Allopurinol attenuates oxidative injury in rat hearts suffered ischemia/reperfusion via suppressing the xanthine oxidase/vascular peroxidase 1 pathway.

Allopurinol, a xanthine oxidase (XO) inhibitor, is reported to alleviate myocardial ischemia/reperfusion (I/R) injury by reducing the production of reactive oxygen species (ROS). As an XO-derived product, H2O2 can act as a substrate of vascular peroxidase 1 (VPO1) to induce the generation of hypochlorous acid (HOCl), a potent oxidant. This study aims to explore whether the XO/VPO1 pathway is involved in the anti-oxidative effects of allopurinol on the myocardial I/R injury. In a rat heart model of I/R, allopurinol alleviated I/R oxidative injury accompanied by decreased XO activity, XO-derived products (H2O2 and uric acid), and VPO1 expression (mRNA and protein). In a cardiac cell model of hypoxia/reoxygenation (H/R), allopurinol or XO siRNA reduced H/R injury concomitant with decreased XO activity, VPO1 expression as well as the XO and VPO1-derived products (H2O2, uric acid, and HOCl). Although knockdown of VPO1 could also exert a beneficial effect on H/R injury, it did not affect XO activity, XO expression, and XO-derived products. Based on these observations, we conclude that the novel pathway of XO/VPO1 is responsible for, at least partly, myocardial I/R-induced oxidative injury, and allopurinol exerted the cardioprotective effects on myocardial I/R injury via inhibiting the XO/VPO1 pathway.

Fasudil ameliorates the ischemia/reperfusion oxidative injury in rat hearts through suppression of myosin regulatory light chain/NADPH oxidase 2 pathway.

Fasudil is a potent Rho-kinase (ROCK) inhibitor and can relax smooth muscle or cardiac muscle contraction through decreasing the phosphorylation level of myosin regulatory light chain (p-MLC20 or p-MLC2v), while p-MLC2v can function as a transcription factor to promote the NADPH oxidase 2 (NOX2) expression in rat hearts subjected to ischemia/reperfusion (I/R). This study aims to explore whether fasudil can protect the rat hearts against I/R oxidative injury through suppressing NOX2 expression via reduction of p-MLC2v level. The SD rat hearts were subjected to 1h-ischemia plus 3h-reperfusion, which showed myocardial injuries (myocardial fiber loss and disarray, increase of creatine kinase release and myocardial infarction/apoptosis), increase in ROCK activity and nuclear p-MLC2v level concomitant with up-regulation of NOX2 and H2O2 production; these phenomena were attenuated by fasudil in a dose-dependent manner. Next, we verified the cardioprotective effect of fasudil and the underlying mechanisms in hypoxia-reoxygenation (H/R) -treated H9c2 cells. Consistent with the results in vivo, the H/R-treated H9c2 cells showed cellular injury (increase in apoptotic ratio), elevation in ROCK activity and nuclear p-MLC2v level, accompanied by up-regulation of NOX2 and H2O2 production; these effects were blocked in the presence of fasudil in a dose-dependent way. Based on these observations, we conclude that beneficial effect of fasudil against myocardial I/R or H/R oxidative injury is related to the suppression of NOX2 expression through decrease of the p-MLC2v level. Our findings also highlight that intervention of MLC2v phosphorylation by drugs may provide a novel strategy to protect heart from I/R oxidative injury.

Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of ß2-AR/PI3K/AKT signaling pathway.

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (ß2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by ß2-AR but not ß1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a ß2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the ß2-AR/PI3K/AKT cascade.

Nuclear cardiac myosin light chain 2 modulates NADPH oxidase 2 expression in myocardium: a novel function beyond muscle contraction.

Recent studies demonstrated that NADPH oxidase 2 (NOX2) expression in myocardium after ischemia-reperfusion (IR) is significantly upregulated. However, the underlying mechanisms remain unknown. This study aims to determine if nuclear cardiac myosin light chain 2 (MYL2), a well-known regulatory subunit of myosin, functions as a transcription factor to promote NOX2 expression following myocardial IR in a phosphorylation-dependent manner. We examined the phosphorylation status of nuclear MYL2 (p-MYL2) in a rat model of myocardial IR (left main coronary artery subjected to 1 h ligation and 3 h reperfusion) injury, which showed IR injury and upregulated NOX2 expression as expected, accompanied by elevated H2O2 and nuclear p-MYL2 levels; these effects were attenuated by inhibition of myosin light chain kinase (MLCK). Next, we explored the functional relationship of nuclear p-MYL2 with NOX2 expression in H9c2 cell model of hypoxia-reoxygenation (HR) injury. In agreement with our in vivo findings, HR treatment increased apoptosis, NOX2 expression, nuclear p-MYL2 and H2O2 levels, and the increases were ameliorated by inhibition of MLCK or knockdown of MYL2. Finally, molecular biology techniques including co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), DNA pull-down and luciferase reporter gene assay were utilized to decipher the molecular mechanisms. We found that nuclear p-MYL2 binds to the consensus sequence AGCTCC in NOX2 gene promoter, interacts with RNA polymerase II and transcription factor IIB to form a transcription preinitiation complex, and thus activates NOX2 gene transcription. Our results demonstrate that nuclear MYL2 plays an important role in IR injury by transcriptionally upregulating NOX2 expression to enhance oxidative stress in a phosphorylation-dependent manner.

A novel pathway of NADPH oxidase/vascular peroxidase 1 in mediating oxidative injury following ischemia-reperfusion.

Vascular peroxidase 1 (VPO1) can utilize reactive oxygen species (ROS) generated from NADPH oxidase (NOX) to catalyze peroxidative reactions. This study was performed to identify a novel pathway of NOX/VPO1 in mediating the oxidative injury following myocardial ischemia reperfusion (IR). In a rat model of myocardial IR, the infarct size, serum creatine kinase (CK) activity, apoptosis, NOX activity, NOX2 and VPO1 expression were measured. In a cell (rat heart-derived H9c2 cells) model of hypoxia/reoxygenation (HR), the apoptosis, NOX activity, NOX2 and VPO1 expression, and H(2)O(2) and HOCl levels were examined. In vivo, IR caused 54.8 ± 1.7 % infarct size in myocardium accompanied by elevated activities of CK, caspase-3 and NOX, up-regulated VPO1 expression and high numbers of myocardial apoptotic cells; these effects were attenuated by pretreatment with the inhibitor of NOX. In vitro, inhibition of NOX or silencing of NOX2 or VPO1 expression significantly suppressed HR-induced cellular apoptosis concomitantly with decreased HOCl production. Inhibition of NOX or silencing of NOX2 led to a decrease in H(2)O(2) production accompanied by a decrease in VPO1 expression and HOCl production. However, silencing of VPO1 expression did not affect NOX2 expression and H(2)O(2) production. H(2)O(2)-induced VPO1 expression was partially reversed by JNK or p38 MAPK inhibitor. Our results demonstrate a novel pathway of NOX2/VPO1 in myocardium, where VPO1 coordinates with NOX2 and amplifies the role of NOX-derived ROS in oxidative injury following IR.

Beneficial effects of capsiate on ethanol-induced mucosal injury in rats are related to stimulation of calcitonin gene-related Peptide release.

Capsiate is a non-pungent analogue of capsaicin from CH-19 Sweet peppers. Capsaicin is reported to trigger calcitonin gene-related peptide (CGRP) release through activation of transient receptor potential vanilloid subfamily member 1 (TRPV1) and produces beneficial effects on gastric mucosa. This study aimed to investigate whether capsiate is able to produce beneficial effects on gastric mucosa and whether the protective effects of capsipate occur through a mechanism involving the activation of TRPV1 and CGRP release. A rat model of gastric mucosal injury was established by the oral administration of acidified ethanol. Gastric tissues were collected for analysis of the gastric ulcer index, cellular apoptosis, activities of caspase-3, catalase and superoxide dismutase (SOD), and levels of CGRP, TNF-α, and malondialdehyde (MDA). Our results show that the acute administration of ethanol significantly increased the gastric ulcer index concomitantly with an increase in cellular apoptosis, caspase-3 activity, and TNF-α and MDA levels, as well as a decrease in the activities of catalase and SOD. Pretreatment with 1 mg/kg capsiate attenuated ethanol-induced gastric mucosal injury and cellular apoptosis accompanied by an increase in CGRP level, catalase, and SOD activities, and a decrease in caspase-3 activity, and TNF-α and MDA levels. The effects of capsiate were inhibited by capsazepine, an antagonist of TRPV1. These results suggest that capsiate is able to produce beneficial effects on ethanol-induced gastric mucosal injury. These effects are related to the stimulation of CGRP release through the activation of TRPV1.

Vanillyl nonanoate protects rat gastric mucosa from ethanol-induced injury through a mechanism involving calcitonin gene-related peptide.

Previous studies have demonstrated that capsaicin-sensitive sensory nerves are involved in the protection of gastric mucosa against damage by various stimuli and calcitoin gene-related peptide (CGRP) is a potential mediator in this process. This study was performed to explore the effect of vanillyl nonanoate, a capsaicin analog, on ethanol-induced gastric mucosal injury and the possible underlying mechanisms. A rat model of gastric mucosal injury was induced by oral administration of acidified ethanol and gastric tissues were collected for analysis of gastric ulcer index, cellular apoptosis, the activities of caspase-3, catalase and superoxide dismutase (SOD), levels of CGRP, TNF-α and malondialdehyde (MDA). The results showed that acute administration of ethanol significantly increased gastric ulcer index concomitantly with increased cellular apoptosis, caspase-3 activity, TNF-α and MDA levels as well as decreased activities of catalase and SOD. Pretreatment with 1mg/kg vanillyl nonanoate significantly attenuated ethanol-induced gastric mucosal injury and cellular apoptosis accompanied by increase of CGRP expression, and SOD activity and decrease of caspase-3 activity, TNF-α and MDA levels. The effects of vanillyl nonanoate were inhibited by capsazepine, an antagonist of capsaicin receptor. Our results suggested that vanillyl nonanoate was able to protect the gastric mucosa against ethanol-induced gastric mucosal injury. The underlying mechanism is related to stimulation of CGRP release and subsequent suppression of ethanol-induced inflammatory reaction, cellular apoptosis and oxidative stress.

The role of the DDAH-ADMA pathway in the protective effect of resveratrol analog BTM-0512 on gastric mucosal injury.

A recent study showed that resveratrol, a polyphenol found in many plant species, exerts dual effects on gastric mucosal injury. By using the model of ethanol-induced gastric mucosal injury in the present study, we explored the effect of trans-3,5,4'-trimethoxystilbene (BTM-0512), a novel analog of resveratrol, on gastric mucosal injury and the possible underlying mechanisms. Gastric mucosal injury in the rat was induced by oral administration of acidified ethanol. The gastric tissues were collected for determination of the gastric ulcer index, asymmetric dimethylarginine (ADMA) and nitric oxide (NO) contents, the activity of dimethylarginine dimethylaminohydrolase (DDAH) and superoxide anion (O2(-)) or hydroxyl radical (OH*) formation. The results showed that acute administration of ethanol significantly increased the gastric ulcer index concomitantly with the decrease in DDAH activity and NO content as well as the increase in ADMA content, effects that were reversed by pretreatment with BTM-0512 (100 mg/kg) or L-arginine (300 mg/kg). Administration of BTM-0512 did not show a significant effect on O2(-) or OH. formation. The results suggest that BTM-0512 could protect the gastric mucosa against ethanol-induced injury, which is mainly related to an increase in DDAH activity and subsequent decrease in ADMA content.