Resveratrol protects primary cortical neuron cultures from transient oxygen‑glucose deprivation by inhibiting MMP‑9

Gao,Dakuan; Huang,Tao; Jiang,Xiaofan; Hu,Shijie; Zhang,Lei; Fei,Zhou

doi:10.3892/mmr.2014.2086

Resveratrol protects primary cortical neuron cultures from transient oxygen‑glucose deprivation by inhibiting MMP‑9

Authors:
- Dakuan Gao
- Tao Huang
- Xiaofan Jiang
- Shijie Hu
- Lei Zhang
- Zhou Fei
View Affiliations

Affiliations: Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, Xi'an, Shaanxi 710032, P.R. China
Published online on: March 28, 2014 https://doi.org/10.3892/mmr.2014.2086
Pages: 2197-2204

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

It was recently shown that resveratrol exerts neuroprotective effects against cerebral ischemia in mice. The aim of the present study was to further confirm these effects in in vitro primary cortical neuron cultures with transient oxygen‑glucose deprivation (OGD), and to investigate whether these effects are due to the inhibition of matrix metalloproteinase‑9 (MMP‑9) and of cell apoptosis. Neuronal primary cultures of cerebral cortex were prepared from BALB/c mice embryos (13‑15 days). Cells from 14- to 16-day cultures were subjected to OGD for 3 h, followed by 21 h of reoxygenation to simulate transient ischemia. Different doses of resveratrol were added into the culture medium during the simulation of transient ischemia. The effect of the extracellular signal‑regulated kinase (ERK) inhibitor U0126 was studied by adding U0126 (5 µg/µl, 4 µl) into the culture medium during transient ischemia; as a control, we used treatment of cells with 50 µM of resveratrol. Cell viability was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Cell apoptosis was assessed by flow cytometry. The effects of resveratrol on the expression of MMP‑9 were analyzed by western blotting and reverse transcription‑polymerase chain reaction (RT‑PCR), while the levels of ERK, phosphorylated (p)‑ERK, cleaved caspase‑3, Bax and Bcl‑2 were measured by western blotting. The results of the MTT assay showed that cell viability is significantly reduced by transient OGD. OGD induced cell apoptosis, the expression of Bax and the activation of caspase‑3 and ERK, inhibited the expression of Bcl‑2 and increased the expression of MMP‑9, while these effects were reversed by treatment with resveratrol. The therapeutic efficacy of resveratrol was shown to be dose‑dependent, with the most suitable dose range determined at 50‑100 µM. Treatment with U0126 inhibited MMP‑9 and Bax expression and caspase‑3 activation, while it further promoted the expression of the anti‑apoptotic molecule Bcl‑2, suggesting that resveratrol inhibits MMP‑9 expression and cell apoptosis by attenuating the activation of ERK1/2. In conclusion, OGD can induce apoptosis through canonical apoptotic signals and by regulating the expression of MMP‑9; the anti‑apoptotic activity of resveratrol and its inhibitory effect on MMP‑9 expression contribute in the reduced activation of ERK.

View Figures

View References

1	Celotti E, Ferrarini R, Zironi R, et al: Resveratrol content of some wines obtained from dried Valpolicella grapes: Recioto and Amarone. J Chromatogr A. 730:47–52. 1996. View Article : Google Scholar : PubMed/NCBI
2	Pany S, Majhi A and Das J: PKC activation by resveratrol derivatives with unsaturated aliphatic chain. PLoS One. 7:e528882012. View Article : Google Scholar : PubMed/NCBI
3	Das J, Pany S and Majhi A: Chemical modifications of resveratrol for improved protein kinase C alpha activity. Bioorg Med Chem. 19:5321–5333. 2011. View Article : Google Scholar : PubMed/NCBI
4	Kesherwani V, Atif F, Yousuf S, et al: Resveratrol protects spinal cord dorsal column from hypoxic injury by activating Nrf-2. Neuroscience. 241:80–88. 2013. View Article : Google Scholar : PubMed/NCBI
5	Huang SS, Tsai MC, Chih CL, et al: Resveratrol reduction of infarct size in Long-Evans rats subjected to focal cerebral ischemia. Life Sci. 69:1057–1065. 2001. View Article : Google Scholar : PubMed/NCBI
6	Wang Q, Xu J, Rottinghaus GE, et al: Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res. 958:439–447. 2002. View Article : Google Scholar : PubMed/NCBI
7	Doyle GA, Pierce RA and Parks WC: Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBP-beta site. J Biol Chem. 272:11840–11849. 1997. View Article : Google Scholar : PubMed/NCBI
8	Lu X, Xu H, Sun B, et al: Enhanced neuroprotective effects of resveratrol delivered by nanoparticles on hydrogen peroxide-induced oxidative stress in rat cortical cell culture. Mol Pharm. 10:2045–2053. 2013. View Article : Google Scholar : PubMed/NCBI
9	de la Torre E, Hovsepian E, Penas FN, et al: Macrophages derived from septic mice modulate nitric oxide synthase and angiogenic mediators in the heart. J Cell Physiol. 228:1584–1593. 2013.PubMed/NCBI
10	Pfefferkorn T and Rosenberg GA: Closure of the blood-brain barrier by matrix metalloproteinase inhibition reduces rtPA-mediated mortality in cerebral ischemia with delayed reperfusion. Stroke. 34:2025–2030. 2003. View Article : Google Scholar
11	Dufour A and Overall CM: Missing the target: matrix metalloproteinase antitargets in inflammation and cancer. Trends Pharmacol Sci. 34:233–242. 2013. View Article : Google Scholar : PubMed/NCBI
12	Saarialho-Kere UK, Welgus HG and Parks WC: Distinct mechanisms regulate interstitial collagenase and 92-kDa gelatinase expression in human monocytic-like cells exposed to bacterial endotoxin. J Biol Chem. 268:17354–17361. 1993.
13	Nagaoka I and Hirota S: Increased expression of matrix metalloproteinase-9 in neutrophils in glycogen-induced peritoneal inflammation of guinea pigs. Inflamm Res. 49:55–62. 2000. View Article : Google Scholar : PubMed/NCBI
14	Nguyen M, Arkell J and Jackson CJ: Human endothelial gelatinases and angiogenesis. Int J Biochem Cell Biol. 33:960–970. 2001. View Article : Google Scholar : PubMed/NCBI
15	Cayabyab FS, Gowribai K and Walz W: Involvement of matrix metalloproteinases-2 and -9 in the formation of a lacuna-like cerebral cavity. J Neurosci Res. 91:920–933. 2013. View Article : Google Scholar : PubMed/NCBI
16	Vu TH, Shipley JM, Bergers G, et al: MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 93:411–422. 1998. View Article : Google Scholar : PubMed/NCBI
17	Gao D, Zhang X, Jiang X, et al: Resveratrol reduces the elevated level of MMP-9 induced by cerebral ischemia-reperfusion in mice. Life Sci. 78:2564–2570. 2006. View Article : Google Scholar : PubMed/NCBI
18	Tauskela JS, Comas T, Hewitt K, et al: Cross-tolerance to otherwise lethal N-methyl-D-aspartate and oxygen-glucose deprivation in preconditioned cortical cultures. Neuroscience. 107:571–584. 2001. View Article : Google Scholar : PubMed/NCBI
19	Gong QH, Wang Q, Shi JS, et al: Inhibition of caspases and intracellular free Ca²⁺ concentrations are involved in resveratrol protection against apoptosis in rat primary neuron cultures. Acta Pharmacol Sin. 28:1724–1730. 2007.PubMed/NCBI
20	Cagnol S and Chambard JC: ERK and cell death: mechanisms of ERK-induced cell death - apoptosis, autophagy and senescence. FEBS J. 277:2–21. 2010. View Article : Google Scholar : PubMed/NCBI
21	Schaller B and Graf R: Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy. J Cereb Blood Flow Metab. 24:351–371. 2004. View Article : Google Scholar : PubMed/NCBI
22	Moskowitz MA, Lo EH and Iadecola C: The science of stroke: mechanisms in search of treatments. Neuron. 67:181–198. 2010. View Article : Google Scholar : PubMed/NCBI
23	Hayashi T and Abe K: Ischemic neuronal cell death and organellae damage. Neurol Res. 26:827–834. 2004. View Article : Google Scholar : PubMed/NCBI
24	Fujimura M, Tominaga T and Chan PH: Neuroprotective effect of an antioxidant in ischemic brain injury: involvement of neuronal apoptosis. Neurocrit Care. 2:59–66. 2005. View Article : Google Scholar : PubMed/NCBI
25	Krause GS, White BC, Aust SD, et al: Brain cell death following ischemia and reperfusion: a proposed biochemical sequence. Crit Care Med. 16:714–726. 1988. View Article : Google Scholar : PubMed/NCBI
26	Lee MK, Kang SJ, Poncz M, et al: Resveratrol protects SH-SY5Y neuroblastoma cells from apoptosis induced by dopamine. Exp Mol Med. 39:376–384. 2007. View Article : Google Scholar : PubMed/NCBI
27	Magnoni S, Baker A, George SJ, et al: Differential alterations in the expression and activity of matrix metalloproteinases 2 and 9 after transient cerebral ischemia in mice. Neurobiol Dis. 17:188–197. 2004. View Article : Google Scholar : PubMed/NCBI
28	Deng X, Zhong Y, Gu L, et al: MiR-21 involve in ERK-mediated upregulation of MMP9 in the rat hippocampus following cerebral ischemia. Brain Res Bull. 94:56–62. 2013. View Article : Google Scholar : PubMed/NCBI
29	Chattopadhyay S and Shubayev VI: MMP-9 controls Schwann cell proliferation and phenotypic remodeling via IGF-1 and ErbB receptor-mediated activation of MEK/ERK pathway. Glia. 57:1316–1325. 2009. View Article : Google Scholar : PubMed/NCBI
30	Wang XJ and Xu JX: Salvianic acid A protects human neuroblastoma SH-SY5Y cells against MPP⁺-induced cytotoxicity. Neurosci Res. 51:129–138. 2005. View Article : Google Scholar : PubMed/NCBI
31	Sawada M, Nakashima S, Banno Y, et al: Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells. Oncogene. 19:3508–3520. 2000. View Article : Google Scholar : PubMed/NCBI
32	Yamakawa H, Ito Y, Naganawa T, et al: Activation of caspase-9 and -3 during H₂O₂-induced apoptosis of PC12 cells independent of ceramide formation. Neurol Res. 22:556–564. 2000.PubMed/NCBI
33	Murphy LO and Blenis J: MAPK signal specificity: the right place at the right time. Trends Biochem Sci. 31:268–275. 2006. View Article : Google Scholar : PubMed/NCBI
34	Tillu DV, Melemedjian OK, Asiedu MN, et al: Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain. Mol Pain. 8:52012. View Article : Google Scholar : PubMed/NCBI