title: PI3K/Akt pathway contributes to neuroprotective effect of Tongxinluo against focal cerebral ischemia and reperfusion injury in rats.
abstract: BACKGROUND
  Tongxinluo (TXL), a compound prescription, is formulated according to the collateral disease doctrine of traditional Chinese medicine, and is widely used for the treatment of cardio-cerebrovascular diseases in China.
  
  
  OBJECTIVE
  We aimed to investigate the neuroprotective effect of TXL on focal cerebral ischemia and reperfusion injury in rats by attenuating its brain damage and neuronal apoptosis, and to assess the potential role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in this protection.
  
  
  METHODS
  Adult Male Sprague-Dawley rats (n=120) were randomly divided into 5 groups: sham, cerebral ischemia and reperfusion (I/R), cerebral ischemia and reperfusion plus TXL (1.6g/kg/day) (TXL1.6), TXL1.6 plus LY294002 and dimethyl sulfoxide (DMSO) (TXL1.6+LY294002), TXL1.6 plus DMSO (TXL1.6+vehicle). Prior to the grouping, TXL1.6 was selected to be the optimal dose of TXL by evaluating the neurological deficits score of five group rats (Sham, I/R, TXL0.4, TXL0.8 and TXL1.6, n=30) at 0, 1, 3, 5, and 7 days after reperfusion. Rats, being subjected to middle cerebral artery occlusion (MCAO) for 90min followed by 24h reperfusion, were the cerebral ischemia/reperfusion models. At 24h after reperfusion, cerebral infarct area was measured via tetrazolium staining and neuronal damage was showed by Nissl staining. The double staining of Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and immunofluorescence labeling with NeuN, was performed to evaluate neuronal apoptosis. Proteins involved in PI3K/Akt pathway were detected by Western blot.
  
  
  RESULTS
  The results showed that TXL markedly improved neurological function, reduced cerebral infarct area, decreased neuronal damage, and significantly attenuated neuronal apoptosis, while these effects were eliminated by inhibition of PI3K/Akt with LY294002. We also found that TXL up-regulated the expression levels of p-PDK1, p-Akt, p-c-Raf, p-BAD and down-regulated Cleaved caspase 3 expression notably, which were partially reversed by LY294002. Additionally, the increment of p-PTEN level on which LY294002 had little effect was also detected in response to TXL treatment.
  
  
  CONCLUSIONS
  These findings demonstrated that TXL provided neuroprotection against cerebral ischemia/reperfusion injury and neuronal apoptosis, and this effect was mediated partly by activation of the PI3K/Akt pathway.
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Zhong-Hai
authors.initials: ZH
authors.lastname: Yu
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Min
authors.initials: M
authors.lastname: Cai
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Xiang
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Zhen-Nian
authors.initials: ZN
authors.lastname: Zhang
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Jing-Si
authors.initials: JS
authors.lastname: Zhang
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Xiao-Ling
authors.initials: XL
authors.lastname: Song
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Bao
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China.
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Li
authors.affiliation: Department of Integrative Medicine, Zhongshan Hospital, Laboratory of Neurology, Institute of Integrative Medicine, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Electronic address: dingfangcai@163.com.
authors.forename: Ding-Fang
authors.initials: DF
authors.lastname: Cai
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002545:Brain Ischemia / Q000188:drug therapy* / Q000378:metabolism; D002681:China; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy / Q000378:metabolism; D008297:Male; D018696:Neuroprotective Agents / Q000494:pharmacology*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000188:drug therapy* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D004365:Drugs, Chinese Herbal; D018696:Neuroprotective Agents; C513941:tongxinluo; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt
keywords: Apoptosis; Cerebral ischemia and reperfusion injury; Neuroprotection; PI3K/Akt; Tongxinluo