Sasanquasaponin promotes cellular chloride efflux and elicits cardioprotection via the PKCε pathway.

Sasanquasaponin (SQS) is an active component of Camellia oleifera Abel. A recent study by our group demonstrated that SQS was able to inhibit ischemia/reperfusion­induced elevation of the intracellular chloride ion concentration ([Cl­]i) and exerted cardioprotective effects; however, the underlying intracellular signal transduction mechanisms have yet to be elucidated. As protein kinase C ε (PKCε) is able to mediate Cl­ homeostasis, the present study investigated its possible involvement in the effects of SQS on cardiomyocytes subjected to ischemia/reperfusion injury. Cardiomyocytes were pre­treated with or without SQS or SQS plus εV1­2, a selective PKCε inhibitor, followed by simulated ischemia/reperfusion (sI/R). The effects on cell viability, PKCε phosphorylation levels, [Cl­]i, mitochondrial membrane potential and reactive oxygen species (ROS) production were assessed using an MTS assay, western blot analysis, colorimetric assays and flow cytometry. The results revealed that treatment with SQS prior to sI/R increased the viability of cardiomyocytes, and efficiently attenuated lactate dehydrogenase and creatine phosphokinase release induced by sI/R. In addition, SQS promoted PKCε phosphorylation and inhibited sI/R­induced elevation of [Cl­]i, paralleled by the attenuation of mitochondrial membrane potential loss and ROS generation. However, when the cardiomyocytes were treated with εV1­2 prior to SQS pre­conditioning, the cardioprotection induced by SQS was reduced and the inhibitory effects of SQS on sI/R­induced elevation of [Cl­]i, production of ROS and loss of mitochondrial membrane potential were also attenuated. These findings indicated that SQS may inhibit sI/R­induced elevation of [Cl­]i through the PKCε signaling pathway to elicit cardioprotection in cultured cardiomyocytes.

Rutaecarpine Reverses the Altered Connexin Expression Pattern Induced by Oxidized Low-density Lipoprotein in Monocytes.

Adhesion of monocytes to the vascular endothelium is crucial in atherosclerosis development. Connexins (Cxs) which form hemichannels or gap junctions, modulate monocyte-endothelium interaction. We previously found that rutaecarpine, an active ingredient of the Chinese herbal medicine Evodia, reversed the altered Cx expression induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells, and consequently decreases the adhesive properties of endothelial cells to monocytes. This study further investigated the effect of rutaecarpine on Cx expression in monocytes exposed to ox-LDL. In cultured human monocytic cell line THP-1, ox-LDL rapidly reduced the level of atheroprotective Cx37 but enhanced that of atherogenic Cx43, thereby inhibiting adenosine triphosphate release through hemichannels. Pretreatment with rutaecarpine recovered the expression of Cx37 but inhibited the upregulation of Cx43 induced by ox-LDL, thereby improving adenosine triphosphate-dependent hemichannel activity and preventing monocyte adhesion. These effects of rutaecarpine were attenuated by capsazepine, an antagonist of transient receptor potential vanilloid subtype 1. The antiadhesive effects of rutaecarpine were also attenuated by hemichannel blocker 18α-GA. This study provides additional evidence that rutaecarpine can modulate Cx expression through transient receptor potential vanilloid subtype 1 activation in monocytes, which contributes to the antiadhesive properties of rutaecarpine.

DJ-1 upregulates anti-oxidant enzymes and attenuates hypoxia/re-oxygenation-induced oxidative stress by activation of the nuclear factor erythroid 2-like 2 signaling pathway.

DJ-1 protein, as a multifunctional intracellular protein, has an important role in transcriptional regulation and anti-oxidant stress. A recent study by our group showed that DJ-1 can regulate the expression of certain anti­oxidant enzymes and attenuate hypoxia/re­oxygenation (H/R)­induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2­like 2 (Nrf2) is an essential transcription factor that regulates the expression of several anti­oxidant genes via binding to the anti­oxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of anti­oxidative enzymes by DJ­1 and contributes to the protective functions of DJ­1 against H/R­induced oxidative stress in H9c2 cells. The results demonstrated that DJ­1­overexpressing H9c2 cells exhibited anti­oxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/R­induced oxidative stress compared with native cells, whereas DJ­1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ­1­mediated anti­oxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ­1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, ARE­binding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ­1­mediated induction of anti­oxidant enzymes and cytoprotection against oxidative stress induced by H/R. In conclusion, these findings indicated that activation of the Nrf2 pathway is a critical mechanism by which DJ-1 upregulates anti-oxidative enzymes and attenuates H/R-induced oxidative stress in H9c2 cells.

DJ-1 Mediates the Delayed Cardioprotection of Hypoxic Preconditioning Through Activation of Nrf2 and Subsequent Upregulation of Antioxidative Enzymes.

We have recently shown that DJ-1 is implicated in the delayed cardioprotective effect of hypoxic preconditioning (HPC) against hypoxia/reoxygenation (H/R) injury as an endogenous protective protein. This study aims to further investigate the underlying mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress. Using a well-characterized cellular model of HPC from rat heart-derived H9c2 cells, we found that HPC promoted nuclear factor erythroid 2-related factor 2 (Nrf2) and its cytoplasmic inhibitor Kelch-like ECH-associated protein-1 (Keap1) dissociation and resulted in increased nuclear translocation, antioxidant response element-binding, and transcriptional activity of Nrf2 24 hours after HPC, with subsequent upregulation of manganese superoxide dismutase (MnSOD) and heme oxygenase-1 (HO-1), which provided delayed protection against H/R-induced oxidative stress in normal H9c2 cells. However, the aforementioned effects of HPC were abolished in DJ-1-knockdown H9c2 cells, which were restored by restoration of DJ-1 expression. Importantly, we showed that inhibition of the Nrf2 pathway in H9c2 cells mimicked the effects of DJ-1 knockdown and abolished HPC-derived induction of antioxidative enzymes (MnSOD and HO-1) and the delayed cardioprotection. In addition, inhibition of Nrf2 also reversed the effects of restored DJ-1 expression on induction of antioxidative enzymes and delayed cardioprotection by HPC in DJ-1-knockdown H9c2 cells. Taken together, this work revealed that activation of Nrf2 pathway and subsequent upregulation of antioxidative enzymes could be a critical mechanism by which DJ-1 mediates the delayed cardioprotection of HPC against H/R-induced oxidative stress in H9c2 cells.

Rutaecarpine prevented dysfunction of endothelial gap junction induced by Ox-LDL via activation of TRPV1.

Gap junctions, which is formed by connexins, has been proved to play an important role in the atherogenesis development. Rutaecarpine was reported to inhibited monocyte migration, which indicates its potential for anti-atherosclerosis activity. This study evaluated the effect of rutaecarpine on endothelial dysfunction, and focused on the regulation of connexin expression in endothelial cells by rutaecarpine. Endothelia damage was induced by exposing HUVEC-12 to Ox-LDL (100mg/l) for 24h, which decreased the expression of protective proteins Cx37 and Cx40, but induced atherogenic Cx43 expression, in both mRNA and protein levels, concomitant with the impaired propidium iodide diffusion through the gap junctions. Pretreatment with rutaecarpine effectively recovered the expression of Cx37 and Cx40, but inhibited Cx43 expression, thereby improving gap junction communication and significantly prevented the endothelial dysfunction. Consequently, the cell viability and nitric oxide production were increased, lactate dehydrogenase production was decreased and monocyte adhesion was inhibited. These protective effects of rutaecarpine were remarkably attenuated by pretreatment with capsazepine, a competitive antagonist of transient receptor potential vanilloid subtype 1 (TRPV1). In summary, this study is the first to report that rutaecarpine prevents endothelial injury and gap junction dysfunction induced by Ox-LDL in vitro, which is related to regulation of connexin expression patterns via TRPV1 activation. These results suggest that rutaecarpine has the potential for use as an anti-atherosclerosis agent with a novel mechanism.

Osteopontin-induced brown adipogenesis from white preadipocytes through a PI3K-AKT dependent signaling.

Recent studies have shown that OPN (osteopontin) plays critical roles in cell survival, differentiation, bio-mineralization, cancer and cardiovascular remodeling. However, its roles in the differentiation of brown adipocytes and the underlying mechanisms remain unclear. Therefore, the aim of this study was to investigate the roles of OPN in the brown adipogenesis and the underlying mechanisms. It was shown that the OPN successfully induced the differentiation of 3T3-L1 white preadipocytes into the PRDM16(+) (PRD1-BF1-RIZ1 homologous domain containing 16) and UCP-1(+) (uncoupling protein-1) brown adipocytes in a concentration and time-dependent manner. Also, activation of PI3K (phosphatidylinositol 3-kinase)-AKT pathway was required for the OPN-induced brown adipogenesis. The findings suggest OPN plays an important role in promoting the differentiation of the brown adipocytes and might provide a potential novel therapeutic approach for the treatment of obesity and related disorders.

Pharmacological activation of PPAR gamma ameliorates vascular endothelial insulin resistance via a non-canonical PPAR gamma-dependent nuclear factor-kappa B trans-repression pathway.

Vascular endothelial insulin resistance (IR) is a critically initial factor in cardiocerebrovascular events resulted from diabetes and is becoming a worldwide public health issue. Thiazolidinediones (TZDs) are clinical insulin-sensitizers acting through a canonical peroxisome proliferator-activated receptor gamma (PPARγ)-dependent insulin trans-activation pathway. However, it remains elusive whether there are other mechanisms. In current study, we investigated whether TZDs improve endothelial IR induced by high glucose concentration or hyperglycemia via a non-canonical PPARγ-dependent nuclear factor-kappa B (NF-κB) trans-repression pathway. Our results showed that pre-treatment with TZDs dramatically decrease the susceptibility of endothelial cell to IR, while post-treatment notably improve the endothelial IR both in vitro and in vivo. Moreover, TZDs substantially increase the levels of endothelial nitric oxide synthase (eNOS) and inhibitory κB alpha (IκBα), whereas decrease those of the phosphorylated inhibitory κB kinase alpha/beta (phosphor-IKKα/ß) and the cytokines including tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cellular adhesion molecule-1 (sVCAM-1), suggesting that TZDs act indeed through a PPARγ-dependent NF-κB trans-repression pathway. These findings highlighted a non-canonical mechanism for TZDs to ameliorate endothelial IR which might provide a potential strategy to prevent and treat the diabetic vascular complications clinically.

A role for diallyl trisulfide in mitochondrial antioxidative stress contributes to its protective effects against vascular endothelial impairment.

Persistent hyperglycemia increases a systemic oxidative stress, causing the onset of vascular endothelial dysfunction and atherosclerosis. Diallyl trisulfide (DAT), a natural organosulfur compound in garlic, has been reported to have actions of dilating blood vessels and antibacteria, etc. In this study, models of obese diabetic rat in vivo and high glucose concentration (HG)-induced endothelial cell injury in vitro were used to investigate the protective effects of DAT on vascular endothelial injury and its underlying mechanisms. In the in vivo model, the obese diabetic rats were injected venously with DAT (5.0 mg kg(-1)d(-1)) and Vitamin E (1.0 mg kg(-1)d(-1)) respectively, once daily for 7 consecutive days. In the in vitro model, HG-injured HUVEC were treated with or without DAT (25 µmol L(-1), 50 µmol L(-1), 100 µmol L(-1)) or Vitamin E (25 µmol L(-1)) respectively for 24h. The extents of vascular endothelial injury and protective effects of DAT were evaluated. The results both in vivo and in vitro displayed that DAT-treatment significantly attenuated the endothelial cell impairments. Besides, DAT-treatment markedly decreased the levels of malondialdehyde (MDA) and reactive oxygen species, whereas elevated the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in mitochondrium. Moreover, DAT-treatment considerably improved mitochondrial respiration function. Taken together, our results suggest that DAT protects vascular endothelium from HG or hyperglycemia induced-injury by reducing mitochondrial oxidative stress. The findings provide a novel insight for DAT to potentially treat the oxidative stress diseases, i.e., atherosclerosis, diabetes, and neurodegenerative diseases.

Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for hypoxic preconditioning-mediated delayed cardioprotection.

It has been well demonstrated that hypoxic preconditioning (HPC) can attenuate hypoxia/reoxygenation (H/R)-induced oxidant stress and elicit delayed cardioprotection by upregulating the expression of multiple antioxidative enzymes such as heme oxygenase-1 (HO-1), manganese superoxide dismutase (MnSOD) and so on. However, the underlying mechanisms of HPC-induced upregulation of antioxidative enzymes are not fully understood. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential transcription factor that regulates expression of several antioxidant genes via binding to the antioxidant response element (ARE) and plays a crucial role in cellular defence against oxidative stress. Here, we wondered whether activation of the Nrf2-ARE pathway is responsible for the induction of antioxidative enzymes by HPC and contributes to the delayed cardioprotection of HPC. Cellular model of HPC from rat heart-derived H9c2 cells was induced 24 h prior to H/R. The results showed that HPC efficiently attenuated H/R-induced viability loss and lactate dehydrogenase leakage. In addition, HPC increased nuclear translocation and ARE binding of Nrf2 during the late phase, upregulated the expression of antioxidative enzymes (HO-1 and MnSOD), inhibited H/R-induced oxidant stress. However, when Nrf2 was specifically knocked down by siRNA, the induction of antioxidative enzymes by HPC was completely abolished and, as a result, the inhibitory effect of HPC on H/R-induced oxidant stress was reversed, and the delayed cardioprotection induced by HPC was also abolished. These results suggest that HPC upregulates antioxidative enzymes through activating the Nrf2-ARE pathway and confers delayed cardioprotection against H/R-induced oxidative stress.

Stable overexpression of DJ-1 protects H9c2 cells against oxidative stress under a hypoxia condition.

It has been well accepted that increased reactive oxygen species (ROS) and the subsequent oxidative stress is one of the major causes of ischemia/reperfusion (I/R) injury. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in regulating cell survival and antioxidant stress. Here, we wondered whether DJ-1 overexpression attenuates simulated ischemia/reperfusion (sI/R)-induced oxidative stress. A rat cDNA encoding DJ-1 was inserted into a mammalian expression vector. After introduction of this construct into H9c2 myocytes, stable clones were obtained. Western blot analysis of the derived clones showed a 2.6-fold increase in DJ-1 protein expressing. Subsequently, the DJ-1 gene-transfected and control H9c2 cells were subjected to sI/R, and then cell viability, lactate dehydrogenase, malondialdehyde, intracellular ROS and antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) were measured appropriately. The results showed that stable overexpression of DJ-1 efficiently attenuated sI/R-induced viability loss and lactate dehydrogenase leakage. Additionally, stable overexpression of DJ-1 inhibited sI/R-induced the elevation of ROS and MDA contents followed by the increase of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) activities and expression. Our data indicate that overexpression of DJ-1 attenuates ROS generation, enhances the cellular antioxidant capacity and prevents sI/R-induced oxidative stress, revealing a novel mechanism of cardioprotection. Importantly, DJ-1 overexpression may be an important part of a protective strategy against ischemia/reperfusion injury.

Hypoxic preconditioning up-regulates DJ-1 protein expression in rat heart-derived H9c2 cells through the activation of extracellular-regulated kinase 1/2 pathway.

Myocardial preconditioning is a powerful phenomenon that can attenuate ischemia/reperfusion-induced oxidant stress and elicit delayed cardioprotection. Its mechanisms involve activation of intracellular signaling pathways and up-regulation of the protective antioxidant proteins. DJ-1 protein, as a multifunctional intracellular protein, plays an important role in attenuating oxidant stress and promoting cell survival. In the present study, we investigated whether DJ-1 is up-regulated during the late phase of hypoxic preconditioning (HP) and the up-regulation of DJ-1 is mediated by extracellular-regulated kinase 1/2 (ERK1/2) signaling pathway. Rat heart-derived H9c2 cells were exposed to HP. Twenty-four hours later cells were subjected to hypoxia/reoxygenation (H/R) and then cell viability, lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS), ERK1/2 phosphorylation, and DJ-1 protein were measured appropriately. The results showed that HP efficiently attenuated H/R-induced viability loss and LDH leakage. In addition, HP promoted ERK1/2 activation, up-regulated DJ-1 protein expression, inhibited H/R induced the elevation of ROS. However, when ERK1/2 phosphorylation was specifically inhibited by U0126, the increase in DJ-1 expression occurring during HP was almost completely abolished and, as a result, the delayed cardioprotection induced by HP was abolished, and the inhibitory effect of HP on H/R-induced oxidant stress was also reversed. Furthermore, knocking down DJ-1 by siRNA attenuated the delayed cardioprotection induced by HP. Our data indicate that HP can up-regulate DJ-1 protein expression through the ERK1/2-dependent signaling pathway. Importantly, DJ-1 might be involved in the delayed cardioprotective effect of HP against H/R injury.

Sasanquasaponin up-regulates anion exchanger 3 expression and elicits cardioprotection via NO/RAS/ERK1/2 pathway.

We have shown recently that sasanquasaponin (SQS) can inhibit ischemia/reperfusion-induced elevation of intracellular Cl(-) concentration ([Cl(-)](i)) and elicit cardioprotection by up-regulating anion exchanger 3 (AE(3)) expression. In the present study, we futher analysed the intracellular signal transduction pathways by which SQS up-regulates AE(3) expression and elicits cardioprotection. Cardiomyocytes were incubated for 24 h with or without 10 µmol/L SQS, followed by simulated ischemia/reperfusion (sI/R). NO formation, Ras activity, and extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation were measured appropriately. We showed that SQS pretreatment efficiently attenuated viability loss and lactate dehydrogenase leakage induced by sI/R in cardiomyocytes. Moreover, SQS induced NO production and promoted Ras activation, which futher promoted extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation. These effects were paralleled by an increase in AE(3) expression. However, when the cardiomyocytes were treated with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (c-PTIO; an NO scavenger), S-trans-trans-farnesylthiosalicylic acid (FTS) (a Ras inhibitor), U0126 (an ERK1/2 inhibitor), respectively, the increase in AE(3) expression occurring during SQS pretreatment was almost completely abolished and, as a result, SQS-induced cardioprotection was prevented. Our findings indicate that SQS might up-regulate AE(3) expression through NO/Ras/ERK1/2 signal pathway to elicit cardioprotection in cultured cardiomyocytes.

Comparison of the effects of cholecalciferol and calcitriol on calcium metabolism and bone turnover in Chinese postmenopausal women with vitamin D insufficiency.

AIM: To compare the effects of cholecalciferol (800 IU/d) and calcitriol (0.25 µg/d) on calcium metabolism and bone turnover in Chinese postmenopausal women with vitamin D insufficiency. METHODS: One hundred Chinese postmenopausal women aged 63.8±7.0 years and with serum 25-hydroxyvitamin D [25(OH)D] concentration <30 ng/mL were recruited. The subjects were divided into 2 groups based on the age and serum 25(OH)D concentration: 50 subjects (group A) received cholecalciferol (800 IU/d), and 50 subjects (group B) received calcitriol (0.25 µg/d) for 3 months. In addition, all the subjects received Caltrate D (calcium plus 125 IU cholecalciferol) daily in the form of one pill. The markers of calcium metabolism and bone turnover, including the serum levels of calcium, phosphorus, alkaline phosphatase, intact parathyroid hormone, 25(OH)D and ß-CrossLaps of type I collagen containing cross-linked C-telopeptide (ß-CTX), were measured before and after the intervention. RESULTS: After the 3-month intervention, the serum 25(OH)D concentration in group A was significantly increased from 16.01 ± 5.0 to 20.02 ± 4.5 ng/mL, while that in group B had no significant change. The serum calcium levels in both the groups were significantly increased (group A: from 2.36 ± 0.1 to 2.45 ± 0.1 mmol/L; group B: from 2.36 ± 0.1 to 2.44 ± 0.1 mmol/L). The levels of serum intact parathyroid hormone in both the groups were significantly decreased (group A: from 48.56 ± 12.8 to 39.59 ± 12.6 pg/mL; group B: from 53.67 ± 20.0 to 40.32 ± 15.4 pg/mL). The serum levels of ß-CTX in both the groups were also significantly decreased (group A: from 373.93 ± 135.3 to 325.04 ± 149.0 ng/L; group B: from 431.00 ± 137.1 to 371.74 ± 185.0 ng/L). CONCLUSION: We concluded that both cholecalciferol (800 IU/d) and calcitriol (0.25 µg/d) plus Caltrate D modifies the serum calcium and bone turnover markers in Chinese postmenopausal women with vitamin D insufficiency. In addition, cholecalciferol (800 IU/d) significantly increased the serum 25(OH)D concentration.

Anion exchanger 3 is required for sasanquasaponin to inhibit ischemia/reperfusion-induced elevation of intracellular Cl- concentration and to elicit cardioprotection.

Recent studies have shown that the cardioprotection of sasanquasaponin (SQS) against ischemia/reperfusion injury is related to inhibiting ischemia/reperfusion-induced elevation of intracellular Cl(-) concentration ([Cl(-) ](i)). However, the mechanism of inhibition remains unclear. Anion exchanger 3 (AE(3)) is an important regulatory protein for [Cl(-)](i). This study investigated whether AE(3) plays the critical role in the inhibitory effect of SQS on elevation of [Cl(-)](i) induced by ischemia/reperfusion and mediates the cardioprotection of SQS in H9c2 cells. Normal and AE(3) -knockdown H9c2 cells were incubated for 24 h with or without various concentrations of SQS (0.1, 1, or 10 µM) followed by simulated ischemia/reperfusion (sI/R). AE(3) expression was detected by Western blot. Flow cytometer analysis was employed to determine [Cl(-)](i,) [Ca(2+)](i) , reactive oxygen species (ROS) production, and cell apoptosis. The results showed that SQS pretreatment concentration-dependently attenuated sI/R-induced viability loss and lactate dehydrogenase leakage in normal H9c2 cells. Additionally, SQS concentration-dependently up-regulated AE(3) protein expression, and inhibited sI/R-induced the elevation of [Cl(-)](i) followed by the attenuation of Ca(2+) overload, ROS production, and cell apoptosis. However, the dose-dependent cardioprotection induced by SQS was abolished in AE(3) -knockdown H9c2 cells, and the inhibitory effects of SQS on [Cl(-)](i), Ca(2+) overload, ROS production, and cell apoptosis were also reversed. Our data indicate that AE(3) mediates the cardioprotective effect of SQS against sI/R injury. Importantly, AE(3) is required for SQS to inhibit sI/R-induced elevation of [Cl(-)](i), which subsequently inhibited sI/R-induced Ca(2+) overload, ROS production, and cell apoptosis.

The protective effects of Polygonum multiflorum stilbeneglycoside preconditioning in an ischemia/reperfusion model of HUVECs.

AIM: To investigate the protective effects of preconditioning human umbilical vein endothelial cells (HUVECs) with Polygonum multiflorum stilbeneglycoside (PMS) under anoxia/reoxygenation (A/R), and the mechanism of protection. METHODS: Prior to A/R, HUVECs were incubated with PMS (0.6 x 10(-11), 1.2 x 10(-11), or 2.4 x 10(-11) mol/L) for 3 h. Cell injury was subsequently evaluated by measuring cell viability with an MTT assay and lactate dehydrogenase (LDH) release, whereas lipid peroxidation was assayed by measuring malondialdehyde (MDA) content. Antioxidant capacity was quantified by superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. Nitric oxide (NO) production was determined by nitrite accumulation. Endothelial NO synthase (eNOS) and inducible NOS (iNOS) protein expression was detected by Western blotting. Guanylate cyclase activity and cyclic GMP (cGMP) activity were assessed by an enzyme immunoassay kit. RESULTS: PMS incubation attenuated A/R-induced injury in a concentration-dependent manner, as evidenced by a decrease in LDH activity and an increase in cell viability. PMS exerted its protective effect by inhibiting the A/R-mediated elevation of MDA content, as well as by promoting the recovery of SOD and GSH-Px activities. Additionally, PMS incubation enhanced NO and cGMP formation by increasing iNOS expression and guanylate cyclase activity. The protective effects of PMS were markedly attenuated by NOS inhibitor L-NAME, soluble guanylate cyclase inhibitor ODQ or PKG inhibitor KT5823. CONCLUSION: PMS preincubation resulted in the enhancement of antioxidant activity and anti-lipid peroxidation. The NO/cGMP/cGMP-dependent protein kinase (PKG) signaling pathway was involved in the effect of PMS on HUVECs.

Involvement of anion exchanger-2 in apoptosis of endothelial cells induced by high glucose through an mPTP-ROS-Caspase-3 dependent pathway.

Excess apoptosis of endothelial cells (EC) plays crucial roles in the onset and progression of vasculopathy in diabetes mellitus. Anion exchanger-2 (AE2) might be involved in the vasculopathy. However, little is known about the molecular mechanisms that AE2 mediated the apoptosis of EC. The purpose of this study was to explore the role of AE2 in the apoptosis of HUVECs induced by high glucose (HG) and its possible mechanisms. First, HUVECs were exposed to different glucose concentrations (5.5, 17.8, 35.6, 71.2 and 142.4 mmol/l, respectively, pH = 7.40) for different time points (12, 24, 48, 72, 120, and 168 h, respectively). Intracellular Cl(-) concentration ([Cl(-)]i), AE2 expression and the apoptosis were assayed. Then, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), Cl(-)-free media or specific RNA interference (RNAi) for AE2 was used to confirm whether AE2 could mediate the apoptosis induced by HG. Finally, the mechanisms of the AE2-mediated apoptosis were investigated by detecting mitochondrial permeability transition pore (mPTP, DeltaPsim) openings, reactive oxygen species (ROS) levels and Caspase-3 activity. We found that HG upregulated the AE2 expression and activity, increased [Cl(-)]i and induced the apoptosis in a time- and concentration-dependent manner. The apoptosis of HUVECs by HG was possibly mediated by AE2 through an mPTP-ROS-Caspase-3 dependent pathway. These findings suggested that AE2 was likely to be a glucose-sensitive transmembrane transporter and a novel potential therapeutic target for diabetic vasculopathy.

Sodium ferulate attenuates anoxia/reoxygenation-induced calcium overload in neonatal rat cardiomyocytes by NO/cGMP/PKG pathway.

Development of intracellular calcium overload is an important pathophysiological factor in myocardial ischemia/reperfusion or anoxia/reoxygenation injury. Recent studies have shown that Sodium Ferulate (SF) stimulates nitric oxide (NO) production and exerts a cardioprotective effect in the ischemia-reperfused heart. However, it has not been determined whether the cardioprotection of SF is associated with suppression of Ca(2+) overload via NO/cyclic GMP (cGMP)/cGMP-dependent protein kinase (PKG) pathway. In this work, after cardiomyocytes were incubated with 100, 200, 400, or 800 microM SF for 3 h, anoxia/reoxygenation injury was induced and intracellular Ca(2+) concentration, NO synthase (NOS) activity, guanylate cyclase activity, NO, and cGMP formation were measured appropriately. The results showed that treatment with SF concentration-dependently inhibited calcium overload induced by anoxia/reoxygenation. We also demonstrated that SF (100-800 microM) concentration dependently enhanced NO and cGMP formation through increasing NOS activity and guanylate cyclase activity in the cardiomyocytes. On the contrary, inhibition of calcium overload by SF was markedly attenuated by addition of an NOS inhibitor, an NO scavenger, an soluble guanylate cyclase inhibitor, and a PKG inhibitor: N(G)-nitro-l-arginine methyl ester (L-NAME, 100 microM), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide (c-PTIO, 1.0 microM), 1H-[1, 2, 4] oxadiazolo [4, 3-alpha] quinoxalin-1-one (ODQ, 20 microM) and KT5823 (0.2 microM), respectively. Our findings indicate that SF significantly attenuates anoxia/reoxygenation-induced Ca(2+) overload and improves cell survival in cultured cardiomyocytes through NO/cGMP/PKG signal pathway.

Hip axis length changes in 10,554 males and females and the association with femoral neck fracture.

Hip axis length (HAL) has been proposed as an independent predictor of hip fracture risk in Caucasian females. There are, however, few data concerning its predictive risk in Chinese. The aim of this study was to investigate the changes of HAL in healthy Chinese population and the relationship between HAL and femoral neck fracture. The study population included 10,554 healthy Chinese people (8665 females, 1889 males) aged 20-97 yrs living in Shanghai. Cases were 106 patients (82 females, 24 males) aged 52 yrs old and over with femoral neck fracture. Controls were 106 age-matched healthy persons. All subjects were measured bone mineral density (BMD) at any site of proximal femur and HAL using dual-energy X-ray absorptiometry. HAL had significantly positive correlations with height and weight. After the adjustment of height and weight, HAL increased with age at 50 yrs of age and over in females, and no difference was found among the age groups in males. Males had longer HAL than females in all age groups. The peak BMD appeared in 30-44 yrs for females and 20-24 yrs for males and decreased thereafter, especially for females at 50 yrs old and over. HAL was similar in both fracture and control groups, whereas the BMD values at proximal femur were significantly lower in fracture group than in controls. There was no evidence that subjects with femoral neck fracture had longer HAL. Because of the limitations of retrospective study and relatively small fracture sample, prospective studies are required to determine the conclusions.

[Protection of camelliasaponin C against injury of myocardial cells by anoxia/reoxygenation and mechanism thereof].

OBJECTIVE: To investigate the protective effects of camelliasaponin C (CS-C) pretreatment on myocardial cell injury induced by anoxia/reoxygenation. METHODS: Myocardial cells were obtained from neonatal SD rats, cultured for 3 to 4 days, added into the wells of a 24-well plate, and were divided randomly into five groups (8 wells for each group): control group, anoxia/reoxygenation (A/R) group, added with anoxia culture fluid for 3 h and then added with imitation reperfusion fluid for 1 h, anoxia preconditioning (AP) group, undergoing anoxia for 10 min before A/R, CS-C pretreated group, added with CS-C 3.75 x 10(-7) mol/L 1 h before A/R, and glibenclamide (Glib) pretreated group, added with CS-C 3.75 x 10(-7) mol/L and Glib 12 microm 1 h before A/R. Trypan blue exclusion was used to detect the viability of the cardiomyocytes, the contents of lactate dehydrogenase (LDH) in the supernatant of culture medium was measured. Electron microscopy was used to observe the ultrastructure of the cardiomyocytes. RESULTS: The contents of LDH of the A/R group was 57.8 U/L +/- 6.4 U/L, significantly higher than that of the control group (12.3 U/L +/- 1.7 U/L, P < 0.01), and the LDH value of the CS-C group was 39.8 U/L +/- 3.9 U/L, significantly lower than that of the A/R group (P < 0.01), not significantly different from that of the AP group (32.4 U/L +/- 5.2 U/L, P > 0.05), but significantly higher than that of the control group. The cardiomyocyte viability of the A/R group was 51.0% +/- 1.9%, significantly lower than that of the control group (92.0% +/- 2.0%, P < 0.01), the cardiomyocyte viability of the CS-C pretreatment group was 76.4% +/- 3.5%, significantly higher than that of the A/R group (P < 0.01), but not significantly different from that of the AP group (78.0% +/- 2.0%, P > 0.05). The cardiomyocyte ultrastructure of the A/R group was significantly changed, and the changes of cardiomyocyte ultrastructure in the CS-C pretreatment group were significantly attenuated compared with the A/R group. The content of LDH of the Glib group was 55.8 U/L +/- 5.0 U/L, significantly higher than that of the CS-C pretreatment group (P < 0.05), and the cardiomyocyte viability of the Glib group was 54.1% +/- 3.7%, significantly lower than that of the CS-C pretreatment group (P < 0.05), and the damage to the cardiomyocyte ultrastructure in the Glib group was more severe than in the CS-C group. CONCLUSION: The cardioprotective effect of CS-C pretreatment is similar to that of anoxia preconditioning and the effective mechanism would be related to the opening of ATP-sensitive K(+) channel. Glib can abolish the cardioprotective effect of CS-C pretreatment.

Sasanquasaponin protects rat cardiomyocytes against oxidative stress induced by anoxia-reoxygenation injury.

Reactive oxygen species can play an important role in the pathogenesis of anoxia-reoxygenation injury. Sasanquasaponin (SQS) is a biologically active ingredient extracted from the Chinese medicinal plant Camellia oleifera Abel. Some studies have shown that SQS possesses potent antioxidant activities. However, it has not been elucidated whether SQS diminishes reactive oxygen species stress induced by anoxia-reoxygenation injury in cardiomyocytes. In this work, neonatal rat cardiomyocytes pretreated with the test compound were subjected to anoxia-reoxygenation. The extent of cellular damage was accessed by cell viability and the amount of released lactate dehydrogenase (LDH). Superoxide dismutase, catalase and glutathione peroxidase activities, reduced (GSH) and oxidized glutathione (GSSG) levels, and malondialdehyde contents were measured by a colorimetric method. The levels of intracellular reactive oxygen species and calcium were determined by flow cytometry. The results showed that SQS reduced LDH release and increased cell viability in a dose-dependent manner up to 10 microM and concomitantly decreased malondialdehyde and GSSG contents, while significantly increased GSH contents and the activities of superoxide dismutase, catalase and glutathione peroxidase. Moreover, treatment with SQS decreased intracellular reactive oxygen species levels and alleviated calcium accumulation in cardiomyocytes undergoing anoxia-reoxygenation. It is suggested that SQS could protect cardiomyocytes against oxidative stress induced by anoxia-reoxygenation by attenuating reactive oxygen species generation and increasing activities of endogenous antioxidants.