title: Pretreatment with PTD-calbindin D 28k alleviates rat brain injury induced by ischemia and reperfusion.
abstract: Calcium toxicity remains the central focus of ischemic brain injury. Calcium channel antagonists have been reported to be neuroprotective in ischemic animal models but have failed in clinical trials. Rather than block the calcium channels, calbindin proteins can buffer excessive intracellular Ca2+, and as a result, maintain the calcium homeostasis. In the present study, we investigated the effect of calbindin D 28k (CaBD) in ischemic brain using the novel technique protein transduction domain (PTD)-mediated protein transduction. We generated PTD-CaBD in Escherichia coli, tested its biologic activity in N-methyl-D-aspartate (NMDA)- and oxygen-glucose deprivation (OGD)-induced hippocampal injury models, and examined the protection of the fusion protein using a rat brain focal ischemia model. Infarct volume was determined using 2,3,5-triphenyl-tetrazolium chloride staining; neuronal injury was examined using terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining and cleaved caspase-3 assay. The results showed that the PTD-CaBD was efficiently delivered into Cos7 cells, hippocampal slice cells, and brain tissue. Pretreatment with PTD-CaBD decreased intracellular free calcium concentration and reduced cell death in NMDA- or OGD-exposed hippocampal slices (P<0.05). Intraperitoneal administration of PTD-CaBD before transient middle cerebral artery occlusion decreased brain infarct volume (280+/-47 versus 166+/-70 mm3, P<0.05), and improved neurologic outcomes compared with the control. Further studies showed that, compared with the control animals, PTD-CaBD decreased TUNEL (58%+/-7% versus 29%+/-3%, P<0.05)- and cleaved caspase-3 (62+/-4/field versus 31+/-6/field, P<0.05)-positive cells in the ischemic boundary zone. These results indicate that systemic administration of PTD-CaBD could attenuate ischemic brain injury, suggesting that PTD-mediated protein transduction might provide a promising and effective approach for the therapies of brain diseases, including cerebral ischemia.
authors.affiliation: Department of Neurology, Institute of Neurology, Hua-Shan Hospital, Fudan University, Shanghai, PR China. yongfengfan@hotmail.com
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Fan
authors.forename: Langfeng
authors.initials: L
authors.lastname: Shi
authors.forename: Yuehua
authors.initials: Y
authors.lastname: Gu
authors.forename: Yanxin
authors.initials: Y
authors.lastname: Zhao
authors.forename: Jun
authors.initials: J
authors.lastname: Xie
authors.forename: Jian
authors.initials: J
authors.lastname: Qiao
authors.forename: Guo-Yuan
authors.initials: GY
authors.lastname: Yang
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Chuan-Zhen
authors.initials: CZ
authors.lastname: Lu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D001921:Brain / Q000473:pathology*; D002545:Brain Ischemia / Q000188:drug therapy* / Q000473:pathology*; D019556:COS Cells; D064026:Calbindins; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D005947:Glucose / Q000172:deficiency; D006624:Hippocampus / Q000378:metabolism / Q000473:pathology; D002534:Hypoxia, Brain / Q000473:pathology; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D020244:Infarction, Middle Cerebral Artery / Q000473:pathology / Q000517:prevention & control; D016202:N-Methylaspartate / Q000494:pharmacology; D018696:Neuroprotective Agents; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D015427:Reperfusion Injury / Q000188:drug therapy* / Q000473:pathology*; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D064026:Calbindins; D018690:Excitatory Amino Acid Agonists; D018696:Neuroprotective Agents; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D016202:N-Methylaspartate; D005947:Glucose; D002118:Calcium