Anti-apoptotic and Neuroprotective Effects of Oxysophoridine on
                    Cerebral Ischemia Both In Vivo and In Vitro

Chen Rui; Li Yuxiang; Jiang Ning; Ma Ningtian; Zhu Qingluan; Hao Yinju; Zhou Ru; Ma Lin; Sun Tao; Yu Jianqiang

doi:10.1055/s-0032-1328705

Planta Medica, Table of Contents

Planta Med 2013; 79(11): 916-923
DOI: 10.1055/s-0032-1328705

Biological and Pharmacological Activity

Original Papers

Georg Thieme Verlag KG Stuttgart · New York

Anti-apoptotic and Neuroprotective Effects of Oxysophoridine on Cerebral Ischemia Both In Vivo and In Vitro

Authors

Chen Rui^*

¹Department of Pharmacology, Ningxia Medical University, Yinchuan, China

²Department of Pharmacy, Women and Childrenʼs Hospital of Jinzhou, Jinzhou, China
Li Yuxiang^*

³College of Nursing, Ningxia Medical University, Yinchuan, China

⁴Shanghai Pudong New Area Gongli Hospital, Shanghai, China
Jiang Ning

⁴Shanghai Pudong New Area Gongli Hospital, Shanghai, China
Ma Ningtian

¹Department of Pharmacology, Ningxia Medical University, Yinchuan, China
Zhu Qingluan

¹Department of Pharmacology, Ningxia Medical University, Yinchuan, China
Hao Yinju

¹Department of Pharmacology, Ningxia Medical University, Yinchuan, China

⁵Ningxia Engineering and Technology Research Center of Hui Medicine Modernization, Yinchuan, China
Zhou Ru

¹Department of Pharmacology, Ningxia Medical University, Yinchuan, China

⁶Ningxia Collaborative Innovation Center of Hui Medicine, Yinchuan, China
Ma Lin

⁷Ningxia Key Lab of Craniocerbral Diseases of Ningxia Hui Autonomous Region, Yinchuan, China
Sun Tao

⁷Ningxia Key Lab of Craniocerbral Diseases of Ningxia Hui Autonomous Region, Yinchuan, China
Yu Jianqiang

¹Department of Pharmacology, Ningxia Medical University, Yinchuan, China

⁸Key Laboratory of Hui Medicine Modernization, Ministry of Education, Yinchuan, China

Abstract

In this study, we investigated the neuroprotective effect of oxysophoridine on ischemia and ischemia-like insults. Protection by oxysophoridine was studied at the in vivo level using a model of middle cerebral artery occlusion in mice and at the in vitro level using primary rat hippocampal neuronal cultures exposed to oxygen-glucose deprivation, a model of ischemia-like injury. The behavioral test was performed by using the neurological scores. The infarction volume of brain was assessed in the brain slices stained with 2,3,5-triphenyl tetrazolium chloride. The neuron apoptosis was evaluated by Hoechst 33342 staining. The morphological change in the neurons was examined using a Transmission Electron Microscope (TEM or EM). To evaluate neuron apoptosis, caspase-3, -9, and − 8 activities were measured using assay kits with an ELISA reader. The Western blotting assay was used to evaluate the release of cytochrome c and expression of caspase-3, Bcl-2, and Bax proteins. The quantitative real-time PCR assay was used to evaluate the release of cytochrome c and the expression of caspase-3 mRNA. Oxysophoridine-treated groups (62.5, 125, 250 mg/kg) markedly reduced neurological deficit scores and infarct volumes. Treatment with oxysophoridine (5, 20, 80 µmol/L) significantly attenuated neuronal damage, with evidence of decreased cell apoptosis and decreased cell morphologic impairment. Furthermore, treatment with oxysophoridine could effectively downregulate the expression of cytochrome c and caspase-3 in both mRNA and protein levels, and Bax in the protein level, and induce an increase of Bcl-2 in the protein level. The caspase-3, -9, and -8 activities were also inhibited. These findings suggested that oxysophoridine may be a potential neuroprotective agent for cerebral ischemia injury.

Key words

oxysophoridine - cerebral ischemia - middle cerebral artery occlusion - oxygen-glucose deprivation - apoptosis - neuroprotection - Siphocampylus verticillatus - Campanulaceae

Full Text

References

References
1 Kato H, Kogure K. Biochemical and molecular characteristics of the brain with developing cerebral infarction. Cell Mol Neurobiol 1999; 19: 93-108
2 Bredesen DE. Keeping neurons alive: the molecular control of apoptosis. Neuroscientist 1996; 2: 211-216
3 Hale AJ, Smith CA, Sutherland LC, Stoneman VEA, Longthorne VL, Culhane AC, Williams GT. Apoptosis: molecular regulation of cell death. Eur J Biochem 1996; 236: 1-26
4 White BC, Sullivan JM, DeGracia DJ, OʼNeil BJ, Neumar RW, Grossman LI, Rafols JA, Krause GS. Brain ischemia and reperfusion: molecular mechanisms of neuronal injury. J Neurol Sci 2000; 179: 1-33
5 Zhu YG, Chen XC, Chen ZZ, Zeng YQ, Shi GB, Su YH, Peng X. Curcumin protects mitochondria from oxidative damage and attenuates apoptosis in cortical neurons. Acta Pharmacol Sin 2004; 25: 1606-1612
6 Lopez-Neblina F, Toledo AH, Toledo-Pereyra LH. Molecular biology of apoptosis in ischemia and reperfusion. J Invest Surg 2005; 18: 335-350
7 Iijima T. Mitochondrial membrane potential and ischemic neuronal death. Neurosci Res 2006; 55: 234-243
8 Zhong R. Research and application of sophora. Ningxia: Peopleʼs Publishing House Press; 1985: 10-20
9 Rodrigues AL, da Silva GL, Mateussi AS, Fernandes ES, Miguel OG, Yunes RA, Calixto JB, Santos AR. Involvement of monoaminergic system in the antidepressant-like effect of the hydroalcoholic extract of Siphocampylus verticillatus . Life Sci 2002; 70: 1347-1358
10 Trentin AP, Santos AR, Miguel OG, Pizzolatti MG, Yunes RA, Calixto JB. Mechanisms involved in the antinociceptive effect in mice of the hydroalcoholic extract of Siphocampylus verticillatus . J Pharm Pharmacol 1997; 49: 567-572
11 Yang G, Gao J, Jia Y, Yan L, Yu J, Jiang Y. Oxysophoridine through intrathecal injection induces antinociception and increases the expression of the GABAAα1 receptor in the spinal cord of mice. Planta Med 2012; 78: 874-880
12 Yu JQ, Jiang YX. Pharmacological research of sophoridine and oxysophoridine. J Ningxia Med Coll 2005; 27: 78-80
13 Zhao J, Li YX, Hao YJ, Chen R, Zhang JZ, Sun T, Yu JQ. Effects of oxysophoridine on rat hippocampal neurons injured by oxygen-glucose deprivation and reperfusion. CNS Neurosci Ther 2013; 19: 138-141
14 Zhang K, Zhao J, Liu H, Wang H, Zhao C, Jiang Y, Yu J. Protective effects of oxysophoridine on acute cerebral ischemia injury in mice. Pharmacol Clin Chin Mater Med 2011; 27: 26-28
15 Macrae IM. New models of focal cerebral ischemia. Br J Clin Pharmacol 1992; 34: 302-308
16 Fein G, Di Sclafani V, Tanabe J, Cardenas V, Weiner MW, Jagust WJ, Reed BR, Norman D, Schuff N, Kusdra L, Greenfield T, Chui H. Hippocampal and cortical atrophy predict dementia in subcortical ischemic vascular disease. Neurology 2000; 55: 1626-1635
17 Zhu XH, Li SJ, Hu HH, Sun LR, Das M, Gao TM. Neuroprotective effects of Xiao-Xu-Ming decoction against ischemic neuronal injury in vivo and in vitro . J Ethnopharmacol 2010; 127: 38-46
18 Qiu J. ‘Back to the futureʼ for Chinese herbal medicines. Nat Rev Drug Discov 2007; 6: 506-507
19 Wang L, Zhou GB, Liu P, Song JH, Liang Y, Yan XJ, Xu F, Wang BS, Mao JH, Shen ZX, Chen SJ, Chen Z. Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia. Proc Natl Acad Sci USA 2008; 105: 4826-4831
20 Kazda S, Towart R. Nimodipine: a new calcium antagonist drug with a preferential cerebrovascular action. Acta Neurochir (Wien) 1982; 63: 259-265
21 Scriabine A, van den Kerckhoff W. Pharmacology of nimodipine. A review. Ann NY Acad Sci 1982; 522: 698-706
22 Towart R, Perzborn E. Nimodipine inhibits carbocyclic thromboxane-induced contractions of cerebral arteries. Eur J Pharmacol 1981; 69: 213-215
23 Towart R, Wehinger E, Meyer H, Kazda S. The effects of nimodipine, its optical isomers and metabolites on isolated vascular smooth muscle. Arzneimittelforschung 1982; 32: 338-346
24 Kazda S. Pharmacology of nimodipine, a calcium antagonist with preferential cerebrovascular activity. Neurochirurgia 1985; 28: 70-73
25 Wadworth AN, McTavish D. Nimodipine. A review of its pharmacological properties, and therapeutic efficacy in cerebral disorders. Drugs Aging 1992; 2: 262-286
26 Liu XQ, Sheng R, Qin ZH. The neuroprotective mechanism of brain ischemic preconditioning. Acta Pharmacol Sin 2009; 30: 1071-1080
27 Chan PH. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem Res 2004; 11: 1943-1949
28 Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol 2007; 35: 495-516
29 Love S. Apoptosis and brain ischaemia. Prog Neuropsychopharmacol Biol Psychiatry 2003; 27: 267-282
30 Sugawara T, Fujimura M, Noshita N, Kim GW, Saito A, Hayashi T, Narasimhan P, Maier CM, Chan PH. Neuronal death/survival signaling pathways in cerebral ischemia. NeuroRx 2004; 1: 17-25
31 Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC, Nicholson DW, Alnemri ES, Green DR, Martin SJ. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 1999; 144: 281-292
32 Merry DE, Korsmeyer SJ. Bcl-2 gene family in the nervous system. Annu Rev Neurosci 1997; 20: 245-267
33 Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for bcl-2 regulation of apoptosis. Science 1997; 275: 1132-1136
34 Ryan KM, Phillips AC, Vousden KH. Regulation and function of the p 53 tumor suppressor protein. Curr Opin Cell Biol 2001; 13: 332-337
35 Zea Longa EL, Weinstein PR, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in the rats. Stroke 1989; 20: 84-91
36 Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986; 17: 472-476
37 Chen R, Li Y, Hao Y, Zhu Q, Wu Y, Zhao Q, Wang H, Ma L, Liu J, Zhao C, Jiang Y, Wang Y, Dai X, Zhang W, Sun T, Yu J. Protective effects of Lycium barbarum polysaccharide on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion. J Mol Histol 2012; 43: 535-542
38 Zhao J, Wu Y, Sun M, Wang J, Li Y, Zhang K, Yu J. Protective effects and mechanisms of OSR on primary cultured hippocampus neurons subjected to anoxic injury in neonatal rat. China J Chin Mater Med 2012; 37: 94-98
39 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(^−ΔΔCT) method. Methods 2011; 25: 402-408