He-Ping Chen

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.

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. Copyright

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.

Cardioprotective effect of sasanquasaponin preconditioning via bradykinin-NO pathway in isolated rat heart.

Sasanquasaponin (SQS) is an effective component of Camellia oleifera Abel. This study was designed to investigate the cardioprotective effect of SQS against ischemia‐reperfusion (I/R) injury and the possible mechanism in isolated rat hearts. These hearts were pretreated by SQS only or SQS and HOE140 in different groups, and then subjected to I/R injury. Hemodynamic parameters, oxidative injury, and NO level were measured. The results showed that SQS preconditioning could decrease the incidences of arrhythmias and improve the heart functions. In addition, SQS preconditioning could protect isolated I/R injured heart against lipid peroxidation, as evidenced by increases in SOD and GSH‐Px activity, and by decreases in contents of MDA, ROS generation. However, HOE140 treatment reversed all these indexes. NO production was significantly decreased after a treatment with HOE140. So we can propose that SQS preconditioning could induce the cardioprotective effects and the possible mechanism was that the activation of bradykinin‐NO system by SQS preconditioning had an inhibition effect on ROS generation in isolated heart. Copyright

DJ-1 is involved in the peritoneal metastasis of gastric cancer through activation of the Akt signaling pathway

Peritoneal metastasis is a major cause of death in patients with advanced gastric carcinoma. DJ-1 is now considered to play an important role in the metastasis of various malignancies. However, it remains largely unclear whether DJ-1 is involved in the development of peritoneal metastasis by gastric carcinoma. In the present study, we showed that the expression of DJ-1 was significantly upregulated in gastric cancer specimens with peritoneal metastasis compared to those without peritoneal metastasis. Knockdown of DJ-1 expression significantly inhibited invasion and migration, in vitro and the in vivo peritoneal metastatic abilities of SGC7901 gastric cancer cells. Moreover, knockdown of DJ-1 also diminished the expression of matrix metallopeptidase (MMP)-2 and MMP-9. All of these effects were reversed by restoration of DJ-1 expression. Following investigation of the pathway through which DJ-1 regulates cell invasion and migration, DJ-1 was found to cause phosphorylation of Akt in SGC7901 gastric cancer cells. Inhibition of the Akt pathway in SGC7901 cells mimicked the effects of DJ-1 knockdown on cell migration, invasion, MMP-2 and MMP-9 expression, and abolished the effects of DJ-1 in promoting SGC7901 cell invasion and migration. Taken together, the present study revealed that DJ-1 plays an important role in the development of peritoneal carcinomatosis from gastric carcinoma, at least partially through activation of the Akt pathway and consequent upregulation of MMP-2 and MMP-9 expression. Thus, DJ-1 may be a potential therapeutic target for peritoneal carcinomatosis of gastric carcinoma.

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.

Upregulation of 14-3-3 isoforms in acute rat myocardial injuries induced by burn and lipopolysaccharide

1 Burn‐induced myocardial injuries can be acute due to loss of body fluid and blood redistribution, and subacute due to pathogenic toxins of infecting bacteria. The goal of this study was to examine expression of 14‐3‐3 in the injured myocardium. 2 Myocardial injury models were created in vivo by subjecting rats to severe burn and administration of lipopolysaccharide. RT‐PCR and Western blotting were employed to assess the expression of 14‐3‐3 proteins and messenger ribonucleic acid (mRNA) for 14‐3‐3h and g in the myocardium, respectively. 3 In the two models, we found that 14‐3‐3 proteins were induced in a time‐dependent fashion. Such a change is at least in part attributed to increases in mRNAs for 14‐3‐3g and h. In contrast to 14‐3‐3z, whose mRNA was not detectable in the heart, mRNA for 14‐3‐3g was found significantly elevated between 24–48 h after burn. 14‐3‐3h mRNA exhibited a marked increase at 3 h continuing to 12 h and then decreased nearly to a normal level after 48 h. In lipopolysaccharide‐treated intact rats, 14‐3‐3g mRNA in myocardium showed a significant increase, reaching a peak at 4 h, followed by a decrease at 6 h. In contrast, 14‐3‐3h mRNA had a slight increase without significance. 4 Our results suggest that 14‐3‐3 may play a role in both acute and subacute (postburn infectious) phases of severe burn.

PRL-3 promotes the peritoneal metastasis of gastric cancer through the PI3K/Akt signaling pathway by regulating PTEN.

Peritoneal metastasis is the most frequent cause of death in patients with advanced gastric carcinoma (GC). The phosphatase of regenerating liver-3 (PRL-3) is recognized as an oncogene and plays an important role in GC peritoneal metastasis. However, the mechanism of how PRL-3 regulates GC invasion and metastasis is unknown. In the present study, we found that PRL-3 presented with high expression in GC with peritoneal metastasis, but phosphatase and tensin homologue (PTEN) was weakly expressed. The p-PTEN/PTEN ratio was also higher in GC with peritoneal metastasis than that in the normal gastric tissues. We also found the same phenomenon when comparing the gastric mucosa cell line with the GC cell lines. After constructing a wild-type and a mutant-type plasmid without enzyme activity and transfecting them into GC SGC7901 cells, we showed that only PRL-3 had enzyme activity to downregulate PTEN and cause PTEN phosphorylation. The results also showed that PRL-3 increased the expression levels of MMP-2/MMP-9 and promoted the migration and invasion of the SGC7901 cells. Knockdown of PRL-3 decreased the expression levels of MMP-2/MMP-9 significantly, which further inhibited the migration and invasion of the GC cells. PRL-3 also increased the expression ratio of p-Akt/Akt, which indicated that PRL-3 may mediate the PI3K/Akt pathway to promote GC metastasis. When we transfected the PTEN siRNA plasmid into the PRL-3 stable low expression GC cells, the expression of p-Akt, MMP-2 and MMP-9 was reversed. In conclusion, our results provide a bridge between PRL-3 and PTEN; PRL-3 decreased the expression of PTEN as well as increased the level of PTEN phosphorylation and inactivated it, consequently activating the PI3K/Akt signaling pathway, and upregulating MMP-2/MMP-9 expression to promote GC cell peritoneal metastasis.

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 anti‑oxidant enzymes and attenuate hypoxia/re‑oxygenation (H/R)‑induced oxidative stress in the cardiomyocyte cell line H9c2; however, the detailed molecular mechanisms have remained to be elucidated. Nuclear factor erythroid 2‑like 2 (Nrf2) is an essential transcription factor that regulates the expression of several anti‑oxidant genes via binding to the anti‑oxidant response element (ARE). The present study investigated whether activation of the Nrf2 pathway is responsible for the induction of anti‑oxidative enzymes by DJ‑1 and contributes to the protective functions of DJ‑1 against H/R‑induced oxidative stress in H9c2 cells. The results demonstrated that DJ‑1‑overexpressing H9c2 cells exhibited anti‑oxidant enzymes, including manganese superoxide dismutase, catalase and glutathione peroxidase, to a greater extent and were more resistant to H/R‑induced oxidative stress compared with native cells, whereas DJ‑1 knockdown suppressed the induction of these enzymes and further augmented the oxidative stress injury. Determination of the importance of Nrf2 in DJ‑1‑mediated anti‑oxidant enzymes induction and cytoprotection against oxidative stress induced by H/R showed that overexpression of DJ‑1 promoted the dissociation of Nrf2 from its cytoplasmic inhibitor Keap1, resulting in enhanced levels of nuclear translocation, ARE‑binding and transcriptional activity of Nrf2. Of note, Nrf2 knockdown abolished the DJ‑1‑mediated induction of anti‑oxidant 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.

Abstract: 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.