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2.6.3 Specific NOX Inhibitors
In contrast to using antioxidants and non-specific NOX inhibitors, specific NOX inhibitors, which can inhibit the relevant sources of ROS in different pathologies, are in dire need. Several researcher groups have already started embarking on the production of specific NOX inhibitors [94].
The triazolo pyrimidine VAS2870, 3-benzyl-7-(2-benzoxazolyl) thio-1,2,3- triazolo (4,5-d) pyrimidine, is a more specific NOX inhibitor that has already shown its strong protective effects in preclinical stroke studies [71, 95, 96]. VAS2870 and its derivate VAS3947 was thought to likely inhibit all NOX isoforms by inhibiting assembly or conformation changes to active NOX complexes [66, 93, 97]. It can inhibit NADPH oxidase activity in platelet-derived growth factor (PDGF)-stimulated primary rat aortic vascular smooth muscle cell and oxLDL-exposed human endo- thelial cell at 10 μM and inhibit the H2O2 generation around wound margin without apparent toxicity in zebrafish larvae [95–97]. It should be noted that the therapeuti- cally relevant time window of intrathecal treatment with VAS2870 is 2 h after cere- bral ischemic reperfusion injury [74].
The pyrazolopyridine derivate GKT136901, 2-(2-chlorophenyl)-4-methyl-5- (pyridin-2-ylmethyl)-1H-pyrazolo [4,3-c] pyridine-3,5(2H,5H)-dione, and its close analogue GKT137831 are recently introduced as a dual NOX1/4 inhibitor [98]. They can inhibit NADPH oxidase activity, TGF-β1/2 and fibronectin induction, and p38 MAP kinase activation in mouse proximal tublar cells at 10 μM and inhibit intracel- lular ROS formation, thrombin-induced CD44, and HAS2 protein and mRNA levels in human aortic smooth muscle cells at 30 μM [99, 100]. Moreover, their oral bioavail- ability and little impact on plasma total triglyceride or cholesterol levels and body weight make these compounds the most suitable for a clinical translation [100].
In addition to VAS2870 and GKT136901, many novel inhibitors, such as Ebselen, S17834, M171, Fulvene-5 were introduced recently by their specific pharmacological functions [101–104]. However, although the first and promising data in vivo of them have been published, more detailed understanding about their functions is still needed.
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breakdown by inducing the influx of substances into brain parenchyma and mediating the degradation of tight juctions in the BBB [107].
3.2 Main Sources of Nitric Oxide
Nitric oxide (NO) is the signaling molecule primitively known as endothelium- derived relaxing factor (EDRF) regulating relaxation of blood vessels [108]. It is a small gaseous molecule synthesised from l-arginine, which controls vascular tone and neurotransmission, induces protein post-translational modification, and regulates mRNA translation and gene transcription [109]. Nitric oxide is synthesized by three isoforms of the enzyme NOS: endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS).
NO derived from eNOS has physiological functions including preserving and maintaining the brain’s microcirculation, reducing smooth muscle proliferation, and inhibiting platelet aggregation [110, 111]. NO derived from nNOS acts as a neurotransmitter that plays a role in neuronal plasticity, transmission of pain signals, memory formation, neurotransmitter release, and regulation of central nervous system blood flow [110, 112]. NO derived from iNOS also has multiple functions including contribution to the neurotoxic actions after ischemic stroke and traumatic brain injury and regulation of cerebral blood flow [113–115]. In contrast to eNOS and nNOS, which are calcium-dependent and produce nanomolar levels of NO, iNOS is calcium- independent and produces micromolar levels of NO [116]. The physiological concentration of NO derived from eNOS is vital to communication of neuron, synaptic transmission, regulation of vascular tone, inflammatory responses, platelet aggregation while the high concentration of NO generated from iNOS and nNOS is detrimental to the ischemic brain [107, 117–119].
3.3 Roles of RNS in Cerebral Ischemic Reperfusion Injury
NO and ONOO− are two common species of RNS that are representative in cerebral ischemic reperfusion injury. In the early phase of ischemic stroke, transient shortage of the blood supply results in the increased eNOS activity that generates low concentration of NO to protect the brain vasculature [120]. At the same time, energy crisis caused by ischemia induces the glutamate accumulation and triggers the acti- vation of calcium channels that can stimulate nNOS to produce NO [121, 122]. In the early stage of reperfusion, there is a transient raise of stable NO metabolites, and the up-regulated expression of iNOS lead to excessive NO generation [121, 122].
Moreover, there are two stage of NO generation after ischemic stroke, and both of stages are correlated with increased iNOS and nNOS respectively [123]. The first stage of NO generation was after 1 h of ischemic stroke while the second stage occurred at 24–48 h of reperfusion after 1 h of ischemic stroke [123]. This increased
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generation of NO, which was derived from nNOS and iNOS, are neurotoxic and contribute to cell death and BBB disruption. In contrast to the high concentration of NO from iNOS and eNOS, the low concentration of NO generated from eNOS exerts neuroprotective effects. Evidence has shown that infarction volumes of eNOS knock- out mice is significantly larger than those of wild-type mice after ischemic stroke [124]. Similarly, using medications, which could increase eNOS activity, has shown the same results in ischemic animal model [124]. The mechanisms of neuroprotec- tive effects by these medications refer to the reduction of thrombosis formation, the enhancement of vasorelaxation, elevation of cerebral blood flow, the suppression of NMDA receptor activation, and the improvement of vasorelaxation [124–126].
Likewise, the overproduction of peroxynitrite also plays a detrimental role in neurons and endothelial cells during ischemic reperfusion injury [127–129]. In physiological status, the reaction between NO and O2− that generates peroxynitrite remains at a diffusion controlled level [130]. During both the ischemic stage and the reperfusion stage, the generation of ONOO− is extremely increased because of the dramatically rapid generation of NO. Coincidently, evidence from blood samples of ischemic stroke has discovered that the increase of ONOO− concentration occurs at 24 and 48 h after ischemic stroke [131, 132]. Because the penetrating capacity of ONOO− across lipid bilayers is 400 times higher than its parent radical superoxide anions approximately, ONOO− is far more neurotoxic than NO. Indeed, the mecha- nisms of cytotoxic effects of ONOO− include lipid membrane peroxidation, protein tyrosine nitration, induction of mitochondrial dysfunction, DNA breakage caused by PARP activation, enzymatic activity inhibition, signal transduction dysfunction, and cytoskeletal disruption by altering protein structure and dysfunction [107, 133–136].
In addition to its neurotoxic effect, the overproduction of ONOO− also leads to the BBB breakdown by mediating the activation of MMPs, the degradation of the tight junction proteins, and the rearrangement of the tight junction proteins [137–141].
3.4 Pharmacological Approaches to Regulate RNS Activity
Because RNS play crucial roles in regulating different functions both in physiologi- cal and pathological states, RNS could be potential drug targets for the treatment of ischemic stroke. Pharmacological approaches by targeting RNS has been well devel- oped in NO-based therapeutic strategies; however, because of the technical limitation of detection, drug development by targeting ONOO− is much slower [107].
The fundamental aim of NO-based therapeutic strategies is to establish balanced concentration of NO by increasing level of NO derived from eNOS and reducing the cytotoxic level of NO derived from iNOS and nNOS. There are three approaches could regulate the NO activity.
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3.4.1 Increasing eNOS Activity
One of the promising agents, which could enhance the eNOS activity, is statins. It can improve expression of eNOS via LDL-dependent and independent pathways [142, 143]. Researches in experimental animal models have shown that statins can reduce both the edema formation and infraction volume after ischemic stroke [144, 145]. Deficiency of the protective effect of statin in eNOS knockout mice has con- firmed that the protective effects of stain are eNOS-dependent [146, 147]. Moreover, based on its beneficial effects, supported by abundant preclinical and clinical studies, statins are one of the recommended drug to prevent stroke [148, 149]. Furthermore, the antioxidant characteristics of statins also contribute to the reduction of oxidative stress in brain after ischemic stroke [150]. However, it has been reported that using stain may lead to increased risk of hemorrhagic stroke and higher incidence of infection [151–153]. Thus, when perform stain treatment, the negative side effects of stain should not be ignored because of its multiple pharmacological activities.
3.4.2 nNOS and iNOS Inhibition
L-NAME, a non-selective NOS inhibitor, has been proven to be beneficial for cerebral ischemic stroke injury in experimental mouse models including prevention of BBB breakdown, reduction of infarction volume, and improvement of the recovery of neuro- logical functions [154, 155]. Moreover, because L-NAME can increase the eNOS activ- ity, it also can exert its protective effects for ischemic stroke via eNOS activation.
Delta-(S-methylisothioureido)-L-norvaline (L-MIN), a specific nNOS inhibitor, has been shown to be able to reduce infraction volume in animal stroke models [156]. Other typical experimental inhibitors of nNOS, such as ARL-17477, tirilazad, 7-nitroindazole, BN80933, and PPBP were also reported to decrease the neurological deficits and infarct volume of animal ischemic stroke models [157–161].
1400 W and aminoguanidine, specific iNOS inhibitors, have been shown that can reduce the infraction volume and attenuate ischemic brain injury [113]. The use of 1400 W and aminoguanidine to inhibit iNOS activity has been proposed to be a valuable approach for human stroke because the expression of iNOS in human brain has been identified after ischemic stroke and the time window for administration of iNOS ihhibitors is longer than other treatments [162].
3.4.3 Increasing Substrates of NO Production
NO donors and substrates of eNOS has been utilized to ameliorate the prognosis of the patients suffering with ischemic stroke for a long time. l-Arginine, a NO precur- sor, has shown to be able to decrease the infract volume, to enhance the blood flow, and to improve the neurological function in rat ischemic stroke models [163, 164].
However, the clinical application of the l-arginine is limited because of the risk of reducing the blood flow to the ischemic penumbra [165].
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