Xingmiao Chen

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

J. Neurochem. (2012) 120, 147–156.

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.

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.

From rapid to delayed and remote postconditioning: the evolving concept of ischemic postconditioning in brain ischemia.

Ischemic postconditioning is a concept originally defined to contrast with that of ischemic preconditioning. While both preconditioning and postconditioning confer a neuroprotective effect on brain ischemia, preconditioning is a sublethal insult performed in advance of brain ischemia, and postconditioning, which conventionally refers to a series of brief occlusions and reperfusions of the blood vessels, is conducted after ischemia/reperfusion. In this article, we first briefly review the history of preconditioning, including the experimentation that initially uncovered its neuroprotective effects and later revealed its underlying mechanisms-of-action. We then discuss how preconditioning research evolved into that of postconditioning--a concept that now represents a broad range of stimuli or triggers, including delayed postconditioning, pharmacological postconditioning, remote postconditioning--and its underlying protective mechanisms involving the Akt, MAPK, PKC and K(ATP) channel cell-signaling pathways. Because the concept of postconditioning is so closely associated with that of preconditioning, and both share some common protective mechanisms, we also discuss whether a combination of preconditioning and postconditioning offers greater protection than preconditioning or postconditioning alone.

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.

Baicalin can scavenge peroxynitrite and ameliorate endogenous peroxynitrite-mediated neurotoxicity in cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE Baicalin is one of the principal flavonoids isolated from the dried root of Scutellaria baicalensis Georgi that has long been used to treat ischemic stroke. However, its neuroprotective mechanisms against cerebral ischemia injury are poorly understood. AIM OF THE STUDY To explore the neuroprotective mechanisms of baicalin against cerebral ischemia reperfusion injury. MATERIAL AND METHODS In chemical systems, we conducted electron paramagnetic resonance (EPR) spin trapping experiments to evaluate the scavenging effects of baicalin on superoxide and nitric oxide, and mass spectrometry (MS) studies on the reaction of baicalin and peroxynitrite. In cellular experiments, we investigated the effects of baicalin against extraneous and endogenous peroxynitrite mediated neurotoxicity in SH-SY5Y cells treated with peroxynitrite donor, synthesized peroxynitrite and exposed to oxygen glucose deprivation and reoxygenation (OGD/RO) in vitro. Moreover, we studied the neuroprotective effects of baicalin by using a rat model of middle cerebral artery occlusion in vivo. FeTMPyP, a peroxynitrite decomposition catalyst, was used as positive control. Cell viability and apoptotic cell death was accessed by MTT assay and TUNEL assay respectively; 3-nitrotyrosine formation and infarction volume were detected by immunostaining experiments and TTC staining respectively. RESULTS Baicalin revealed strong antioxidant ability by directly scavenging superoxide and reacting with peroxynitrite. Baicalin protected the neuronal cells from extraneous and endogenous peroxynitrite-induced neurotoxicity. In ischemia-reperfused brains, baicalin inhibited the formation of 3-nitrotyrosine, reduced infarct size and attenuated apoptotic cell death, whose effects were similar to FeTMPyP. CONCLUSIONS Baicalin can directly scavenge peroxynitrite and the peroxynitrite-scavenging ability contributes to its neuroprotective mechanisms against cerebral ischemia reperfusion injury.

Calycosin-7-O-β-d-glucoside regulates nitric oxide /caveolin-1/matrix metalloproteinases pathway and protects blood–brain barrier integrity in experimental cerebral ischemia–reperfusion injury

ETHNOPHARMACOLOGY RELEVANCE Astragali Radix (AR) has been used for thousands years to treat ischemic stroke. Calycosin and its glycoside form calycosin-7-O-β-D-glucoside (CG) are two representative isoflavones in Astragali Radix. However, its neurological effects and related molecular mechanisms are largely unknown. The present study aims to evaluate the neuroprotective effects of CG on blood-brain barrier (BBB) integrity of ischemic brain tissue and explore the relevant signaling mechanisms. MATERIAL AND METHOD Male adult Sprague-Daweley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus 24 h or 14 days of reperfusion. CG (26.8 mg/kg) was intraperitoneally administered into the rats at 15 min before onset of ischemia. The neuroprotective effects of CG were evaluated by measuring infarct volume, histological damage and BBB permeability. Furthermore, the effects of CG on scavenging nitric oxide (NO), and modulating matrix metalloproteinases (MMPs) and caveolin-1 (cav-1) were investigated with in vitro cultured brain microvascular endothelial cells treated with NO donor or oxygen-glucose deprivation (OGD) and/or in vivo rat model of MCAO cerebral ischemia-reperfusion injury. RESULTS CG treatment significantly reduced infarct volume, histological damage and BBB permeability in the in vivo MCAO ischemia-reperfusion rat model. CG treatment remarkably inhibited the expression and activities of MMPs, and secured the expression of cav-1 and tight junction proteins in the microvessels isolated from ischemic rat cortex. Furthermore, CG was revealed to scavenge NO, inhibit the activities of MMP-2 and MMP-9, and attenuate cell death in the in vitro cultured brain microvascular endothelial cells under OGD condition. CONCLUSION CG could protect BBB integrity in experimental cerebral ischemia-reperfusion injury via regulating NO/cav-1/MMPs pathway.

Beyond water channel: Aquaporin-4 in adult neurogenesis

Aquaporin-4 (AQP4) is a key molecule for maintaining water and ion homeostasis associated with neuronal activity in the central nervous system, but the roles of AQP4 in adult neurogenesis remain largely unexplored. Based on preliminary evidences over the past years, AQP4 appears to emerge as an important player regulating the multiple steps of adult neurogenesis. In this mini-review, we discuss the recent findings that reveal a specific functional role of AQP4 in regulating the adult neurogenesis, including proliferation of neural progenitors/neural stem cells, fate specification and differentiation, neuronal migration, and the potential mechanisms. Further studies on the regulation of AQP4 in promoting neurogenesis will lead to better understanding of the signaling mechanisms of adult neurogenesis and potentially provide an opportunity to develop AQP4 as new drug target for neurogenesis.

Pros and cons of current approaches for detecting peroxynitrite and their applications

Peroxynitrite, a representative of reactive nitrogen species, plays important roles in the physiological and pathological processes of many oxidative stress-related diseases. It is generated from the reaction of nitric oxide (NO) and superoxide (O2·–) and is far more active than its precursors. Peroxynitrite can be further decomposed into other cytotoxic reactive species. Peroxynitrite and its derivatives can interact with various biomolecules such as DNA and proteins. Due to its high reactivity and short lifetime, accurate detection of peroxynitrite in biological systems is a challenge task. In the last decade, huge efforts have been made to develop reliable techniques to assess the generation of peroxynitrite in various cellular and animal experiments. There are three major approaches for peroxynitrite detection, including electrochemical sensors, detection of nitrotyrosine formation, and fluorescent probes. Particularly, progress has been made in developing novel fluorescent probes to detect peroxynitrite with relatively high sensitivity and specificity. Herein, we review the recent progress made in peroxynitrite detection methods and discuss the advantages and disadvantages of these methods. The development of these techniques will offer new opportunities for understanding the roles of peroxynitrite in the oxidative stress-related physiological and pathological conditions and provide platforms for drug discovery targeting peroxynitrite and other free radicals for therapeutic purposes.

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

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. (Hepatology 2014;60:687–699)