title: Protective effects of Tongxinluo on cerebral ischemia/reperfusion injury related to Connexin 43/Calpain II/Bax/Caspase-3 pathway in rat.
abstract: BACKGROUND
  Tongxinluo (TXL) is a multifunctional traditional Chinese medicine and has been widely used in the treatment of cardiovascular and cerebrovascular diseases. Numerous studies demonstrate that TXL is a novel neuroprotective drug, however, the mechanisms are largely unknown.
  
  
  OBJECTIVE
  we aimed to demonstrate the protective effect of TXL on cerebral ischemia/reperfusion (I/R) injury and provide the evidence for the involvement of Connexin 43/Calpain II/ Bax/Caspase-3 pathway in TXL-mediated neuroprotection.
  
  
  METHODS
  Focal cerebral I/R injury were induced by transient middle cerebral artery occlusion (MCAO, for 90min) in adult male Sprague-Dawley rats. We estimated the effects of TXL on I/R injury including neurological deficit assessment and cerebral infarct volume measurement via TTC staining, and detected the protein expression of Connexin 43 (Cx43) by western blot. Furthermore, after the intracerebroventricular injection of carbenoxolone (CBX, the inhibitor of Cx43) at 30min before MCAO surgery, Calpain II, Bax and cleaved Caspased-3 immunoreactivity in ischemic penumbra region was detected by immunofluorescent staining, and cell apoptosis was detected by TUNEL staining.
  
  
  RESULTS
  TXL treatment greatly improved neurological deficit and reduced the infarction volume compared to MCAO with buffer treatment (P<0.05), and TXL pre-post treatment showed better results than TXL pre-treatment. TXL pre-post treatment significantly up-regulated Cx43 protein expression at 3d, 7d and 14d post-injury compared to MCAO with buffer treatment (P<0.05). Meanwhile, the immunoreactivity of Calpain II, Bax and cleaved Caspase-3 in ischemic penumbra region was obviously decreased by TXL pre-post treatment compared to MCAO group (P<0.05). However, with the treatment of the Cx43 inhibitor, CBX, the down-regulated effect of TXL on Calpain II, Bax and cleaved Caspase-3 immunoreactivity was abolished (P<0.05). Moreover, the protective effect of TXL against neuron apoptosis in penumbra region was conteracted by CBX (P<0.05).
  
  
  CONCLUSIONS
  TXL could effectively protect against I/R injury and reduced cell death via Cx43/Calpain II/Bax/Caspase-3 pathway, which contribute to I/R injury prevention and therapy.
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. Electronic address: chengxiaolucky@126.com.
authors.forename: Xiao
authors.initials: X
authors.lastname: Cheng
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China; Medical Experimental Center, Nanyang Institute of Technology, Nanyang 473004, P.R. China. Electronic address: jiyun0728@163.com.
authors.forename: Zijun
authors.initials: Z
authors.lastname: Hou
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. Electronic address: 1534873641@qq.com.
authors.forename: Jingbo
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. Electronic address: 1440124821@qq.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. Electronic address: 747635117@qq.com.
authors.forename: Lixin
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. Electronic address: 278137393@qq.com.
authors.forename: Ziyi
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: Department of Anatomy, Zhong Shan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China. Electronic address: 554776504@qq.com.
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Zhou
authors.affiliation: Department of Neurology, Guangdong Provincial Hospital of Traditional Chinese Medicine, Guangzhou 510120, China; The Second Institute of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. Electronic address: hzdxliyan0719@163.com.
authors.forename: Yefeng
authors.initials: Y
authors.lastname: Cai
mesh_terms: D000818:Animals; D002545:Brain Ischemia / Q000188:drug therapy*; D002154:Calpain / Q000032:analysis / Q000502:physiology*; D053148:Caspase 3 / Q000032:analysis / Q000502:physiology*; D018031:Connexin 43 / Q000032:analysis / Q000502:physiology*; D004365:Drugs, Chinese Herbal / Q000627:therapeutic use*; D008297:Male; D018696:Neuroprotective Agents / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000517:prevention & control*; D015398:Signal Transduction / Q000502:physiology*; D051028:bcl-2-Associated X Protein / Q000032:analysis / Q000502:physiology*
chemical_list: C494828:Bax protein, rat; D018031:Connexin 43; D004365:Drugs, Chinese Herbal; D018696:Neuroprotective Agents; D051028:bcl-2-Associated X Protein; C513941:tongxinluo; D002154:Calpain; D053148:Caspase 3
keywords: Cell apoptosis; Cerebral ischemia/reperfusion injury; Connexin43; Ischemic penumbra region; Tongxinluo capsule