High Expression Levels of NADPH Oxidase 3 in the Cerebrum of Ten-Week-Old Stroke-Prone Spontaneously Hypertensive Rats

Abstract

We previously demonstrated that the high levels of oxidative stress in the brains of ten-week-old stroke-prone hypertensive rats (SHRSP) were attributable to intrinsic, not extrinsic factors (Biol. Pharm. Bull., 33, 2010, Michihara et al.). The aim of the present study was to determine whether increases in the enzymes producing reactive oxygen species (ROS), reductions in the enzymes and proteins removing ROS, or increases in an enzyme and transporter removing antioxidants promoted oxidative stress in the SHRSP cerebrum. No significant decreases were observed in the mRNA levels of enzymes that remove ROS between SHRSP and normotensive Wistar Kyoto rats. The activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and the protein and mRNA levels of NOX3, an enzyme that produces ROS, were significantly increased in the SHRSP cerebrum. These results suggested that the high expression levels of NOX3 increased oxidative stress in the SHRSP cerebrum.

Reactive oxygen species (ROS), including superoxide, hydrogen peroxide, and the hydroxyl radical, are byproducts of oxygen metabolism in cells, and are produced in all oxygen-utilizing organs. Oxidative stress is known to occur when an imbalance exists between the production and removal of ROS by the antioxidant system.¹⁾ Oxidative stress has been implicated in the development of several diseases such as cancer, Alzheimer’s disease, cardiovascular disease, hypertension, and stroke.^2,3) The main cause of stroke is hypertension. An epidemiological study identified lower serum cholesterol levels as a cause of cerebral hemorrhage.⁴⁾ On the other hand, ROS have been shown to play a role in the development of brain injury following cerebral hemorrhage,⁵⁾ and secondary brain injury after cerebral hemorrhage has also been associated with oxidative stress.⁶⁾ These findings suggest that not only hypertension and lower serum cholesterol levels, but also oxidative stress is responsible for the development of stroke.

Oxidative stress is generally caused by an increase in the levels of enzymes producing ROS and free radicals, or a reduction in antioxidant levels or the enzymes that remove ROS and free radicals. Superoxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide (H₂O₂), which is then reduced to H₂O and O₂ by catalase (CAT), the glutathione system, comprising glutathione peroxidase (GPX) and glutathione reductase (GR),⁷⁾ or the thioredoxin system, which consists of thioredoxin (TXN), peroxiredoxin (PRX), and thioredoxin reductase (TXNR).⁸⁾ A previous study reported that the main enzymes detoxifying H₂O₂ were the glutathione system in the brain, the TXN system in mitochondria, and catalase in peroxisomes.⁹⁾ Uncoupling protein 2 (UCP2), which is a mitochondrial inner membrane carrier protein, acts as a negative regulator of ROS production.¹⁰⁾ Uric acid, which is a natural antioxidant, was previously reported to be effluxed by ATP-binding cassette subfamily G member 2 (ABCG2).¹¹⁾ Urate oxidase (UOX) catalyzes the oxidation of urate to allantoin.¹²⁾ Enzymes such as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), xanthine oxidase (XO), and nitric oxide synthase (NOS) are known to be upregulated by increases in ROS and free radicals.¹³⁾ The NOX family generates large amounts of superoxide.¹⁴⁾ XO produces urate and ROS (superoxide and hydrogen peroxide).¹⁵⁾ Furthermore, xanthine and XO have been shown to generate hydrogen peroxide instead of superoxide.¹⁶⁾ Nitric oxide (NO) is synthesized by three nitric oxide synthases.^17,18) The NO–superoxide interaction not only decreases the bioavailability of NO, but also produces the reactive peroxynitrate (a ROS),¹⁹⁾ which disrupts endothelial membrane caveolae, cholesterol-rich membrane lipid rafts.

Spontaneously hypertensive rats (stroke-prone) (SHRSP) develop severe hypertension with stroke.^20,21) Serum cholesterol levels were previously shown to be lower in these rats than in normotensive Wistar Kyoto rats (WKY).²²⁾ Oxidized protein levels in the aorta, heart, and kidney were also found to be markedly higher in SHRSP than in WKY.²³⁾ Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG: a biomarker of systemic oxidative stress) levels were high in 14–17-week-old SHRSP, while ROS levels were elevated in the brains of 16-week-old SHRSP.²⁴⁾ A previous study proposed uric acid (antioxidant) content in the cerebral cortex as a cause of cerebral injury because the administration of an inhibitor of XO led to markedly lower uric acid levels and more severe cerebral injury in the treated group than in the control group.²⁵⁾ Superoxide, NOX activity, and SOD protein levels in the brains of 15-week-old SHRSP were significantly higher than those in the brains of WKY.²⁶⁾ We previously demonstrated that oxidized proteins were significantly increased in the brains, but not kidneys or serum of 10-week-old SHRSP, indicating that high levels of oxidative stress in the brains of these rats were a direct effect of oxidative stress in the brain itself, and not a secondary effect of oxidative stress in serum or other tissues including the kidneys.²⁷⁾ However, it currently remains unclear whether all NOX family members increase oxidative stress, increase the levels of enzymes producing ROS, or decrease the levels of enzymes removing ROS in the brains of 10-week-old SHRSP, or if these events in the cerebrum are actually associated with stroke.

In the present study, we investigated whether the expression levels of enzymes producing or removing ROS or free radicals increased oxidative stress in the cerebrum of 10-week-old SHRSP. The results obtained suggested that elevated NOX activity due to the upregulation of NOX3, strongly influenced the increase observed in oxidative stress in the SHRSP cerebrum.

MATERIALS AND METHODS

Materials

The Quick Gene RNA tissue kit SII was purchased from FUJIFILM (Tokyo, Japan), the SYBR Ex Script reverse transcription polymerase chain reaction (RT-PCR) kit from TaKaRa (Shiga, Japan), BioMasher^® from Nippi, Inc. (Tokyo, Japan), rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) immunoglobulin G (IgG) or rabbit anti-NOX3 IgG from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.), rabbit anti-NOX2 IgG from Abcam, Inc. (Tokyo, Japan), goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP) from Invitrogen Corporation (Carlsbad, CA, U.S.A.), and the ECL Western blotting detection kit from GE Healthcare (Tokyo, Japan). All other chemicals were of reagent grade and purchased commercially.

Animals

Inbred male (10 weeks old) SHRSP/Izm (average body weight: 220±10 g, average cerebrum weight 0.8 g) and WKY/Izm (average body weight: 270±10 g, average cerebrum weight 0.7 g) were obtained from the Disease Model Co-operative Research Association, Japan. All rats were sacrificed using previously described methods.²⁷⁾ The experimental protocol was reviewed and approved by the Animal Care and Use Committee of Fukuyama University and complied with guidelines for the care and use of animals by the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Real-Time PCR

The cerebrum (15 mg) was homogenized with a Biomasher (tissue homogenizer; Asist, Tokyo, Japan). The extraction of total RNA (17 µg) from tissues, the reverse transcriptase reaction, and real-time PCR analysis of enzymes producing and removing ROS were performed according to previously described methods.²⁷⁾ As shown in Table 1, the primer pairs of real-time PCR were designed using the TaKaRa perfect real-time support system or UCSC Genome Bioinformatics. A part of the primer was also quoted from ref. 28. GAPDH was used as an internal control.

Table 1. Real-Time PCR Primer Sequences

Genes	Primer sequence
Superoxide dismutase 1 (SOD1)	F: 5′-CGTCATTCACTTCGAGCAGA-3′
	R: 5′-AAAATGAGGTCCTGCAGTGG-3′
Superoxide dismutase 2 (SOD2)	F: 5′-GGCCAAGGGAGATGTTACAA-3′
	R: 5′-GCTTGATAGCCTCCAGCAAC-3′
Superoxide dismutase 3 (SOD3)	F: 5′-TCAGAGGCTCTTTCTCAGGC-3′
	R: 5′-CTGCTAAGTCGACACCGGAC-3′
Catalase (CAT)	F: 5′-ACATGGTCTGGGACTTCTGG-3′
	R: 5′-CAAGTTTTTGATGCCCCTGGT-3′
Thioredoxin (TXN)	F: 5′-AGCTGATCGAGAGCAAGGAA-3′
	R: 5′-TCAAGGAACACCACATTGGA-3′
Thioredoxin reductase 1 (TXNR 1)	F: 5′-TCAAGGTGACCGCTAAGTCC-3′
	R: 5′-TCTTCCCGGTCTTTTCATTG-3′
Thioredoxin reductase 2 (TXNR 2)	F: 5′-CAGTTATGTGGCCCTGGAGT-3′
	R: 5′-TCGGGAGTTTTCTGATGAGG-3′
Thioredoxin reductase 3 (TXNR 3)	F: 5′-GGGAGTTTGTGGAACTGCAT-3′
	R: 5′-GGCAGAGAGAACAGGTCGTC-3′
Peroxiredoxin (PRX)	F: 5′-CTGAGACTAGTCCAGGCCTTCC-3′
	R: 5′-CTGGCTGCTCAAAGCTGTCTG-3′
Glutathione peroxidase 1 (GPX 1)	F: 5′-CAGTTCGGACATCAGGAGAAT-3′
	R: 5′-AGAGCGGGTGAGCCTTCT-3′
Glutathione peroxidase 3 (GPX3)	F: 5′-GGCTTTGTGCCTAATTTCCA-3′
	R: 5′-CCCACCAGGAACTTCTCAAA-3′
Glutathione reductase (GR)	F: 5′-GGGCAAAGAAGATTCCAGGTT-3′
	R: 5′-GGACGGCTTCATCTTCAGTGA-3′
Uncoupling protein 2 (UCP 2)	F: 5′-TCATCACTTTCCCTCTAGACACC-3′
	R: 5′-AAGCTCATCTGGCGCTGTAG-3′
ATP binding cassette transporter (ABCG2)	F: 5′-TCCAAGGTTGGAACTCAGTTTA-3′
	R: 5′-AAGATGGAATATCGAGGCTG-3′
Urate oxidase (UOX)	F: 5′-CTACCAGAATCGGGACGTGGA-3′
	R: 5′-CTCTGTCATAGGGCCCAGCAA-3′
NADPH oxidase 1 (NOX 1)	F: 5′-CACTGTGGCTTTGGTTCTA-3′
	R: 5′-TGAGGACTCCTGCAACTCCT-3′
NADPH oxidase 2 (NOX 2)	F: 5′-GTGGAGTGGTGTGAATGC-3′
	R: 5′-TTTGGTGGAGGATGTGATGA-3′
NADPH oxidase 3 (NOX 3)	F: 5′-GACCCAACTGGAATGAGGAA-3′
	R: 5′-AATGAACGACCCTAGGATCT-3′
NADPH oxidase 4 (NOX 4)	F: 5′-CTGTACAACCAAGGGCCAGAA-3′
	R: 5′-TGCAGTTGAGGTTCAGGACAGA-3′
Xanthine oxidase (XO)	F: 5′-GCATGCCAGACCATACTGAA-3′
	R: 5′-AAATCCAGTTGCGGACAAAC-3′
Neuronal nitric oxide synthase (nNOS)	F: 5′-CCGGAATTCGAATACCAGCCTGATC-3′
	R: 5′-CGAATTCCTCCAGGAGGGTGTCCACCGCATG-3′
Inducible nitric oxide synthase (iNOS)	F: 5′-CCTTGTTCAGCTAGCCCTTC-3′
	R: 5′-GGTATGCCCGAGTTCTTTCA-3′
Endothelial nitric oxide synthase (eNOS)	F: 5′-TGGCAGCCCTAAGACCTATG-3′
	R: 5′-AGTCCGAAAATGTCCTCGTG-3′
GAPDH	F: 5′-GGCACAGTCAAGGCTGAGAATG-3′
	R: 5′-ATGGTGGTGAAGACGCCAGTA-3′

NADPH Oxidase Activity

The lucigenin-enhanced chemiluminescence assay was measured.²⁹⁾ Rat cerebrum (15 mg) were homogenized with five strokes in a Teflon homogenizer in three volumes of homogenate buffer containing 50 mM phosphate buffer (pH 7.0), 1 µM leupeptin, 1 µM pepstatin A. The homogenate of the cerebrum of SHRSP or WKY was incubated for 30 min at 37°C in homogenate buffer containing 1 mM CaCl₂, 5 µM lucigenin, and 100 µM NADPH. Light emission was recorded for 15 min using a luminometer. NOX activity was calculated from the ratio of mean light units to total protein levels and expressed as arbitrary units.

Protein Assay

Protein concentrations were measured as described by Lowry et al. using bovine serum albumin (BSA) as the standard.³⁰⁾

Preparation of the Crude Extract and Immunoblot Analysis

Rat cerebrum (15 mg) were homogenized with five strokes in a Teflon homogenizer in three volumes of homogenate buffer containing 0.5 µM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100, and 1% sodium dodecyl sulfate (SDS). SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed according to the method of Laemmli and Towbin et al., respectively.^31,32) NOX2, NOX3, or GAPDH on the nylon membrane was detected by incubating with the rabbit anti-NOX2 antibody, the rabbit anti-NOX3 antibody, or the rabbit anti-GAPDH antibody, washing five times, and then incubating with goat anti-rabbit IgG conjugated to HRP. After the membrane was washed five more times, positive bands were detected and quantified using previously described methods.²⁷⁾ GAPDH was used as an internal control.

Statistical Analysis

The significance of differences was evaluated using the Student’s t-test (Microsoft Excel, Microsoft Japan Co., Ltd.). Values of p<0.05 were regarded as statistically significant. Data are presented as the mean±standard deviation (S.D.).

RESULTS

Comparison of mRNA Levels of Enzymes and Proteins Removing ROS, or the Enzyme and Transporter Removing Antioxidants between SHRSP and WKY

In order to determine whether decreases in the levels of enzymes and protein removing ROS or urate, which is an antioxidant, occurred in the cerebrum of SHRSP, we investigated the mRNA levels of these enzymes using real-time PCR. SOD1 and SOD3 mRNA levels were higher in the SHRSP cerebrum than in the WKY cerebrum; however, no significant differences were observed in those of SOD3 (Table 2). Furthermore, no significant differences were noted in the mRNA levels of SOD2, CAT, TXN, TXNR1, TXNR2, TXNR3, PRX, GPX1, GPX3, GR, and UCP2, which are involved in the removal of ROS, between the SHRSP cerebrum and WKY cerebrum (Table 2). mRNA levels of UOX, which participates in the oxidation of urate, were higher in the SHRSP cerebrum, but not significantly so (Table 3). mRNA levels of the urate removal transporter ABCG2 in the cerebrum were similar between SHRSP and WKY (Table 3).

Table 2. Comparison of mRNA Levels of Enzymes and Proteins Removing ROS in the Cerebrum of WKY and SHRSP

Enzymes	WKY	SHRSP
Superoxide dismutase 1 (SOD1)	1.42±0.44	2.25±0.47*
Superoxide dismutase 2 (SOD2)	1.12±0.09	1.18±0.17
Superoxide dismutase 3 (SOD3)	1.38±0.45	2.54±1.02
Catalase (CAT)	1.34±0.33	1.12±0.13
Thioredoxin (TXN)	0.89±0.33	0.73±0.15
Thioredoxin reductase 1 (TXNR 1)	0.89±0.12	0.62±0.15
Thioredoxin reductase 2 (TXNR 2)	1.24±0.18	1.13±0.22
Thioredoxin reductase 3 (TXNR 3)	1.08±0.77	1.20±0.25
Peroxiredoxin (PRX)	0.83±0.18	0.97±0.39
Glutathione peroxidase 1 (GPX 1)	1.36±0.41	1.35±0.32
Glutathione peroxidase 3 (GPX3)	0.79±0.29	0.66±0.16
Glutathione reductase (GR)	1.36±0.32	1.20±0.11
Uncoupling protein 2 (UCP 2)	0.63±0.26	0.41±0.12

Values are the mean±S.D. of four to six independent experiments. Statistical analyses were performed using the Student’s t-test. * p<0.05.

Table 3. Comparison of mRNA Levels of the Transporter and Enzyme Removing Urate in the Cerebrum of WKY and SHRSP

Enzymes	WKY	SHRSP
ATP binding cassette transporter G2 (ABCG2)	1.71±0.63	1.80±0.40
Urate oxidase (UOX)	0.82±0.30	1.60±0.97

Values are the mean±S.D. of four independent experiments. Statistical analyses were performed using the Student’s t-test.

Comparison of mRNA Levels of Enzymes Producing ROS between SHRSP and WKY

In order to determine whether increases in the levels of enzymes producing ROS occurred in the cerebrum of SHRSP, we examined the expression levels of these enzymes using real-time PCR (Table 4). No significant differences were observed in the mRNA levels of NOX1, NOX4, neuronal nitric oxide synthase (nNOS), and endothelial nitric oxide synthase (eNOS), which are involved in the production of ROS, between the SHRSP cerebrum and the WKY cerebrum. NOX2 and NOX3 mRNA levels were markedly higher in the SHRSP cerebrum than in the WKY cerebrum. NOX5 mRNA was not detected by real-time PCR even though the primer pairs of NOX5 were designed using UCSC Genome Bioinformatics (data not shown). The mRNA levels of XO and inducible nitric oxide synthase (iNOS) were significantly lower in the SHRSP cerebrum than in the WKY cerebrum.

Table 4. Comparison of mRNA Levels of Enzymes Producing ROS in the Cerebrum of WKY and SHRSP

Enzymes	WKY	SHRSP
NADPH oxidase 1 (NOX 1)	0.87±0.20	1.00±0.37
NADPH oxidase 2 (NOX 2)	0.48±0.08	2.85±0.79*
NADPH oxidase 3 (NOX 3)	1.43±0.99	7.72±3.05*
NADPH oxidase 4 (NOX 4)	0.63±0.35	1.27±0.89
Xanthine oxidase (XO)	0.89±0.12	0.59±0.21*
Neuronal nitric oxide synthase (nNOS)	0.97±0.05	1.27±0.66
Inducible nitric oxide synthase (iNOS)	1.06±0.06	0.77±0.04*
Endothelial nitric oxide synthase (eNOS)	0.64±0.29	0.49±0.25

Values are the mean±S.D. of four to six independent experiments. Statistical analyses were performed using the Student’s t-test. * p<0.05.

Comparison of NOX Activity between SHRSP and WKY

We investigated whether NOX activity as well as the mRNA levels of NOX2 and NOX3 increased in the SHRSP cerebrum. NOX activity was significantly higher in the SHRSP cerebrum than in the WKY cerebrum (Fig. 1). These results strongly suggested that the high levels of oxidative stress observed in the cerebrum of SHRSP were caused by elevated superoxide levels due to increases in NOX2 and NOX3. However, the ratio of the increase in NOX activity was not proportional to that in the mRNA levels of NOX2 or NOX3.

Fig. 1. NOX Activity in the Cerebrum of SHRSP and WKY

NOX activity was measured in the cerebrum of SHRSP and WKY as described in Materials and Methods. Values are the mean±S.D. of four independent experiments. Statistical analyses were performed using the Student’s t-test. Significant differences: * p<0.05.

Comparison of NOX2 and NOX3 Protein Levels between SHRSP and WKY

We determined whether the ratio of the increase in the protein levels of NOX2 and NOX3 as well as that in NOX activity was higher in the SHRSP cerebrum. Although NOX3 protein levels were significantly higher in SHRSP than in WKY, no significant differences were observed in NOX2 protein levels between SHRSP and WKY (Fig. 2). These results indicated that the high NOX activity levels observed in the SHRSP cerebrum were caused by increases in NOX3 protein levels.

Fig. 2. NOX2 and NOX3 Protein Levels in the Cerebrum of SHRSP and WKY

Samples (A: NOX2 and NOX3; 30 µg, GAPDH; 5 µg) were subjected to immunoblotting using the anti-NOX2 antibody, the anti-NOX3 antibody, or the anti-GAPDH antibody, and signals were measured using an Intelligent Quantifier (B). Relative protein levels were quantified using GAPDH as an internal control. Values are the mean±S.D. of four independent experiments. Statistical analyses were performed using the Student’s t-test. Significant differences: * p<0.05.

Comparison of Nucleotide Sequences of Exons in NOX3 Genes between SHRSP and WKY

In order to establish whether changes in the amino acid sequence in NOX3 occurred between SHRSP and WKY, we compared the nucleotide sequences of all exons in the NOX3 genes of WKY and SHRSP using the complete genome sequence data of WKY and SHRSP, as described previously (purchased from the Society for Hypertension-related Disease Model research).³³⁾ A mutation was not detected in any of the exons of NOX3 between WKY and SHRSP (data not shown). These results suggest that reductions in NOX activity by the mutation of an amino acid in NOX3 did not occur in SHRSP. It was also considered that the ability of NOX3 activity in SHRSP is similar to that in WKY. Therefore, the upregulation of NOX activity in the SHRSP cerebrum may have been due to higher NOX3 protein levels.

DISCUSSION

A previous study demonstrated that urate content was lower in young SHRSP than in WKY.¹²⁾ In the present study, we found that XO mRNA levels were significantly lower in SHRSP (Table 4). Reductions in urate content due to a decrease in XO mRNA level may also be partly involved in the high levels of oxidative stress observed in the SHRSP cerebrum.

Although the interaction between superoxide and NO synthesized by NOS reduces superoxide levels, reactive peroxynitrate, which is a stronger ROS than superoxide, is produced by this reaction. Reductions in iNOS levels (Table 4) increased superoxide levels, but inhibited the production of peroxynitrate; therefore, this decrease in iNOS did not appear to play an important role in the higher levels of oxidative stress observed in the SHRSP cerebrum.

We previously reported that SOD and CAT levels were high in the SHRSP brain.²⁷⁾ In the present study, no significant differences were observed in the mRNA levels of SOD2 (in mitochondria), SOD3 (outside mitochondria), and CAT between the SHRSP cerebrum and WKY cerebrum; however, SOD1 mRNA levels (in the cytosol) were higher in SHRSP cerebrum, as described previously. These results suggested that the expression levels of SOD2, SOD3, and CAT may differ in cell types or at each site of the brain.

Superoxide levels and NOX activity were previously shown to be significantly higher in the brains of 15-week-old SHRSP than in their WKY counterparts.¹³⁾ However, it currently remains unclear which of the NOX members increases NOX activity. NOX2 protein levels in the cerebrum were similar between SHRSP and WKY, whereas NOX2 mRNA levels were markedly higher in SHRSP. NOX3 protein and mRNA levels in the cerebrum were significantly higher in SHRSP than in WKY. The ratio of the increase observed in NOX3 protein levels (2.3-fold) in the SHRSP cerebrum was proportional to that in NOX activity levels (2.6-fold), but not that in NOX3 mRNA levels (5.4-fold). The mutation of an amino acid in NOX3 was not observed between SHRSP and WKY. Therefore, high NOX activity levels in the SHRSP cerebrum were mainly caused by increases in NOX3 protein levels; however, the influence of the upregulation of another NOX member other than NOX2 may also be involved in the increase observed in NOX activity. Furthermore, the difference in the ratio between the protein and mRNA levels of NOX2 or NOX3 in the cerebrum of SHRSP may have occurred due to the accelerated degradation of proteins or another regulation mechanism involving the upregulation of microRNA (miRNA). miRNA is known to accelerate the degradation of mRNA or reduce protein levels by inhibiting its synthesis from mRNA.^34,35)

Superoxide levels and NOX activity were lower in the brains of SHRSP fed standard laboratory rat chow containing pioglitazone (1 mg/kg/d), which is an agonist of peroxisome proliferator-activated receptor γ, than in the brains of SHRSP fed standard laboratory rat chow containing 0.5% carboxymethylcellulose, and this reduction was independent of blood pressure.¹³⁾ These findings indicated that pioglitazone influenced stroke in SHRSP by reducing brain and vascular superoxide levels via the suppression of NOX activity. NOX is known to be distributed not only in phagocytes, but also in a large number of tissues.³⁶⁾ NOX1 (in smooth muscle and the endothelium), NOX2 (in smooth muscle, the endothelium, microglia,³⁷⁾ and neurons), NOX3 (in the brain), NOX4 (in smooth muscle, the endothelium, and neurons), and NOX5 (in smooth muscle and the endothelium) were previously shown to be expressed in the brain. NOX2 was found to be activated not only in the plasma membrane, but also in granule membranes, indicating that the production of superoxide by NOX occurs either in an extracellular or intracellular area. Although superoxide does not permeate the plasma membrane, it has the ability to pass through anion channels. Hydrogen peroxide produced from superoxide by SOD readily permeates the plasma membrane. In the present study, we detected strong NOX activity due to the high expression levels of NOX3 in the cerebrum of 10-week-old SHRSP. Based on these results, we proposed the following mechanisms for the high levels of oxidative stress observed in the SHRSP cerebrum. 1) The high extracellular and intracellular levels of superoxide caused by NOX3 may have enhanced oxidative stress in the SHRSP cerebrum by continuously acting on/affecting the plasma membrane. 2) The higher levels of superoxide increased the production of hydrogen peroxide intracellularly and extracellularly, thereby promoting the synthesis of hydroxyl radicals. Extracellular and intracellular hydroxyl radicals, which are more reactive than superoxide and hydrogen peroxide, may have increased oxidative stress in the SHRSP cerebrum by acting on/affecting the plasma membrane. 3) Extracellular superoxide levels (caused by NOX3) passed through anion channels, were changed to hydrogen peroxide by SOD in the cell, which then reacted with the high levels of superoxide in the cell (produced by NOX3), resulting in the synthesis of hydroxyl radicals. Furthermore, the increase in hydroxyl radicals in the cell may have enhanced oxidative stress in the SHRSP cerebrum by the acting on/affecting the plasma membrane. 4) After extracellular superoxide (caused by NOX3) was changed to hydrogen peroxide by SOD extracellularly, hydrogen peroxide readily permeated the plasma membrane. Hydroxyl radicals were synthesized when hydrogen peroxide reacted with superoxide in the cell (generated by NOX3), thereby enhancing oxidative stress in the SHRSP cerebrum by acting on/affecting the plasma membrane. Therefore, the enhanced production of superoxide and the hydroxyl radical due to increases in NOX3 may disrupt the BBB (blood–brain barrier) and induce angionecrosis in the plasma membrane of vascular cells of the SHRSP cerebrum.

In conclusion, the results of the present study indicated that the higher levels of oxidative stress in the SHRSP cerebrum were strongly influenced by the upregulation of NOX3. Further studies are needed to elucidate mechanisms responsible for the increase in NOX3 expression levels in the SHRSP brain.

Acknowledgment

This work was supported by Fukuyama University Grant for Academic Research Projects (GARP).

Conflict of Interest

The authors declare no conflict of interest.

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