Anti-inflammatory effects of Edaravone and Scutellarin in activated microglia in experimentally induced ischemia injury in rats and in BV-2 microglia

BMC Neuroscience

Chunyun Wu²  Eng-Ang Ling¹  Parakalan Rangarajan¹  Hao Zha²  Yun Yuan¹ 

[1] Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Blk MD10, 4 Medical Drive, Singapore 117597, Singapore;Department of Anatomy and Histology/Embryology, Faculty of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Kunming 650500, People’s Republic of China

关键词: Anti-inflammation;    Scutellarin;    Edaravone;    Cerebral ischemia;    Activated microglia;   

Others  :  1090577
DOI  :  10.1186/s12868-014-0125-3

 received in 2014-09-26, accepted in 2014-11-10,  发布年份 2014

【 摘 要 】

Background

In response to cerebral ischemia, activated microglia release excessive inflammatory mediators which contribute to neuronal damage. Therefore, inhibition of microglial over-activation could be a therapeutic strategy to alleviate various microglia-mediated neuroinflammation. This study was aimed to elucidate the anti-inflammatory effects of Scutellarin and Edaravone given either singly, or in combination in activated microglia in rats subjected to middle cerebral artery occlusion (MCAO), and in lipopolysaccharide (LPS)-induced BV-2 microglia. Expression of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and inducible nitric oxide synthase (iNOS) was assessed by immunofluorescence staining and Western blot. Reactive oxygen species (ROS) and nitric oxide (NO) levels were determined by flow cytometry and fluorescence microscopy, respectively.

Results

In vivo, both Edaravone and Scutellarin markedly reduced the infarct cerebral tissue area with the latter drug being more effective with the dosage used; furthermore, when used in combination the reduction was more substantial. Remarkably, a greater diminution in distribution of activated microglia was observed with the combined drug treatment which also attenuated the immunoexpression of TNF-α, IL-1β and iNOS to a greater extent as compared to the drugs given separately. In vitro, both drugs suppressed upregulated expression of inflammatory cytokines, iNOS, NO and ROS in LPS-induced BV-2 cells. Furthermore, Edaravone and Scutellarin in combination cumulatively diminished the expression levels of the inflammatory mediators being most pronounced for TNF-α as evidenced by Western blot.

Conclusion

The results suggest that Edaravone and Scutellarin effectively suppressed the inflammatory responses in activated microglia, with Scutellarin being more efficacious within the dosage range used. Moreover, when both drugs were used in combination, the infarct tissue area was reduced more extensively; also, microglia-mediated inflammatory mediators notably TNF-α expression was decreased cumulatively.

【 授权许可】

   
2014 Yuan et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Lambertsen KL, Biber K, Finsen B: Inflammatory cytokines in experimental and human stroke. J Cereb Blood Flow Metab 2012, 32(9):1677-1698.
• [2]Ling EA, Ng YK, Wu CH, Kaur C: Microglia: its development and role as a neuropathology sensor. Prog Brain Res 2001, 132:61-79.
• [3]Dheen ST, Kaur C, Ling EA: Microglial activation and its implications in the brain diseases. Curr Med Chem 2007, 14(11):1189-1197.
• [4]Neumann H, Kotter MR, Franklin RJ: Debris clearance by microglia: an essential link between degeneration and regeneration. Brain 2009, 132(Pt 2):288-295.
• [5]Jin R, Yang G, Li G: Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol 2010, 87(5):779-789.
• [6]Zhang P, Li W, Li L, Wang N, Li X, Gao M, Zheng J, Lei S, Chen X, Lu H, Liu Y: Treatment with edaravone attenuates ischemic brain injury and inhibits neurogenesis in the subventricular zone of adult rats after focal cerebral ischemia and reperfusion injury. Neuroscience 2012, 201:297-306.
• [7]Kaur C, Ling EA: Antioxidants and neuroprotection in the adult and developing central nervous system. Curr Med Chem 2008, 15(29):3068-3080.
• [8]Wu CY, Zha H, Xia QQ, Yuan Y, Liang XY, Li JH, Guo ZY, Li JJ: Expression of angiotensin II and its receptors in activated microglia in experimentally induced cerebral ischemia in the adult rats. Mol Cell Biochem 2013, 382(1–2):47-58.
• [9]Zhang HF, Hu XM, Wang LX, Xu SQ, Zeng FD: Protective effects of scutellarin against cerebral ischemia in rats: evidence for inhibition of the apoptosis-inducing factor pathway. Planta Med 2009, 75(2):121-126.
• [10]Hong H, Liu GQ: Protection against hydrogen peroxide-induced cytotoxicity in PC12 cells by scutellarin. Life Sci 2004, 74(24):2959-2973.
• [11]Liu H, Yang X, Tang R, Liu J, Xu H: Effect of scutellarin on nitric oxide production in early stages of neuron damage induced by hydrogen peroxide. Pharmacol Res 2005, 51(3):205-210.
• [12]Ma JY, Jiang WW, Zhou ZT, Li JM, Wang HY: The promoting angiogenesis and anti-inflammation effect of scutellarin on polyglycolic acid scaffold of balb/c mice model. J Asian Nat Prod Res 2008, 10(11–12):1147-1153.
• [13]Luo P, Tan ZH, Zhang ZF, Zhang H, Liu XF, Mo ZJ: Scutellarin isolated from Erigeron multiradiatus inhibits high glucose-mediated vascular inflammation. Yakugaku Zasshi 2008, 128(9):1293-1299.
• [14]Wang S, Wang H, Guo H, Kang L, Gao X, Hu L: Neuroprotection of Scutellarin is mediated by inhibition of microglial inflammatory activation. Neuroscience 2011, 185:150-160.
• [15]Chen X, Shi X, Zhang X, Lei H, Long S, Su H, Pei Z, Huang R: Scutellarin attenuates hypertension-induced expression of brain toll-like receptor 4/nuclear factor kappa B. Mediators Inflamm 2013, 2013:432623.
• [16]Czeh M, Gressens P, Kaindl AM: The yin and yang of microglia. Dev Neurosci 2011, 33(3–4):199-209.
• [17]Guillot-Sestier MV, Town T: Innate immunity in Alzheimer’s disease: a complex affair. CNS Neurol Disord Drug Targets 2013, 12(5):593-607.
• [18]Politis M, Pavese N, Tai YF, Kiferle L, Mason SL, Brooks DJ, Tabrizi SJ, Barker RA, Piccini P: Microglial activation in regions related to cognitive function predicts disease onset in Huntington’s disease: a multimodal imaging study. Hum Brain Mapp 2011, 32(2):258-270.
• [19]Qian L, Flood PM, Hong JS: Neuroinflammation is a key player in Parkinson’s disease and a prime target for therapy. J Neural Transm 2010, 117(8):971-979.
• [20]Patel AR, Ritzel R, McCullough LD, Liu F: Microglia and ischemic stroke: a double-edged sword. Int J Physiol Pathophysiol Pharmacol 2013, 5(2):73-90.
• [21]Kaur C, Ling EA: Periventricular white matter damage in the hypoxic neonatal brain: role of microglial cells. Prog Neurobiol 2009, 87(4):264-280.
• [22]Zhang N, Komine-Kobayashi M, Tanaka R, Liu M, Mizuno Y, Urabe T: Edaravone reduces early accumulation of oxidative products and sequential inflammatory responses after transient focal ischemia in mice brain. Stroke 2005, 36(10):2220-2225.
• [23]Abe K, Yuki S, Kogure K: Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988, 19(4):480-485.
• [24]Uno M, Kitazato KT, Suzue A, Matsuzaki K, Harada M, Itabe H, Nagahiro S: Inhibition of brain damage by edaravone, a free radical scavenger, can be monitored by plasma biomarkers that detect oxidative and astrocyte damage in patients with acute cerebral infarction. Free Radic Biol Med 2005, 39(8):1109-1116.
• [25]Dohi K, Satoh K, Mihara Y, Nakamura S, Miyake Y, Ohtaki H, Nakamachi T, Yoshikawa T, Shioda S, Aruga T: Alkoxyl radical-scavenging activity of edaravone in patients with traumatic brain injury. J Neurotrauma 2006, 23(11):1591-1599.
• [26]Itoh T, Satou T, Nishida S, Tsubaki M, Imano M, Hashimoto S, Ito H: Edaravone protects against apoptotic neuronal cell death and improves cerebral function after traumatic brain injury in rats. Neurochem Res 2010, 35(2):348-355.
• [27]Wang GH, Jiang ZL, Li YC, Li X, Shi H, Gao YQ, Vosler PS, Chen J: Free-radical scavenger edaravone treatment confers neuroprotection against traumatic brain injury in rats. J Neurotrauma 2011, 28(10):2123-2134.
• [28]Sivakumar V, Ling EA, Lu J, Kaur C: Role of glutamate and its receptors and insulin-like growth factors in hypoxia induced periventricular white matter injury. Glia 2010, 58(5):507-523.
• [29]Tang H, Tang Y, Li N, Shi Q, Guo J, Shang E, Duan JA: Neuroprotective effects of scutellarin and scutellarein on repeatedly cerebral ischemia-reperfusion in rats. Pharmacol Biochem Behav 2014, 118:51-59.
• [30]Lehnardt S, Lehmann S, Kaul D, Tschimmel K, Hoffmann O, Cho S, Krueger C, Nitsch R, Meisel A, Weber JR: Toll-like receptor 2 mediates CNS injury in focal cerebral ischemia. J Neuroimmunol 2007, 190(1–2):28-33.
• [31]Lalancette-Hebert M, Phaneuf D, Soucy G, Weng YC, Kriz J: Live imaging of toll-like receptor 2 response in cerebral ischaemia reveals a role of olfactory bulb microglia as modulators of inflammation. Brain 2009, 132(Pt 4):940-954.
• [32]Ziegler G, Freyer D, Harhausen D, Khojasteh U, Nietfeld W, Trendelenburg G: Blocking TLR2 in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke. J Cereb Blood Flow Metab 2011, 31(2):757-766.
• [33]Bohacek I, Cordeau P, Lalancette-Hebert M, Gorup D, Weng YC, Gajovic S, Kriz J: Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury. J Neuroinflammation 2012, 9:191. BioMed Central Full Text
• [34]Ziegler G, Harhausen D, Schepers C, Hoffmann O, Rohr C, Prinz V, Konig J, Lehrach H, Nietfeld W, Trendelenburg G: TLR2 has a detrimental role in mouse transient focal cerebral ischemia. Biochem Biophys Res Commun 2007, 359(3):574-579.
• [35]McCoy MK, Tansey MG: TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation 2008, 5:45. BioMed Central Full Text
• [36]Gregersen R, Lambertsen K, Finsen B: Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab 2000, 20(1):53-65.
• [37]Pettigrew LC, Kindy MS, Scheff S, Springer JE, Kryscio RJ, Li Y, Grass DS: Focal cerebral ischemia in the TNFalpha-transgenic rat. J Neuroinflammation 2008, 5:47. BioMed Central Full Text
• [38]Lambertsen KL, Clausen BH, Babcock AA, Gregersen R, Fenger C, Nielsen HH, Haugaard LS, Wirenfeldt M, Nielsen M, Dagnaes-Hansen F, Bluethmann H, Faergeman NJ, Meldgaard M, Deierborg T, Finsen B: Microglia protect neurons against ischemia by synthesis of tumor necrosis factor. J Neurosci 2009, 29(5):1319-1330.
• [39]Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP: A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997, 385(6618):729-733.
• [40]Vidal PM, Lemmens E, Avila A, Vangansewinkel T, Chalaris A, Rose-John S, Hendrix S: ADAM17 is a survival factor for microglial cells in vitro and in vivo after spinal cord injury in mice. Cell Death Dis 2013, 4:e954.
• [41]Pantoni L, Sarti C, Inzitari D: Cytokines and cell adhesion molecules in cerebral ischemia: experimental bases and therapeutic perspectives. Arterioscler Thromb Vasc Biol 1998, 18(4):503-513.
• [42]Hosomi N, Ban CR, Naya T, Takahashi T, Guo P, Song XY, Kohno M: Tumor necrosis factor-alpha neutralization reduced cerebral edema through inhibition of matrix metalloproteinase production after transient focal cerebral ischemia. J Cereb Blood Flow Metab 2005, 25(8):959-967.
• [43]Cao Q, Li P, Lu J, Dheen ST, Kaur C, Ling EA: Nuclear factor-kappaB/p65 responds to changes in the notch signaling pathway in murine BV-2 cells and in amoeboid microglia in postnatal rats treated with the gamma-secretase complex blocker DAPT. J Neurosci Res 2010, 88(12):2701-2714.
• [44]Cao Q, Lu J, Kaur C, Sivakumar V, Li F, Cheah PS, Dheen ST, Ling EA: Expression of notch-1 receptor and its ligands jagged-1 and delta-1 in amoeboid microglia in postnatal rat brain and murine BV-2 cells. Glia 2008, 56(11):1224-1237.
• [45]Yao L, Kan EM, Kaur C, Dheen ST, Hao A, Lu J, Ling EA: Notch-1 signaling regulates microglia activation via NF-kappaB pathway after hypoxic exposure in vivo and in vitro. PLoS One 2013, 8(11):e78439.
• [46]Wu YP, Tan CK, Ling EA: Expression of Fos-like immunoreactivity in the brain and spinal cord of rats following middle cerebral artery occlusion. Exp Brain Res 1997, 115(1):129-136.
• [47]Zgavc T, Ceulemans AG, Hachimi-Idrissi S, Kooijman R, Sarre S, Michotte Y: The neuroprotective effect of post ischemic brief mild hypothermic treatment correlates with apoptosis, but not with gliosis in endothelin-1 treated rats. BMC Neurosci 2012, 13:105. BioMed Central Full Text
• [48]Ceulemans AG, Zgavc T, Kooijman R, Hachimi-Idrissi S, Sarre S, Michotte Y: Mild hypothermia causes differential, time-dependent changes in cytokine expression and gliosis following endothelin-1-induced transient focal cerebral ischemia. J Neuroinflammation 2011, 8:60. BioMed Central Full Text