4. NADPH Oxidase Inhibitors and Experimental Models of PD
It is well documented that NADPH oxidase is upregulated in PD [13]. Reports reveal that NADPH-oxidase activation plays a critical role in the degeneration of dopaminergic neurons and inactivation of this enzyme may be a promising target for PD treatment [13, 55, 61]. Reports also suggest that microglial toxin, LPS-induced loss of nigral dopaminergic neurons in vivo and in vitro, was significantly less pronounced in NADPH-oxidase-deficient mice, when compared to normal control mice [62]. In the normal CNS, NADPH oxidase is quiescent, but, in patients with PD and in MPTP-intoxicated mice, NADPH oxidase is clearly expressed and activated in glial cells in the ventral midbrain. Thus, agents that inhibit NADPH oxidase activation may be ideal therapeutic agents for the management of PD. Here, we summarize a list of recently published and patented compounds which act as NADPH-oxidase-derived ROS inhibitors in experimental animal models focusing on PD pathology. 
Ethyl pyruvate (EP, Figure 1(a)) is an effective scavenger of ROS, especially hydrogen peroxide, by virtue of its nonenzymatic oxidative decarboxylation reaction. EP has been reported to exert pharmacological effects, such as scavenging of ROS, suppression of inflammation, inhibition of apoptosis, and support of cellular ATP synthesis. Recently, EP has been shown to rescue nigrostriatal dopaminergic neurons by regulating glial activation in an MPTP intoxicated mouse model of PD. A single injection of EP (10, 25, and 50 mg/kg body weight) per day administered to mice into the peritoneum at 12 h after the last MPTP injection exerted neuroprotection which was associated with suppression of NADPH-oxidase-derived ROS production by activated microglia [63].
Aminoethyl-benzenesulfonylfluoride (AEBSF, Figure 1(b)), an NADPH oxidase inhibitor, was reported to be useful in ameliorating oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells. AEBSF at 300 μM significantly blocked 1-methyl-4-phenylpyridinium ion-(MPP+-) induced ROS production for over 45 min in N27 cells in a dose-dependent manner and rescued the cells from apoptotic cell death. The study supported that NADPH oxidase play a critical role in the oxidative damage in PD and inhibiting this enzyme activation by AEBSF may lead to novel therapy for PD [61].
Apocynin (4-hydroxy-3-methoxyacetophenone, Figure 1(c)), a selective inhibitor of NADPH-oxidase, can block the production of superoxide and oxygen-free radicals that typically accompany inflammation. Apocynin is a compound originally isolated from the medicinal plant Picrorhiza kurroa, which inhibited both intracellular and extracellular ROS production by interfering with the phagosomal association of the cytosolic protein p47phox. Apocynin has been used to prevent oxidative-stress-mediated cell damage in several disease models. Recently, apocynin has been proved to improve neurological function and can be used as a neuroprotective agent [64–68]. In the presence of microglial NADPH oxidase activation in vitro, apocynin was able to reduce extracellular ROS and cellular damage. Apocynin pretreatment (0.5 mM for 30 min) also attenuated the rotenone-induced release of superoxides from activated microglia mediated by NADPH oxidase [55].
In another study, exposure to paraquat (50 μM), a herbicide with a structure similar to the neurotoxin MPP+, has been shown to produce PD-like symptoms and generated ROS (including superoxide anions) in BV-2 cells. Paraquat-induced cytotoxicity in BV-2 cells was accompanied by translocation of the p67phox cytosolic subunit of NADPH oxidase to the membrane. Paraquat-induced ROS production was significantly inhibited by apocynin (1 mM) [69]. Data from the studies above indicated that apocynin may inhibit the release of microglial NADPH-oxidase-mediated superoxide in microglia-enhanced degeneration of dopaminergic neurons.
Diphenyliodonium (DPI, Figure 1(d)) is a widely used selective NADPH oxidase inhibitor which was documented to act as a neuroprotective agent due to its potent anti-inflammatory properties mediated through the inhibition of ROS. At very low concentrations, DPI inhibited both extracellular superoxide and intracellular ROS production in dopaminergic neurons from LPS-induced degeneration and in LPS-treated microglial activation [70]. In a report by Ma and Zhou, DPI at 3–30 μM concentrations inhibited intrinsic NADPH oxidase activity in N27 cells. Furthermore, DPI blocked MPP+-induced ROS production. DPI also promoted the survival of primary striatal neurons [71] and protected against glutamate-induced apoptosis in dopaminergic SH-SY5Y cells [72]. In addition, several in vivo studies demonstrated that DPI delivered protection against global cerebral ischemia [64], rotenone- [55], paraquat- [73], 6-OHDA- [74], MPTP- [59], and IFN-γ/LPS- [75] induced dopaminergic degeneration. In a recent study, Gao et al. [76] developed a two-hit (neuroinflammation and mutant α-synuclein (α-syn) overexpression) animal model to investigate mechanisms through which mutant α-syn and inflammation work in concert to mediate chronic PD neurodegeneration. Results revealed that LPS stimulation within the brain to transgenic mice over expressing human A53T mutant α-syn developed persistent neuroinflammation, chronic progressive degeneration of the nigrostriatal dopamine pathway, accumulation of aggregated, nitrated α-syn, and formation of Lewy body-like inclusions in nigral neurons. Continuous inhibition of NADPH oxidase by 4-week infusion of DPI (5 μg/kg/h) blocked α-syn pathology and nigral neurodegeneration. These studies provide a potential role of DPI in the treatment of PD.
Dextromethorphan (DM, Figure 1(e)) is neuroprotective through inhibition of microglial activation and NADPH oxidase activation. Earlier reports reveal that DM protects dopaminergic neurons against inflammation-mediated degeneration induced by LPS, through inhibition of superoxide radicals [77]. Furthermore, Li et al. [78] reported that femto- (10−13 and 10−14 M) and micromolar (10−5 to 10−7 M) concentrations of DM (both pre- and posttreatment) showed equal efficacy in protecting LPS-induced dopaminergic neuron death in midbrain neuron-glia cultures. These studies indicated that the neuroprotective effect elicited by femtomolar concentrations of DM is mediated through the inhibition of LPS-induced proinflammatory mediators, especially superoxide. In another study, DM was reported to elicit neuroprotective effects in the MPTP-intoxicated PD model in mice. DM significantly reduced the MPTP-induced production of both extracellular superoxide free radicals and intracellular ROS in both in vitro and in vivo experiments [79].
Recently, Ramanathan et al. [80] revealed that DM increased superoxide dismutase (SOD) and catalase, reduced thiobarbituric acid reactive substances in the hippocampal and striatal regions of monosodium glutamate-induced neurodegeneration in rats, and improved neuroprotection based on its antioxidant properties. Another recent report also indicated that increased ROS production in activated BV-2 microglial cells by LPS was associated with increased expression of NADPH oxidase (NOX)-2, a subunit component of NADPH oxidase and DM significantly suppressed the upregulation of NOX-2 as well as subsequent ROS production in activated BV-2 cells [81]. In light of this, DM may form the potential therapeutic strategy for the treatment of PD. The metabolite of DM, 3-hydroxymorphinan (Figure 2(a)), also protected the nigrostriatal pathway against MPTP-elicited damage both in vivo and in vitro by reducing MPTP-elicited reactive microgliosis as evidenced by the decreased production of ROS due to its potent neuroprotection in PD [82].
Resveratrol (3, 4, 5-trihydroxy-trans-stilbene, Figure 2(b)), a nonflavonoid polyphenol naturally found in red wine and grapes, has been found to possess antioxidant, anticancer, and anti-inflammatory properties. Recent results from Zhang et al. [83] clearly demonstrated that resveratrol pretreatment (15–60 μM) for 30 min stimulated with LPS (10 ng/mL) protected dopaminergic neurons against LPS-induced neurotoxicity in concentration and time-dependent manners through the inhibition of microglial activation and the subsequent reduction in release of proinflammatory factors. The authors showed that resveratrol reduced NADPH-oxidase-mediated generation of ROS and inhibited the LPS-induced translocation of NADPH oxidase cytosolic subunit p47phox to the cell membrane. The most important finding is that resveratrol failed to exhibit neuroprotection in cultures from NADPH-oxidase-deficient mice. This data indicates that NADPH oxidase may be a major player in resveratrol-mediated neuroprotection in the models of PD.
The epigallocatechin (EGCG, Figure 2(c)), a catechin polyphenol, was reported to reduce neuronal NADPH expression in rats exposed to acute hypoxia [84]. EGCG was also reported to inhibit cytosolic subunits of NADPH oxidase from translocating into the membrane, suggesting that inhibition of NADPH oxidase activity may prevent oxidative stress. In a recent report, the effects of EGCG on dichlorodiphenyltrichloroethane (DDT), a pesticide which is believed to play a causative role in the etiology of PD, was studied. It was found that EGCG concentration dependently (1 μM, 3 μM, and 10 μM) reduced DDT-induced cell death in dopaminergic SH-SY5Y cells. Reports also indicated that EGCG was capable of reducing dopaminergic neurotoxin 6-hydroxydopamine-(6-OHDA-) induced cell death in SH-SY5Y cells [85] and MPTP-induced neurotoxicity in mice [86]. The authors suggest that consumption of green tea, which contains high concentrations of EGCG, may provide potential prophylactic effects in reducing the risk of developing PD.
Few neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) 38, PACAP 27, and its internal peptide, Gly-Ile-Phe (GIF), were reported to be neuroprotective at 10−13 M against LPS-induced dopaminergic neurotoxicity. PACAP is widely distributed in the peripheral and central nervous system, where PACAP release is reported to serve as a neuronal survival factor [87, 88]. PACAP is reported to have diverse functions and has been shown to act as a neurotransmitter/neuromodulator [89] and a neuroprotectant [90, 91]. PACAP 38 and GIF also protected against MPTP-induced neurotoxicity in animal models. The polypeptides significantly ameliorated the production of microglia-derived ROS, where both LPS- and phorbol 12-myristate 13-acetate-induced superoxide and intracellular ROS were inhibited. The study showed that PACAP38 and GIF were neuroprotective only in normal cultures and not in NADPH oxidase deficient cultures, proving the important role of NADPH oxidase for GIF and PACAP 38s neuroprotection [92].
The steroid hormone, 17β-estradiol (E2, Figure 2(d)), is released into the blood where it can exert trophic or regulatory effects on many different target tissues such as the breast, ovary, uterus, bone, and brain. Reports revealed that E2 treatment (0.025 mg; 14–21 day release via minipumps) strongly attenuated the elevation of NADPH oxidase activity in the hippocampal CA1 region following cerebral ischemia in brain, which correlated with its suppression of O2 − levels and its neuroprotective effect [93]. Moreover, E2 inhibited activation of the GTPase, Rac1, in an Akt-dependent manner following cerebral ischemia, which is critical for NOX-2 activation. Due to its neuroprotective effect and potent role in inhibiting NADPH oxidase expression, E2 may be further developed for the treatment of PD [94].
Transforming growth factor (TGF)-β1 is a pleiotropic cytokine that plays a critical role in the control of cell growth, differentiation, inflammation, cell chemotaxis, apoptosis, and hematopoiesis. Studies have shown that TGF-β1 can protect neurons from cell death induced by oxidative injury [95]. A recent report by Qian and Flood [96] revealed that the neuroprotective effects of TGF-β1 are mainly attributed to its ability to inhibit the production of ROS from microglia during their activation or reactivation. TGF-β1 inhibited LPS-induced NADPH oxidase subunit p47phox translocation from the cytosol to the membrane in microglia, thereby exerting potent anti-inflammatory and neuroprotective properties.
Sinomenine (Figure 2(e)), a natural dextrorotatory morphinan analog, was reported to possess anti-inflammatory and neuroprotective properties by the inhibition of microglial NADPH oxidase. Sinomenine pretreatment for 30 min at micromolar (10−6–10−5 M) and subpicomolar concentrations (10−14–10−13 M) showed equivalent efficacy in protecting against dopaminergic neuron death in rat midbrain neuron-glial cultures. Furthermore, sinomenine suppressed LPS-induced extracellular ROS production via the inhibition of NADPH cytosolic subunit p47phox translocation to the cell membrane. These findings strongly suggest that the protective effects of sinomenine are most likely mediated through the inhibition of microglial NADPH oxidase activity [70].
N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide (FLZ, Figure 3(a)) is a squamosamide derivative reported to mediate anti-inflammatory and neuroprotective effects in both LPS and MPTP-intoxicated models of PD [97]. For in vivo studies, FLZ (75 mg/kg, p.o.) was administered 30 min before every MPTP injection (15 mg/kg, s.c.) for 6 consecutive days. For LPS (2 ng/mL) stimulation, 10 μM of FLZ was pretreated for 1 h. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-α (TNF-α), nitric oxide (NO), and prostaglandin E2 (PGE2). Findings from this study revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase, the key microglial superoxide-producing enzyme [97].
Phycocyanobilin (PCB, Figure 3(b)), a chromophore derived from biliverdin, plays an essential light-harvesting role in many blue-green algae and cyanobacteria. It constitutes up to 1% of the dry weight of spirulina. Recently, it was reported that C-phycocyanin administered orally (the spirulina holoprotein that includes PCB) suppresses the neurotoxic impact of the excitotoxin kainite in rats, and a diet high in spirulina ameliorates the loss of dopaminergic neurons in the MPTP-induced Parkinsonian syndrome in mice. The central physiological effects of PCB may also reflect inhibition of neuronal NADPH oxidase, which is known to have a modulatory impact on neuron function, and can mediate neurotoxicity in certain neurodegenerative diseases [98]. PCB has been shown to be a potent inhibitor of NADPH oxidase activity in various human cell cultures at micromolar concentrations. PCB may thus have versatile potential for preserving the healthy function of the CNS in advanced old age patients suffering from neuroinflammatory diseases including PD.
In a recent study, Santiago et al. [99] investigated the effect of simvastatin (Figure 3(c)) a commonly used, cholesterol-lowering drug, in LPS and MPTP neurodegenerative models to identify its neuroprotective effects for PD. The study suggested that simvastatin (5 mg/kg body weight, i.p.) could prevent neurotoxic damage by LPS stimulation in microglial cells. Studies by Brenneman et al. [90] also indicated that simvastatin is associated with a reduced incidence of dementia and PD in elderly patients. Simvastatin treatment (10 μM) blocked the rac1-dependent activation of NADPH oxidase and O2 − production and significantly diminished microglial CC chemokine ligand 5 (a major chemo attractant of inflammatory cells) expression induced by interferon-β alone or by a combination of interferon-β/TNF-α, thereby exerting its suppressive effects on inflammation in the CNS [100]. Furthermore, simvastatin inhibited NADPH oxidase and the production of ROS in microglia [101]. A recent study showed that simvastatin protects dopaminergic neurodegeneration in in vivo parkinsonian models [102]. Further, simvastatin was also reported to attenuate superoxide generation by NADPH oxidase activation, protecting the endothelial cell barrier [103]. All these data suggest the protective effect of simvastatin against the degeneration of dopaminergic neurons and may be developed as a promising drug to provide neuroprotection in PD.
Minocycline (Figure 3(d)), a well-known semisynthetic tetracycline derivative, is neuroprotective in several animal models of neurodegeneration, including PD [104, 105]. Studies have demonstrated that the neuroprotective actions of minocycline are attributable to inhibition of microglial activation accompanied by oxidative stress. Choi et al. [106] reported that minocycline (25 or 50 mg/kg) exerted neuroprotection by significantly attenuating thrombin-induced neurotoxic effects through inhibition of NADPH oxidase activation and ROS production from activated microglia.
Several patents for various categories of compounds have been claimed for selectively inhibiting NADPH oxidase by proving to be useful in the treatment and/or prevention of inflammatory conditions in neurodegenerative diseases. Patented compounds published over the last five years for selectively inhibiting NADPH oxidase were collectively described in our earlier review [107]. The most recent relevant patents showing a possible role in ameliorating neurodegenerative diseases such as PD include the pyrazolo pyridine derivatives [108], tetrahydroindole derivatives [109], imipramine blue analogs [110], quinolone derivatives [111], and hesperidin and hesperetin analogs [112]. These compounds were shown to selectively inhibit and downregulate the expression of NADPH oxidase by suppressing ROS generation, consequently proving their importance as novel therapies in ameliorating neuroinflammatory degenerative diseases including PD.