Journal of Brain & Neuroscience Research Category: Clinical Type: Research Article
FGF-2 Enhances Generation of ApoE-Containing HDL along with FGF-1 in Rat Astrocytes under Oxidative Stress
- Mariko Hoshikawa1, Shinji Yokoyama2, Hideki Hida3, Makoto Michikawa4, Jin-ichi Ito5*
- 1 Department Of Biochemistry, Department Of Neurobiology And Anatomy, Graduate School Of Medical Sciences, Nagoya City University, Nagoya, Japan
- 2 Nutritional Health Science Research Center, Chubu University, Kasugai, Japan
- 3 Department Of Neurophysiology And Brain Science, Graduate School Of Medical Sciences, Nagoya City University, Nagoya, Japan
- 4 Department Of Biochemistry, Graduate School Of Medical Sciences, Nagoya City University, Nagoya, Japan
- 5 Department Of Nutrition, Faculty Of Health And Nutrition, Shubun University, Ichinomiya, Japan
*Corresponding Author:Jin-ichi Ito
Department Of Nutrition, Faculty Of Health And Nutrition, Shubun University, Ichinomiya, Japan
Received Date: Feb 22, 2018 Accepted Date: May 01, 2018 Published Date: May 18, 2018
MATERIALS AND METHODS
Analysis of proteins in whole cell and Conditioned Medium (CM) by western blotting
Analysis of CM by sucrose-density gradient centrifugation
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Biosynthesis and release of lipid
Isolation of FGF-1, FGF-2, and apoE using Heparin-Sepharose
RESULTS AND DISCUSSION
Figure 1: Effects of FGF-2 and FGF-1 on production and release of apoE. W/W cells were treated with or without 100 µM H2O2 or tBH for 10 min and washed. The cells were incubated in 0.1% BSA/F-10 or 2% FCS/F-10 for 24 h to analyze mRNA expression of FGF-1, FGF-2, apoE, GAPDH, and ?-actin (A). W/W and W/M cells were treated with or without 100 µM H2O2 and incubated in fresh 0.02% BSA/F-10 for 24 h. The CM was incubated with Heparin-Sepharose for analysis of the release of FGF-1, FGF-2, and apoE, and without Heparin-Sepharose for HSP90 by Western blotting (B). W/W cells were incubated with or without 50 or 100 ng/ml of FGF-1 (SIGMA) or FGF-2 (WAKO) for 24 hand then in a fresh 0.02% BSA/F-10 for 16 h for analysis of apoE secretion by Western blotting (C). W/W cells were treated with FGF-1 or FGF-2 (0 or 100 ng/ml) for 24 h and washed. RT-PCR was performed for the determination of mRNA expression of apoE, HMG CoA-R, and ?-actin by using specific probes (D).
Figure 3: : Effects of FGF-1 and FGF-2 on oxidative stress-induced release of cytosolic proteins. W/W (A) or W/M cells (B) were treated with or without 100 ng/ml of FGF-1, FGF-2, insulin (Ins), (SIGMA), epidermal growth factor (EGF, SIGMA), or insulin-like growth factor 1 (IGF1, WAKO) for 24 h. W/W cells were then treated with or without 100 µM H2O2. The CM was analyzed for the release of HSP110, HSP90, HSP70, ?-actin, or apoE. Total mRNA was extracted from W/W cells treated with or without FGF-1 or FGF-2 and analyzed by RT-PCR for FGF-1, FGF-2, GAPDH, or ??actin (C). W/W cells were treated with or without 100 µM H2O2, washed, and incubated with or without 100 ng/ml of FGF-1 for 24 h. The cells were treated by sonication in 0.02M Tris buffer, pH 7.5, containing protease inhibitors cocktail (Sigma) for 20 sec, followed by centrifugation at 367,000 × g for 30 min. The supernatant obtained from 1 mg protein of cells was incubated with Heparin-Sepharose for analysis of FGF-1, FGF-2, and apoE (D).
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Citation:Hoshikawa M, Yokoyama S, Hida H, Michikawa M, Ito J (2018) FGF-2 Enhances Generation of ApoE-Containing HDL along with FGF-1 in Rat Astrocytes under Oxidative Stress. J Brain Neursci 2: 002.
Copyright: © 2018 Mariko Hoshikawa, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.