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.

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.

Treatment Effects of Acetazolamide on Ischemic Stroke: A Meta-Analysis and Systematic Review.

BACKGROUND: Ischemic stroke significantly contributes to high mortality and disability rates. Cerebral edema is a common consequence of ischemic stroke and can lead to aggravation or even death. Current treatment strategies are limited to decompressive craniectomy and the intravascular administration of hypertonic drugs, which have significant side effects. Acetazolamide (ACZ) plays a therapeutic role in cerebral edema by inhibiting aquaporin-4 (AQP-4) and improving collateral circulation. This study aimed to perform a meta-analysis and systematic review of ACZ therapy for ischemic stroke and evaluate its efficacy in animal models. METHODS: We searched Embase, Cochrane Library, PubMed, Web of Science, Chinese National Knowledge Infrastructure, Wanfang Database, and Chinese Biomedical Literature Database until April 2023 for studies on ACZ in ischemic animal models. The quality of the animal trials was assessed using the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Stroke. RESULTS: After screening 376 articles, only 5 studies were included. We found that ACZ reduced brain edema in cerebral ischemia 24 hours after onset (standard mean difference, -2.00; 95% confidence interval, -3.57 to -0.43, P = 0.01). ACZ also inhibited AQP-4 expression 24 hours after onset (standard mean difference-1.46; 95% confidence interval, -2.01 to -0.91, P < 0.001). Brain edema and AQP-4 expression also showed a declining trend on the third day after onset, although there were not enough data to support this. The effect of ACZ on brain ischemia in animals' neurological function is uncertain because of the limited research data. CONCLUSIONS: ACZ inhibited AQP-4 and alleviated brain edema after ischemic stroke in the early stages but seemingly could not improve the neurological function.

Intensity-modulated radiation therapy versus para-aortic field radiotherapy to treat para-aortic lymph node metastasis in cervical cancer: prospective study.

AIM: To compare dosimetry, efficacy, and toxicity of intensity-modulated radiation therapy (IMRT) with para-aortic field radiotherapy in patients with para-aortic lymph node (PALN) metastasis of cervical cancer. METHODS: This prospective study examined 60 patients with cervical cancer with PALN metastasis who underwent whole-pelvis radiotherapy followed by brachytherapy between November 1, 2004 and May 31, 2008. After 3 cycles of chemotherapy, patients were serially allocated into two groups and treated with IMRT or para-aortic field RT at doses of 58-68 Gy and 45-50 Gy, respectively. Treatment response was evaluated and toxicities were assessed. Patients in the IMRT group were treated with both para-aortic field RT and IMRT in order to compare the exposure dose of organs at risk. RESULTS: In the IMRT group, the mean dose delivered to the planning target volume was 67.5 Gy. At least 99% of the gross tumor volume received effective coverage and radical dose (median, 63.5 Gy; range, 54.5-66) during treatment. IMRT plans yielded better dose conformity to the target and better sparing of the spinal cord and small intestine than para-aortic field RT. The IMRT patients experienced less acute and chronic toxicities. The IMRT group also had higher 2- and 3-year survival rates than the para-aortic RT group (2-year, 58.8% vs 25.0%, P = 0.019; 3-year, 36.4% vs 15.6%, P = 0.016). However, no significant difference was found in 1-year survival (67.7% vs 51.3%, P =0.201). The median survival in the IMRT group was 25 months (range, 3 to 37 months). The actuarial overall survival, disease-free survival, and locoregional control rates at 2 years were 67%, 77%, and 88%, respectively, in the IMRT group. CONCLUSIONS: IMRT provides better clinical outcomes than para-aortic field radiotherapy in patients with PALN metastasis. However, cervical local and distal recurrence remain a problem. Long-term follow-up and studies involving more patients are needed to confirm our results.

Optic Nerve Sheath Measurements by Computed Tomography to Predict Intracranial Pressure and Guide Surgery in Patients with Traumatic Brain Injury.

BACKGROUND: Research has shown that the optic nerve sheath diameter (ONSD) is a good predictor of intracranial pressure (ICP) and may predict the need for surgery in patients with head injury. The objective was to test the value of ONSD in predicting the requirement for surgery in patients with traumatic brain injury (TBI). METHODS: In this retrospective cohort study, we first verified the correlation between ICP and ONSD using data from 62 patients with TBI who had undergone ICP monitoring. Second, we analyzed head computed tomography images from patients with TBI who were admitted to the emergency department where patients had been divided into surgery or conservative treatment groups, dependent on the assessment of a neurosurgeon. The correlation between ICP and ONSD was measured using linear regression analysis. Biologistic and receiver operating characteristic curve analyses were used to test the diagnostic value of ONSD to predict surgery. RESULTS: ONSD was significantly correlated with ICP (r = 0.606; P < 0.01), and there was a significant linear regression equation (y = 0.071 × ICP + 3.533; P < 0.01), with ONSD predicting the requirement for surgery in patients with TBI (area under the curve, 0.920; P < 0.01; 95% confidence interval, 0.877-0.962). CONCLUSIONS: ONSD measured via head computed tomography correlates with ICP and can predict the requirement for surgery in patients with TBI following admission to the emergency department.

Exogenous Tetranectin Protects Against 1-Methyl-4-Phenylpyridine-Induced Neurotoxicity by Inhibiting Apoptosis and Autophagy Through Ribosomal Protein S6 Kinase Beta-1.

BACKGROUND: Tetranectin is a secreted homotrimeric protein belonging to the C-type lectin family. Our previous studies found that tetranectin was not only related to, but also played a protective role in, Parkinson disease. In this study, we aim to illustrate the molecular mechanism of the secreted tetranectin. METHODS: We used exogenous tetranectin to investigate the function and molecular mechanism of secreted tetranectin in a 1-methyl-4-phenylpyridine (MPP+)-induced SH-SY5Y cell model. Cell viability and reactive oxygen species were measured to assess the protective effects of tetranectin against MPP+. Apoptosis was measured in several aspects, including Bcl-2/Bax expression, caspase-3/7 activity, annexin V staining, and nuclear morphology. Autophagy was measured as LC3 expression and autophagy flux. Moreover, we used cell immunofluorescence to detect the transport of tetranectin. Western blotting was performed to measure the phosphorylation level of ribosomal protein S6 kinase beta-1 (p70S6K1), and co-immunoprecipitation was applied to confirm the interaction between tetranectin and p70S6K1. RESULTS: The data showed exogenous tetranectin alleviated MPP+-induced toxicity, high reactive oxygen species levels, apoptosis, and autophagy and changed the phosphorylation level of p70S6K1. Immunofluorescence images suggested exogenous tetranectin could be taken into SH-SY5Y cells, and the co-immunoprecipitation experiment indicated tetranectin interacted with p70S6K1. CONCLUSIONS: Exogenous tetranectin protects against MPP+-induced neurotoxicity by promoting p70S6K1 phosphorylation once taken into SH-SY5Y cells.

Involvement of DJ­1 in ischemic preconditioning­induced delayed cardioprotection in vivo.

DJ­1 protein, as a multifunctional intracellular protein, has been demonstrated to serve a critical role in regulating cell survival and oxidative stress. To provide in vivo evidence that DJ­1 is involved in the delayed cardioprotection induced by ischemic preconditioning (IPC) against oxidative stress caused by ischemia/reperfusion (I/R), the present study subjected male Sprague­Dawley rats to IPC (3 cycles of 5­min coronary occlusion/5­min reperfusion) 24 h prior to I/R (30­min coronary occlusion/120­min reperfusion). A lentiviral vector containing short hairpin RNA was injected into the left ventricle three weeks prior to IPC, to knockdown DJ­1 in situ. Lactate dehydrogenase (LDH) and creatine kinase­MB (CK­MB) release, infarct size, cardiac function, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities, malondialdehyde (MDA), intracellular reactive oxygen species (ROS), and DJ­1 protein expression levels were assessed. IPC caused a significant increase in the expression levels of DJ­1 protein. In addition, IPC reduced LDH and CK­MB release, attenuated myocardial infarct size, improved cardiac function following I/R, and inhibited the elevation of ROS and MDA and the decrease in activities of the antioxidant enzymes SOD, CAT and GPx. However, in situ knockdown of DJ­1 attenuated the IPC­induced delayed cardioprotection, and reversed the inhibitory effect of IPC on I/R­induced oxidative stress. The present study therefore provided novel evidence that DJ­1 is involved in the delayed cardioprotection of IPC against I/R injury in vivo. Notably, DJ­1 is required for IPC to inhibit I/R­induced oxidative stress.

Mortality and Outcome Comparison Between Brain Tissue Oxygen Combined with Intracranial Pressure/Cerebral Perfusion Pressure-Guided Therapy and Intracranial Pressure/Cerebral Perfusion Pressure-Guided Therapy in Traumatic Brain Injury: A Meta-Analysis.

BACKGROUND: The combination of brain tissue oxygen and standard intracranial pressure (ICP)/cerebral perfusion pressure (CPP)-guided therapy is thought to improve traumatic brain injury (TBI) prognosis compared with standard ICP/CPP-guided therapy. However, related results of previous observational studies and recently published cohort studies and randomized controlled trials (RCTs) remain controversial. The objective of this study was to compare the effect of the combined therapy with that of standard ICP/CPP-guided therapy on mortality rate, favorable outcome, ICP/CPP, and length of stay (LOS). METHODS: We systematically searched PubMed, Embase, Cochrane Library, ClinicalTrials.gov, and Web of Science in July 2016 for studies comparing the combined therapy and standard ICP/CPP-guided therapy. Random-effect and fixed-effect models were used for pooled analyses. RESULTS: After screening 362 studies, 8 cohort studies and 1 RCT were included. Primary outcomes were mortality and favorable outcome. The overall mortality risk ratio showed no obvious advantages between the 2 groups (risk ratio [RR], 0.76; 95% confidence interval [CI], 0.54-1.06) and discharge mortality (RR, 1.01; 95% CI, 0.80-1.26) and 3-month mortality (RR, 0.77; 95% CI, 0.53-1.12). Compared with the ICP/CPP group, the combined group was more likely to achieve better outcome during the 6 months after TBI (RR, 1.26; 95% CI, 1.04-1.52) or exactly at 6 months (RR, 1.34; 95% CI, 1.07-1.68), whereas ICP (standardized mean difference [SMD], -0.19; 95% CI, -0.43 to 0.05), CPP (SMD, 0.13; 95% CI, -0.09 to 0.35), and LOS (SMD, 0.13; 95% CI, -0.11 to 0.37) showed no obvious differences. CONCLUSIONS: Compared with standard ICP/CPP-guided therapy, brain tissue oxygen combined with ICP/CPP-guided therapy improved long-term outcomes without any effects on mortality, ICP/CPP, or LOS.

Sasanquasaponin induces increase of Cl­/HCO3­ exchange of anion exchanger 3 and promotes intracellular Cl­ efflux in hypoxia/reoxygenation cardiomyocytes.

Anion exchanger 3 (AE3) is known to serve crucial roles in maintaining intracellular chloride homeostasis by facilitating the reversible electroneutral exchange of Cl­ for HCO3­ across the plasma membrane. Our previous studies reported that sasanquasaponin (SQS) can inhibit hypoxia/reoxygenation (H/R)­induced elevation of intracellular Cl­ concentration ([Cl­]i) and elicit cardioprotection by favoring Cl­/HCO3­ exchange of AE3. However, the molecular basis for SQS­induced increase of Cl­/HCO3­ exchange of AE3 remains unclear. The present study demonstrated that SQS activates protein kinase Cε (PKCε) and stimulates the phosphorylation of AE3 in H9c2 cells. Notably, SQS­induced AE3 phosphorylation was blocked by the PKCε selective inhibitor εV1­2, and a S67A mutation of AE3, indicating that SQS could promote phosphorylation of Ser67 of AE3 via a PKCε­dependent regulatory signaling pathway. Additionally, both inhibition of PKCε by εV1­2 and S67A mutation of AE3 eradicated the SQS­induced increase of AE3 activity, reversed the inhibitory effect of SQS on H/R­induced elevation of [Cl­]i, Ca2+ overload and generation of reactive oxygen species, and eliminated SQS­induced cardioprotection. In conclusion, PKCε­dependent phosphorylation of serine 67 on AE3 may be responsible for the increase of Cl­/HCO3­ exchange of AE3 and intracellular chloride efflux by SQS, and contributes to the cardioprotection of SQS against H/R in H9c2 cells.

Sasanquasaponin-induced cardioprotection involves inhibition of mPTP opening via attenuating intracellular chloride accumulation.

Sasanquasaponin (SQS) has been reported to elicit cardioprotection by suppressing hypoxia/reoxygenation (H/R)-induced elevation of intracellular chloride ion concentration ([Cl-]i). Given that the increased [Cl-]i is involved to modulate the mitochondrial permeability transition pore (mPTP), we herein sought to further investigate the role of mPTP in the cardioprotective effect of SQS on H/R injury. H9c2 cells were incubated for 24h with or without 10µM SQS followed by H/R. The involvement of mPTP was determined with a specific mPTP agonist atractyloside (ATR). The results showed that SQS attenuated H/R-induced the elevation of [Cl-]i, accompanied by reduction of lactate dehydrogenase release and increase of cell viability. Moreover, SQS suppressed mPTP opening, and protected mitochondria, as indicated by preserved mitochondrial membrane potential and respiratory chain complex activities, decreased mitochondrial reactive oxygen species generation, and increased ATP content. Interestingly, extracellular Cl--free condition created by replacing Cl- with equimolar gluconate resulted in a decrease in [Cl-]i and induced protective effects similar to SQS preconditioning, whereas pharmacologically opening of the mPTP with ATR abolished all the protective effects induced by SQS or Cl--free, including suppression of mPTP opening, maintenance of mitochondrial membrane potential, and subsequent improvement of mitochondrial function. The above results allow us to conclude that SQS-induced cardioprotection may be mediated by preserving the mitochondrial function through preventing mPTP opening via inhibition of H/R-induced elevation of [Cl-]i.

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.

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 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.