Andrographolide Inhibits PI3K/AKT-Dependent NOX2 and iNOS Expression Protecting Mice against Hypoxia/Ischemia-Induced Oxidative Brain Injury

Abstract

This study aimed to explore the mechanisms by which andrographolide protects against hypoxia-induced oxidative/nitrosative brain injury provoked by cerebral ischemic/reperfusion (CI/R) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone CI/R injury with andrographolide (10–100 µg/kg, i. v.) at 1 h after hypoxia ameliorated CI/R-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. CI/R induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (NOX2), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b cells due to activation of nuclear factor-kappa B (NF-κB) and hypoxia-inducible factor 1-alpha (HIF-1α). All these changes were significantly diminished by andrographolide. In BV-2 cells, OGD induced ROS and nitric oxide production by upregulating NOX2 and iNOS via the phosphatidylinositol-3-kinase (PI3K)/AKT-dependent NF-κB and HIF-1α pathways, and these changes were suppressed by andrographolide and LY294002. Our results indicate that andrographolide reduces NOX2 and iNOS expression possibly by impairing PI3K/AKT-dependent NF-κB and HIF-1α activation. This compromises microglial activation, which then, in turn, mediates andrographolide's protective effect in the CI/R mice.

Key words

andrographolide - HIF-1α - hypoxia - iNOS - gp91NADPH oxidase (NOX2) - phosphatidylinositol-3-kinase (PI3K)/AKT

References

• 1 Lo E H, Dalkara T, Moskowitz M A. Mechanisms, challenges and opportunities in stroke.  Nat Rev Neurosci. 2003;  4 399-415
• 2 Shen Y C, Wang Y H, Chou Y C, Liou K T, Yen J C, Wang W Y, Liao J F. Dimemorfan protects rats against ischemic stroke through activation of sigma-1 receptor-mediated mechanisms by decreasing glutamate accumulation.  J Neurochem. 2008;  104 558-572
• 3 Argaw A T, Zhang Y, Snyder B J, Zhao M L, Kopp N, Lee S C, Raine C S, Brosnan C F, John G R. IL-1beta regulates blood-brain barrier permeability via reactivation of the hypoxia-angiogenesis program.  J Immunol. 2006;  177 5574-5584
• 4 Nagel S, Genius J, Heiland S, Horstmann S, Gardner H, Wagner S. Diphenyleneiodonium and dimethylsulfoxide for treatment of reperfusion injury in cerebral ischemia of the rat.  Brain Res. 2007;  1132 210-217
• 5 Borutaite V, Hope H, Brown G C. Arachidonate and NADPH oxidase synergise with iNOS to induce death in macrophages: mechanisms of inflammatory degeneration.  Pharmacol Rep. 2006;  58 (Suppl.) 96-102
• 6 Harari O A, Liao J K. NF-κB and innate immunity in ischemic stroke.  Ann NY Acad Sci. 2010;  1207 32-40
• 7 Semenza G L. HIF-1: mediator of physiological and pathophysiological responses to hypoxia.  J Appl Physiol. 2000;  88 1474-1480
• 8 Sharp F R, Bernaudin M. HIF1 and oxygen sensing in the brain.  Nat Rev Neurosci. 2004;  5 437-448
• 9 Chen W, Ostrowski R P, Obenaus A, Zhang J H. Prodeath or prosurvival: two facets of hypoxia inducible factor-1 in perinatal brain injury.  Exp Neurol. 2009;  216 7-15
• 10 Correia S C, Moreira P I. Hypoxia-inducible factor 1: a new hope to counteract neurodegeneration?.  J Neurochem. 2010;  112 1-12
• 11 Mi Z, Rapisarda A, Taylor L, Brooks A, Creighton-Gutteridge M, Melillo G, Varesio L. Synergystic induction of HIF-1alpha transcriptional activity by hypoxia and lipopolysaccharide in macrophages.  Cell Cycle. 2008;  7 232-241
• 12 Treins C, Giorgetti-Peraldi S, Murdaca J, Semenza G L, van Obberghen E. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway.  J Biol Chem. 2002;  277 27975-27981
• 13 Lu D Y, Liou H C, Tang C H, Fu W M. Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1alpha.  Biochem Pharmacol. 2006;  72 992-1000
• 14 Shen Y C, Chen C F, Chiou W F. Suppression of rat neutrophil reactive oxygen species production and adhesion by the diterpenoid lactone andrographolide.  Planta Med. 2000;  66 314-317
• 15 Shen Y C, Chen C F, Chiou W F. Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect.  Br J Pharmacol. 2002;  135 399-406
• 16 Chiou W F, Lin J J, Chen C F. Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide.  Br J Pharmacol. 1998;  125 327-334
• 17 Chao W W, Kuo Y H, Lin B F. Anti-inflammatory activity of new compounds from Andrographis paniculata by NF-kappaB transactivation inhibition.  J Agric Food Chem. 2010;  58 2505-2512
• 18 Chan S J, Wong W S, Wong P T, Bian J S. Neuroprotective effects of andrographolide in a rat model of permanent cerebral ischaemia.  Br J Pharmacol. 2010;  161 668-679
• 19 Kim H H, Sawada N, Soydan G, Lee H S, Zhou Z, Hwang S K, Waeber C, Moskowitz M A, Liao J K. Additive effects of statin and dipyridamole on cerebral blood flow and stroke protection.  J Cereb Blood Flow Metab. 2008;  28 1285-1293
• 20 Kofler J, Otsuka T, Zhang Z, Noppens R, Grafe M R, Koh D W, Dawson V L, de Murcia J M, Hurn P D, Traystman R J. Differential effect of PARP-2 deletion on brain injury after focal and global cerebral ischemia.  J Cereb Blood Flow Metab. 2006;  26 135-141
• 21 Shen Y C, Sung Y J, Chen C F. Magnolol inhibits Mac-1 (CD11b/CD18)-dependent neutrophil adhesion: relationship with its antioxidant effect.  Eur J Pharmacol. 1998;  343 79-86
• 22 Woo A Y, Waye M M, Tsui S K, Yeung S T, Cheng C H. Andrographolide up-regulates cellular-reduced glutathione level and protects cardiomyocytes against hypoxia/reoxygenation injury.  J Pharmacol Exp Ther. 2008;  325 226-235
• 23 Margaill I, Allix M, Boulu R G, Plotkine M. Dose- and time-dependence of L-NAME neuroprotection in transient focal cerebral ischaemia in rats.  Br J Pharmacol. 1997;  120 160-163
• 24 Mander P, Borutaite V, Moncada S, Brown G C. Nitric oxide from inflammatory-activated glia synergizes with hypoxia to induce neuronal death.  J Neurosci Res. 2005;  79 208-215
• 25 Wang Y H, Wang W Y, Chang C C, Liou K T, Sung Y J, Liao J F, Chen C F, Chang S, Hou Y C, Chou Y C, Shen Y C. Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its anti-oxidative effect and modulation of NF-kappa B activation.  J Biomed Sci. 2006;  13 127-141
• 26 Li J, Luo L, Wang X, Liao B, Li G. Inhibition of NF-kappaB expression and allergen-induced airway inflammation in a mouse allergic asthma model by andrographolide.  Cell Mol Immunol. 2009;  6 381-385
• 27 Antonov A S, Antonova G N, Munn D H, Mivechi N, Lucas R, Catravas J D, Verin A D. AlphaVbeta3 integrin regulates macrophage inflammatory responses via PI3-kinase/Akt-dependent NF-kappaB activation.  J Cell Physiol. 2011;  226 469-476
• 28 Ardeshna K M, Pizzey A R, Devereux S, Khwaja A. The PI3 kinase, p 38 SAP kinase, and NFkappaB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells.  Blood. 2000;  96 1039-1046
• 29 Monaghan-Benson E, Burridge K. The regulation of vascular endothelial growth factor-induced microvascular permeability requires Rac and reactive oxygen species.  J Biol Chem. 2009;  284 25602-25611
• 30 Wang W, Wang J, Dong S F, Liu C H, Italiani P, Sun S H, Xu J, Boraschi D, Ma S P, Qu D. Immunomodulatory activity of andrographolide on macrophage activation and specific antibody response.  Acta Pharmacol Sin. 2010;  31 191-201
• 31 Lin H H, Tsai C W, Chou F P, Wang C J, Hsuan S W, Wang C K, Chen J H. Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells.  Toxicol Appl Pharmacol. 2011;  250 336-345
• 32 Minet E, Ernest I, Michel G, Roland I, Remacle J, Raes M, Michiels C. HIF1A gene transcription is dependent on a core promoter sequence encompassing activating and inhibiting sequences located upstream from the transcription initiation site and cis elements located within the 5′UTR.  Biochem Biophys Res Commun. 1999;  261 534-540

1 These authors contributed equally to this study.