Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats

We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. l-Nitroarginine methyl ester (l-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.     

Omentin, a novel adipokine, induces vasodilation in rat isolated blood vessels

Omentin is a recently identified adipose tissue-derived cytokine and is implicated in obesity-related cardiovascular disorders. In the present study, we tested the hypothesis that omentin could directly affect vascular reactivity of isolated blood vessels. In endothelium-intact rat isolated aorta, pretreatment with omentin (300 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 μM)-induced concentration-dependent contraction. In NA (100 nM)-pre-contracted aorta, omentin (1-300 ng/ml) directly induced an endothelium-dependent relaxation. While a nitric oxide (NO) synthase (NOS) inhibitor, N^G-nitro-l-arginine methyl ester (100 μM, 30 min) inhibited the relaxation, a PI3 K/Akt inhibitor, LY294002 (10 μM, 30 min) or a tyrosine kinase inhibitor, genistein (30 μM, 30 min) was ineffective. Omentin (300 ng/ml, 5 min) induced a phosphorylation of endothelial NOS at serine 1177 but not a phosphorylation of Akt at serine 473. Omentin (1-300 ng/ml) also relaxed NA pre-contracted mesenteric artery. Present study for the first time demonstrated that omentin has a vasodilating effect on isolated blood vessels, which is mediated through endothelium-derived NO.     

FERM domain promotes resveratrol-induced apoptosis in endothelial cells via inhibition of NO production

Focal adhesion kinase (FAK) consists of an N-terminal band 4.1; ezrin, radixin, moesin (FERM) domain; tyrosine kinase domain; and C-terminal FA targeting domain. Here we show that ectopically expressed FERM is largely located in the cytosolic fraction under quiescent conditions. We further found that this ectopically expressed FERM domain aggravates endothelial cell apoptosis triggered by 100 μM resveratrol, whereas FERM had no effect on apoptosis induced by TNF-α. We determined that resveratrol at low doses (<20 μM) promotes phosphorylation (S1177) of eNOS via an AMPK-dependent pathway. The presence of the FERM domain blocked this resveratrol-stimulated eNOS phosphorylation and NO production. Thus, the pro-apoptotic activity of cytosolic FERM domain is at least partially mediated by down-regulation of NO, a critical cell survival factor. Consistently, we found that the apoptosis induced by cytosolic FERM in the presence of resveratrol was reversed by an NO donor, SNAP. In conclusion, FERM located in the cytosolic fraction plays a pivotal role in aggravating cell apoptosis through diminishing NO production.     

Glyoxal causes inflammatory injury in human vascular endothelial cells

To explore mechanisms of diabetes-associated vascular endothelial cells (ECs) injury, human umbilical vein ECs were treated for 24 h with high glucose (HG; 26 mM), advanced glycation end-products (AGEs; 100 μg/ml) or their intermediate, glyoxal (GO: 50-5000 μM). HG and AGEs had no effects on ECs morphology and inflammatory states as measured by vascular cell adhesion molecule (VCAM)-1 and cyclooxygenase (COX)-2 expressions. GO (500 μM, 24 h) induced cytotoxic morphological changes and protein expression of COX-2 but not VCAM-1. GO (500 μM, 24 h) activated ERK but not JNK, p38 or NF-κB. However, ERK inhibitor PD98059 was ineffective to GO-induced COX-2. While EUK134, synthetic combined superoxide dismutase/catalase mimetic, had no effect on GO-mediated inflammation, sodium nitroprusside inhibited it. The present results indicate that glyoxal, a metabolite of glucose might be a more powerful inducer for vascular ECs inflammatory injury. Nitric oxide but not anti-oxidant is preventive against GO-mediated inflammatory injury.     

Heteroalicyclic carboxamidines as inhibitors of inducible nitric oxide synthase; the identification of (2R)-2-pyrrolidinecarboxamidine as a potent and selective haem-co-ordinating inhibitor

Heteroalicyclic carboxamidines were synthesised and evaluated as inhibitors of nitric oxide synthases. (2R)-2-Pyrrolidinecarboxamidine, in particular, was shown to be a highly potent in vitro (IC₅₀ = 0.12 μM) and selective iNOS inhibitor (>100-fold vs both eNOS and nNOS), with probable binding to the key anchoring glutamate residue and co-ordination to the haem iron.     

Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase

Peroxynitrite (ONOO(-)), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O(2)()) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817-826). Here we studied how ONOO(-) influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO(-) increased basal and agonist-stimulated Ser(1179) phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO(-) strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser(1179). Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation. In contrast to Akt, ONOO(-) significantly activated 5'-AMP-activated kinase (AMPK), as evidenced by its increased Thr(172) phosphorylation as well as increased Ser(92) phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O(2)(), ONOO(-) significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO(-) up-regulated eNOS-Ser(P)(1179). In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation as well as O(2)() release. We conclude that ONOO(-) inhibits Akt and increases AMPK-dependent Ser(1179) phosphorylation of eNOS resulting in enhanced O(2)() release.     

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD⁺-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.     

Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor

We demonstrated that ginsenoside-Re (Re), a pharmacological active component of ginseng, is a functional ligand of glucocorticoid receptor (GR) using competitive ligand-binding assay (IC₅₀ = 156.6 nM; K_d = 49.7 nM) and reporter gene assay. Treatment with Re (1 μM) raises intracellular Ca²⁺ ([Ca²⁺]_i) and nitric oxide (NO) levels in human umbilical vein endothelial cells as measured using fura-2 and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, respectively. Western blot analysis shows that Re increased phosphorylation of endothelial nitric oxide synthase. These effects were abolished by GR antagonist RU486, siRNA targeting GR, non-selective cation channel blocker 2-aminoethyldiphenylborate, or in the absence of extracellular Ca²⁺, indicating Re is indeed an agonistic ligand for the GR and the activated GR induces rapid Ca²⁺ influx and NO production in endothelial cells.     

Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression

It is unclear whether metformin, one of the anti-hyperglycemic agents commonly used for type 2 diabetes, could affect bone formation through activation of AMP-activated protein kinase (AMPK). In order to clarify this issue, we investigated the effects of metformin on the differentiation and mineralization of osteoblastic MC3T3-E1 cells as well as intracellular signal transduction. Metformin (50 μM) significantly increased collagen-I and osteocalcin mRNA expression, stimulated alkaline phosphatase activity, and enhanced cell mineralization. Moreover, metformin significantly activated AMPK in dose- and time-dependent manners, and induced endothelial nitric oxide synthase (eNOS) and bone morphogenetic protein-2 (BMP-2) expressions. Supplementation of Ara-A (0.1 mM), a specific AMPK inhibitor, significantly reversed the metformin-induced eNOS and BMP-2 expressions. Our findings suggest that metformin can induce the differentiation and mineralization of osteoblasts via activation of the AMPK signaling pathway, and that this drug might be beneficial for not only diabetes but also osteoporosis by promoting bone formation.     

Inhibitory effect of schisandrin on spontaneous contraction of isolated rat colon

This study examined the effect of schisandrin, one of the major lignans isolated from Schisandra chinensis, on spontaneous contraction in rat colon and its possible mechanisms. Schisandrin produced a concentration-dependent inhibition (EC₅₀ = 1.66 μM) on the colonic spontaneous contraction. The relaxant effect of schisandrin could be abolished by the neuronal Na⁺ channel blocker tetrodotoxin (1 μM) but not affected by propranolol (1 μM), phentolamine (1 μM), atropine (1 μM) or nicotine desensitization, suggesting possible involvement of non-adrenergic non-cholinergic (NANC) transmitters released from enteric nerves. N^ω-nitro-l-arginine methyl ester (100-300 μM), a nitric oxide synthase inhibitor, attenuated the schisandrin response. The role of nitric oxide (NO) was confirmed by an increase in colonic NO production after schisandrin incubation, and the inhibition on the schisandrin responses by soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-α]-quinoxalin-1-one (1-30 μM). Non-nitrergic NANC components may also be involved in the action of schisandrin, as suggested by the significant inhibition of apamin on the schisandrin-induced responses. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (100 μM), a selective P2 purinoceptor antagonist, markedly attenuated the responses to schisandrin. In contrast, neither 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A₁ receptors, nor chymotrypsin, a serine endopeptidase, affected the responses. All available results have demonstrated that schisandrin produced NANC relaxation on the rat colon, with the involvement of NO and acting via cGMP-dependent pathways. ATP, but not adenosine and VIP, likely plays a role in the non-nitrergic, apamin-sensitive component of the response.     

Naringenin, a citrus flavonoid, increases muscle cell glucose uptake via AMPK

Naringenin, a flavonoid found in high concentrations in grapefruit, has been reported to have antioxidant, antiatherogenic, and anticancer effects. Effects on lipid and glucose metabolism have also been reported. Naringenin is structurally similar to the polyphenol resveratrol, that has been reported to activate the SIRT1 protein deacetylase and to have antidiabetic properties. In the present study we examined the direct effects of naringenin on skeletal muscle glucose uptake and investigated the mechanism involved. Naringenin stimulated glucose uptake in L6 myotubes in a dose- and time-dependent manner. Maximum stimulation was seen with 75 μM naringenin for 2 h (192.8 ± 24%, p < 0.01), a response comparable to maximum insulin response (190.1 ± 13%, p < 0.001). Similar to insulin, naringenin did not increase glucose uptake in myoblasts indicating that GLUT4 glucose transporters may be involved in the naringenin-stimulated glucose uptake. In addition, naringenin did not have a significant effect on basal or insulin-stimulated Akt phosphorylation while significantly increased AMPK phosphorylation/activation. Furthermore, silencing of AMPK, using siRNA approach, abolished the naringenin-stimulated glucose uptake. The SIRT1 inhibitors nicotinamide and EX527 did not have an effect on naringenin-stimulated AMPK phosphorylation and glucose uptake. Our data show that naringenin increases glucose uptake by skeletal muscle cells in an AMPK-dependent manner.     

Tetrahydrobiopterin attenuates homocysteine induced endothelial dysfunction

Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH₄) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH₄ (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH₄ (10 M). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH₄. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH₄ (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH₄ with resultant uncoupling of nitric oxide synthase. BH₄ may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia.     

The acute-phase protein serum amyloid A induces endothelial dysfunction that is inhibited by high-density lipoprotein

The acute-phase protein serum amyloid A (SAA) is elevated during inflammation and may be deposited in atheroma where it promotes atherosclerosis. We investigated the proatherogenic effects of SAA on the vascular endothelium and their regulation by high-density lipoprotein (HDL). Exposure of human aortic endothelial cells (HAEC) to SAA (0.25-25 μg/ml) decreased nitric oxide (^•NO) synthesis/bioavailability, although the endothelial NO synthase monomer-to-dimer ratio was unaffected. SAA (10 μg/ml) stimulated a Ca²⁺ influx linked to apocynin-sensitive superoxide radical anion (O₂^•−) production. Gene expression for arginase-1, nuclear factor κB (NF-κB), interleukin-8, and tissue factor (TF) increased within 4 h of SAA stimulation. Enzymatically active Arg-1/2 was detected in HAEC cultured with SAA for 24 h. Therefore, in addition to modulating ^•NO bioavailability by stimulating O₂^•− production in the endothelium, SAA modulated vascular l-Arg bioavailability. SAA also diminished relaxation of preconstricted aortic rings induced by acetylcholine, and added superoxide dismutase restored the vascular response. Preincubation of HAEC with HDL (100 or 200, but not 50, μg/ml) before (not after) SAA treatment ameliorated the Ca²⁺ influx and O₂^•− production; decreased TF, NF-κB, and Arg-1 gene expression; and preserved overall vascular function. Thus, SAA may promote endothelial dysfunction by modulating ^•NO and l-Arg bioavailability, and HDL pretreatment may be protective. The relative HDL to SAA concentrations may regulate the proatherogenic properties of SAA on the vascular endothelium.     

Visfatin causes endothelium-dependent relaxation in isolated blood vessels

Visfatin is a novel adipocyte-derived cytokine. We hypothesized that visfatin could directly affect vascular reactivity. To test the hypothesis, effects of visfatin on contraction of isolated blood vessels were examined. In endothelium-intact rat aorta, pretreatment with visfatin (100 ng/ml, 30 min) inhibited noradrenaline (NA; 1 nM-1 μM)-induced contraction. In NA (100 nM)-pre-contracted aorta, visfatin (1-100 ng/ml) directly induced a relaxation. Although an N^G-Nitro-l-arginine methyl ester (300 μM, 15 min) inhibited the relaxation, an insulin receptor inhibitor, AGL2263 (10 μM, 20 min) was ineffective. Visfatin (100 ng/ml, 20 min) induced a phosphorylation of eNOS at serine 1177 and a de-phosphorylation of eNOS at threonine 495. Visfatin also induced a phosphorylation of Akt at serine 473 and a substrate of cGMP-dependent protein kinase, vasodilator stimulated phosphoprotein at serine 239. Present study revealed for the first time that visfatin has a vasodilating effect on isolated blood vessels, which is mediated via endothelium-derived NO.     

SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol

Endothelial nitric oxide synthase (eNOS) plays a crucial role in endothelial cell functions. SIRT1, a NAD⁺-dependent deacetylase, is shown to regulate endothelial function and hence any alteration in endothelial SIRT1 will affect normal vascular physiology. Cigarette smoke (CS)-mediated oxidative stress is implicated in endothelial dysfunction. However, the role of SIRT1 in regulation of eNOS by CS and oxidants are not known. We hypothesized that CS-mediated oxidative stress downregulates SIRT1 leading to acetylation of eNOS which results in reduced nitric oxide (NO)-mediated signaling and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) and H₂O₂ showed decreased SIRT1 levels, activity, but increased phosphorylation concomitant with increased eNOS acetylation. Pre-treatment of endothelial cells with resveratrol significantly attenuated the CSE- and oxidant-mediated SIRT1 levels and eNOS acetylation. These findings suggest that CS- and oxidant-mediated reduction of SIRT1 is associated with acetylation of eNOS which have implications in endothelial dysfunction.     

A protective role of ciglitazone in ox‐LDL‐induced rat microvascular endothelial cells via modulating PPARγ‐dependent AMPK/eNOS pathway

Thiazolidinediones, the antidiabetic agents such as ciglitazone, has been proved to be effective in limiting atherosclerotic events. However, the underlying mechanism remains elucidative. Ox‐LDL receptor‐1 (LOX‐1) plays a central role in ox‐LDL‐mediated atherosclerosis via endothelial nitric oxide synthase (eNOS) uncoupling and nitric oxide reduction. Therefore, we tested the hypothesis that ciglitazone, the PPARγ agonist, protected endothelial cells against ox‐LDL through regulating eNOS activity and LOX‐1 signalling. In the present study, rat microvascular endothelial cells (RMVECs) were stimulated by ox‐LDL. The impact of ciglitazone on cell apoptosis and angiogenesis, eNOS expression and phosphorylation, nitric oxide synthesis and related AMPK, Akt and VEGF signalling pathway were observed. Our data showed that both eNOS and Akt phosphorylation, VEGF expression and nitric oxide production were significantly decreased, RMVECs ageing and apoptosis increased after ox‐LDL induction for 24 hrs, all of which were effectively reversed by ciglitazone pre‐treatment. Meanwhile, phosphorylation of AMP‐activated protein kinase (AMPK) was suppressed by ox‐LDL, which was also prevented by ciglitazone. Of interest, AMPK inhibition abolished ciglitazone‐mediated eNOS function, nitric oxide synthesis and angiogenesis, and increased RMVECs ageing and apoptosis. Further experiments showed that inhibition of PPARγ significantly suppressed AMPK phosphorylation, eNOS expression and nitric oxide production. Ciglitazone‐mediated angiogenesis and reduced cell ageing and apoptosis were reversed. Furthermore, LOX‐1 protein expression in RMVECs was suppressed by ciglitazone, but re‐enhanced by blocking PPARγ or AMPK. Ox‐LDL‐induced suppression of eNOS and nitric oxide synthesis were largely prevented by silencing LOX‐1. Collectively, these data demonstrate that ciglitazone‐mediated PPARγ activation suppresses LOX‐1 and moderates AMPK/eNOS pathway, which contributes to endothelial cell survival and function preservation.     

Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum

The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N₂O using NADH as the direct reductant. The K_M for NO was determined to be 20 μM and the k_cat to be 5000 min⁻¹. Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO.     

同型半胱氨酸对内皮细胞一氧化氮合酶系统的损伤机制及叶酸的拮抗效应

本实验探讨同型半胱氨酸(Hcy)对人脐静脉内皮细胞(HUVEC)一氧化氮合酶(eNOS)的损伤机制及叶酸(FA)的拮抗效应。HUVEC原代培养,传至第3代后,将其与不同浓度Hcv(10μmol/L、30μmol/L、100μmol/L和300μmol/L)、FA(100μmol/L)或两者联合共同培养72h,用RT-PCR和免疫组织化学技术分别估测细胞eNOS mRNA水平及eNOS蛋白质量;高效液相色谱测定细胞内不对称二甲基精氨酸(ADMA)含量;并分别测定二甲基精氨酸二甲胺水解酶(DDAH)、eNOS活性及一氧化氮(NO)含量。HUVEC与不同浓度Hcy培养72h后,eNOS mRNA和蛋白质表达皆受到抑制;eNOS活性降低;NO生成减少。同时,DDAH活性降低;细胞内ADMA含量呈剂量依赖性增加。加入FA后,eNOS蛋白质水平上调;eNOS活性增强;NO生成增多。同时,DDAH活性增强,ADMA蓄积减少;但eNOS mRNA表达没有改变。Hcy对内皮细胞eNOS的损伤机制涉及eNOS酶蛋白和eNOS的基因表达两个层面,其对eNOS酶蛋白的抑制机制可能通过DDAH-ADMA通路,FA可拮抗Hcy对eNOS酶蛋白的抑制作用,显示出对HHcy有一定的保护作用。但FA对HHcy所导致的eNOS基因表达的抑制无保护效应。     

Neuroprotective effect of allicin against traumatic brain injury via Akt/endothelial nitric oxide synthase pathway-mediated anti-inflammatory and anti-oxidative activities

Allicin, one of the main biologically active compounds derived from garlic, has been shown to exert various anti-oxidative and anti-inflammatory activities in in vitro and in vivo studies. Here, we sought to investigate the potential neuroprotective effects of allicin against traumatic brain injury (TBI) in rats. We found that allicin treatment (10 and 50 mg/kg, not 1 mg/kg) significantly reduced brain edema and motor functional deficits, as well as apoptotic neuronal cell death in injured cortex. These protective effects could be observed even if the administration was delayed to 4 h after injury. Moreover, allicin treatment decreased the expression levels of MDA and protein carbonyl, preserved the endogenous antioxidant enzyme activities, and suppressed the expression of inflammatory cytokines. The results of Western blot analysis showed that allicin increased the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Blocking Akt/eNOS pathway activation by specific inhibitor LY294002 (10 μL, 10 mmol/L) or L-NIO (0.5 mg/kg) partly reversed the protective effects of allicin and its anti-inflammatory activities. The allicin induced anti-oxidative activity was partly prevented by LY294002, but not L-NIO. In summary, our data strongly suggested that allicin treatment at an appropriate dose can exert protective effect against TBI through Akt/eNOS pathway-mediated anti-inflammatory and anti-oxidative activities.