title: A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-κB and the PKCα/PI3-K/Akt pathways.
abstract: BACKGROUND
  The natural product tetramethylpyrazine (TMP) has a variety of biologic activities, including neuroprotection. Nitrones are powerful free radical scavengers. We have designed and synthesized a TMP derivative, TN-2, which is armed with two nitrone moieties.
  
  
  OBJECTIVE
  In this study, we investigated the neuroprotective effect of TN-2 against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro and in zebrafish.
  
  
  METHODS
  PC12 cells, zebrafish and rats were exposed to 6-OHDA challenge. MTT assay, LDH release, Hoechst staining, DAF-FM staining, luciferase reporter construct transfection, and western blotting were applied to detect cell viability, apoptosis, intracellular nitric oxide (NO), NF-κB transcriptional activity and proteins expression. In zebrafish, whole-mount staining and real-time PCR were performed to quantify dopaminergic neurons and mRNA expression. Hematoxylin and eosin staining and immunohistochemistry for glial fibrillary acidic protein were used to detect the astrocyte activation in the unilateral 6-OHDA rat model.
  
  
  RESULTS
  TN-2 but not TMP exhibited potent neuroprotective effect against 6-OHDA-induced apoptosis in PC12 cells. Moreover, TN-2 prevented dopaminergic neuron loss and suppressed mRNA expression of pro-inflammatory genes, including IL-1β, TNF-α and COX-2, in 6-OHDA-treated zebrafish. TN-2 remarkably attenuated microglial/astrocyte activation in the unilateral 6-OHDA rat model. The mechanistic study demonstrated that TN-2 inhibited over-production of intracellular NO and protein expression of inducible nitric oxide synthase through down-regulating NF-κB activity. Additionally, the PKCα/PI3-K/Akt pathway was also involved in the neuroprotection of TN-2.
  
  
  CONCLUSIONS
  These results suggest that TN-2 protected against 6-OHDA-induced neurotoxicity via modulating the NF-κB-medicated neuroinflammation and PKCα/PI3-K/Akt pathways.
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenue Padre Tomás Pereira S.J., Macao, China; Department of Applied Biology and Chemical Technology, Institute of Modern Chinese Medicine, Hong Kong Polytechnic University, Hong Kong, China.
authors.forename: Da-Ping
authors.initials: DP
authors.lastname: Xu
authors.affiliation: Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenue Padre Tomás Pereira S.J., Macao, China; Department of Applied Biology and Chemical Technology, Institute of Modern Chinese Medicine, Hong Kong Polytechnic University, Hong Kong, China; Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China. Electronic address: zaijunzhang@163.com.
authors.forename: Zai-Jun
authors.initials: ZJ
authors.lastname: Zhang
authors.affiliation: Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China.
authors.forename: Ye-Wei
authors.initials: YW
authors.lastname: Sun
authors.affiliation: Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China.
authors.forename: Bao-Jian
authors.initials: BJ
authors.lastname: Guo
authors.affiliation: Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China.
authors.forename: Yu-Qiang
authors.initials: YQ
authors.lastname: Wang
authors.affiliation: Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China.
authors.forename: Pui-Man
authors.initials: PM
authors.lastname: Hoi
authors.affiliation: Department of Applied Biology and Chemical Technology, Institute of Modern Chinese Medicine, Hong Kong Polytechnic University, Hong Kong, China.
authors.forename: Yi-Fan
authors.initials: YF
authors.lastname: Han
authors.affiliation: State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Avenue Padre Tomás Pereira S.J., Macao, China. Electronic address: simonlee@umac.mo.
authors.forename: Simon Ming-Yuen
authors.initials: SM
authors.lastname: Lee
mesh_terms: D000818:Animals; D008297:Male; D016328:NF-kappa B / Q000502:physiology*; D018696:Neuroprotective Agents / Q000494:pharmacology; D016627:Oxidopamine / Q000633:toxicity*; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D051571:Protein Kinase C-alpha / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology*; D011719:Pyrazines / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015027:Zebrafish
chemical_list: D016328:NF-kappa B; D018696:Neuroprotective Agents; D011719:Pyrazines; D016627:Oxidopamine; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051571:Protein Kinase C-alpha; C017953:tetramethylpyrazine
keywords: 6-Hydroxydopamine; Neuroinflammation; Neuroprotection; Tetramethylpyrazine derivative