Objective: The present study evaluated the ability of rosmarinic acid (RA) to inhibit microglia activation induced by lipopolysaccharide (LPS) in the N9 murine microglial cell line, and investigated the putative mechanisms involved in this process. Methods: In all tests, N9 murine microglial cells were pretreated with RA (0.1, 1.0, and 10 μM) for 20 h and exposed to LPS (1 μM/mL) for 4 h. Cell viability was measured by Trypan blue exclusion assay. Flow cytometry was used to detect reactive oxygen species (ROS), quantify cleaved caspase-3, and analyze the mitochondrial electrochemical potential. iNOS, Arg-1, TNF-α, IL-1β, and IL-6 proteins were analyzed by Western blotting, and their antigens were detected using the chemiluminescence technique. The effect of RA on DNA was evaluated by the Comet assay. Results: RA attenuated the expression of the M1 marker iNOS and the levels of proinflammatory factors, including TNF-α, IL-1β, and IL-6; it increased the expression of the M2 marker Arg-1, and inhibited, at least in part, ROS generation and loss of mitochondrial outer membrane permeabilization through the inhibition of cleaved caspase-3 activation. RA also inhibited DNA damage, reassuring cell protection. Conclusions: The results suggested a protective effect of RA through downregulation of inflammatory cytokines and cleaved caspase-3.

Briggs SW, Galanopoulou AS: Altered GABA signaling in early life epilepsies. Neural Plast 2011;2011:527605.
Rassendren F, Audinat E: Purinergic signaling in epilepsy. J Neurosci Res 2016;94:781-793.
Scharfman HE: Neuroscience: metabolic control of epilepsy. Science 2015;347:1312-1313.
Zaccara G, Schmidt D: Do traditional anti-seizure drugs have a future? A review of potential anti-seizure drugs in clinical development. Pharmacol Res 2016;104:38-48.
Aguiar CC, Almeida AB, Araújo PV, de Abreu RN, Chaves EM, do Vale OC, Macêdo DS, Woods DJ, Fonteles MM, Vasconcelos SM: Oxidative stress and epilepsy: literature review. Oxid Med Cell Longev 2012;79: 52-59.
Devinsky O, Vezzani A, Najjar S, De Lanerolle NC, Rogawski MA: Glia and epilepsy: excitability and inflammation. Trends Neurosci 2013;36:174-184.
Najjar S, Pearlman DM, Devinsky O, Najjar A, Zagzag D: Neurovascular unit dysfunction with blood-brain barrier hyperpermeability contributes to major depressive disorder: a review of clinical and experimental evidence. J Neuroinflammation 2013;10:142.
Najjar S, Pearlman DM, Hirsch S, Friedman K, Strange J, Reidy J, Khoukaz M, Ferrell RB, Devinsky O, Najjar A, Zagzag D: Brain biopsy findings link major depressive disorder to neuroinflammation, oxidative stress, and neurovascular dysfunction: a case report. Biol Psychiatry 2014;75:23-26.
Rowles J, Olsen M: Perspectives on the development of antioxidant antiepileptogenic agents. Mini Rev Med Chem 2012;12:1015-1027.
Eggen BJ, Raj D, Hanisch UK, Boddeke HW: Microglial phenotype and adaptation. J Neuroimmune Pharmacol 2013;8:807-823.
Gertig U, Hanisch UK: Microglial diversity by responses and responders. Front Cell Neurosci 2014;8:101.
Kettenmann H, Hanisch UK, Noda M, Verkhratsky A: Physiology of microglia. Physiol Rev 2011;91:461-553.
Kettenmann H, Kirchhoff F, Verkhratsky A: Microglia: new roles for the synaptic stripper. Neuron 2013;77:10-18.
Bosch ME, Kielian T: Neuroinflammatory paradigms in lysosomal storage diseases. Front Neurosci 2015;9:417.
Stoll G, Jander S: The role of microglia and macrophages in the pathophysiology of the CNS. Prog Neurobiol 1999;58:233-247.
Zuchero JB, Barres BA: Glia in mammalian development and disease. Development 2015;142:3805-3809.
Ojo JO, Rezaie P, Gabbott PL, Stewart MG: Impact of age-related neuroglial cell responses on hippocampal deterioration. Front Aging Neurosci 2015;7:57.
Pedata F, Dettori I, Coppi E, Melani A, Fusco I, Corradetti R, Pugliese AM: Purinergic signalling in brain ischemia. Neuropharmacology 2016;104:105-130.
Tang Y, Le W: Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol 2016;53:1181-1194.
Ghosh M, Xu Y, Pearse DD: Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines. J Neuroinflammation 2016;13:9.
Vezzani A, Viviani B: Neuromodulatory properties of inflammatory cytokines and their impact on neuronal excitability. Neuropharmacology 2015;96:70-82.
Marin I, Kipnis J: Learning and memory and the immune system. Learn Mem 2013;20:601-606.
Najjar S, Bernbaum M, Lai G, Devinsky O: Immunology and epilepsy. Rev Neurol Dis 2008;5:109-116.
Najjar S, Pearlman D, Miller DC, Devinsky O: Refractory epilepsy associated with microglial activation. Neurologist 2011;17:249-254.
Shan Y, Wang DD, Xu YX, Wang C, Cao L, Liu YS, Zhu CQ: Aging as a precipitating factor in chronic restraint stress-induced Tau aggregation pathology, and the protective effects of rosmarinic acid. J Alzheimers Dis 2016;49:829-844.
Amoah SK, Sandjo LP, Kratz JM, Biavatti MW: Rosmarinic acid - pharmaceutical and clinical aspects. Planta Med 2016;82:388-406.
Coelho VR, Vieira CG, de Souza LP, Moysés F, Basso C, Papke DK, Pires TR, Siqueira IR, Picada JN, Pereira P: Antiepileptogenic, antioxidant and genotoxic evaluation of rosmarinic acid and its metabolite caffeic acid in mice. Life Sci 2015;122:65-71.
Kelsey NA, Wilkins HM, Linseman DA: Nutraceutical antioxidants as novel neuroprotective agents. Molecules 2010;15:7792-7814.
Luan H, Kan Z, Xu Y, Lv C, Jiang W: Rosmarinic acid protects against experimental diabetes with cerebral ischemia: relation to inflammation response. J Neuroinflammation 2013;10:28.
Nabavi SF, Tenore GC, Daglia M, Tundis R, Loizzo MR, Nabavi SM: The cellular protective effects of rosmarinic acid: from bench to bed side. Curr Neurovasc Res 2015;12:98-105.
Seo JS, Choi J, Leem YH, Han PL: Rosmarinic acid alleviates neurological symptoms in the G93A-SOD1 transgenic mouse model of amyotrophic lateral sclerosis. Exp Neurobiol 2015;24:341-350.
Yamada M, Ono K, Hamaguchi T, Noguchi-Shinohara M: Natural phenolic compounds as therapeutic and preventive agents for cerebral amyloidosis. Adv Exp Med Biol 2015;863:79-94.
Coelho VR, Vieira CG, de Souza LP, da Silva LL, Pflüger P, Regner GG, Papke DK, Picada JN, Pereira P: Behavioral and genotoxic evaluation of rosmarinic and caffeic acid in acute seizure models induced by pentylenetetrazole and pilocarpine in mice. Naunyn Schmiedebergs Arch Pharmacol 2016;389:1195-1203.
Grigoletto J, Oliveira CV, Grauncke AC, Souza TL, Souto NS, Freitas ML, Furian AF, Santos AR, Oliveira MS: Rosmarinic acid is anticonvulsant against seizures induced by pentylenetetrazol and pilocarpine in mice. Epilepsy Behav 2017;62:27-34.
Khamse S, Sadr SS, Roghani M, Hasanzadeh G, Mohammadian M: Rosmarinic acid exerts a neuroprotective effect in the kainite rat model of temporal lobe epilepsy: underlying mechanisms. Pharm Biol 2015;53:1818-1825.
Viau CM, Guecheva TN, Sousa FG, Pungartnik C, Brendel M, Saffi J, Henriques JA: SnCl2-induced DNA damage and repair inhibition of MMS-caused lesions in V79 Chinese hamster fibroblasts. Arch Toxicol 2009;83:769-775.
Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M: Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 1983;130:1910-1917.
Singh NP, McCoy MT, Tice RR, Schneider EL: A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988;175:184-191.
Nadin SB, Vargas-Roig LM, Ciocca DR: A silver staining method for single-cell gel assay. J Histochem Cytochem 2001;49:1183-1186.
Collins AR, Ma AG, Duthie SJ: The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mut Res 1995;336:69-77.
Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A: Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. Mutat Res 2007;627:31-35.
Emadi A, Ross AE, Cowan KM, Fortenberry YM, Vuica-Ross M: A chemical genetic screen for modulators of asymmetrical 2,2'-dimeric naphthoquinones cytotoxicity in yeast. PLoS One 2010;26:10846.
Viau CM, Moura DJ, Facundo VA, Saffi J: The natural triterpene 3β,6β,16β-trihydroxy-lup-20(29)-ene obtained from the flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cells. BMC Complement Altern Med 2014;14:280.
Vezzani A: Anti-inflammatory drugs in epilepsy: does it impact epileptogenesis? Expert Opin Drug Saf 2015;14:583-592.
Tang Y, Le W: Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol 2016;53:1181-1194.
Mikita J, Dubourdieu-Cassagno N, Deloire MS, Vekris A, Biran M, Raffard G, Brochet B, Canron MH, Franconi JM, Boiziau C, Petry KG: Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis: a melioration of clinical status by M2 activated monocyte administration. Mult Scler 2011;17:2-15.
Xu F, Huang J, He Z, Chen J, Tang X, Song Z, Guo Q, Huang C: Microglial polarization dynamics in dorsal spinal cord in the early stages following chronic sciatic nerve damage. Neurosci Lett 2016;617:6-13.
Asano S, Chantler PD, Barr TL: Gene expression profiling in stroke: relevance of blood-brain interaction. Curr Opin Pharmacol 2016;26:80-86.
Jenkinson CP, Grody WW, Cederbaum SD: Comparative properties of arginases. J Neuroinflammation 1996;10:28.
Ma L, Jia J, Liu X, Bai F, Wang Q, Xiong L: Activation of murine microglial N9 cells is attenuated through cannabinoid receptor CB2 signaling. Biochem Biophys Res Commun 2015;458:92-97.
Benson MJ, Manzanero S, Borges K: Complexalterations in microglial M1/M2 markers during the development of epilepsy in two mouse models. Epilepsia 2015;56:895-905.
Ghasemzadeh Rahbardar M, Amin B, Mehri S, Mirnajafi-Zadeh SJ, Hosseinzadeh H: Anti-inflammatory effects of ethanolic extract of Rosmarinus officinalis L. and rosmarinic acid in a rat model of neuropathic pain. Biomed Pharmacoter 2017;86:441-449.
Gogvadze V, Orrenius S, Zhivotovsky B: Analysis of mitochondrial dysfunction during cell death. Methods Mol Biol 2015;1264:385-393.
Man SM, Kanneganti TD: Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat Rev Immunol 2016;16:7-21.
Perez-Pinzon MA, Stetler RA, Fiskum G: Novel mitochondrial targets for neuroprotection. J Cereb Blood Flow Metab 2012;32:1362-1376.
Otera H, Mihara K: Mitochondrial dynamics: functional link with apoptosis. Int J Cell Biol 2012;82:1676.
Otera H, Ohsakaya S, Nagaura Z, Ishihara N, Mihara K: Export of mitochondrial AIF in response to proapoptotic stimuli depends on processing at the intermembrane space. EMBO J 2005;24:1375-1386.
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