treatment reduced GLUT5, p47/p67-phox, NADPH oxidase 4 (NOX4) and receptor for advanced glycation end products (RAGE) expressions. On the contrary, metformin or resveratrol inhibited p47-phox, GLUT5, and SGLT2 expressions, but not nuclear factor erythroid 2-related factor 2 (NRF2). In summary, dapagliflozin, metformin or resveratrol down-regulated p47-phox expression through SGLT2 inactivation and ROS reduction. These important findings imply that SGLT2 can be blocked to ameliorate oxidative stress in the cataracts of DM patients.

µ-Opioid Receptor-Mediated AT1R-TLR4 Crosstalk Promotes Microglial Activation to Modulate Blood Pressure Control in the Central Nervous System.

Opioids, a kind of peptide hormone involved in the development of hypertension, cause systemic and cerebral inflammation, and affects regions of the brain that are important for blood pressure (BP) control. A cause-and-effect relationship exists between hypertension and inflammation; however, the role of blood pressure in cerebral inflammation is not clear. Evidence showed that AT1R and µOR heterodimers' formation in the NTS might lead to the progression of hypertension. In this study, we investigated the formation of the µOR/AT1R heterodimer, determined its correlation with µORs level in the NTS, and explored the role of TLR4-dependent inflammation in the development of hypertension. Results showed that Ang II increased superoxide and Iba-1 (microgliosis marker: ionized calcium-binding adaptor molecule (1) levels in the NTS of spontaneously hypertensive rats (SHRs). The AT1R II inhibitor, losartan, significantly decreased BP and abolished superoxide, Iba-1, TLR4 expression induced by Ang II. Furthermore, losartan significantly increased nNsOSS1416 phosphorylation. Administration of a µOR agonist or antagonist in the NTS of WKY and SHRs increased endogenous µ-opioids, triggered the formation of µOR/AT1R heterodimers and the TLR4-dependent inflammatory pathway, and attenuated the effect of depressor nitric oxide (NO). These results imply an important link between neurotoxicity and superoxides wherein abnormal increases in NTS endogenous µ-opioids promote the interaction between Ang II and µOR, the binding of Ang II to AT1R, and the activation of microglia. In addition, the interaction between Ang II and µOR enhanced the formation of the AT1R and µOR heterodimers, and inactivated nNOS-derived NO, leading to the development of progressive hypertension.

Mechanical Stretching-Induced Traumatic Brain Injury Is Mediated by the Formation of GSK-3ß-Tau Complex to Impair Insulin Signaling Transduction.

Traumatic brain injury confers a significant and growing public health burden. It is a major environmental risk factor for dementia. Nonetheless, the mechanism by which primary mechanical injury leads to neurodegeneration and an increased risk of dementia-related diseases is unclear. Thus, we aimed to investigate the effect of stretching on SH-SY5Y neuroblastoma cells that proliferate in vitro. These cells retain the dopamine-ß-hydroxylase activity, thus being suitable for neuromechanistic studies. SH-SY5Y cells were cultured on stretchable membranes. The culture conditions contained two groups, namely non-stretched (control) and stretched. They were subjected to cyclic stretching (6 and 24 h) and 25% elongation at 1 Hz. Following stretching at 25% and 1 Hz for 6 h, the mechanical injury changed the mitochondrial membrane potential and triggered oxidative DNA damage at 24 h. Stretching decreased the level of brain-derived neurotrophic factors and increased amyloid-ß, thus indicating neuronal stress. Moreover, the mechanical injury downregulated the insulin pathway and upregulated glycogen synthase kinase 3ß (GSK-3ß)S9/p-Tau protein levels, which caused a neuronal injury. Following 6 and 24 h of stretching, GSK-3ßS9 was directly bound to p-TauS396. In contrast, the neuronal injury was improved using GSK-3ß inhibitor TWS119, which downregulated amyloid-ß/p-Taus396 phosphorylation by enhancing ERK1/2T202/Y204 and AktS473 phosphorylation. Our findings imply that the neurons were under stress and that the inactivation of the GSK3ß could alleviate this defect.

3H-1,2-Dithiole-3-Thione Protects Lens Epithelial Cells against Fructose-Induced Epithelial-Mesenchymal Transition via Activation of AMPK to Eliminate AKR1B1-Induced Oxidative Stress in Diabetes Mellitus.

Studies demonstrated that the receptor of advanced glycation end products (RAGE) induced epithelial-mesenchymal transition (EMT) formation in the lens epithelial cells (LECs) of diabetic cataracts. This work investigated how 3H-1,2-dithiole-3-thione (D3T) reduces EMT formation in LECs of the fructose-induced diabetes mellitus (DM). LECs were isolated during cataract surgery from patients without DM or with DM. In a rat model, fructose (10% fructose, eight weeks) with or without D3T (10 mg/kg/day) treatment induced DM, as verified by blood pressure and serum parameter measurements. We observed that the formation of advanced glycation end products (AGEs) was significantly higher in epithelial human lens of DM (+) compared to DM (-) cataracts. Aldose reductase (AKR1B1), AcSOD2, and 3-NT were significantly enhanced in the rat lens epithelial sections of fructose-induced DM, however, the phosphorylation level of AMPKT172 showed a reversed result. Interestingly, administration of D3T reverses the fructose-induced effects in LECs. These results indicated that AMPKT172 may be required for reduced superoxide generation and the pathogenesis of diabetic cataract. Administration of D3T reverses the fructose-induced EMT formation the LECs of fructose-induced DM. These novel findings suggest that the D3T may be a candidate for the pharmacological prevention of cataracts in patients with DM.

Exercise prevents the impairment of learning and memory in prenatally phthalate-exposed male rats by improving the expression of plasticity-related proteins.

Regular exercise has been identified to facilitate neuroplasticity that maximize functional outcome after brain injuries. Brain-derived neurotrophic factor (BDNF) has emerged as a key facilitator of neuroplasticity after exercise. The activity-regulated cytoskeleton associated protein (Arc) is induced by BDNF and N-methyl-d-aspartic acid receptor (NMDAR), contributing to functional modification of neuroplasticity in the hippocampus. Meanwhile, early-life exposure to neuroendocrine disruptor di-(2-ethylhexyl)-phthalate (DEHP) is a risk factor for behavioral deficits, but the mechanisms responsible for DEHP-induced neurotoxicity are not well understood. The purpose of this study is to investigate whether hippocampal Arc expression is impaired by DEHP exposure and to examine the protective role of exercise in the prenatally DEHP-exposed male rats. Sprague Dawley dams were fed with vehicle or DEHP during gestation. The male offspring were trained to treadmill running for 5 weeks followed by examination of behavioral and biochemical outcomes. The results showed that DEHP-exposed rats exhibited impairment of spatial learning and memory as well as down-regulations of BDNF, NMDAR, Arc, and synaptophysin. Importantly, aerobic exercise during childhood-adolescence prevented the impairment of learning and memory by recovering the expressions of BDNF, NMDAR, Arc, and synaptophysin. These findings suggest that exercise may provide beneficial effects on ameliorating the impairment of neuroplasticity in the prenatally DEHP-exposed male rats at late adolescence.

CX3CR1-microglia mediates neuroinflammation and blood pressure regulation in the nucleus tractus solitarii of fructose-induced hypertensive rats.

BACKGROUND: Inflammation is a common pathophysiological trait found in both hypertension and cardiac vascular disease. Recent evidence indicates that fractalkine (FKN) and its receptor CX3CR1 have been linked to inflammatory response in the brain of hypertensive animal models. Here, we investigated the role of CX3CR1-microglia in nitric oxide (NO) generation during chronic inflammation and systemic blood pressure recovery in the nucleus tractus solitarii (NTS). METHODS: The hypertensive rat model was used to study the role of CX3CR1-microglia in NTS inflammation following hypertension induction by oral administration of 10% fructose water. The systolic blood pressure was measured by tail-cuff method of non-invasive blood pressure. The CX3CR1 inhibitor AZD8797 was administered intracerebroventricularly (ICV) in the fructose-induced hypertensive rat. Using immunoblotting, we studied the nitric oxide synthase signaling pathway, NO concentration, and the levels of FKN and CX3CR1, and pro-inflammatory cytokines were analyzed by immunohistochemistry staining. RESULTS: The level of pro-inflammatory cytokines IL-1ß, IL-6, TNF-α, FKN, and CX3CR1 were elevated two weeks after fructose feeding. AZD8797 inhibited CX3CR1-microglia, which improved the regulation of systemic blood pressure and NO generation in the NTS. We also found that IL-1ß, IL-6, and TNF-α levels were recovered by AZD8797 addition. CONCLUSION: We conclude that CX3CR1-microglia represses the nNOS signaling pathway and promotes chronic inflammation in fructose-induced hypertension. Collectively, our results reveal the role of chemokines such as IL-1ß, IL-6, and TNF-α in NTS neuroinflammation with the involvement of FKN and CX3CR1.

Vitamin D Attenuates Loss of Endothelial Biomarker Expression in Cardio-Endothelial Cells.

Vitamin D is associated with cardiovascular health through activating the vitamin D receptor that targets genes related to cardiovascular disease (CVD). The human cardiac microvascular endothelial cells (HCMECs) were used to develop mechanically and TGF-ß1-induced fibrosis models, and the rat was used as the isoproterenol (ISO)-induced fibrosis model. The rats were injected with ISO for the first five days, followed by vitamin D injection for the consecutive three weeks before being sacrificed on the fourth week. Results showed that mechanical stretching reduced endothelial cell marker CD31 and VE-cadherin protein expressions, as well as increased α-smooth muscle actin (α-SMA) and fibronectin (FN). The transforming growth factor-ß1 (TGF-ß1) reduced CD31, and increased α-SMA and FN protein expression levels. Vitamin D presence led to higher protein expression of CD31, and lower protein expressions of α-SMA and FN compared to the control in the TGF-ß1-induced fibrosis model. Additionally, protein expression of VE-cadherin was increased and fibroblast-specific protein-1 (FSP1) was decreased after vitamin D treatment in the ISO-induced fibrosis rat. In conclusion, vitamin D slightly inhibited fibrosis development in cell and animal models. Based on this study, the beneficial effect of vitamin D may be insignificant; however, further investigation of vitamin D's effect in the long-term is required in the future.

Mechanism of a methylxanthine drug theophylline-induced Ca2+ signaling and cytotoxicity in AML12 mouse hepatocytes.

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca2+ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca2+ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca2+ concentration ([Ca2+]i) was measured by the Ca2+-sensitive fluorescent dye fura-2. Theophylline (25-125 µM) induced [Ca2+]i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished theophylline-induced [Ca2+]i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca2+]i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca2+]i rises. In Ca2+-containing medium, modulators of store-operated Ca2+ channels inhibited 30% of the [Ca2+]i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca2+-associated cytotoxicity and induced Ca2+ entry through PLC-independent Ca2+ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca2+ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity.

Mechanisms underlying the effect of an oral antihyperglycaemic agent glyburide on calcium ion (Ca2+ ) movement and its related cytotoxicity in prostate cancer cells.

Glyburide is an agent commonly used to treat type 2 diabetes and also affects various physiological responses in different models. However, the effect of glyburide on Ca2+ movement and its related cytotoxicity in prostate cancer cells is unclear. This study examined whether glyburide altered Ca2+ signalling and viability in PC3 human prostate cancer cells and investigated those underlying mechanisms. Intracellular Ca2+ concentrations ([Ca2+ ]i ) in suspended cells were measured by using the fluorescent Ca2+ -sensitive dye fura-2. Cell viability was examined by WST-1 assay. Glyburide at concentrations of 100-1000 µM induced [Ca2+ ]i rises. Ca2+ removal reduced the signal by approximately 60%. In Ca2+ -containing medium, glyburide-induced Ca2+ entry was inhibited by 60% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and inhibitor (GF109203X), and modulators of store-operated Ca2+ channels (nifedipine, econazole and SKF96365). Furthermore, glyburide induced Mn2+ influx suggesting of Ca2+ entry. In Ca2+ -free medium, inhibition of phospholipase C (PLC) with U73122 significantly inhibited glyburide-induced [Ca2+ ]i rises. Treatment with the endoplasmic reticulum (ER) Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished glyburide-evoked [Ca2+ ]i rises. Conversely, treatment with glyburide abolished BHQ-evoked [Ca2+ ]i rises. Glyburide at 100-500 µM decreased cell viability, which was not reversed by pretreatment with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, glyburide induced [Ca2+ ]i rises by Ca2+ entry via PKC-sensitive store-operated Ca2+ channels and Ca2+ release from the ER in a PLC-dependent manner. Glyburide also caused Ca2+ -independent cell death. This study suggests that glyburide could serve as a potential agent for treatment of prostate cancer.

Exploring the impact of a naturally occurring sapogenin diosgenin on underlying mechanisms of Ca2+ movement and cytotoxicity in human prostate cancer cells.

Literature has shown that diosgenin, a naturally occurring sapogenin, inducedcytotoxic effects in many cancer models. This study investigated the effect of diosgenin on intracellular Ca2+ concentration ([Ca2+ ]i) and cytotoxicity in PC3 human prostate cancer cells. Diosgenin (250-1000 µM) caused [Ca2+ ]i rises which was reduced by Ca2+ removal. Treatment with thapsigargin eliminated diosgenin-induced [Ca2+ ]i increases. In contrast, incubation with diosgeninabolished thapsigargin-caused [Ca2+ ]i increases. Suppression of phospholipase C with U73122 eliminated diosgenin-caused [Ca2+ ]i increases. Diosgenin evoked Mn2+ influx suggesting that diosgenin induced Ca2+ entry. Diosgenin-induced Ca2+ influx was suppressed by PMA, GF109203X, and nifedipine, econazole, or SKF96365. Diosgenin (250-600 µM) concentration-dependently decreased cell viability. However, diosgenin-induced cytotoxicity was not reversed by chelation of cytosolic Ca2+ with BAPTA/AM. Together, diosgenin evoked [Ca2+ ]i increases via Ca2+ release and Ca2+ influx, and caused Ca2+ -non-associated deathin PC3 cells. These findings reveal a newtherapeutic potential of diosgenin for human prostate cancer.

Angiotensin II inhibits DDAH1-nNOS signaling via AT1R and µOR dimerization to modulate blood pressure control in the central nervous system.

G protein-coupled receptors (GPCRs) are important drug targets. Blocking angiotensin II (Ang II) type 1 receptor signaling alleviates hypertension and improves outcomes in patients with heart failure. Changes in structure and trafficking of GPCR, and desensitization of GPCR signaling induce pathophysiological processes. We investigated whether Ang II, via induction of AT1R and µ-opioid receptor (µOR) dimerization in the nucleus tractus solitarius (NTS), leads to progressive hypertension. Ang II signaling increased µOR and adrenergic receptor α2A (α2A-AR) heterodimer levels and decreased expression of extracellular signal-regulated kinases 1/2T202/Y204, ribosomal protein S6 kinaseT359/S363, and nNOSS1416 phosphorylation. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression was abolished in the NTS of adult spontaneously hypertensive rats (SHRs). Endomorphin-2 was overexpressed in NTS of adult SHRs compared with that in 6-week-old Wistar-Kyoto rats (WKY). Administration of µOR agonist into the NTS of WKY increased blood pressure (BP), decreased nitric oxide (NO) production, and decreased DDAH1 activity. µOR agonist significantly reduced the activity of DDAH1 and decreased neuronal NO synthase (nNOS) phosphorylation. The AT1R II inhibitor, losartan, significantly decreased BP and abolished AT1R-induced formation of AT1R and µOR, and α2A-AR and µOR, heterodimers. Losartan also significantly increased the levels of nNOSS1416 phosphorylation and DDAH1 expression. These results show that Ang II may induce expression of endomorphin-2 and abolished DDAH1 activity by enhancing the formation of AT1R and µOR heterodimers in the NTS, leading to progressive hypertension.

Dapagliflozin Prevents NOX- and SGLT2-Dependent Oxidative Stress in Lens Cells Exposed to Fructose-Induced Diabetes Mellitus.

PURPOSE: Cataracts in patients with diabetes mellitus (DM) are a major cause of blindness in developed and developing countries. This study aims to examine whether the generation of reactive oxygen species (ROS) via the increased expression of glucose transporters (GLUTs) and the receptor for advanced glycation end products (RAGE) influences the cataract development in DM. METHODS: Lens epithelial cells (LECs) were isolated during cataract surgery from patients without DM or with DM, but without diabetic retinopathy. In a rat model, fructose (10% fructose, 8 or 12 weeks) with or without dapagliflozin (1.2 mg/day, 2 weeks) treatment did induce DM, as verified by blood pressure and serum parameter measurements. Immunofluorescence stainings and immunoblottings were used to quantify the protein levels. Endogenous O2˙¯ production in the LECs was determined in vivo with dihydroethidium stainings. RESULTS: We investigated that GLUT levels in LECs differed significantly, thus leading to the direct enhancement of RAGE-associated superoxide generation in DM patients with cataracts. Superoxide production was significantly higher in LECs from rats with fructose-induced type 2 DM, whereas treatment with the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin prevented this effect in fructose-fed rats. Protein expression levels of the sodium/glucose cotransporter 2 (SGLT2), GLUT1, GLUT5, the nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase subunit p67-phox, NOX2/4 and RAGE were upregulated in fructose-fed animals, whereas dapagliflozin treatment reversed these effects. CONCLUSIONS: In rats with fructose-induced DM, dapagliflozin downregulates RAGE-induced NADPH oxidase expression in LECs via the inactivation of GLUTs and a reduction in ROS generation. These novel findings suggest that the SGLT2 inhibitor dapagliflozin may be a candidate for the pharmacological prevention of cataracts in patients with DM.

Closer to Nature Through Dynamic Culture Systems.

Mechanics in the human body are required for normal cell function at a molecular level. It is now clear that mechanical stimulations play significant roles in cell growth, differentiation, and migration in normal and diseased cells. Recent studies have led to the discovery that normal and cancer cells have different mechanosensing properties. Here, we discuss the application and the physiological and pathological meaning of mechanical stimulations. To reveal the optimal conditions for mimicking an in vivo microenvironment, we must, therefore, discern the mechanotransduction occurring in cells.

Exploration of the effect of the alkaloid colchicine on Ca2+ handling and its related physiology in human oral cancer cells.

OBJECTIVE: Colchicine, extracted from plants of the genus Colchicum, is a commonly prescribed drug for inflammatory diseases. It has been shown that colchicine affected various physiological responses in different models. However, the effect of colchicine on cytosolic free Ca2+ levels ([Ca2+]i) and its related physiology in human oral cancer cells is unknown. This study examined whether colchicine altered Ca2+ homeostasis and caused cytotoxicity in OC2 human oral cancer cells. METHODS: The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. RESULTS: Colchicine at concentrations of 250-650 µM induced [Ca2+]i rises concentration-dependently. The response was reduced by approximately 40% by removing extracellular Ca2+. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited colchicine-evoked [Ca2+]i rises. Conversely, treatment with colchicine inhibited thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished colchicine-induced Ca2+ release. In Ca2+-containing medium, colchicine-induced Ca2+ entry was supported by Mn2+-caused quenching of fura-2 fluorescence and the entry was partly inhibited by protein kinase C (PKC) modulators (phorbol 12-myristate 13 acetate, PMA; and GF109203X) and by three modulators of store-operated Ca2+ channels (nifedipine, econazole and SKF96365). Colchicine at 250-650 µM decreased cell viability, which was not reversed by pretreatment with the Ca2+chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). CONCLUSIONS: In OC2 cells, colchicine induced [Ca2+]i rises by evoking PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Furthermore, colchicine caused cell death that was not triggered by preceding [Ca2+]i rises.