Inhibition of TIM‐4 protects against cerebral ischaemia‐reperfusion injury

TIM‐4 plays an important role in ischaemia‐reperfusion injury of liver and kidney; however, the effects of TIM‐4 on cerebral ischaemia‐reperfusion injury (IRI) are unknown. The purpose of the present study was to investigate the potential role of TIM‐4 in experimental brain ischaemia‐reperfusion injury. In this study, cerebral ischaemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1 hour in C57/BL6 mice. The TIM‐4 expression was detected in vivo or vitro by real‐time quantitative polymerase chain reaction, Western blot and flow cytometric analysis. In vivo, the administration of anti‐TIM‐4 antibodies significantly suppressed apoptosis, inhibited inflammatory cells and enhanced anti‐inflammatory responses. In vitro, activated microglia exhibited reduced cellular proliferation and induced IRI injury when co‐cultured with neurons; these effects were inhibited by anti‐TIM‐4 antibody treatment. Similarly, microglia transfected with TIM‐4 siRNA and stimulated by LPS + IFN‐γ alleviated the TIM‐4‐mediated damage to neurons. Collectively, our data indicate that the inhibition of TIM‐4 can improve the inflammatory response and exerts a protective effect in cerebral ischaemia‐reperfusion injury.     

CRISPR/Cas9‐mediated whole genomic wide knockout screening identifies mitochondrial ribosomal proteins involving in oxygen‐glucose deprivation/reperfusion resistance

Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia‐reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK‐N‐BE(2) cells to oxygen‐glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome‐wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia‐reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine‐type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR‐induced apoptosis. We also demonstrated that OGDR down‐regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome‐scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia‐reperfusion damage.     

Circular RNA YAP1 acts as the sponge of microRNA‐21‐5p to secure HK‐2 cells from ischaemia/reperfusion‐induced injury

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion‐stimulated renal injury. AKI model was established by treating HK‐2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R‐treated HK‐2 cells was evaluated via RT‐qPCR. CCK‐8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR‐21‐5p was verified by RT‐qPCR and luciferase assay. The functions of miR‐21‐5p in I/R‐triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down‐regulated circYAP1 was observed in AKI blood samples and I/R‐treated HK‐2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R‐disposed cells. Besides, we found circYAP1 could sponge to miR‐21‐5p. Interestingly, miR‐21‐5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR‐21‐5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK‐2 cells from I/R injury via sponging miR‐21‐5p.     

MicroRNA‐140‐5p elevates cerebral protection of dexmedetomidine against hypoxic–ischaemic brain damage via the Wnt/β‐catenin signalling pathway

Hypoxia–ischaemia (HI) remains a major cause of foetal brain damage presented a scarcity of effective therapeutic approaches. Dexmedetomidine (DEX) and microRNA‐140‐5p (miR‐140‐5p) have been highlighted due to its potentially significant role in the treatment of cerebral ischaemia. This study was to investigate the role by which miR‐140‐5p provides cerebral protection using DEX to treat hypoxic–ischaemic brain damage (HIBD) in neonatal rats via the Wnt/β‐catenin signalling pathway. The HIBD rat models were established and allocated into various groups with different treatment plans, and eight SD rats into sham group. The learning and memory ability of the rats was assessed. Apoptosis and pathological changes in the hippocampus CA1 region and expressions of the related genes of the Wnt/β‐catenin signalling pathway as well as the genes responsible of apoptosis were detected. Compared with the sham group, the parameters of weight, length growth, weight ratio between hemispheres, the rate of reaching standard, as well as Bcl‐2 expressions, were all increased. Furthermore, observations of increased levels of cerebral infarction volume, total mortality rate, response times, total response duration, expressions of Wnt1, β‐catenin, TCF‐4, E‐cadherin, apoptosis rate of neurons, and Bax expression were elevated. Following DEX treatment, the symptoms exhibited by HIBD rats were ameliorated. miR‐140‐5p and si‐Wnt1 were noted to attenuate the progression of HIBD. Our study demonstrates that miR‐140‐5p promotes the cerebral protective effects of DEX against HIBD in neonatal rats by targeting the Wnt1 gene through via the negative regulation of the Wnt/β‐catenin signalling pathway.     

Long noncoding RNA TUG1 contributes to cerebral ischaemia/reperfusion injury by sponging mir‐145 to up‐regulate AQP4 expression

Emerging studies have shown that long noncoding RNA (lncRNA) TUG1 (taurine‐up‐regulated gene 1) plays critical roles in multiple biological processes. However, the expression and function of lncRNA TUG1 in cerebral ischaemia/reperfusion injury have not been reported yet. In this study, we found that LncRNA TUG1 expression was significantly up‐regulated in cultured MA‐C cells exposed to OGD/R injury, while similar results were also observed in MCAO model. Mechanistically, knockdown of TUG1 decreased lactate dehydrogenase levels and the ratio of apoptotic cells and promoted cell survival in vitro. Moreover, knockdown of TUG1 decreased AQP4 (encoding aquaporin 4) expression to attenuate OGD/R injury. TUG1 could interact directly with miR‐145, and down‐regulation of miR‐145 could efficiently reverse the function of TUG1 siRNA on AQP4 expression. Finally, the TUG1 shRNA reduced the infarction area and cell apoptosis in I/R mouse brains in vivo. In summary, our results suggested that lncRNA TUG1 may function as a competing endogenous RNA (ceRNA) for miR‐145 to induce cell damage, possibly providing a new therapeutic target in cerebral ischaemia/reperfusion injury.     

Renalase contributes to the renal protection of delayed ischaemic preconditioning via the regulation of hypoxia‐inducible factor‐1α

Ischaemic preconditioning (IPC) attenuates acute kidney injury (AKI) from renal ischaemia reperfusion. Renalase, an amine oxidase secreted by the proximal tubule, not only degrades circulating catecholamines but also protects against renal ischaemia reperfusion injury. Here, it has been suggested that the renoprotective effect of renal IPC is partly mediated by renalase. In a model of brief intermittent renal IPC, the increased cortex renalase expression was found to last for 48 hrs. IPC significantly reduced renal tubular inflammation, necrosis and oxidative stress following renal ischaemia reperfusion injury. Such effects were attenuated by blocking renalase with an anti‐renalase monoclonal antibody. We further demonstrated that renalase expression was up‐regulated by hypoxia in vitro via an hypoxia‐inducible factor (HIF)‐1α mechanism. The IPC‐induced up‐regulation of renalase in vivo was also reduced by pre‐treatment with an HIF‐1α inhibitor, 3‐(5′‐Hydroxymethyl‐2′‐furyl)‐1‐benzyl indazole. In summary, the renoprotective effect of IPC is partly dependent on the renalase expression, which may be triggered by hypoxia via an HIF‐1α mechanism. Endogenous renalase shows potential as a therapeutic agent for the prevention and treatment of AKI.     

Methyl‐isobutyl amiloride reduces brain Lac/NAA,cell death and microglial activation in a perinatal asphyxia model

Na⁺/H⁺ exchanger (NHE) blockade attenuates the detrimental consequences of ischaemia and reperfusion in myocardium and brain in adult and neonatal animal studies. Our aim was to use magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry to investigate the cerebral effects of the NHE inhibitor, methyl isobutyl amiloride (MIA) given after severe perinatal asphyxia in the piglet. Eighteen male piglets (aged < 24 h) underwent transient global cerebral hypoxia‐ischaemia and were randomized to (i) saline placebo; or (ii) 3 mg/kg intravenous MIA administered 10 min post‐insult and 8 hourly thereafter. Serial phosphorus‐31 (³¹P) and proton (¹H) MRS data were acquired before, during and up to 48 h after hypoxia‐ischaemia and metabolite‐ratio time‐series Area under the Curve (AUC) calculated. At 48 h, histological and immunohistochemical assessments quantified regional tissue injury. MIA decreased thalamic lactate/N‐acetylaspartate and lactate/creatine AUCs (both p < 0.05) compared with placebo. Correlating with improved cerebral energy metabolism, transferase mediated biotinylated d‐UTP nick end‐labelling (TUNEL) positive cell density was reduced in the MIA group in cerebral cortex, thalamus and white matter (all p < 0.05) and caspase 3 immunoreactive cells were reduced in pyriform cortex and caudate nucleus (both p < 0.05). Microglial activation was reduced in pyriform and midtemporal cortex (both p < 0.05). Treatment with MIA starting 10 min after hypoxia‐ischaemia was neuroprotective in this perinatal asphyxia model.     

Folic acid deficiency enhanced microglial immune response via the Notch1/nuclear factor kappa B p65 pathway in hippocampus following rat brain I/R injury and BV2 cells

Recent studies revealed that folic acid deficiency (FD) increased the likelihood of stroke and aggravated brain injury after focal cerebral ischaemia. The microglia‐mediated inflammatory response plays a crucial role in the complicated pathologies that lead to ischaemic brain injury. However, whether FD is involved in the activation of microglia and the neuroinflammation after experimental stroke and the underlying mechanism is still unclear. The aim of the present study was to assess whether FD modulates the Notch1/nuclear factor kappa B (NF‐κB) pathway and enhances microglial immune response in a rat middle cerebral artery occlusion‐reperfusion (MCAO) model and oxygen‐glucose deprivation (OGD)‐treated BV‐2 cells. Our results exhibited that FD worsened neuronal cell death and exaggerated microglia activation in the hippocampal CA1, CA3 and Dentate gyrus (DG) subregions after cerebral ischaemia/reperfusion. The hippocampal CA1 region was more sensitive to ischaemic injury and FD treatment. The protein expressions of proinflammatory cytokines such as tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 were also augmented by FD treatment in microglial cells of the post‐ischaemic hippocampus and in vitro OGD‐stressed microglia model. Moreover, FD not only dramatically enhanced the protein expression levels of Notch1 and NF‐κB p65 but also promoted the phosphorylation of pIkBα and the nuclear translocation of NF‐κB p65. Blocking of Notch1 with N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester partly attenuated the nuclear translocation of NF‐κB p65 and the protein expression of neuroinflammatory cytokines in FD‐treated hypoxic BV‐2 microglia. These results suggested that Notch1/NF‐κB p65 pathway‐mediated microglial immune response may be a molecular mechanism underlying cerebral ischaemia‐reperfusion injury worsened by FD treatment.     

Poly (I:C) therapy decreases cerebral ischaemia/reperfusion injury via TLR3‐mediated prevention of Fas/FADD interaction

Toll‐like receptor (TLR)‐mediated signalling plays a role in cerebral ischaemia/reperfusion (I/R) injury. Modulation of TLRs has been reported to protect against cerebral I/R injury. This study examined whether modulation of TLR3 with poly (I:C) will induce protection against cerebral I/R injury. Mice were treated with or without Poly (I:C) (n = 8/group) 1 hr prior to cerebral ischaemia (60 min.) followed by reperfusion (24 hrs). Poly (I:C) pre‐treatment significantly reduced the infarct volume by 57.2% compared with untreated I/R mice. Therapeutic administration of Poly (I:C), administered 30 min. after cerebral ischaemia, markedly decreased infarct volume by 34.9%. However, Poly (I:C)‐induced protection was lost in TLR3 knockout mice. In poly (I:C)‐treated mice, there was less neuronal damage in the hippocampus compared with untreated I/R mice. Poly (I:C) treatment induced IRF3 phosphorylation, but it inhibited NF‐κB activation in the brain. Poly (I:C) also decreased I/R‐induced apoptosis by attenuation of Fas/FasL‐mediated apoptotic signalling. In addition, Poly (I:C) treatment decreased microglial cell caspase‐3 activity. In vitro data showed that Poly (I:C) prevented hypoxia/reoxygenation (H/R)‐induced interaction between Fas and FADD as well as caspase‐3 and ‐8 activation in microglial cells. Importantly, Poly (I:C) treatment induced co‐association between TLR3 and Fas. Our data suggest that Poly (I:C) decreases in cerebral I/R injury via TLR3 which associates with Fas, thereby preventing the interaction of Fas and FADD, as well as microglial cell caspase‐3 and ‐8 activities. We conclude that TLR3 modulation by Poly (I:C) could be a potential approach for protection against ischaemic stroke.     

Tat‐antioxidant 1 protects against stress‐induced hippocampal HT‐22 cells death and attenuate ischaemic insult in animal model

Oxidative stress‐induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat‐Atox1 and examined the roles of Tat‐Atox1 in oxidative stress‐induced hippocampal HT‐22 cell death and an ischaemic injury animal model. Tat‐Atox1 effectively transduced into HT‐22 cells and it protected cells against the effects of hydrogen peroxide (H₂O₂)‐induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat‐Atox1 regulated cellular survival signalling such as p53, Bad/Bcl‐2, Akt and mitogen‐activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat‐Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat‐Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat‐Atox1 protects against oxidative stress‐induced HT‐22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat‐Atox1 has potential as a therapeutic agent for the treatment of oxidative stress‐induced ischaemic damage.     

Inhibition of autophagy-dependent pyroptosis attenuates cerebral ischaemia/reperfusion injury

Autophagy is closely associated with cerebral ischaemia/reperfusion injury, but the underlying mechanisms are unknown. We investigated whether Spautin-1 ameliorates cerebral ischaemia/reperfusion injury by inhibiting autophagy and whether its derived pyroptosis is involved in this process. We explored the mechanism of Spautin-1 in cerebral ischaemia/reperfusion. To answer these questions, healthy male Sprague-Dawley rats were exposed to middle cerebral artery occlusion for 60 minutes followed by reperfusion for 24 hours. We found that cerebral ischaemia/reperfusion increased the expression levels of autophagy and pyroptosis-related proteins. Treatment with Spautin-1 reduced the infarct size and water content and restored some neurological functions. In vitro experiments were performed using oxygen-glucose deprivation/reoxygenation to model PC12 cells. The results showed that PC12 cells showed a significant decrease in cell viability and a significant increase in ROS and autophagy levels. Spautin-1 treatment reduced autophagy and ROS accumulation and attenuated NLRP3 inflammasome-dependent pyroptosis. However, these beneficial effects were greatly blocked by USP13 overexpression, which significantly counteracted the inhibition of autophagy and NLRP3 inflammasome-dependent ferroptosis by Spautin-1. Together, these results suggest that Spautin-1 may ameliorate cerebral ischaemia-reperfusion injury via the autophagy/pyroptosis pathway. Thus, inhibition of autophagy may be considered as a promising therapeutic approach for cerebral ischaemia-reperfusion injury.     

Knockdown of lncRNA AK139328 alleviates myocardial ischaemia/reperfusion injury in diabetic mice via modulating miR‐204‐3p and inhibiting autophagy

This study was aimed at investigating the effects of lncRNA AK139328 on myocardial ischaemia/reperfusion injury (MIRI) in diabetic mice. Ischaemia/reperfusion (I/R) model was constructed in normal mice (NM) and diabetic mice (DM). Microarray analysis was utilized to identify lncRNA AK139328 overexpressed in DM after myocardial ischaemia/reperfusion (MI/R). RT‐qPCR assay was utilized to investigate the expressions of lncRNA AK139328 and miR‐204‐3p in cardiomyocyte and tissues. Left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and fractioning shortening (FS) were obtained by transthoracic echocardiography. Haematoxylin‐eosin (HE) staining and Masson staining were utilized to detect the damage of myocardial tissues degradation of myocardial fibres and integrity of myocardial collagen fibres. Evans Blue/TTC staining was used to determine the myocardial infarct size. TUNEL staining was utilized to investigate cardiomyocyte apoptosis. The targeted relationship between lncRNA AK139328 and miR‐204‐3p was confirmed by dual‐luciferase reporter gene assay. MTT assay was used for analysis of cardiomyocyte proliferation. Western blot was utilized to investigate the expression of alpha smooth muscle actin (α‐SMA), Atg7, Atg5, LC3‐II/LC3‐I and p62 marking autophagy. Knockdown of lncRNA AK139328 relieved myocardial ischaemia/reperfusion injury in DM and inhibited cardiomyocyte autophagy as well as apoptosis of DM. LncRNA AK139328 modulated miR‐204‐3p directly. MiR‐204‐3p and knockdown of lncRNA AK139328 relieved hypoxia/reoxygenation injury via inhibiting cardiomyocyte autophagy. Silencing lncRNA AK139328 significantly increased miR‐204‐3p expression and inhibited cardiomyocyte autophagy, thereby attenuating MIRI in DM.     

Dexmedetomidine attenuates neuronal injury after spinal cord ischaemia‐reperfusion injury by targeting the CNPY2‐endoplasmic reticulum stress signalling

Dexmedetomidine (Dex) has been proven to exert protective effects on multiple organs in response to ischaemia‐reperfusion injury, but the specific mechanism by which this occurs has not been fully elucidated. The purpose of this study was to investigate whether Dex attenuates spinal cord ischaemia‐reperfusion injury (SCIRI) by inhibiting endoplasmic reticulum stress (ERS). Our team established a model of SCIRI and utilized the endoplasmic reticulum agonist thapsigargin. Dex (25 g/kg) was intraperitoneally injected 30 minutes before spinal cord ischaemia. After 45 minutes of ischaemia, the spinal cord was reperfused for 24 hours. To evaluate the neuroprotective effect of Dex on SCIRI, neurological function scores were assessed in rats and apoptosis of spinal cord cells was determined by TUNEL staining. To determine whether the endoplasmic reticulum apoptosis pathway CNPY2‐PERK was involved in the neuroprotective mechanism of Dex, the expression levels of related proteins (CNPY2, GRP78, PERK, CHOP, caspase‐12, caspase‐9 and caspase‐3) were detected by western blot analysis and RT‐PCR. We observed that Dex significantly increased the neurological function scores after SCIRI and decreased apoptosis of spinal cord cells. The expression of ERS‐related apoptosis proteins was significantly increased by SCIRI but was significantly decreased in response to Dex administration. Taken together, the results of this study indicate that Dex may attenuate SCIRI by inhibiting the CNPY2‐ERS apoptotic pathway.     

Pre‐conditions for eliminating mitochondrial dysfunction and maintaining liver function after hepatic ischaemia reperfusion

The liver, the largest organ with multiple synthesis and secretion functions in mammals, consists of hepatocytes and Kupffer, stem, endothelial, stellate and other parenchymal cells. Because of early and extensive contact with the external environment, hepatic ischaemia reperfusion (IR) may result in mitochondrial dysfunction, autophagy and apoptosis of cells and tissues under various pathological conditions. Because the liver requires a high oxygen supply to maintain normal detoxification and synthesis functions, it is extremely susceptible to ischaemia and subsequent reperfusion with blood. Consequently, hepatic IR leads to acute or chronic liver failure and significantly increases the total rate of morbidity and mortality through multiple regulatory mechanisms. An increasing number of studies indicate that mitochondrial structure and function are impaired after hepatic IR, but that the health of liver tissues or liver grafts can be effectively rescued by attenuation of mitochondrial dysfunction. In this review, we mainly focus on the subsequent therapeutic interventions related to the conservation of mitochondrial function involved in mitigating hepatic IR injury and the potential mechanisms of protection. Because mitochondria are abundant in liver tissue, clarification of the regulatory mechanisms between mitochondrial dysfunction and hepatic IR should shed light on clinical therapies for alleviating hepatic IR‐induced injury.     

β‐arrestin 2 negatively regulates NOD2 signalling pathway through association with TRAF6 in microglia after cerebral ischaemia/reperfusion injury

We previously reported that nucleotide‐binding oligomerization domain‐containing protein (NOD) 2 was involved in the inflammatory responses to cerebral ischaemia/reperfusion (I/R) insult. However, the mechanism by which NOD2 participates in brain ischaemic injury and the regulation of NOD2 in the process are still obscure. Increased β‐arrestin 2 (ARRB2) expression was observed in microglia following cerebral I/R in wild‐type mice besides the up‐regulation of NOD2 and TRAF6. Stimulation of NOD2 by muramyl dipeptide (MDP) in BV2 cells induced the activation of NF‐κB by the phosphorylation of p65 subunit and the degradation of IκBα. Meanwhile, the protein level of Cyclooxygenase‐2 (COX‐2), the protein expression and activity of MMP‐9 were significantly increased in BV2 cells after administration of MDP. Furthermore, overexpression of ARRB2 significantly suppressed the inflammation induced by MDP, silence of ARRB2 significantly enhanced the inflammation induced by MDP in BV2 cells. In addition, we observed endogenous interaction of TRAF6 and ARRB2 after stimulation of MDP or cerebral I/R insult, indicating ARRB2 negatively regulates NOD2‐triggered inflammatory signalling pathway by associating with TRAF6 in microglia after cerebral I/R injury. Finally, the in vivo study clearly confirmed that ARRB2 negatively regulated NOD2‐induced inflammatory response, as ARRB2 deficiency exacerbated stroke outcomes and aggravated the NF‐κB signalling pathway induced by NOD2 stimulation after cerebral I/R injury. These findings revealed ARRB2 negatively regulated NOD2 signalling pathway through the association with TRAF6 in cerebral I/R injury.     

Neuroprotective effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on ischaemia/reperfusion‐induced neuronal injury by activating the Nrf2/HO‐1 pathway

1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a lipophilic phenolic agent, has an antioxidant activity and reactive oxygen species (ROS) scavenging property. However, the role of HTHQ on cerebral ischaemic/reperfusion (I/R) injury and the underlying mechanisms remain poorly understood. In the present study, we demonstrated that HTHQ treatment ameliorated cerebral I/R injury in vivo, as demonstrated by the decreased infarct volume ration, neurological deficits, oxidative stress and neuronal apoptosis. HTHQ treatment increased the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) and its downstream antioxidant protein, haeme oxygenase‐1 (HO‐1). In addition, HTHQ treatment decreases oxidative stress and neuronal apoptosis of PC12 cells following hypoxia and reperfusion (H/R) in vitro. Moreover, we provided evidence that PC12 cells were more vulnerable to H/R‐induced oxidative stress after si‐Nrf2 transfection, and the HTHQ‐mediated protection was lost in PC12 cells transfected with siNrf2. In conclusion, these results suggested that HTHQ possesses neuroprotective effects against oxidative stress and apoptosis after cerebral I/R injury via activation of the Nrf2/HO‐1 pathway.     

Time Course of Peripheral Oxidative Stress as Consequence of Global Ischaemic Brain Injury in Rats

Free radicals play an important role in the pathogenesis of brain injury. This study evaluates the potential relationship between ischaemia/reperfusion (I/R)-induced brain injury, peripheral oxidative stress (lymphocyte DNA damage), plasma antioxidant potential and uric acid levels. We observed that 15 min of ischaemia were sufficient to significantly increase lymphocyte DNA damage that remained elevated at the end of early (3 h) reperfusion and at later (72 h) reperfusion time; this parameter was not significantly increased, when compared to preoperated levels. In parallel, antioxidant potential was elevated after 15 min of ischaemia, remained high at early (3 h) reperfusion and decreased again with longer (72 h) reperfusion. A close association between the plasma antioxidant status and the uric acid content has been confirmed by findings that changes in TRAP values positively correlate with uric acid concentration in rat plasma after ischaemic injury. Moreover, results of in vitro experiments with extra uric acid addition to control plasma have shown that uric acid contributes to a greater part of TRAP values. These results indicate a similar time course of brain I/R-associated oxidative stress and peripheral antioxidant defence status and/or oxidative stress in animal experiments.     

A key role for Mg(2+) in TRPM7's control of ROS levels during cell stress

The TRPM7 (transient receptor potential melastatin 7) channel has been shown to play a pivotal role in cell survival during brain ischaemia as well as in the survival of other cell types challenged with apoptotic stimuli. Ca(2+) is thought to be central to the channel's ability to regulate ROS (reactive oxygen species) production. However, channel-mediated entry of Mg(2+) and Zn(2+) have also been implicated in cell death. In the present study, we show that depletion of TRPM7 by RNA interference in fibroblasts increases cell resistance to apoptotic stimuli by decreasing ROS levels in an Mg(2+)-dependent manner. Depletion of TRPM7 lowered cellular Mg(2+), decreased the concentration of ROS and lessened p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) activation as well as decreased caspase 3 activation and PARP [poly(ADP-ribose) polymerase] cleavage in response to apoptotic stimuli. Re-expression of TRPM7 or of a kinase-inactive mutant of TRPM7 in TRPM7-knockdown cells increased cellular Mg(2+) and ROS levels, as did expression of the Mg(2+) transporter SLC41A2 (solute carrier family 41 member 2). In addition, expression of SLC41A2 increased the sensitivity of TRPM7-knockdown cells to apoptotic stimuli and boosted ROS generation in response to cell stress. Taken together, these data uncover an essential role for Mg(2+) in TRPM7's control of cell survival and in the regulation of cellular ROS levels.