The Neuroprotection of Puerarin Against Cerebral Ischemia is Associated with the Prevention of Apoptosis in Rats

Abstract

Previous work has shown that puerarin (Pur), extracted from the dried root of Pueraria lobata (Wild) Ohwi, increases cerebral blood flow in dogs and attenuates cerebral and spinal cord injury resulting from ischemia and reperfusion in rats and rabbits. The present study further demonstrates the neuroprotective effects of Pur on cerebral ischemic injury in rats and the mechanisms underlying the protective effects. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAo) for 50 min followed by reperfusion for 24 h. Pur (50, 100 mg/kg, i.p) was administered at the onset of MCAo. Twenty-four hours after reperfusion, neurological deficits were evaluated in Pur- and vehicle-treated rats. The infarct volume and edema ratios were assessed from stained brain slices. The results showed that Pur (100 mg/kg) markedly decreased the infarct volume by 34 % (P < 0.01) in cerebral cortex and improved the neurological functions (P < 0.05) after MCAo. Furthermore, flow cytometric analysis of annexin-V and PI labeling cells showed that the percentages of apoptosis and necrosis in the dorsolateral cortex were significantly reduced by 38.6 % and 28.5 % (P < 0.01 and P < 0.05) following treatment with Pur (100 mg/kg) in MCAo rats. Caspase-3 activity, a biochemical marker of apoptosis, was significantly inhibited after treatment with Pur in the dorsolateral cortex. In agreement with this result, the expression of the X-chromosome-linked inhibitor of apoptosis protein (XIAP) was obviously up-regulated after administration of Pur (100 mg/kg), while caspase-3 gene was down-regulated in the dorsolateral cortex. These results suggest that the neuroprotection of puerarin against cerebral ischemia is associated with anti-apoptosis.

Key words

Puerarin - neuroprotection - cerebral ischemia - apoptosis - XIAP - caspase-3

References

• 1 Fan L L, Sun L H, Li J, Yue X H, Yu H X, Wang S Y. The protective effect of puerarin against myocardial reperfusion injury: study on cardiac function.  J Chin Med. 1992;  105 11-7
• 2 Xuan B, Zhou Y H, Yang R L, Li N, Min Z D, Chiou G C. Improvement of ocular blood flow and retinal functions with puerarin analogs.  J Ocular Pharmacol and Therapeutics,. 1999;  15 207-16
• 3 Chen L, Chai Q, Zhao A, Chai X. Effect of puerarin on cerebral blood flow in dogs.  Chin Materia Med J. 1995;  20 560-2
• 4 Wang L, Zhao A, Wang F, Chai Q, Chai X. Protective effect of puerarin on acute cerebral ischemia in rats.  Chin Materia Med J. 1997;  22 752-4
• 5 Sang H F, Zhang Y M, Xu L X, Wang Q, Ji G L, Wu M C. Protective effect of puerarin on spinal cord injury resulting from ischemia and reperfusion in rabbits.  Fourth Mil Med Univ J. 2001;  22 414-17
• 6 Dong L P, Wang T Y. Effects of puerarin against glutamate excitotoxicity on cultured mouse cerebral cortical neurons.  Acta Pharmacol Sin. 1998;  19 339-42
• 7 Dong L P, Han M, Yuan F, Xu L X, Wang T Y. The protective effect of puerarin on cultured rat cerebral cortical astrocytes.  Chin Appl Physicol J. 2001;  17 141-3
• 8 Onteniente B, Couriaud C, Braudeau J, Benchoua A, Guegan C. The mechanisms of cell death in focal cerebral ischemia highlight neuroprotective perspectives by anti-caspase therapy.  Biochemical Pharmacol. 2003;  66 1643-9
• 9 Trapp T, Korhonen L, Besselmann M, Martinez R, Mercer E A, Lindholm D. Transgenic mice overexpressing XIAP in neurons show better outcome after transient cerebral ischemia.  Mol Cell Neurosci. 2003;  23 302-13
• 10 Le D A, Wu Y, Huang Z, Matsushita K, Plesnila N, Augustinack J C, Hyman B T. et al . Caspase activation and neuroprotection in caspase-3-deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation.  Proc Natl Acad Sci USA. 2002;  99 15 188-93
• 11 LeBlanc A. Natural cellular inhibitors of caspases.  Progress in Neuro-Psychopharmacol & Biological Psychiatry. 2003;  27 215-29
• 12 Korhonen L, Belluardo N, Lindholm D. Regulation of X chromosome-linked inhibitor of apoptosis protein in kainic acid induced neuronal death in the rat hippocampus.  Mol Cell Neurosci. 2001;  17 364-72
• 13 Xu D, Bureau Y, McIntyre D C, Nicholson D W, Liston P, Zhu Y, Fong W G. et al . Attenuation of ischemia-induced cellular and behavioral deficits by X-chromosome-linked inhibitor of apoptosis protein overexpression in the rat hippocampus.  J Neurosci. 1999;  19 5026-33
• 14 Longa E Z, Weinstein P R, Carlson S, Cummins R. Reversible middle cerebral artery occlusion without craniectomy in rats.  Stroke. 1989;  20 84-91
• 15 Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th Edition Academic Press San Diego; 1998
• 16 Hatfield R H, Gill R, Brazell C. The dose-response relationship and therapeutic window for dizocilpine (MK-801) in a rat focal ischemia model.  Eur J Pharmacol. 1992;  216 1-7
• 17 Prakasa B P, Yoshida Y, Su M, Segura M, Kawamura S, Yasui N. Immunohistochemical expression of Bcl-2, Bax and cytochrome c following focal cerebral ischemia and effect of hypothermia in rat.  Neurosci Lett. 2000;  291 196-200
• 18 Boldyrev A, Song R, Lawrence D, Carpenter O. Carnosine protects against excitotoxic cell death independently of effects on reactive oxygen species.  Neurosci. 1999;  94 571-7
• 19 Lee S H, Kim M, Kim Y J, Kim Y A, Chi J G, Roh J K, Yoon B W. Ischemic intensity influences the distribution of delayed infarction and apoptotic cell death following transient focal cerebral ischemia in rats.  Brain Res. 2002;  956 14-23
• 20 Mcphail L T, Vanderluit J L, Mcbride C B, Oschipok L W, Crocker S J, Xu D, Thompson C S. et al . Endogenous expression of inhibitor of apoptosis proteins in facial motoneurons of neonatal and adult rats following axotomy.  Neurosci. 2003;  117 567-75
• 21 Benchoua A, Guegan C, Couriaud C, Hosseini H, Sampaio N, Morin D, Onteniente B. Specific caspase pathways are activated in two stages of cerebral infarction.  J Neurosci. 2001;  21 7127-7134
• 22 Yang J, Klaidman L K, Chang M L, Kem S, Sugawara T, Chan P, Adams J D. Nicotinamide therapy protects against both necrosis and apoptosis in a stroke model.  Pharmacol Biochem Behav. 2002;  73 901-910
• 23 Gillardon F, Kiprianova I, Sandkuhler J, Hossmann K A, Spranger M. Inhibition of caspases prevents cell death of hippocampal CA1 neurons, but not impairment of hippocampal long-term potentiation following global ischemia.  Neurosci Lett. 1999;  93 1219-22
• 24 Love S. Apoptosis and brain ischemia.  Progress in Neuro-Psychopharmacol Biological Psychiatry. 2003;  27 267-82
• 25 Xu X H, Zhao T Q. Effects of puerarin on memory behavior and lipid peroxidation in aging mice.  Acta Pharmacol Sin. 2002;  23 587-590