Caveolin-1 Is Critical for Lymphocyte Trafficking into Central Nervous System during Experimental Autoimmune Encephalomyelitis.

UNLABELLED: Multiple sclerosis (MS) is a progressive autoimmune disease of the CNS with its underlying mechanisms not fully understood. In the present study, we tested the hypothesis that caveolin-1, a major membrane scaffolding protein, plays a critical role in the pathogenesis of experimental autoimmune encephalomyelitis, a laboratory murine model of MS. We found increased expression of caveolin-1 in serum and spinal cord tissues in association with disease incidence and severity in wild-type mice with active encephalomyelitis. After immunization, Cav-1 knock-out mice showed remarkable disease resistance with decreased incidence and clinical symptoms. Furthermore, Cav-1 knock-out mice had alleviated encephalitogenic T cells trafficking into the CNS with decreased expressions of adhesion molecules ICAM-1 and VCAM-1 within the lesions. In agreement with in vivo studies, in vitro knockdown of caveolin-1 compromised the upregulation of ICAM-1 in endothelial cells, leading to the amelioration of the transendothelial migration of pathogenic TH1 and TH17 cells. Together, those results indicate that caveolin-1 serves as an active modulator of CNS-directed lymphocyte trafficking and could be a therapeutic target for neuroinflammatory diseases, such as multiple sclerosis. SIGNIFICANCE STATEMENT: The hallmark feature of neuroinflammatory diseases is the massive infiltrations of encephalitogenic leukocytes into the CNS parenchyma, a process that remains largely unclear. Our study demonstrates the critical contribution of caveolin-1 to encephalomyelitis pathogenesis and CNS-directed lymphocyte trafficking by modulation of adhesion molecules ICAM-1 and VCAM-1, highlighting the pathological involvement of caveolin-1 in neuroinflammatory diseases.

Fluorescent Probe HKSOX-1 for Imaging and Detection of Endogenous Superoxide in Live Cells and In Vivo.

Superoxide anion radical (O2(•-)) is undoubtedly the most important primary reactive oxygen species (ROS) found in cells, whose formation and fate are intertwined with diverse physiological and pathological processes. Here we report a highly sensitive and selective O2(•-) detecting strategy involving O2(•-) cleavage of an aryl trifluoromethanesulfonate group to yield a free phenol. We have synthesized three new O2(•-) fluorescent probes (HKSOX-1, HKSOX-1r for cellular retention, and HKSOX-1m for mitochondria-targeting) which exhibit excellent selectivity and sensitivity toward O2(•-) over a broad range of pH, strong oxidants, and abundant reductants found in cells. In confocal imaging, flow cytometry, and 96-well microplate assay, HKSOX-1r has been robustly applied to detect O2(•-) in multiple cellular models, such as inflammation and mitochondrial stress. Additionally, our probes can be efficiently applied to visualize O2(•-) in intact live zebrafish embryos. These probes open up exciting opportunities for unmasking the roles of O2(•-) in health and disease.

Caveolin-1 is essential for protecting against binge drinking-induced liver damage through inhibiting reactive nitrogen species.

UNLABELLED: Caveolin-1 (Cav-1) is known to participate in many diseases, but its roles in alcoholic liver injury remain unknown. In the present study, we aimed to explore the roles of Cav-1 in protecting hepatocytes from ethanol-mediated nitrosative injury. We hypothesized that Cav-1 could attenuate ethanol-mediated nitrosative stress and liver damage through regulating epidermal growth factor receptor/signal transducer and activator of transcription 3/inducible nitric oxide synthase (EGFR/STAT3/iNOS)-signaling cascades. Ethanol-fed mice had time- and dose-dependent increases of Cav-1 in serum and liver with peak increase at 12 hours. Compared to wild-type mice, Cav-1 deficiency mice revealed higher expression of iNOS, higher levels of nitrate/nitrite and peroxynitrite, and had more serious liver damage, accompanied with higher levels of cleaved caspase-3 and apoptotic cell death in liver, and higher levels of alanine aminotransferase and aspartate aminotransferase in serum. Furthermore, the results revealed that the ethanol-mediated Cav-1 increase was in an extracellular signal-regulated kinase-dependent manner, and Cav-1 protected hepatocytes from ethanol-mediated apoptosis by inhibiting iNOS activity and regulating EGFR- and STAT3-signaling cascades. In agreement with these findings, clinical trials in human subjects revealed that serum Cav-1 level was time dependently elevated and peak concentration was observed 12 hours after binge drinking. Alcohol-induced liver lesions were negatively correlated with Cav-1 level, but positively correlated with nitrate/nitrite level, in serum of binge drinkers. CONCLUSIONS: Cav-1 could be a cellular defense protein against alcoholic hepatic injury through inhibiting reactive nitrogen species and regulating EGFR/STAT3/iNOS-signaling cascades.

Molecular imaging of peroxynitrite with HKGreen-4 in live cells and tissues.

Peroxynitrite (ONOO(-)), the product of a radical combination reaction of nitric oxide and superoxide, is a potent biological oxidant involved in a broad spectrum of physiological and pathological processes. Herein we report the development, characterization, and biological applications of a new fluorescent probe, HKGreen-4, for peroxynitrite detection and imaging. HKGreen-4 utilizes a peroxynitrite-triggered oxidative N-dearylation reaction to achieve an exceptionally sensitive and selective fluorescence turn-on response toward peroxynitrite in chemical systems and biological samples. We have thoroughly evaluated the utility of HKGreen-4 for intracellular peroxynitrite imaging and, more importantly, demonstrated that HKGreen-4 can be efficiently employed to visualize endogenous peroxynitrite generated in Escherichia coli-challenged macrophages and in live tissues from a mouse model of atherosclerosis. This probe should serve as a powerful molecular imaging tool to explore peroxynitrite biology under a variety of physiological and pathological contexts.

Targeting reactive nitrogen species: a promising therapeutic strategy for cerebral ischemia-reperfusion injury.

Ischemic stroke accounts for nearly 80% of stroke cases. Recanalization with thrombolysis is a currently crucial therapeutic strategy for re-building blood supply, but the thrombolytic therapy often companies with cerebral ischemia-reperfusion injury, which are mediated by free radicals. As an important component of free radicals, reactive nitrogen species (RNS), including nitric oxide (NO) and peroxynitrite (ONOO(-)), play important roles in the process of cerebral ischemia-reperfusion injury. Ischemia-reperfusion results in the production of nitric oxide (NO) and peroxynitrite (ONOO(-)) in ischemic brain, which trigger numerous molecular cascades and lead to disruption of the blood brain barrier and exacerbate brain damage. There are few therapeutic strategies available for saving ischemic brains and preventing the subsequent brain damage. Recent evidence suggests that RNS could be a therapeutic target for the treatment of cerebral ischemia-reperfusion injury. Herein, we reviewed the recent progress regarding the roles of RNS in the process of cerebral ischemic-reperfusion injury and discussed the potentials of drug development that target NO and ONOO(-) to treat ischemic stroke. We conclude that modulation for RNS level could be an important therapeutic strategy for preventing cerebral ischemia-reperfusion injury.

SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B.

Insulin resistance is often characterized as the most critical factor contributing to the development of type 2 diabetes. SIRT1 has been reported to be involved in the processes of glucose metabolism and insulin secretion. However, whether SIRT1 is directly involved in insulin sensitivity is still largely unknown. Here we show that SIRT1 is downregulated in insulin-resistant cells and tissues and that knockdown or inhibition of SIRT1 induces insulin resistance. Furthermore, increased expression of SIRT1 improved insulin sensitivity, especially under insulin-resistant conditions. Similarly, resveratrol, a SIRT1 activator, enhanced insulin sensitivity in vitro in a SIRT1-dependent manner and attenuated high-fat-diet-induced insulin resistance in vivo at a dose of 2.5 mg/kg/day. Further studies demonstrated that the effect of SIRT1 on insulin resistance is mediated by repressing PTP1B transcription at the chromatin level. Taken together, the finding that SIRT1 improves insulin sensitivity has implications toward resolving insulin resistance and type 2 diabetes.

Caveolin-1 regulates nitric oxide-mediated matrix metalloproteinases activity and blood-brain barrier permeability in focal cerebral ischemia and reperfusion injury.

The roles of caveolin-1 (cav-1) in regulating blood-brain barrier (BBB) permeability are unclear yet. We previously reported that cav-1 was down-regulated and the production of nitric oxide (NO) induced the loss of cav-1 in focal cerebral ischemia and reperfusion injury. The present study aims to address whether the loss of cav-1 impacts on BBB permeability and matrix metalloproteinases (MMPs) activity during cerebral ischemia-reperfusion injury. We found that focal cerebral ischemia-reperfusion down-regulated the expression of cav-1 in isolated cortex microvessels, hippocampus, and cortex of ischemic brain. The down-regulation of cav-1 was correlated with the increased MMP-2 and -9 activities, decreased tight junction (TJ) protein zonula occludens (ZO)-1 expression and enhanced BBB permeability. Treatment of N(G) -nitro-L-arginine methyl ester [L-NAME, a non-selective nitric oxide synthase (NOS) inhibitor] reserved the expression of cav-1, inhibited MMPs activity, and reduced BBB permeability. To elucidate the roles of cav-1 in regulating MMPs and BBB permeability, we used two approaches including cav-1 knockdown in cultured brain microvascular endothelial cells (BMECs) in vitro and cav-1 knockout (KO) mice in vivo. Cav-1 knockdown remarkably increased MMPs activity in BMECs. Meanwhile, with focal cerebral ischemia-reperfusion, cav-1 deficiency mice displayed higher MMPs activities and BBB permeability than wild-type mice. Interestingly, the effects of L-NAME on MMPs activity and BBB permeability was partly reversed in cav-1 deficiency mice. These results, when taken together, suggest that cav-1 plays important roles in regulating MMPs activity and BBB permeability in focal cerebral ischemia and reperfusion injury. The effects of L-NAME on MMPs activity and BBB permeability are partly mediated by preservation of cav-1.

APPL2 Negatively Regulates Olfactory Functions by Switching Fate Commitments of Neural Stem Cells in Adult Olfactory Bulb via Interaction with Notch1 Signaling.

Adult olfactory neurogenesis plays critical roles in maintaining olfactory functions. Newly-generated neurons in the subventricular zone migrate to the olfactory bulb (OB) and determine olfactory discrimination, but the mechanisms underlying the regulation of olfactory neurogenesis remain unclear. Our previous study indicated the potential of APPL2 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 2) as a modulating factor for neurogenesis in the adult olfactory system. In the present study, we report how APPL2 affects neurogenesis in the OB and thereby mediates olfactory discrimination by using both in vitro neural stem cells (NSCs) and an in vivo animal model-APPL2 transgenic (Tg) mice. In the in vitro study, we found that APPL2 overexpression resulted in NSCs switching from neuronal differentiation to gliogenesis while APPL2 knockdown promoted neurogenesis. In the in vivo study, APPL2 Tg mice had a higher population of glial cells and dampened neuronal production in the olfactory system, including the corpus callosum, OB, and rostral migratory stream. Adult APPL2 Tg mice displayed impaired performance in olfactory discrimination tests compared with wild-type mice. Furthermore, we found that an interaction of APPL2 with Notch1 contributed to the roles of APPL2 in modulating the neurogenic lineage-switching and olfactory behaviors. In conclusion, APPL2 controls olfactory discrimination by switching the fate choice of NSCs via interaction with Notch1 signaling.

Neuroprotective Effects and Hepatorenal Toxicity of Angong Niuhuang Wan Against Ischemia-Reperfusion Brain Injury in Rats.

Angong Niuhuang Wan (AGNHW) is a classic prescription in traditional Chinese medicine (TCM) used for stroke treatment, but its efficacies remain to be confirmed. With its arsenic- and mercury-containing materials, the application of AGNHW raises great safety concerns. Herein, we aim to explore the neuropharmacological effects against cerebral ischemia-reperfusion injury and evaluate the toxicological effects of AGNHW for better use. Male SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) and following 22 h of reperfusion. AGNHW (257 mg/kg, 1× AGNHW) were orally administered for pharmacological effects and 257, 514, and 1,028 mg/kg (equivalent to 1×, 2×, 4× AGNHW) were used for the toxicological study. The results revealed that AGNHW treatment reduced the infarct size and protected the blood-brain barrier (BBB) integrity in the MCAO rat ischemic stroke model. AGNHW treatment up-regulated bcl-2 expression and down-regulated the expressions of Bax, p47phox, inducible nitric oxide synthase (iNOS), and 3-nitrotyrosine (3-NT), and inhibited the expressions and activities of matrix metalloproteinase-2 (MMP-2), MMP-9, and reserved tight junction protein zonula occludens-1 (ZO-1) and claudin-5 in the ischemic brains. These results indicated that the neuroprotective mechanisms of AGNHW could be associated with its antioxidant properties by inhibiting oxidative/nitrative stress-mediated MMP activation and protecting tight junction proteins in the ischemic brains. Administration of 1× AGNHW for 7 days would not induce the accumulation of mercury in blood, liver, and kidney at day 14. Administration of 2× AGNHW and 4× AGNHW for 7 days increased the level of mercury in the kidney. For arsenic level, administration of 1× AGNHW for 7 days would increase the level of arsenic in the liver and blood without increase of arsenic in the kidney at day 14. Administration of 2× AGNHW and 4× AGNHW for 7 days would further increase the level of arsenic in the liver and blood. There is no influence on body weight, organ index, histological structures, and renal and liver functions. These results suggest that short-term treatment of AGNHW within 1 week should be safe and has neuroprotective effects against cerebral ischemia-reperfusion injury.

[Research on the soluble solids content of pear internal quality index by near-infrared diffuse reflectance spectroscopy].

The objectives of the present study were to establish the relationships between the near-infrared diffuse reflectance (NIR) spectra and the soluble solids content (SSC) of internal quality index of pear fruit, and to evaluate the value of NIR spectrometry in measuring the SSC of internal quality index of pear fruit. NIR spectroscopy in the 350-1 800 nm range was used to analyze the SSC of internal quality index of pear fruit with multi-linear regression (MLR), principal component regression (PCR) and partial least square (PLS) regression. Meanwhile, the best combinations of different positions at pear fruit, the logarithms of the reflectance reciprocal log(1/R), its first derivative D1 log(1/R) and second derivative D2 log(1/R) were investigated. The best prediction results, based on the comparative analysis, were obtained with the PLS model and D1 log(1/R) at equatorial position of pear fruit. The results show that the predictions with PLS models, based on D1 log(1/R) at equatorial position of pear fruit, are correlation coefficients (R(p)) of 0.851 7 and root mean standard error of prediction (RMSEP) of 0.879 3 for SSC. The preliminary research on the built models indicates that NIR spectroscopy could provide an accurate, reliable and nondestructive method for assessing the SSC of internal quality index of pear fruit.

[Nondestructive measurement of vitamin C in Nanfeng tangerine by visible/near-infrared diffuse reflectance spectroscopy].

The present paper reports the study on visible/near-infrared (Vis/NIR) diffuse reflectance spectroscopy for nondestructive measurement of vitamin C in tangerine (Nanfeng, Jiangxi). In the wavelength range of 350-1800 nm, principal component analysis (PCA) and partial least squares (PLS) were used to build mathematic models with respect to pre-processing spectra. The principal components (PCs) and wavelength selection for calibration model were determined based on full cross validation and regression coefficients. The results, based on prediction for 10 unknown samples, are 0.813, 2.112 mg (100 g)(-1) and -0.810 mg (100 g)(-1) for prediction correlation coefficient, root mean square error of prediction, and prediction deviation, respectively. The analysis methods and conclusions of this experiment will provide some reference values for non-destructive measurement of vitamin C and other quality indices in Nanfeng tangerine using Vis/NIR diffuse reflectance spectrometric technique.

Adaptor Protein APPL2 Affects Adult Antidepressant Behaviors and Hippocampal Neurogenesis via Regulating the Sensitivity of Glucocorticoid Receptor.

Adaptor proteins containing the pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPLs) are multifunctional adaptor proteins involved in regulating many biological activities and processes. The newly identified metabolic factor APPL2 showed the potentials to modulate cell growth, but whether APPL2 could affect adult neurogenesis and animal mood behaviors remains unknown. In the present study, APPL2 transgenic (Tg) mice and wild-type littermates were used for testing our hypothesis that APPL2 could affect glucocorticoid receptor (GR) signaling and modulate hippocampal neurogenesis, which contributes to depressive and anxiety behaviors. Compared with WT littermates, APPL2 Tg mice had enhanced GR phosphorylation under basic condition but had no different plasma corticosterone (CORT) level and GR phosphorylation under stress stimulation. APPL2 Tg mice had decreased hippocampal neurogenesis that was reversed by GR antagonist RU486. APPL2 Tg mice also showed the impaired hippocampal neurogenesis and presented the depressive and anxiety behaviors. In conclusion, APPL2 could be an important regulator for adult neurogenesis. APPL2 overexpression could blunt the activation of glucocorticoid receptor when undergoing environmental stress. Our study suggests that APPL2 might be a new therapeutic target for mental disorders.

Inhibition of Peroxynitrite-Induced Mitophagy Activation Attenuates Cerebral Ischemia-Reperfusion Injury.

Activated autophagy/mitophagy has been intensively observed in ischemic brain, but its roles remain controversial. Peroxynitrite (ONOO-), as a representative of reactive nitrogen species, is considered as a critical neurotoxic factor in mediating cerebral ischemia-reperfusion (I/R) injury, but its roles in autophagy/mitophagy activation remain unclear. Herein, we hypothesized that ONOO- could induce PINK1/Parkin-mediated mitophagy activation via triggering dynamin-related protein 1 (Drp1) recruitment to damaged mitochondria, contributing to cerebral I/R injury. Firstly, we found PINK1/Parkin-mediated mitophagy activation was predominant among general autophagy, leading to rat brain injury at the reperfusion phase after cerebral ischemia. Subsequently, increased nitrotyrosine was found in the plasma of ischemic stroke patients and ischemia-reperfused rat brains, indicating the generation of ONOO- in ischemic stroke. Moreover, in vivo animal experiments illustrated that ONOO- was dramatically increased, accompanied with mitochondrial recruitment of Drp1, PINK1/Parkin-mediated mitophagy activation, and progressive infarct size in rat ischemic brains at the reperfusion phase. FeTMPyP, a peroxynitrite decomposition catalyst, remarkably reversed mitochondrial recruitment of Drp1, mitophagy activation, and brain injury. Intriguingly, further study revealed that ONOO- induced tyrosine nitration of Drp1 peptide, which might contribute to mitochondrial recruitment of Drp1 for mitophagy activation. In vitro cell experiments yielded consistent results with in vivo animal experiments. Taken together, all above findings support the hypothesis that ONOO--induced mitophagy activation aggravates cerebral I/R injury via recruiting Drp1 to damaged mitochondria.

Naringin Attenuates Cerebral Ischemia-Reperfusion Injury Through Inhibiting Peroxynitrite-Mediated Mitophagy Activation.

Excessive autophagy/mitophagy plays important roles during cerebral ischemia-reperfusion (I/R) injury. Peroxynitrite (ONOO-), a representative reactive nitrogen species, mediates excessive mitophagy activation and exacerbates cerebral I/R injury. In the present study, we tested the hypothesis that naringin, a natural antioxidant, could inhibit ONOO--mediated mitophagy activation and attenuate cerebral I/R injury. Firstly, we demonstrated that naringin possessed strong ONOO- scavenging capability and also inhibited the production of superoxide and nitric oxide in SH-SY5Y cells exposed to 10 h oxygen-glucose-deprivation plus 14 h of reoxygenation or ONOO- donor 3-morpholinosydnonimine conditions. Naringin also inhibited the expression of NADPH oxidase subunits and iNOS in rat brains subjected to 2 h ischemia plus 22 h reperfusion. Next, we found that naringin was able to cross the blood-brain barrier, and naringin decreased neurological deficit score, reduced infarct size, and attenuated apoptotic cell death in the ischemia-reperfused rat brains. Furthermore, naringin reduced 3-nitrotyrosine formation, decreased the ratio of LC3-II to LC3-I in mitochondrial fraction, and inhibited the translocation of Parkin to the mitochondria. Taken together, naringin could be a potential therapeutic agent to prevent the brain from I/R injury via attenuating ONOO--mediated excessive mitophagy.

Peroxynitrite enhances self-renewal, proliferation and neuronal differentiation of neural stem/progenitor cells through activating HIF-1α and Wnt/ß-catenin signaling pathway.

Hypoxic/ischemic stimulation could mediate growth and differentiation of neural stem/progenitor cells (NSCs) into mature neurons but its underlying mechanisms are largely unclear. Peroxynitrite formation is considered as a crucial pathological process contributing to cerebral ischemia-reperfusion injury. In the present study, we tested the hypothesis that peroxynitrite at low concentration could function as redox signaling to promote the growth of NSCs under hypoxic/ischemic conditions. Increased NSCs proliferation was accompanied by peroxynitrite production in the rat brains with 1 h of ischemia plus 7 days of reperfusion in vivo. Cell sorting experiments revealed that endogenous peroxynitrite level affected the capacity of proliferation and self-renewal in NSCs in vitro. Hypoxia stimulated peroxynitrite production and promoted NSCs self-renewal, proliferation and neuronal differentiation whereas treatments of peroxynitrite decomposition catalysts (PDCs, FeTMPyP and FeTPPS) blocked the changes in NSCs self-renewal, proliferation and neuronal differentiation. Exogenous peroxynitrite treatment revealed similar effects to promote NSCs proliferation, self-renewal and neuronal differentiation. Furthermore, the neurogenesis-promoting effects of peroxynitrite were partly through activating HIF-1α correlated with enhanced Wnt/ß-catenin signaling pathway. In conclusion, peroxynitrite could be a cellular redox signaling for promoting NSCs proliferation, self-renewal and neuronal differentiation and peroxynitrite production could contribute to neurogenesis in ischemic/hypoxic NSCs.

Baicalin Attenuates Blood-Brain Barrier Disruption and Hemorrhagic Transformation and Improves Neurological Outcome in Ischemic Stroke Rats with Delayed t-PA Treatment: Involvement of ONOO--MMP-9 Pathway.

Tissue plasminogen activator (t-PA) has a restrictive therapeutic window within 4.5 h after ischemic stroke with the risk of hemorrhagic transformation (HT) and neurotoxicity when it is used beyond the time window. In the present study, we tested the hypothesis that baicalin, an active compound of medicinal plant, could attenuate HT in cerebral ischemia stroke with delayed t-PA treatment. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 4.5 h and then continuously received t-PA infusion (10 mg/kg) for 0.5 h and followed by 19-h reperfusion. Baicalin (50, 100, 150 mg/kg) was administrated via femoral vein at 4.5 h after MCAO cerebral ischemia. Delayed t-PA infusion significantly increased the mortality rate, induced HT, blood-brain barrier (BBB) damage, and apoptotic cell death in the ischemic brains and exacerbated neurological outcomes in cerebral ischemia-reperfusion rats at 24 h after MCAO cerebral ischemia. Co-treatment of baicalin significantly reduced the mortality rates, ameliorated the t-PA-mediated BBB disruption and HT. Furthermore, baicalin showed to directly scavenge peroxynitrite and inhibit MMP-9 expression and activity in the ischemic brains with the delayed t-PA treatment. Baicalin had no effect on the t-PA fibrinolytic function indicated by t-PA activity assay. Taken together, baicalin could attenuate t-PA-mediated HT and improve the outcomes of ischemic stroke treatment possibly via inhibiting peroxynitrite-mediated MMP-9 activation.

[Near infrared diffuse reflectance spectroscopy for rapid analysis of soluble solids content in navel orange].

The potential of using visible and near infrared diffuse reflectance spectroscopy to assess soluble solids content (SSC) of intact navel orange was examined. A total 40 samples were used to develop the calibration and prediction models. NIR spectral data were collected in the spectral region between 350 and 2 500 nm and their second derivative spectra were used for the present study. Different scattering correction algorithms (no preprocessing and multiplicative scattering correction (MSC)) were compared. Camibration models based on different spectral ranges, different derivatives and different kinds of statistical models including partial least square (PLS) and principal component regression (PCR) were also compared. The best results of PLS models with the second derivative spectra are r = 0.929, RMSEC = 0.517 and RMSEP = 0.592 in the wavelength range of 361-2 488 nm. The results show that this method is feasible for non destructive assessing of SSC of the navel orange.

Reactive nitrogen species as therapeutic targets for autophagy: implication for ischemic stroke.

INTRODUCTION: Roles of autophagy/mitophagy activation in ischemic stroke remain controversial. To elucidate potential reasons, we analyze the factors responsible for divergent results in literatures. Reactive nitrogen species (RNS) are important cytotoxic factors in ischemic stroke. Herein, we particularly discuss the roles played by RNS in autophagy/mitophagy and ischemic brain injury. Areas covered: Following factors should be considered in the studies on autophagy/mitophagy in ischemic stroke: (1) Protocols for administration of autophagy regulators including administration time points, routes and doses, etc.; (2) Specificity of autophagy regulators; (3) Animal models of cerebral ischemia with or without reperfusion. In the underlying mechanisms of autophagy/mitophagy, we particularly discuss the potential roles of RNS in mediating excessive autophagy/mitophagy during cerebral ischemia/reperfusion injury. Expert opinion: Emphasis should be given to the following aspects in future studies: (1) Targeting RNS and related cellular signaling pathways in the regulation of autophagy/mitophagy might be a promising strategy for developing novel drugs as well as combined therapy for thrombolytic treatment to reach better outcomes for ischemic stroke; (2) Developing circulating plasma biomarkers linking RNS-mediated autophagy/mitophagy to the magnitude of ischemic brain injury will benefit for stroke treatment. Subsequently, RNS could be dominant therapeutic targets to regulate autophagy/mitophagy for ischemic stroke.