1. Hackett PH, Roach RC. High-altitude illness. N Engl J Med 2001; 345:107-114.
2. Flück M. Functional, structural and molecular plasticity of mammalian skeletal muscle in response to exercise stimuli. J Exp Biol 2006; 209:2239-2248.
3. Kleszczyński K, Zillikens D, Fischer TW. Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK). J Pineal Res 2016; 61:187-197.
4. Ramanathan L, Gozal D, Siegel JM. Antioxidant responses to chronic hypoxia in the rat cerebellum and pons. J Neurochem 2005; 93:47-52.
5. Niki E. Assessment of antioxidant capacity in vitro and in vivo. Free Radic Biol Med 2010; 49:503-515.
6. Ciarlone GE, Dean JB. Acute hypercapnic hyperoxia stimulates reactive species production in the caudal solitary complex of rat brain slices but does not induce oxidative stress. Am J Physiol Cell Physiol 2016; 311:C1027-C1039.
7. Cabrerizo S, De La Cruz JP, López-Villodres JA, Muñoz-Marín J, Guerrero A, Reyes JJ, et al. Role of the inhibition of oxidative stress and inflammatory mediators in the neuroprotective effects of hydroxytyrosol in rat brain slices subjected to hypoxia reoxygenation. J Nutr Biochem 2013; 24:2152-2157.
8. Lu Q, Rau TF, Harris V, Johnson M, Poulsen DJ, Black SM. Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures. Eur J Neurosci 2011; 34:1093-1101.
9. DeFeudis FV, Drieu K. Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications. Curr Drug Targets 2000; 1:25-58.
10. Li L, Zhang QG, Lai LY, Wen XJ, Zheng T, Cheung CW, et al. Neuroprotective effect of ginkgolide B on bupivacaine-induced apoptosis in SH-SY5Y cells. Oxid Med Cell Longev 2013; 2013:159864.
11. Nada SE, Shah ZA. Preconditioning with Ginkgo biloba (EGb 761®) provides neuroprotection through HO1 and CRMP2. Neurobiol Dis 2012; 46:180-189.
12. Shah ZA, Nada SE, Doré S. Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection. Neuroscience 2011; 180:248-255.
13. Oh JH, Oh J, Togloom A, Kim SW, Huh K. Effects of Ginkgo biloba extract on cultured human retinal pigment epithelial cells under chemical hypoxia. Curr Eye Res 2013; 38:1072-1082.
14. Ahlemeyer B, Möwes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents. Eur J Pharmacol 1999; 367:423-430.
15. Huang M, Qian Y, Guan T, Huang L, Tang X, Li Y. Different neuroprotective responses of Ginkgolide B and bilobalide, the two Ginkgo components, in ischemic rats with hyperglycemia. Eur J Pharmacol 2012; 677: 71-76.
16. Gao J, Chen T, Zhao D, Zheng J, Liu Z. Ginkgolide B exerts cardioprotective properties against doxorubicin-induced cardiotoxicity by regulating reactive oxygen species, Akt and Calcium signaling pathways in vitro and in vivo. PLoS One 2016; 11:e0168219.
17. Botao Y, Ma J, Xiao W, Xiang Q, Fan K, Hou J, et al. Protective effect of ginkgolide B on high altitude cerebral edema of rats. High Alt Med Biol 2013; 14:61-64.
18. Yang W, Zhang X, Wang N, et al. Effects of acute systemic hypoxia and hypercapnia on brain damage in a rat model of hypoxia-ischemia. PLoS One 2016; 11:e0167359.
19. Lee CH, Park JH, Ahn JH, Won MH. Effects of melatonin on cognitive impairment and hippocampal neuronal damage in a rat model of chronic cerebral hypoperfusion. Exp Ther Med 2016; 11:2240-2246
20. Zhu B, Wang ZG, Ding J, Tan J, Fang X, Wang Q, et al. Chronic lipopolysaccharide exposure induces cognitive dysfunction without affecting BDNF expression in the rat hippocampus. Exp Ther Med 2014; 7: 750-754.
21. Maitra I, Marcocci L, Droy-Lefaix MT, Packer L. Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761. Biochem Pharmacol 1995; 49:1649-1655.
22. Ahlemeyer B, Krieglstein J. Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer’s disease. Pharmacopsychiatry 2003; 36:S8-14.
23. Bate C, Salmona M, Williams A. Ginkgolide B inhibits the neurotoxicity of prions or amyloid-beta1-42. J Neuroinflammation 2004; 1:4.
24. Pincemail J, Thirion A, Dupuis M, Braquet P, Drieu K, Deby C. Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes. Experientia 1987; 43:181-184.
25. Zhang S, Chen B, Wu W, Bao L, Qi R. Ginkgolide B reduces inflammatory protein expression in oxidized low-density lipoprotein-stimulated human vascular endothelial cells. J Cardiovasc Pharmacol 2011; 57:721-727.
26. Li R, Chen B, Wu W, Bao L, Li J, Qi R. Ginkgolide B suppresses intercellular adhesion molecule-1 expression via blocking nuclear factor-kappaB activation in human vascular endothelial cells stimulated by oxidized low-density lipoprotein. J Pharmacol Sci 2009; 110:362-369.
27. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 2005; 12:1161-1208.
28. Bastianetto S, Ramassamy C, Doré S, Christen Y, Poirier J, Quirion R. The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid. Eur J Neurosci 2000; 12:1882-1890.
29. Smith JV, Luo Y. Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol Biotechnol 2004; 64:465-472.
30. Zhou T, You WT, Ma ZC, Liang QD, Tan HL, Xiao CR, et al. Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation. Acta Pharmacol Sin 2016; 37:177-186.
TATCCAATCCTGTGCTGCTAT-3′ and 5′-CTCTTGCGGAGTATTTGTGC-3′ for Bcl-2, 5′-TCTGACGGCAACTTCAACTG-3′ and 5′-AGGAAAACGCATTATAGACCAC-3′ for Bax, 5′-TGACTGGAAAGCCGAAACTCCGAAACTC- 3′ and 5′- AGCCTCCACCGGTATCTTCT-3′ for caspase-3. The thermal cycling conditions were as follows: 1 cycle at 95 °C for 10 min; 30 cycles at 94 °C for 15 sec, 55 °C for 30 sec, and 72 °C for 30 sec; and 1 cycle at 72 °C for 10 min. The PCR reactions were performed with a SYBR green-based system (TaKaRa, Cat.RR82LR), and the gene fold changes were calculated using the (2−ΔΔCt) method.
Oxidative stress index test
Animals of all experimental groups were sacrificed by cervical dislocation after intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg), and the hippocampus was resected. The hippocampus was homogenized with cold normal saline (NS), centrifuged (4 °C, 12,500 g, 10 min), and the supernatant was collected for assays.
MDA (Beyotime, cat.S0131), SOD (Solarbio, cat. BC0175), catalase (Beyotime, cat.S0051), and GSH (Beyotime, cat.S0053) assays were performed using assay kits, according to manufacturers’ instructions.
Statistical analysis
All values were presented as mean±SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) by SPSS software (version 15.0; Chicago, IL, USA). An effect was considered statistically significant if the P-value was less than 0.05 (P< 0.05).
Results
The protective effect of GB on hippocampal neuron damage induced by acute hypoxia
HE staining showed that, in rats of the untreated control group (group I), neurons and neural tissue were intact and had normal morphology (Figure 1). These cells had integrity, regular structure, high cell-density, and were aligned in the CA1, CA2, and dentate gyrus (DG) areas. The pyramidal cells had round nuclei, prominent nucleoli, and clear cytoplasm. However, in rats exposed to 6000 m high altitude (group II), neuronal cells showed morphological changes including irregular cell contour, edema, low cell-density, irregular nuclei with unclear nucleolus and loose chromatin. Hypoxia-induced injury to neuronal cells was ameliorated by prior GB treatment (group III).
Nissl staining yielded similar results. As shown in Figure 1, after acute exposure to 6000 m simulated altitude, pronounced neuronal loss was observed in the hippocampal CA1 area of rats of group II. This damage could be mitigated by treatment with GB, and visible Nissl bodies were observed in animals of group III.
These results indicate that acute hypoxia exposure induced neuronal damage in the hippocampus. However, prior intragastric administration of GB (12 mg/kg) could protect hippocampal cells from hypoxia-induced damage.
Anti-apoptotic effect of GB on hippocampal neuron damage induced by acute hypoxia
Using TUNEL staining, apoptosis in the brains of rats in the different groups was observed as follows (Figure 2A): the number of apoptotic cells was increased in the high-altitude group; the GB group had fewer apoptotic cells in the CA1 area. When comparing the AI at the same time point among different groups (Figure 2B), the AI of the GB group was significantly lower than that of the high-altitude group (P < 0.05).
We also tested the mRNA level of the apoptosis indices including Bax, Bcl-2, and caspase-3 (Figure 2C). Compared to the control group, Bax and caspase-3 expression increased, and Bcl-2 expression decreased (P < 0.05) in the high-altitude group. There was no significant difference in apoptotic index expression (P > 0.1) between the control and GB groups. These results indicate that acute hypoxia exposure leads to apoptosis in hippocampal nerve cells, and that GB may have an anti-apoptotic effect by which it could protect hypoxia-induced brain damage.
GB treatment protects against oxidative stress in hippocampal neurons exposed to acute hypoxia
As shown in Figures 3A-D, the activity of SOD and catalase and the concentration of GSH decreased significantly, and the concentration of MDA increased significantly in the high-altitude group compared to the control group (P < 0.05). In addition, SOD and catalase activity, and GSH concentration increased, and MDA concentration decreased in the GB group compared to the high altitude group (P < 0.05). Thus, GB likely prevents oxidative stress.
Discussion
In modern times, more people visit plateaus with minimal conditioning, for work or tourism. The development of medicines to protect the brain under hypoxic conditions is therefore of clinical significance. In this study, we established a mammalian model to simulate high-altitude and hypoxia environments to study the neuroprotective effect of GB on the brain. There are 3 major subfields of the hippocampus that include DG, CA1, and CA3. Previous studies have shown that the CA1 region is an important area that correlates with spatial memory impairment (19, 20). Therefore in this study, the CA1 region was chosen as a major region to identify the apoptosis and neuronal survival conditions in this test.
GB exerts antioxidant effects by scavenging peroxy radicals (21-23) and lowering ROS and MDA levels (15, 24), and can suppress oxidized low-density lipoprotein (LDL)-induced inflammatory protein expression and inhibit nuclear factor-κB (NF-κB) activation in human endothelial cells (25, 26). Our results similarly suggest that augmentation of the antioxidant defense system by GB eventually leads to quenching of free radicals and reduction of ROS and lipid peroxidation. Oxidative stress can cause mitochondrial damage, complete caspase activation, and ultimately apoptosis in neuronal cells (27). Therefore, the neuroprotective effect of GB is likely mediated by its antioxidant function.
The effective suppression of apoptosis and rescue of germ cells by GB extract in testes exposed to doxorubicin (Dox) has been reported; however, the mechanism responsible for this process remains unclear. GB extract suppresses apoptosis induced by beta-amyloid in neuronal cells by influencing mitochondrial membrane potentials, regulating Bcl-2 family members, and repressing caspase-3 activity (28, 29). GB induced the expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1) in a pregnane X receptor (PXR)-dependent manner to exert anti-apoptotic and anti-inflammatory effects in endothelial cells (30). Our results showed an anti-apoptotic effect of GB consistent with those described in the aforementioned studies. We found that GB could reduce the expression of Bax and caspase-3, which are unregulated in apoptotic cells. Therefore, the anti-apoptotic property of GB could also play a role in GB-mediated neuroprotection.
This study has some limitations. First, the animal decompression chamber had to be reset to normal altitude conditions because of daily feeding. The animal model therefore failed to simulate the exact conditions of the human body on a plateau. Second, we used only one dose of GB in our experiment, and did not carry out a dose-response study. Third, we did not investigate the influence of GB on the molecular pathways of apoptosis and oxidative stress. We intend to address these limitations in future studies.
Conclusion
In summary, we successfully established an animal model for plateau hypoxia, which showed marked neuronal cell damage in brain slices. We showed that GB treatment could protect against hippocampal injury. Finally, we showed that GB could protect neuronal cells against acute hypoxia by mechanisms that include anti-oxidative and anti-apoptotic effects. Our results could provide a basis for the development of novel therapeutic strategies to ensure neuroprotection under acute hypoxia at high altitudes.
Conflicts of Interest
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to subject of this article.
Acknowledgment
The study was sponsored by National Nature Science Fund of China (81371444). No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.
References
1. Hackett PH, Roach RC. High-altitude illness. N Engl J Med 2001; 345:107-114.
2. Flück M. Functional, structural and molecular plasticity of mammalian skeletal muscle in response to exercise stimuli. J Exp Biol 2006; 209:2239-2248.
3. Kleszczyński K, Zillikens D, Fischer TW. Melatonin enhances mitochondrial ATP synthesis, reduces reactive oxygen species formation, and mediates translocation of the nuclear erythroid 2-related factor 2 resulting in activation of phase-2 antioxidant enzymes (γ-GCS, HO-1, NQO1) in ultraviolet radiation-treated normal human epidermal keratinocytes (NHEK). J Pineal Res 2016; 61:187-197.
4. Ramanathan L, Gozal D, Siegel JM. Antioxidant responses to chronic hypoxia in the rat cerebellum and pons. J Neurochem 2005; 93:47-52.
5. Niki E. Assessment of antioxidant capacity in vitro and in vivo. Free Radic Biol Med 2010; 49:503-515.
6. Ciarlone GE, Dean JB. Acute hypercapnic hyperoxia stimulates reactive species production in the caudal solitary complex of rat brain slices but does not induce oxidative stress. Am J Physiol Cell Physiol 2016; 311:C1027-C1039.
7. Cabrerizo S, De La Cruz JP, López-Villodres JA, Muñoz-Marín J, Guerrero A, Reyes JJ, et al. Role of the inhibition of oxidative stress and inflammatory mediators in the neuroprotective effects of hydroxytyrosol in rat brain slices subjected to hypoxia reoxygenation. J Nutr Biochem 2013; 24:2152-2157.
8. Lu Q, Rau TF, Harris V, Johnson M, Poulsen DJ, Black SM. Increased p38 mitogen-activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures. Eur J Neurosci 2011; 34:1093-1101.
9. DeFeudis FV, Drieu K. Ginkgo biloba extract (EGb 761) and CNS functions: basic studies and clinical applications. Curr Drug Targets 2000; 1:25-58.
10. Li L, Zhang QG, Lai LY, Wen XJ, Zheng T, Cheung CW, et al. Neuroprotective effect of ginkgolide B on bupivacaine-induced apoptosis in SH-SY5Y cells. Oxid Med Cell Longev 2013; 2013:159864.
11. Nada SE, Shah ZA. Preconditioning with Ginkgo biloba (EGb 761®) provides neuroprotection through HO1 and CRMP2. Neurobiol Dis 2012; 46:180-189.
12. Shah ZA, Nada SE, Doré S. Heme oxygenase 1, beneficial role in permanent ischemic stroke and in Gingko biloba (EGb 761) neuroprotection. Neuroscience 2011; 180:248-255.
13. Oh JH, Oh J, Togloom A, Kim SW, Huh K. Effects of ginkgo biloba extract on cultured human retinal pigment epithelial cells under chemical hypoxia. Curr Eye Res 2013; 38:1072-1082.
14. Ahlemeyer B, Möwes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents. Eur J Pharmacol 1999; 367:423-430.
15. Huang M, Qian Y, Guan T, Huang L, Tang X, Li Y. Different neuroprotective responses of Ginkgolide B and bilobalide, the two Ginkgo components, in ischemic rats with hyperglycemia. Eur J Pharmacol 2012; 677: 71-76.
16. Gao J, Chen T, Zhao D, Zheng J, Liu Z. Ginkgolide B Exerts Cardioprotective Properties against Doxorubicin-Induced Cardiotoxicity by Regulating Reactive Oxygen Species, Akt and Calcium Signaling Pathways In Vitro and In Vivo. PLoS One 2016; 11:e0168219.
17. Botao Y, Ma J, Xiao W, Xiang Q, Fan K, Hou J, et al. Protective effect of ginkgolide B on high altitude cerebral edema of rats. High Alt Med Biol 2013; 14:61-64.
18. Yang W, Zhang X, Wang N, et al. Effects of Acute Systemic Hypoxia and Hypercapnia on Brain Damage in a Rat Model of Hypoxia-Ischemia. PLoS One 2016; 11:e0167359.
19. Lee CH, Park JH, Ahn JH, Won MH. Effects of melatonin on cognitive impairment and hippocampal neuronal damage in a rat model of chronic cerebral hypoperfusion. Exp Ther Med 2016; 11:2240-2246
20. Zhu B, Wang ZG, Ding J, Tan J, Fang X, Wang Q, et al. Chronic lipopolysaccharide exposure induces cognitive dysfunction without affecting BDNF expression in the rat hippocampus. Exp Ther Med 2014; 7: 750-754.
21. Maitra I, Marcocci L, Droy-Lefaix MT, Packer L. Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761. Biochem Pharmacol 1995; 49:1649-1655.
22. Ahlemeyer B, Krieglstein J. Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer’s disease. Pharmacopsychiatry 2003; 36:S8-14.
23. Bate C, Salmona M, Williams A. Ginkgolide B inhibits the neurotoxicity of prions or amyloid-beta1-42. J Neuroinflammation 2004; 1:4.
24. Pincemail J, Thirion A, Dupuis M, Braquet P, Drieu K, Deby C. Ginkgo biloba extract inhibits oxygen species production generated by phorbol myristate acetate stimulated human leukocytes. Experientia 1987; 43:181-184.
25. Zhang S, Chen B, Wu W, Bao L, Qi R. Ginkgolide B reduces inflammatory protein expression in oxidized low-density lipoprotein-stimulated human vascular endothelial cells. J Cardiovasc Pharmacol 2011; 57:721-727.
26. Li R, Chen B, Wu W, Bao L, Li J, Qi R. Ginkgolide B suppresses intercellular adhesion molecule-1 expression via blocking nuclear factor-kappaB activation in human vascular endothelial cells stimulated by oxidized low-density lipoprotein. J Pharmacol Sci 2009; 110:362-369.
27. Valko M, Morris H, Cronin MT. Metals, toxicity and oxidative stress. Curr Med Chem 2005; 12:1161-1208.
28. Bastianetto S, Ramassamy C, Doré S, Christen Y, Poirier J, Quirion R. The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid. Eur J Neurosci 2000; 12:1882-1890.
29. Smith JV, Luo Y. Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol Biotechnol 2004; 64:465-472.
30. Zhou T, You WT, Ma ZC, Liang QD, Tan HL, Xiao CR, et al. Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation. Acta Pharmacol Sin 2016; 37:177-186.
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