Brain Research Bulletin 100 (2014) 14–21
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Allicin protects rat cortical neurons against mechanical trauma injury by regulating nitric oxide synthase pathways Yue-fei Zhou a,1 , Wen-tao Li b,1 , Hong-cheng Han c,1 , Da-kuan Gao a , Xiao-sheng He a , Liang Li a , Jin-ning Song b,1 , Zhou Fei a,∗,1 a
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China Department of Neurosurgery, The First Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China c Department of Cadre Ward, Xi’an Central Hospital, Xi’an, Shaanxi 710000, China b
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Article history: Received 11 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Traumatic brain injury Nitric oxide synthase ERK Akt
a b s t r a c t Allicin, a small molecule that is responsible for the typical smell and most of the functions of garlic, possesses a broad spectrum of pharmacological activities and is considered to have therapeutic potential in many pathologic conditions. In the present study, we investigated the potential protective effect of allicin in an in vitro model of traumatic brain injury (TBI) using primary cultured rat cortical neurons. We found that allicin treatment significantly reduced mechanical trauma-induced lactate dehydrogenase (LDH) release and inhibited apoptotic neuronal death in a dose-dependent manner. These protective effects were observed even if allicin treatment was delayed to 2 h after injury. Allicin significantly decreased the expression of inducible nitric oxide synthase (iNOS) and increased the phosphorylation of endothelial NOS (eNOS) but had no effect on neuronal NOS (nNOS) expression. Allicin-induced protection in cortical neurons was augmented by iNOS and nNOS antagonists and was partly reversed by blocking eNOS phosphorylation. In addition, allicin treatment inhibited the TBI-induced activation of ERK and further enhanced the phosphorylation of Akt in TBI-injured neurons. The Akt inhibitor LY294002 attenuated the allicin-induced increase in eNOS expression and phosphorylation, whereas the ERK inhibitor PD98059 had opposite effects on the expression of iNOS and eNOS. Pretreatment with LY294002 or PD98059 partly prevented or further enhanced allicin-induced neuroprotection, respectively. Collectively, these data demonstrate that allicin treatment may be an effective therapeutic strategy for traumatic neuronal injury and that the potential underlying mechanism involves Akt- and ERK-mediated regulation of NOS pathways. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Traumatic brain injury (TBI), also known as brain trauma, is defined as a blow or jolt to the head or a penetrating head injury that disrupt the function of the brain. TBI can be classified based on severity (mild, moderate or severe), mechanism (closed or penetrating head injury) or other features (occurring in a specific location or over a widespread area) (Hukkelhoven et al., 2003; Topolovec-Vranic et al., 2012). TBI is a major cause of injury-related hospitalization, disability and death worldwide, especially in children and young adults. According to the literature, an average of 634 000 incidents of TBI, either alone or in conjunction with other injuries, occur each year among children in the United States (Langlois et al., 2006). In spite of dramatic improvements in the
∗ Corresponding author. Tel.: +86 29 84775330; fax: +86 29 84775331. E-mail addresses: [email protected] (J.-n. Song), [email protected] (Z. Fei). 1 These authors contributed equally to this work. 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.10.013
management of TBI, the ability to improve patient outcomes is still inadequate (Andriessen et al., 2010). Garlic, also known as Allium sativum, is widely used around the world for its pungent flavor as a seasoning or condiment. Animal studies and clinical trials in humans have suggested the possible medicinal use and health benefits of garlic, which has been used for thousands of years in traditional Chinese medicine for treating various ailments (Ginter and Simko, 2010). Allicin (diallyl thiosulfinate), one of the most biologically active compounds in garlic, is produced during the crushing of garlic cloves and is responsible for the typical smell and most of the functions of garlic (Lawson and Gardner, 2005). Previous in vitro and in vivo studies have shown that allicin possess a variety of biological effects, such as anti-inflammatory, antimicrobial, antifungal, antiparasitic, antihypertensive and anticancer activities (Hunter et al., 2005). An in vitro study indicated that allicin decreases ROS generation and increases the level of glutathione through its antioxidative ability in endothelial cells (Horev-Azaria et al., 2009). A recent study also demonstrated that allicin attenuates spinal cord
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ischemia–reperfusion injury via an improvement of mitochondrial function (Zhu et al., 2012). Based on the above observations, we hypothesized that allicin may have neuroprotective effects against mechanical trauma-induced neuronal injury in cultured rat cortical neurons. In the present study, mechanical trauma injury in cultured cortical neurons, a standard in vitro model of TBI, was used to investigate the potential protective effect of allicin with a focus on nitric oxide synthase pathways.
2. Materials and methods
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immediate neuronal death under the blade followed by a secondary insult to other cells. 2.4. LDH assay Neuronal injury was quantitatively assessed by measuring the release of LDH, a cytoplasmic enzyme released from injured cells after injury. LDH release into the culture medium was detected using a diagnostic kit according to the manufacturer’s instructions (Chen et al., 2011). The results were normalized to the maximal LDH release, which was determined by treating control wells for 60 min with 1% Triton X-100 to lyse all cells.
2.1. Subjects 2.5. TUNEL staining All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). All efforts were made to minimize animal number and suffering. Allicin (purity > 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 2.2. Cortical cell culture Cortical neurons were cultured from Sprague-Dawley rats using a method reported previously (Chen et al., 2012). Briefly, pregnant rats were sacrificed and embryonic day 16–18 cortices were removed. The isolated cortices were dissociated via mechanical trituration and suspended in plating medium containing Neurobasal medium, 2% B27 supplement and 0.5 mM l-glutamine. The culture plates were coated with poly-d-lysine (PLL, 50 g/ml) at room temperature overnight, and neurons were plated at a density of 3 × 105 cells/cm2 . Glial growth was suppressed by treatment with 5-fluoro2-deoxyuridine and uridine on the third day after plating. Neurons were maintained at 37 ◦ C in a humidified 5% CO2 incubator, and half of the culture medium was changed every other day. 2.3. In vitro TBI model To mimic TBI in vitro, a mechanical trauma injury model in primary cultured cortical neurons was employed as previously described (Mori et al., 2002). Briefly, mechanical injury was induced using a sterile 21-gauge needle to draw parallel scratches across the circular wells of culture plates (9 × 9 scratches in six-well plates and 6 × 6 scratches in 24-well plates). These scratches caused
To investigate the effect of allicin on traumatic neuronal injury induced apoptosis, TUNEL staining was performed using an in situ cell death detection kit. Briefly, cortical neurons were seeded on PLL-coated glass slides at a density of 3 × 105 cells/cm2 , and were subjected to mechanical trauma and various treatments. Then, the neurons were fixed by immersing slides in freshly prepared 4% methanol-free formaldehyde solution in PBS for 20 min at room temperature. The neurons were then permeabilized with 0.2% Triton X-100 for 5 min. Cells were labeled with fluorescein TUNEL reagent mixture for 60 min at 37 ◦ C according to the manufacturer’s suggested protocol. Subsequently, the slides were examined by fluorescence microscopy and the number of TUNEL-positive (apoptotic) cells was counted. Hoechst 33342 (10 g/ml) was used to stain the nucleus. 2.6. Western blot analysis Cultured cortical neurons were washed with ice-cold PBS, and harvested in lysis buffer containing protease inhibitors. The protein content was determined using a BCA protein assay kit. An amount of 40 g protein was resolved on 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: cleaved caspase-3, iNOS, p-eNOS, eNOS, nNOS, p-Akt, Akt, p-ERK, ERK and -actin. Membranes were then washed and incubated for 1 h at room temperature with secondary antibodies. The Image J analysis software was used to quantify the optical density of each band. 2.7. Statistical analysis Statistical analysis was performed using the SPSS 16.0 software package. Statistical evaluation of the data was performed
Fig. 1. Allicin attenuates mechanical trauma-induced neuronal injury. Cultured cortical neurons were pretreated with allicin at different concentrations (1 M, 10 M, 50 M, 100 M and 200 M) 1 h before injury, and LDH release was assayed 24 h later (A). After pretreatment with 50 M allicin 1 h before injury, LDH release was assayed at 3 h, 6 h, 12 h, 24 h, 36 h and 48 h after injury (B). The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the control group and *P < 0.05 vs. the vehicle group.
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by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Allicin attenuates mechanical trauma-induced neuronal injury To test whether allicin has a neuroprotective effect in traumatic neuronal injury, we first treated cultured rat cortical neurons with allicin at different concentrations (1 M, 10 M, 50 M, 100 M or 200 M) 1 h before injury. The increased LDH release induced by traumatic neuronal injury was significantly reduced by allicin at 10, 50 and 100 M in a dose-dependent manner, whereas allicin at 1 M or 200 M had no effect (Fig. 1A). As shown in Fig. 1B, 50 M allicin treatment showed a remarkable protective effect against traumatic neuronal injury from 6 h to 48 h after injury. 3.2. Allicin inhibits mechanical trauma induced apoptosis To investigate whether allicin induced neuroprotection in our in vitro TBI model was mediated through anti-apoptotic activity, we examined the apoptotic morphology of cortical neurons by TUNEL staining (Fig. 2A). There were no obvious TUNEL-positive neurons in the control group, whereas the number of TUNEL-positive cells increased after traumatic injury, and this effect was significantly decreased by allicin treatment at both 12 h and 24 h after injury. As shown in Fig. 2C, allicin also inhibited the expression of cleavedcaspase-3 compared with neurons in the vehicle group. 3.3. Therapeutic window of allicin in cortical neurons Protection induced by pretreatment is of very little relevance to clinical therapy for TBI patients. Therefore, we next determined the time window of allicin-induced protective effects. Mechanical trauma-injured neurons were treated with 50 M allicin at different time points (1 h before TBI, at TBI initiation, and at 1 h, 2 h or 4 h after TBI), and LDH release and neuronal apoptosis were assayed 24 h later. As shown in Fig. 3A, a significant reduction in LDH release was observed when allicin was administered 0 h, 1 h or 2 h after traumatic injury, but not when the administration was delayed by 4 h. Consistent with these results, a significant anti-apoptotic effect was still detected when allicin was added 0 h or 1 h after injury as measured by TUNEL staining (Fig. 3B). 3.4. Effect of allicin on the expression of nitric oxide synthases (NOS) We then attempted to determine the potential underlying molecular mechanisms responsible for allicin-induced neuroprotection. The expressions of the three isoforms of NOS were assayed by western blotting (Fig. 4A), and the results showed that the expression of iNOS was significantly increased by traumatic injury and was partly reversed by allicin treatment. The expression of peNOS in the vehicle group was increased slightly compared with control neurons (Fig. 4B). Allicin treatment not only increased the expression of eNOS (P < 0.05 vs. control and P < 0.05 vs. vehicle) but also further elevated p-eNOS expression compared with vehicle group. In addition, neither traumatic neuronal injury nor allicin administration had an effect on the expression of nNOS (P > 0.05). 3.5. Effects of NOS antagonists on allicin induced neuroprotection To further confirm the roles of the three NOS isoforms in allicin-induced protection against traumatic neuronal injury, the
non-specific NOS inhibitor L-NAME (100 M), the eNOS antagonist L-NIO (10 M), the iNOS antagonist 1400W (10 M) or the nNOS antagonist NPLA (5 M) was administered 30 min before injury to block the activation of NOS (Fig. 5). The neurons treated with L-NAME in the presence of allicin exhibited further declines in LDH release and apoptotic rate compared with neurons treated with allicin alone. The selective eNOS antagonist L-NIO partly reversed allicin- induced decreases in LDH release and apoptosis, while 1400W and NPLA further enhanced allicin induced neuroprotection. These data indicate that allicin differentially regulate the expression and activation of the three NOS isoforms, which might contribute to its protective effect against traumatic neuronal injury. 3.6. Effects of allicin on the activation of ERK and Akt Because the Akt and ERK pathways have been reported to play important roles in traumatic neuronal injury, we hypothesized that allicin-induced protection might be partly mediated by the Akt and ERK pathways. Western blot analysis revealed that the expression of p-Akt was slightly elevated by traumatic injury (P < 0.05 vs. the control group) and further increased by allicin treatment (P < 0.05 vs. the vehicle group) (Fig. 6A and B). The expression of p-ERK was significantly increased by injury and was partly abolished by allicin treatment (Fig. 6A and C). In addition, neither traumatic injury nor allicin had any effect on the total protein levels of Akt and ERK1/2. 3.7. Effects of ERK and Akt antagonists on allicin induced NOS expression and neuroprotection To further investigate the involvement of the Akt and ERK pathways in the allicin-induced regulation of NOS expression and protection against traumatic neuronal injury, the selective Akt antagonist LY294002 (20 M) and ERK antagonist PD98059 (40 M) were used 30 min before injury to block the activation of Akt and ERK, respectively. Western blot analysis revealed that LY294002 prevented the increase in p-eNOS and eNOS expression but had no effect on the expression of iNOS and nNOS. Blocking ERK activation by PD98059 further decreased the expression of iNOS (P < 0.05 vs. the allicin group) and increased the expression of eNOS (P < 0.05 vs. the allicin group), but had no effects on the expression of p-eNOS and nNOS (Fig. 7). In addition, the results showed that the allicin induced decrease of LDH release was partly reversed by LY294002, but further reduced by PD98059 (Fig. 8A). As shown in Fig. 8B, similar results for apoptotic cell death were also observed. 4. Discussion In the present study, we investigated the effect of allicin, the most biologically active compound in garlic, in an experimental traumatic neuronal injury model in primary cultured cortical neurons. Treatment with allicin led to significantly lower LDH release after in vitro TBI insult and a reduction in apoptotic neuronal cell death in a dose-dependent manner. The therapeutic time window is very important for the investigation of neuroprotective agents, and several compounds or agents have been demonstrated to be ineffective for patients in clinical trials because their efficacy in animals lasts for only a limited time (Villmann and Becker, 2007). In the present study, we found that treatment with allicin, even 2 h after insult, resulted in decreased LDH release and reduced apoptosis, indicating that the therapeutic time window of allicin is relevant to clinical practice. Therefore, our results suggest that allicin is therapeutically effective for modulating the pathological process after TBI; however, further in vivo experiments are needed. Nitric oxide (NO) is one of the few known gaseous signaling molecules and plays important roles in many physiological and
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Fig. 2. Allicin inhibits mechanical trauma-induced apoptosis. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The apoptotic neuronal death was detected by TUNEL staining (A) and calculated (B) at 12 h and 24 h after injury. The expression of cleaved-caspase-3 was measured by western blotting at 12 h and 24 h after injury (C). The data are represented as the means ± SD from five experiments. *P < 0.05.
pathological processes (Hou et al., 1999). It is biosynthesized endogenously from l-arginine, oxygen and the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) by various nitric oxide synthase (NOS) enzymes, including constitutive calcium-dependent NOS (endothelial NOS, eNOS and neuronal NOS, nNOS) and inducible calcium-independent NOS (iNOS) (Alderton et al., 2001). The three isoforms of NOS have been shown to be important players in posttraumatic secondary brain injury for many years. It was first demonstrated in 1998 that NOS is generated as early as 5 min after injury and remains increased for at least a week in a rat lateral fluid percussion TBI model (Wada et al., 1998a, 1998b). An increase in nNOS activation at three
days after injury was also observed and was considered to contribute to the histological damage induced by TBI (Wada et al., 1998a). Moreover, the induction of iNOS in both the acute and late phases of TBI was reported both in humans and in experimental animal models (Gahm et al., 2002; Louin et al., 2006; Lu et al., 2003). Consistent with previous findings, in the present study, we found that the expression levels of iNOS and eNOS, as well as the phosphorylation level of eNOS, were all significantly increased by traumatic neuronal injury. The expression of nNOS was not altered in injured neurons when compared with control cells, which may be explained by the differences between in vitro and in vivo conditions.
Fig. 3. Therapeutic window of allicin in cortical neurons. After treatment with 50 M allicin at different time points (1 h before TBI, at TBI initiation, 1 h, 2 h or 4 h after TBI initiation), LDH release (A) and apoptotic rate (B) were assayed to determine the time window of the allicin-induced protective effects. The data are represented as the means ± SD from five experiments. *P < 0.05 vs. the vehicle group.
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Fig. 4. Effect of allicin on the expression of three isoforms of NOS. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The expression levels of iNOS, p-eNOS, eNOS and nNOS were determined by western blotting (A) and calculated (B) 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the control group and *P < 0.05 vs. the vehicle group.
Fig. 5. Effects of NOS antagonists on allicin-induced neuroprotection. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The nonspecific NOS inhibitor L-NAME (100 M), the eNOS antagonist L-NIO (10 M), the iNOS antagonist 1400 W (10 M) or the nNOS antagonist NPLA (5 M) was added 30 min before injury to block the activation of the isoforms of NOS. LDH release (A) and apoptotic rate (B) were assayed 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the vehicle group and *P < 0.05 vs. the allicin treated group.
Although it is suggested that the three isoforms of NOS are important regulators of neuronal function in injured brain tissue, the exact roles of eNOS, nNOS and iNOS in neuronal survival and death are complicated and may depend on the nature of the insults or other conditions. The excessive amount of nNOS-derived NO has been demonstrated to be a neurotoxic factor (Zhou and Zhu, 2009), and pretreatment with the selective nNOS inhibitor 7-nitroindazole
reduces contusion volume and improve behavioral recovery in experimental TBI models (Wada et al., 1999). The eNOS isoform is constitutively expressed, and several neuroprotective agents have been shown to protect against neuronal injury via enhancing the phosphorylation level of eNOS (Asahi et al., 2005; Liu et al., 2012; Wang et al., 2013). While eNOS and nNOS have been shown to be beneficial and detrimental, respectively, as mentioned above, the
Fig. 6. Effects of allicin on the activation of Akt and ERK. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The expression levels of p-Akt, Akt, p-ERK and ERK were determined by western blotting (A) and calculated (B) and (C) 24 h after injury. The data ware represented as the means ± SD from five experiments. # P < 0.05 vs. the control group and *P < 0.05 vs. the vehicle group.
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Fig. 7. Effects of Akt and ERK antagonists on the expression of NOS. Cultured cortical neurons were pretreated with the Akt antagonist LY294002 (LY, 20 M) or the ERK antagonist PD98059 (PD, 40 M) in the presence or absence of 50 M allicin. The expression levels of iNOS, p-eNOS, eNOS and nNOS were determined by western blotting (A) and calculated (B) 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the vehicle group and *P < 0.05 vs. the allicin group.
role of iNOS in TBI appears to be disputable. The knockdown of iNOS expression through gene deletion was reported to be protective by reducing the post-TBI lesion volume and improving neurological recovery (Jones et al., 2004). However, another study indicated that iNOS inhibition increased neuronal cell loss and worsened neurological recovery (Sinz et al., 1999). In the present study, we found that the selective nNOS inhibitor 1400W further enhanced the protective effect of allicin, indicating a detrimental role of post-TBI iNOS activation in our in vitro neuronal trauma model. Furthermore, the eNOS inhibitor L-NIO and the nNOS inhibitor NPLA partly reversed and further enhanced, respectively, allicin-induced neuroprotection in traumatic neuronal injury. These data strongly support the important role of the NOS pathway in traumatic neuronal injury. Furthermore, the neuroprotective effect of allicin is dependent on different regulatory effects on the three isoforms of NOS. TBI not only triggers a series of deleterious biochemical events including oxidative stress, activation of pro-inflammatory mediators and up-regulation of proteases, but also activates some compensatory protective mechanisms, such as increased expression of anti-apoptotic proteins and activation of several prosurvival signaling pathways (Nortje and Menon, 2004; Stoica and Faden, 2010; Zhao et al., 2007). Akt, also known as protein kinase B (PKB), is a serine/threonine kinase with pro-survival activities in several neuronal injury models (Brunet et al., 2001; Zhao et al., 2006). Furthermore, another important deleterious kinase found in brain injury, ERK, a mitogenic activated protein (MAP) kinase, mediates signal transduction from noxious stimuli to downstream pro-apoptotic or necrotic cascades (Shioda et al., 2009;
Subramaniam and Unsicker, 2010). In the present study, allicin treatment inhibited TBI-induced activation of ERK, while further enhanced the phosphorylation of Akt in TBI-injured neurons. The allicin mediated protection against traumatic neuronal injury was augmented by PD98059 but partly prevented by LY294002, confirming the opposite effects of Akt and ERK activation in TBI. In addition, divergent regulatory effects of the Akt and ERK pathways have been reported in previous studies. For example, ROS induced by acute exercise plays an important role in activating eNOS through the Akt and AMPK pathways (Barbosa et al., 2013). The activation of the PI3K/Akt/eNOS pahway has been demonstrated to be involved in the neuroprotection against ischemia reperfusion injury induced by recombinant human erythropoietin or remote ischemic post-conditioning (Fu et al., 2013; Peng et al., 2012). A previous study in human respiratory epithelial cells indicated that epimagnolin and fargesin inhibit iNOS expression and decrease the production of NO via the ERK pathway (Baek et al., 2009). The data from our present study show that LY294002 attenuated the allicin-induced increase in eNOS expression and phosphorylation, whereas PD98059 has opposite effects on the expression of iNOS and eNOS. These data suggest that TBI induced ERK phosphorylation can aggravate neuronal cell death through iNOS activation and eNOS inhibition and that the allicin-induced increase in the expression and phosphorylation of eNOS is differentially modulated by the Akt and ERK pathways. In summary, we have shown that a natural product, allicin, protects primary cultured cortical neurons against mechanical trauma injury by regulating three isoforms of NOS. The allicin-induced protection and regulation of the NOS pathways are differentially
Fig. 8. Effects of Akt and ERK antagonists on allicin induced neuroprotection. Cultured cortical neurons were pretreated with the Akt antagonist LY294002 (LY, 20 M) or the ERK antagonist PD98059 (PD, 40 M) in the presence or absence of 50 M allicin. LDH release (A) and apoptotic rate (B) were assayed 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the vehicle group and *P < 0.05 vs. the allicin group.
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Fig. 9. A diagram summarizing the findings of allicin-induced neuroprotection against traumatic neuronal injury.
mediated by Akt and ERK (Fig. 9). However, our study also has limitations. First, neuronal death induced by traumatic injury is not mediated only by apoptosis, but neuronal autophagy or necrosis was not determined in the present study. Second, animal models are more complicated systems than in vitro systems, and in vivo studies of allicin, its therapeutic potential and the underlying mechanisms are needed. Acknowledgments The work was supported by the National Natural Science Foundation of China (No. 30930093), the National Science & Technology Pillar Program (No. 2012BAI11B02) and the Research Foundation of PLA (No. 2010gxjs078 and No. AWS11J008). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.brainresbull. 2013.10.013. References Alderton, W.K., Cooper, C.E., Knowles, R.G., 2001. Nitric oxide synthases: structure, function and inhibition. The Biochemical Journal 357, 593–615. Andriessen, T.M., Jacobs, B., Vos, P.E., 2010. Clinical characteristics and pathophysiological mechanisms of focal and diffuse traumatic brain injury. Journal of Cellular and Molecular Medicine 14, 2381–2392. Asahi, M., Huang, Z., Thomas, S., Yoshimura, S., Sumii, T., Mori, T., Qiu, J., AminHanjani, S., Huang, P.L., Liao, J.K., et al., 2005. Protective effects of statins involving both eNOS and tPA in focal cerebral ischemia. Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism 25, 722–729. Baek, J.A., Lee, Y.D., Lee, C.B., Go, H.K., Kim, J.P., Seo, J.J., Rhee, Y.K., Kim, A.M., Na, D.J., 2009. Extracts of Magnoliae flos inhibit inducible nitric oxide synthase via ERK in human respiratory epithelial cells. Nitric Oxide: Biology and Chemistry/Official Journal of the Nitric Oxide Society 20, 122–128. Barbosa, V.A., Luciano, T.F., Marques, S.O., Vitto, M.F., Souza, D.R., Silva, L.A., Santos, J.P., Moreira, J.C., Dal-Pizzol, F., Lira, F.S., et al., 2013. Acute exercise induce endothelial nitric oxide synthase phosphorylation via Akt and AMP-activated protein kinase in aorta of rats: role of reactive oxygen species. International Journal of Cardiology 167, 2983–2988. Brunet, A., Datta, S.R., Greenberg, M.E., 2001. Transcription-dependent and independent control of neuronal survival by the PI3K-Akt signaling pathway. Current Opinion in Neurobiology 11, 297–305. Chen, T., Fei, F., Jiang, X.F., Zhang, L., Qu, Y., Huo, K., Fei, Z., 2012. Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons. Free Radical Biology & Medicine 52, 208–217.
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Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Allicin protects rat cortical neurons against mechanical trauma injury by regulating nitric oxide synthase pathways Yue-fei Zhou a,1 , Wen-tao Li b,1 , Hong-cheng Han c,1 , Da-kuan Gao a , Xiao-sheng He a , Liang Li a , Jin-ning Song b,1 , Zhou Fei a,∗,1 a
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China Department of Neurosurgery, The First Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China c Department of Cadre Ward, Xi’an Central Hospital, Xi’an, Shaanxi 710000, China b
a r t i c l e
i n f o
Article history: Received 11 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Traumatic brain injury Nitric oxide synthase ERK Akt
a b s t r a c t Allicin, a small molecule that is responsible for the typical smell and most of the functions of garlic, possesses a broad spectrum of pharmacological activities and is considered to have therapeutic potential in many pathologic conditions. In the present study, we investigated the potential protective effect of allicin in an in vitro model of traumatic brain injury (TBI) using primary cultured rat cortical neurons. We found that allicin treatment significantly reduced mechanical trauma-induced lactate dehydrogenase (LDH) release and inhibited apoptotic neuronal death in a dose-dependent manner. These protective effects were observed even if allicin treatment was delayed to 2 h after injury. Allicin significantly decreased the expression of inducible nitric oxide synthase (iNOS) and increased the phosphorylation of endothelial NOS (eNOS) but had no effect on neuronal NOS (nNOS) expression. Allicin-induced protection in cortical neurons was augmented by iNOS and nNOS antagonists and was partly reversed by blocking eNOS phosphorylation. In addition, allicin treatment inhibited the TBI-induced activation of ERK and further enhanced the phosphorylation of Akt in TBI-injured neurons. The Akt inhibitor LY294002 attenuated the allicin-induced increase in eNOS expression and phosphorylation, whereas the ERK inhibitor PD98059 had opposite effects on the expression of iNOS and eNOS. Pretreatment with LY294002 or PD98059 partly prevented or further enhanced allicin-induced neuroprotection, respectively. Collectively, these data demonstrate that allicin treatment may be an effective therapeutic strategy for traumatic neuronal injury and that the potential underlying mechanism involves Akt- and ERK-mediated regulation of NOS pathways. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Traumatic brain injury (TBI), also known as brain trauma, is defined as a blow or jolt to the head or a penetrating head injury that disrupt the function of the brain. TBI can be classified based on severity (mild, moderate or severe), mechanism (closed or penetrating head injury) or other features (occurring in a specific location or over a widespread area) (Hukkelhoven et al., 2003; Topolovec-Vranic et al., 2012). TBI is a major cause of injury-related hospitalization, disability and death worldwide, especially in children and young adults. According to the literature, an average of 634 000 incidents of TBI, either alone or in conjunction with other injuries, occur each year among children in the United States (Langlois et al., 2006). In spite of dramatic improvements in the
∗ Corresponding author. Tel.: +86 29 84775330; fax: +86 29 84775331. E-mail addresses: [email protected] (J.-n. Song), [email protected] (Z. Fei). 1 These authors contributed equally to this work. 0361-9230/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.brainresbull.2013.10.013
management of TBI, the ability to improve patient outcomes is still inadequate (Andriessen et al., 2010). Garlic, also known as Allium sativum, is widely used around the world for its pungent flavor as a seasoning or condiment. Animal studies and clinical trials in humans have suggested the possible medicinal use and health benefits of garlic, which has been used for thousands of years in traditional Chinese medicine for treating various ailments (Ginter and Simko, 2010). Allicin (diallyl thiosulfinate), one of the most biologically active compounds in garlic, is produced during the crushing of garlic cloves and is responsible for the typical smell and most of the functions of garlic (Lawson and Gardner, 2005). Previous in vitro and in vivo studies have shown that allicin possess a variety of biological effects, such as anti-inflammatory, antimicrobial, antifungal, antiparasitic, antihypertensive and anticancer activities (Hunter et al., 2005). An in vitro study indicated that allicin decreases ROS generation and increases the level of glutathione through its antioxidative ability in endothelial cells (Horev-Azaria et al., 2009). A recent study also demonstrated that allicin attenuates spinal cord
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ischemia–reperfusion injury via an improvement of mitochondrial function (Zhu et al., 2012). Based on the above observations, we hypothesized that allicin may have neuroprotective effects against mechanical trauma-induced neuronal injury in cultured rat cortical neurons. In the present study, mechanical trauma injury in cultured cortical neurons, a standard in vitro model of TBI, was used to investigate the potential protective effect of allicin with a focus on nitric oxide synthase pathways.
2. Materials and methods
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immediate neuronal death under the blade followed by a secondary insult to other cells. 2.4. LDH assay Neuronal injury was quantitatively assessed by measuring the release of LDH, a cytoplasmic enzyme released from injured cells after injury. LDH release into the culture medium was detected using a diagnostic kit according to the manufacturer’s instructions (Chen et al., 2011). The results were normalized to the maximal LDH release, which was determined by treating control wells for 60 min with 1% Triton X-100 to lyse all cells.
2.1. Subjects 2.5. TUNEL staining All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). All efforts were made to minimize animal number and suffering. Allicin (purity > 98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). 2.2. Cortical cell culture Cortical neurons were cultured from Sprague-Dawley rats using a method reported previously (Chen et al., 2012). Briefly, pregnant rats were sacrificed and embryonic day 16–18 cortices were removed. The isolated cortices were dissociated via mechanical trituration and suspended in plating medium containing Neurobasal medium, 2% B27 supplement and 0.5 mM l-glutamine. The culture plates were coated with poly-d-lysine (PLL, 50 g/ml) at room temperature overnight, and neurons were plated at a density of 3 × 105 cells/cm2 . Glial growth was suppressed by treatment with 5-fluoro2-deoxyuridine and uridine on the third day after plating. Neurons were maintained at 37 ◦ C in a humidified 5% CO2 incubator, and half of the culture medium was changed every other day. 2.3. In vitro TBI model To mimic TBI in vitro, a mechanical trauma injury model in primary cultured cortical neurons was employed as previously described (Mori et al., 2002). Briefly, mechanical injury was induced using a sterile 21-gauge needle to draw parallel scratches across the circular wells of culture plates (9 × 9 scratches in six-well plates and 6 × 6 scratches in 24-well plates). These scratches caused
To investigate the effect of allicin on traumatic neuronal injury induced apoptosis, TUNEL staining was performed using an in situ cell death detection kit. Briefly, cortical neurons were seeded on PLL-coated glass slides at a density of 3 × 105 cells/cm2 , and were subjected to mechanical trauma and various treatments. Then, the neurons were fixed by immersing slides in freshly prepared 4% methanol-free formaldehyde solution in PBS for 20 min at room temperature. The neurons were then permeabilized with 0.2% Triton X-100 for 5 min. Cells were labeled with fluorescein TUNEL reagent mixture for 60 min at 37 ◦ C according to the manufacturer’s suggested protocol. Subsequently, the slides were examined by fluorescence microscopy and the number of TUNEL-positive (apoptotic) cells was counted. Hoechst 33342 (10 g/ml) was used to stain the nucleus. 2.6. Western blot analysis Cultured cortical neurons were washed with ice-cold PBS, and harvested in lysis buffer containing protease inhibitors. The protein content was determined using a BCA protein assay kit. An amount of 40 g protein was resolved on 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk and incubated with the following primary antibodies: cleaved caspase-3, iNOS, p-eNOS, eNOS, nNOS, p-Akt, Akt, p-ERK, ERK and -actin. Membranes were then washed and incubated for 1 h at room temperature with secondary antibodies. The Image J analysis software was used to quantify the optical density of each band. 2.7. Statistical analysis Statistical analysis was performed using the SPSS 16.0 software package. Statistical evaluation of the data was performed
Fig. 1. Allicin attenuates mechanical trauma-induced neuronal injury. Cultured cortical neurons were pretreated with allicin at different concentrations (1 M, 10 M, 50 M, 100 M and 200 M) 1 h before injury, and LDH release was assayed 24 h later (A). After pretreatment with 50 M allicin 1 h before injury, LDH release was assayed at 3 h, 6 h, 12 h, 24 h, 36 h and 48 h after injury (B). The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the control group and *P < 0.05 vs. the vehicle group.
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by one-way analysis of variance (ANOVA). A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Allicin attenuates mechanical trauma-induced neuronal injury To test whether allicin has a neuroprotective effect in traumatic neuronal injury, we first treated cultured rat cortical neurons with allicin at different concentrations (1 M, 10 M, 50 M, 100 M or 200 M) 1 h before injury. The increased LDH release induced by traumatic neuronal injury was significantly reduced by allicin at 10, 50 and 100 M in a dose-dependent manner, whereas allicin at 1 M or 200 M had no effect (Fig. 1A). As shown in Fig. 1B, 50 M allicin treatment showed a remarkable protective effect against traumatic neuronal injury from 6 h to 48 h after injury. 3.2. Allicin inhibits mechanical trauma induced apoptosis To investigate whether allicin induced neuroprotection in our in vitro TBI model was mediated through anti-apoptotic activity, we examined the apoptotic morphology of cortical neurons by TUNEL staining (Fig. 2A). There were no obvious TUNEL-positive neurons in the control group, whereas the number of TUNEL-positive cells increased after traumatic injury, and this effect was significantly decreased by allicin treatment at both 12 h and 24 h after injury. As shown in Fig. 2C, allicin also inhibited the expression of cleavedcaspase-3 compared with neurons in the vehicle group. 3.3. Therapeutic window of allicin in cortical neurons Protection induced by pretreatment is of very little relevance to clinical therapy for TBI patients. Therefore, we next determined the time window of allicin-induced protective effects. Mechanical trauma-injured neurons were treated with 50 M allicin at different time points (1 h before TBI, at TBI initiation, and at 1 h, 2 h or 4 h after TBI), and LDH release and neuronal apoptosis were assayed 24 h later. As shown in Fig. 3A, a significant reduction in LDH release was observed when allicin was administered 0 h, 1 h or 2 h after traumatic injury, but not when the administration was delayed by 4 h. Consistent with these results, a significant anti-apoptotic effect was still detected when allicin was added 0 h or 1 h after injury as measured by TUNEL staining (Fig. 3B). 3.4. Effect of allicin on the expression of nitric oxide synthases (NOS) We then attempted to determine the potential underlying molecular mechanisms responsible for allicin-induced neuroprotection. The expressions of the three isoforms of NOS were assayed by western blotting (Fig. 4A), and the results showed that the expression of iNOS was significantly increased by traumatic injury and was partly reversed by allicin treatment. The expression of peNOS in the vehicle group was increased slightly compared with control neurons (Fig. 4B). Allicin treatment not only increased the expression of eNOS (P < 0.05 vs. control and P < 0.05 vs. vehicle) but also further elevated p-eNOS expression compared with vehicle group. In addition, neither traumatic neuronal injury nor allicin administration had an effect on the expression of nNOS (P > 0.05). 3.5. Effects of NOS antagonists on allicin induced neuroprotection To further confirm the roles of the three NOS isoforms in allicin-induced protection against traumatic neuronal injury, the
non-specific NOS inhibitor L-NAME (100 M), the eNOS antagonist L-NIO (10 M), the iNOS antagonist 1400W (10 M) or the nNOS antagonist NPLA (5 M) was administered 30 min before injury to block the activation of NOS (Fig. 5). The neurons treated with L-NAME in the presence of allicin exhibited further declines in LDH release and apoptotic rate compared with neurons treated with allicin alone. The selective eNOS antagonist L-NIO partly reversed allicin- induced decreases in LDH release and apoptosis, while 1400W and NPLA further enhanced allicin induced neuroprotection. These data indicate that allicin differentially regulate the expression and activation of the three NOS isoforms, which might contribute to its protective effect against traumatic neuronal injury. 3.6. Effects of allicin on the activation of ERK and Akt Because the Akt and ERK pathways have been reported to play important roles in traumatic neuronal injury, we hypothesized that allicin-induced protection might be partly mediated by the Akt and ERK pathways. Western blot analysis revealed that the expression of p-Akt was slightly elevated by traumatic injury (P < 0.05 vs. the control group) and further increased by allicin treatment (P < 0.05 vs. the vehicle group) (Fig. 6A and B). The expression of p-ERK was significantly increased by injury and was partly abolished by allicin treatment (Fig. 6A and C). In addition, neither traumatic injury nor allicin had any effect on the total protein levels of Akt and ERK1/2. 3.7. Effects of ERK and Akt antagonists on allicin induced NOS expression and neuroprotection To further investigate the involvement of the Akt and ERK pathways in the allicin-induced regulation of NOS expression and protection against traumatic neuronal injury, the selective Akt antagonist LY294002 (20 M) and ERK antagonist PD98059 (40 M) were used 30 min before injury to block the activation of Akt and ERK, respectively. Western blot analysis revealed that LY294002 prevented the increase in p-eNOS and eNOS expression but had no effect on the expression of iNOS and nNOS. Blocking ERK activation by PD98059 further decreased the expression of iNOS (P < 0.05 vs. the allicin group) and increased the expression of eNOS (P < 0.05 vs. the allicin group), but had no effects on the expression of p-eNOS and nNOS (Fig. 7). In addition, the results showed that the allicin induced decrease of LDH release was partly reversed by LY294002, but further reduced by PD98059 (Fig. 8A). As shown in Fig. 8B, similar results for apoptotic cell death were also observed. 4. Discussion In the present study, we investigated the effect of allicin, the most biologically active compound in garlic, in an experimental traumatic neuronal injury model in primary cultured cortical neurons. Treatment with allicin led to significantly lower LDH release after in vitro TBI insult and a reduction in apoptotic neuronal cell death in a dose-dependent manner. The therapeutic time window is very important for the investigation of neuroprotective agents, and several compounds or agents have been demonstrated to be ineffective for patients in clinical trials because their efficacy in animals lasts for only a limited time (Villmann and Becker, 2007). In the present study, we found that treatment with allicin, even 2 h after insult, resulted in decreased LDH release and reduced apoptosis, indicating that the therapeutic time window of allicin is relevant to clinical practice. Therefore, our results suggest that allicin is therapeutically effective for modulating the pathological process after TBI; however, further in vivo experiments are needed. Nitric oxide (NO) is one of the few known gaseous signaling molecules and plays important roles in many physiological and
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Fig. 2. Allicin inhibits mechanical trauma-induced apoptosis. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The apoptotic neuronal death was detected by TUNEL staining (A) and calculated (B) at 12 h and 24 h after injury. The expression of cleaved-caspase-3 was measured by western blotting at 12 h and 24 h after injury (C). The data are represented as the means ± SD from five experiments. *P < 0.05.
pathological processes (Hou et al., 1999). It is biosynthesized endogenously from l-arginine, oxygen and the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) by various nitric oxide synthase (NOS) enzymes, including constitutive calcium-dependent NOS (endothelial NOS, eNOS and neuronal NOS, nNOS) and inducible calcium-independent NOS (iNOS) (Alderton et al., 2001). The three isoforms of NOS have been shown to be important players in posttraumatic secondary brain injury for many years. It was first demonstrated in 1998 that NOS is generated as early as 5 min after injury and remains increased for at least a week in a rat lateral fluid percussion TBI model (Wada et al., 1998a, 1998b). An increase in nNOS activation at three
days after injury was also observed and was considered to contribute to the histological damage induced by TBI (Wada et al., 1998a). Moreover, the induction of iNOS in both the acute and late phases of TBI was reported both in humans and in experimental animal models (Gahm et al., 2002; Louin et al., 2006; Lu et al., 2003). Consistent with previous findings, in the present study, we found that the expression levels of iNOS and eNOS, as well as the phosphorylation level of eNOS, were all significantly increased by traumatic neuronal injury. The expression of nNOS was not altered in injured neurons when compared with control cells, which may be explained by the differences between in vitro and in vivo conditions.
Fig. 3. Therapeutic window of allicin in cortical neurons. After treatment with 50 M allicin at different time points (1 h before TBI, at TBI initiation, 1 h, 2 h or 4 h after TBI initiation), LDH release (A) and apoptotic rate (B) were assayed to determine the time window of the allicin-induced protective effects. The data are represented as the means ± SD from five experiments. *P < 0.05 vs. the vehicle group.
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Fig. 4. Effect of allicin on the expression of three isoforms of NOS. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The expression levels of iNOS, p-eNOS, eNOS and nNOS were determined by western blotting (A) and calculated (B) 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the control group and *P < 0.05 vs. the vehicle group.
Fig. 5. Effects of NOS antagonists on allicin-induced neuroprotection. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The nonspecific NOS inhibitor L-NAME (100 M), the eNOS antagonist L-NIO (10 M), the iNOS antagonist 1400 W (10 M) or the nNOS antagonist NPLA (5 M) was added 30 min before injury to block the activation of the isoforms of NOS. LDH release (A) and apoptotic rate (B) were assayed 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the vehicle group and *P < 0.05 vs. the allicin treated group.
Although it is suggested that the three isoforms of NOS are important regulators of neuronal function in injured brain tissue, the exact roles of eNOS, nNOS and iNOS in neuronal survival and death are complicated and may depend on the nature of the insults or other conditions. The excessive amount of nNOS-derived NO has been demonstrated to be a neurotoxic factor (Zhou and Zhu, 2009), and pretreatment with the selective nNOS inhibitor 7-nitroindazole
reduces contusion volume and improve behavioral recovery in experimental TBI models (Wada et al., 1999). The eNOS isoform is constitutively expressed, and several neuroprotective agents have been shown to protect against neuronal injury via enhancing the phosphorylation level of eNOS (Asahi et al., 2005; Liu et al., 2012; Wang et al., 2013). While eNOS and nNOS have been shown to be beneficial and detrimental, respectively, as mentioned above, the
Fig. 6. Effects of allicin on the activation of Akt and ERK. Cultured cortical neurons were pretreated with 50 M allicin 1 h before mechanical trauma. The expression levels of p-Akt, Akt, p-ERK and ERK were determined by western blotting (A) and calculated (B) and (C) 24 h after injury. The data ware represented as the means ± SD from five experiments. # P < 0.05 vs. the control group and *P < 0.05 vs. the vehicle group.
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Fig. 7. Effects of Akt and ERK antagonists on the expression of NOS. Cultured cortical neurons were pretreated with the Akt antagonist LY294002 (LY, 20 M) or the ERK antagonist PD98059 (PD, 40 M) in the presence or absence of 50 M allicin. The expression levels of iNOS, p-eNOS, eNOS and nNOS were determined by western blotting (A) and calculated (B) 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the vehicle group and *P < 0.05 vs. the allicin group.
role of iNOS in TBI appears to be disputable. The knockdown of iNOS expression through gene deletion was reported to be protective by reducing the post-TBI lesion volume and improving neurological recovery (Jones et al., 2004). However, another study indicated that iNOS inhibition increased neuronal cell loss and worsened neurological recovery (Sinz et al., 1999). In the present study, we found that the selective nNOS inhibitor 1400W further enhanced the protective effect of allicin, indicating a detrimental role of post-TBI iNOS activation in our in vitro neuronal trauma model. Furthermore, the eNOS inhibitor L-NIO and the nNOS inhibitor NPLA partly reversed and further enhanced, respectively, allicin-induced neuroprotection in traumatic neuronal injury. These data strongly support the important role of the NOS pathway in traumatic neuronal injury. Furthermore, the neuroprotective effect of allicin is dependent on different regulatory effects on the three isoforms of NOS. TBI not only triggers a series of deleterious biochemical events including oxidative stress, activation of pro-inflammatory mediators and up-regulation of proteases, but also activates some compensatory protective mechanisms, such as increased expression of anti-apoptotic proteins and activation of several prosurvival signaling pathways (Nortje and Menon, 2004; Stoica and Faden, 2010; Zhao et al., 2007). Akt, also known as protein kinase B (PKB), is a serine/threonine kinase with pro-survival activities in several neuronal injury models (Brunet et al., 2001; Zhao et al., 2006). Furthermore, another important deleterious kinase found in brain injury, ERK, a mitogenic activated protein (MAP) kinase, mediates signal transduction from noxious stimuli to downstream pro-apoptotic or necrotic cascades (Shioda et al., 2009;
Subramaniam and Unsicker, 2010). In the present study, allicin treatment inhibited TBI-induced activation of ERK, while further enhanced the phosphorylation of Akt in TBI-injured neurons. The allicin mediated protection against traumatic neuronal injury was augmented by PD98059 but partly prevented by LY294002, confirming the opposite effects of Akt and ERK activation in TBI. In addition, divergent regulatory effects of the Akt and ERK pathways have been reported in previous studies. For example, ROS induced by acute exercise plays an important role in activating eNOS through the Akt and AMPK pathways (Barbosa et al., 2013). The activation of the PI3K/Akt/eNOS pahway has been demonstrated to be involved in the neuroprotection against ischemia reperfusion injury induced by recombinant human erythropoietin or remote ischemic post-conditioning (Fu et al., 2013; Peng et al., 2012). A previous study in human respiratory epithelial cells indicated that epimagnolin and fargesin inhibit iNOS expression and decrease the production of NO via the ERK pathway (Baek et al., 2009). The data from our present study show that LY294002 attenuated the allicin-induced increase in eNOS expression and phosphorylation, whereas PD98059 has opposite effects on the expression of iNOS and eNOS. These data suggest that TBI induced ERK phosphorylation can aggravate neuronal cell death through iNOS activation and eNOS inhibition and that the allicin-induced increase in the expression and phosphorylation of eNOS is differentially modulated by the Akt and ERK pathways. In summary, we have shown that a natural product, allicin, protects primary cultured cortical neurons against mechanical trauma injury by regulating three isoforms of NOS. The allicin-induced protection and regulation of the NOS pathways are differentially
Fig. 8. Effects of Akt and ERK antagonists on allicin induced neuroprotection. Cultured cortical neurons were pretreated with the Akt antagonist LY294002 (LY, 20 M) or the ERK antagonist PD98059 (PD, 40 M) in the presence or absence of 50 M allicin. LDH release (A) and apoptotic rate (B) were assayed 24 h after injury. The data are represented as the means ± SD from five experiments. # P < 0.05 vs. the vehicle group and *P < 0.05 vs. the allicin group.
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Fig. 9. A diagram summarizing the findings of allicin-induced neuroprotection against traumatic neuronal injury.
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