Down-regulation of NOX4 by betulinic acid protects against cerebral ischemia-reperfusion in mice

Abstract

Ischemic stroke leads to high potentiality of mortality and disability. The current treatment for ischemic stroke is mainly focused on intravenous thrombolytic therapy. However, ischemia/ reperfusion induces neuronal damage, which significantly influences the outcome of patients with ischemic stroke, and the exact mechanism implicated in ischemia/reperfusion injury remains unclear, although evidence shows that oxidative stress is likely to be involved. Betulinic acid is mainly known for its anti-tumor and anti-inflammatory activities. Our previous study showed that betulinic acid could decrease the reactive oxygen species (ROS) production by regulating the expression of NADPH oxidase. Thus, we hypothesized that betulinic acid may protect against brain ischemic injury in the animal model of stroke. Focal cerebral ischemia was achieved by using the standard intraluminal occlusion method and reperfusion enabled after 2 h ischemia. Neurological deficits were scored. Infarct size was determined with 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and the mRNA expression of NADPH oxidase 4 (NOX4) was determined by RT-PCR in infarct tissue. ROS generation and apoptosis in ischemic tissue were analyzed by measuring the oxidative conversion of cell permeable 2’,7’-dichloro-fluorescein diacetate (DCF-DA) to fluorescent dichlorofluorescein (DCF) in fluorescence microplate reader and TUNEL assay, respectively. In Kunming mice, 2 h of middle cerebral artery (MCA) occlusion followed by 24 or 72 h of reperfusion led to an enhanced NOX4 expression in the ischemic hemisphere. This was associated with elevated levels of ROS generation and neuronal apoptosis. Pre-treatment with betulinic acid (50 mg/kg/day for 7 days via gavage) prior to MCA occlusion prevented the ischemia/reperfusion-induced up-regulation of NOX4 and ROS production. In addition, treatment with betulinic acid could markedly blunt the ischemia/reperfusion-induced neuronal apoptosis. Finally, betulinic acid reduced infarct volume and ameliorated the neurological deficit in this stroke mouse model. Our results suggest that betulinic acid protects against cerebral ischemia/reperfusion injury in mice and the down-regulation of NOX4 may represent a mechanism contributing to this effect.

Keywords

betulinic acid / cerebral ischemia/reperfusion / NOX4 / oxidative stress

References

[1] EshahNF. Lifestyle and health promoting behaviours in jordanian subjects without prior history of coronary heart disease. Int J Nurs Pract, 2011, 17(1): 27-35 PMID: 21251151

[2] SudaS, KatsumataT, OkuboS, et al. . Low serum n-3 polyunsaturated fatty acid/n-6 polyunsaturated fatty acid ratio predicts neurological deterioration in Japanese patients with acute ischemic stroke. Cerebrovasc Dis, 2013, 36(5-6): 388-393 PMID: 24248098

[3] MargaillI, PlotkineM, LerouetD. Antioxidant strategies in the treatment of stroke. Free Radic Biol Med, 2005, 39(4): 429-443 PMID: 16043015

[4] AppelrosP, JonssonF, AsbergS, et al. . Trends in stroke treatment and outcome between 1995 and 2010: Observations from riks-stroke, the swedish stroke register. Cerebrovasc Dis, 2014, 37(1): 22-29 PMID: 24355999

[5] KogaM, ArihiroS, MiyashitaF, et al. . Factors associated with early recanalization failure following intravenous rt-pa therapy for ischemic stroke. Cerebrovasc Dis, 2013, 36(4): 299-305 PMID: 24135558

[6] ThomasSR, WittingPK, DrummondGR. Redox control of endothelial function and dysfunction: Molecular mechanisms and therapeutic opportunities. Antioxid Redox Signal, 2008, 10(10): 1713-1765 PMID: 18707220

[7] ChrissobolisS, FaraciFM. The role of oxidative stress and nadph oxidase in cerebrovascular disease. Trends Mol Med, 2008, 14(11): 495-502 PMID: 18929509 PMCID: 3140460

[8] BedardK, KrauseKH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev, 2007, 87(1): 245-313 PMID: 17237347

[9] ChenH, YoshiokaH, KimGS, et al. . Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection. Antioxid Redox Signal, 2011, 14(8): 1505-1517 PMID: 20812869 PMCID: 3061196

[10] NayerniaZ, JaquetV, KrauseKH. New insights on NOX enzymes in the central nervous system. Antioxid Redox Signal, 2014, 20(17): 2815-2837 PMID: 24206089 PMCID: 4026375

[11] McCannSK, RoulstonCL. NADPH oxidase as a therapeutic target for neuroprotection against ischaemic stroke: future perspectives. Brain Sci, 2013, 3(2): 561-598 PMID: 24961415 PMCID: 4061864

[12] SerranoF, KolluriNS, WientjesFB, et al. . Nadph oxidase immunoreactivity in the mouse brain. Brain Res, 2003, 988(1-2): 193-198 PMID: 14519542

[13] QinYY, LiM, FengX, et al. . Combined NADPH and the NOX inhibitor apocynin provides greater anti-inflammatory and neuroprotective effects in a mouse model of stroke. Free Radic Biol Med, 2017, 104: 333-345 PMID: 28132925

[14] WalderCE, GreenSP, DarbonneWC, et al. . Ischemic stroke injury is reduced in mice lacking a functional nadph oxidase. Stroke, 1997, 28(11): 2252-2258 PMID: 9368573

[15] RadermacherKA, WinglerK, LanghauserF, et al. . Neuroprotection after stroke by targeting nox4 as a source of oxidative stress. Antioxid Redox Signal, 2013, 18(12): 1418-1427 PMID: 22937798 PMCID: 3603500

[16] KleinschnitzC, GrundH, WinglerK, et al. . Post-stroke inhibition of induced nadph oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol, 2010, 8(9): e1000479 PMID: 20877715 PMCID: 2943442

[17] MullauerFB, KesslerJH, MedemaJP. Betulinic acid, a natural compound with potent anticancer effects. Anticancer Drugs, 2010, 21(3): 215-227 PMID: 20075711

[18] LuQ, XiaN, XuH, et al. . Betulinic acid protects against cerebral ischemia-reperfusion injury in mice by reducing oxidative and nitrosative stress. Nitric Oxide, 2011, 24(3): 132-138 PMID: 21292018

[19] JiaoS, ZhuH, HeP, et al. . Betulinic acid protects against cerebral ischemia/reperfusion injury by activating the PI3K/Akt signaling pathway. Biomed Pharmacother, 2016, 84: 1533-1537 PMID: 27876208

[20] FörstermannU, LiH. Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling. Br J Pharmacol, 2011, 164(2): 213-223 PMID: 21198553 PMCID: 3174401

[21] AsahiM, AsahiK, WangX, et al. . Reduction of tissue plasminogen activator-induced hemorrhage and brain injury by free radical spin trapping after embolic focal cerebral ischemia in rats. J Cereb Blood Flow Metab, 2000, 20(3): 452-457 PMID: 10724108

[22] BedersonJB, PittsLH, TsujiM, et al. . Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke, 1986, 17(3): 472-476 PMID: 3715945

[23] KahlesT, LuedikeP, EndresM, et al. . Nadph oxidase plays a central role in blood-brain barrier damage in experimental stroke. Stroke, 2007, 38(11): 3000-3006 PMID: 17916764

[24] ZhangX, HeF, YangJ, et al. . Protective effects of epigallocatechin-3-gallate on intestinal ischemia reperfusion injury through enhanced activation of PI3K/Akt pathway in rats. J Huazhong Univ Sci Technolog Med Sci, 2015, 35(3): 378-38325 PMID: 26072077

[25] InfangerDW, SharmaRV, DavissonRL. Nadph oxidases of the brain: Distribution, regulation, and function. Antioxid Redox Signal, 2006, 8(9-10): 1583-1596 PMID: 16987013

[26] CarboneF, TeixeiraPC, BraunersreutherV, et al. . Pathophysiology and treatments of oxidative injury in ischemic stroke: Focus on the phagocytic NADPH oxidase 2. Antioxid Redox Signal, 2015, 23(5): 460-489 PMID: 24635113 PMCID: 4545676

[27] McCartyMF. NADPH oxidase activity in cerebral arterioles is a key mediator of cerebral small vessel disease-Implications for prevention. Healthcare (Basel), 2015, 3(2): 233-251

[28] TakacI, SchroderK, ZhangL, et al. . The e-loop is involved in hydrogen peroxide formation by the nadph oxidase nox4. J Biol Chem, 2011, 286(15): 13304-13313 PMID: 21343298 PMCID: 3075677

[29] MillerAA, DrummondGR, SchmidtHH, et al. . Nadph oxidase activity and function are profoundly greater in cerebral versus systemic arteries. Circ Res, 2005, 97(10): 1055-1062 PMID: 16210546

[30] AldieriE, RigantiC, PolimeniM, et al. . Classical inhibitors of noxnad(p)h oxidases are not specific. Curr Drug Metab, 2008, 9(8): 686-696 PMID: 18855607

[31] GraySP, JhaJC, KennedyK, et al. . Combined NOX1/4 inhibition withGKT137831 in mice provides dose-dependent reno-and atheroprotection even in established microand macrovascular disease. Diabetologia, 2017, 60(5): 927-937 PMID: 28160092

[32] Steinkamp-FenskeK, BollingerL, VollerN, et al. . Ursolic acid from the chinese herb danshen (salvia miltiorrhiza l.) upregulates enos and downregulates nox4 expression in human endothelial cells. Atherosclerosis, 2007, 195(1): e104-111 PMID: 17481637

[33] ValletP, CharnayY, StegerK, et al. . Neuronal expression of the nadph oxidase nox4, and its regulation in mouse experimental brain ischemia. Neuroscience, 2005, 132(2): 233-238 PMID: 15802177

[34] MaMW, WangJ, ZhangQ, et al. . NADPH oxidase in brain injury and neurodegenerative disorders. Mol Neurodegener, 2017, 12(1): 7 PMID: 28095923 PMCID: 5240251

[35] AlakurttiS, MakelaT, KoskimiesS, et al. . Pharmacological properties of the ubiquitous natural product betulin. Eur J Pharm Sci, 2006, 29(1): 1-13 PMID: 16716572