title: rLj-RGD3, a Novel Recombinant Toxin Protein from Lampetra japonica, Protects against Cerebral Reperfusion Injury Following Middle Cerebral Artery Occlusion Involving the Integrin-PI3K/Akt Pathway in Rats.
abstract: BACKGROUND
  The RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the Lampetra japonica fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model. We also explored the mechanism by which rLj-RGD3 acts by measuring protein and mRNA expression levels, with an emphasis on the FAK and integrin-PI3K/Akt anti-apoptosis pathways.
  
  
  METHODS
  rLj-RGD3 was obtained from the buccal secretions of Lampetra japonica using gene recombination technology. Sprague Dawley (SD) rats were randomly divided into the following seven groups: a sham group; a vehicle-treated (VT) group; 100.0 μg·kg-1, 50.0 μg·kg-1 and 25.0 μg·kg-1 dose rLj-RGD3 groups; and two positive controls, including 1.5 mg·kg-1 Edaravone (ED) and 100.0 μg·kg-1 Eptifibatide (EP). MCAO was induced using a model consisting of 2 h of ischemia and 24 h of reperfusion. Behavioral changes were observed in the normal and operation groups after focal cerebral ischemia/reperfusion was applied. In addition, behavioral scores were evaluated at 4 and 24 h after reperfusion. Brain infarct volumes were determined based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining. Moreover, neuronal apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. We determined the expression levels of focal adhesion kinase (FAK), phosphatidyl inositol 3-kinase (PI3K), protein kinase B (Akt, PKB), caspase-3 and Bcl-2 in the brain using western blot analysis and RT-PCR assays. The research protocol was approved by the Institutional Ethics Committee of Dalian Medical University.
  
  
  RESULTS
  The behavioral scores and cerebral infarct volumes of the rLj-RGD3 groups were markedly lower at 4 and 24 h/RF. The rLj-RGD3 protein significantly ameliorated pathological changes in the brain and reduced the number of apoptotic neurons. Furthermore, the FAK and PI3K/Akt pathways were activated. rLj-RGD3 significantly increased the expression of FAK, p-FAK and Bcl-2 proteins. In contrast, caspase-3 expression was inhibited.
  
  
  CONCLUSIONS
  We conclude that recombinant Lampetra japonica RGD-peptide (rLj-RGD3) exerts a protective effect against cerebral ischemia/reperfusion injury in the brain. In addition, the mechanism of this protection is associated with the activation of the integrin-PI3K/Akt pathway. These results provide a theoretical foundation and an experimental basis for using RGD peptides as novel drugs for treating ischemic cerebral vascular diseases in addition to promoting the research and development of marine biotechnology drugs.
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.|College of Basic Medicine, Jilin Medical College, Jilin, Jilin Province, China, 132013.
authors: Qian
authors: Q
authors: Lu
authors: School of Life Sciences, Liaoning Normal University, Dalian, Liaoning Province, China, 116029.|Key Laboratory of Biotechnology and Drug Discovery of Liaoning Province, Dalian, Liaoning Province, China, 116029.
authors: Jihong
authors: J
authors: Wang
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.
authors: Junshu
authors: J
authors: Jiang
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.
authors: Shengnan
authors: S
authors: Wang
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.
authors: Qilan
authors: Q
authors: Jia
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.
authors: Yue
authors: Y
authors: Wang
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.
authors: Weiping
authors: W
authors: Li
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.
authors: Qin
authors: Q
authors: Zhou
authors: Department of Pharmacology, Dalian Medical University, Dalian, Liaoning Province, China, 116044.|School of Life Sciences, Liaoning Normal University, Dalian, Liaoning Province, China, 116029.
authors: Li
authors: L
authors: Lv
authors: School of Life Sciences, Liaoning Normal University, Dalian, Liaoning Province, China, 116029.|Key Laboratory of Biotechnology and Drug Discovery of Liaoning Province, Dalian, Liaoning Province, China, 116029.
authors: Qingwei
authors: Q
authors: Li
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000517:prevention & control*; D005397:Fish Venoms / Q000494:pharmacology*; D016023:Integrins / Q000378:metabolism*; D007798:Lampreys / Q000378:metabolism*; D008297:Male; D020768:Middle Cerebral Artery / Q000473:pathology*; D009842:Oligopeptides / Q000494:pharmacology*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000517:prevention & control*
chemical_list: D005397:Fish Venoms; D016023:Integrins; D009842:Oligopeptides; C574387:RGD3 protein, Lampetra japonica; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt