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Enance medium (80 DMEM/20 M199) containing five horse serum and 1 fetal bovine serum. H9c2 rat myocyte-like cells were obtained from ATCC (Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle Medium supplemented with ten fetal calf serum, 100 U/ml of penicillin and 100 g/ml of streptomycin, at 37 in a humidified atmosphere with 5 CO2. Cells cultured to 80?0 confluence were used for experiments. The following agents had been utilised for cell treatments: TBHQ, 4-hydroxy-2-nonenal (4-HNE) (from Cayman Chemicals, Ann Arbor, MI, USA), wortmannin (from Merck Millipore) and Akt1/2 Kinase Inhibitor (Akti) (from Sigma, St. Louis, MO, USA). Histology and immunohistochemistry analyses. Hearts have been perfuse-fixed in situ at one hundred mmHg for 5 min and additional fixed in four paraformaldehyde overnight, dehydrated, and embedded in paraffin. Short-axis sections of 5 m thickness have been all cut in the horizontal plane in the degree of the papillary muscle as described34. Sections were stained with hematoxylin and eosin (H E), picrosirius red, or Masson's trichrome. For measurements on myocyte cross-sectional area, 100 cells in 5 random 400?fields from every heart have been analyzed. Immunohistochemical staining for 4-HNE was performed utilizing a rabbit polyclonal antibody (from Abcam, Cambridge, UK) and VECTASTAIN Elite ABC Kit (from Vector Laboratories, Burlingame, CA, USA). Damaging handle slides have been incorporated in all staining experiments by replacing the primary antibody with non-immune IgG to exclude the presence of non-specific background. To quantify the histological and immuno-stained images, we randomly chosen ten fields for every single sample, and used Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA) for morphometric evaluation. The application was operated by an independent investigator in a blind manner. Hydroxyproline assay. Hydroxyproline assay was performed to quantify the collagen content material in LV as used previously62, applying a industrial kit from Jiancheng Bioengineering Institute (Nanjing, China) based on the manufacturer's directions. Western blotting. Western blotting was performed as described63. The following major antibodies had been utilised: Nrf2, AMP-activated protein kinase (AMPK ), Poor (all from Abcam), Akt, p-Akt, extracellular signal-regulated kinase (ERK), p-ERK, caspase 3, cleaved caspase 3, p-AMPK (all from Cell Signaling Technologies, Beverley, MA, USA), and p-Bad (from Novus Biologicals, Littleton, CO, USA). The Image J software (NIH) was used for densitometry measurement of western blots, which was operated by an independent investigator inside a blind manner.approach described previously with some modifications64. Protein homogenates on the left ventricular tissue had been ready in cold lysis buffer (100 mM NaCl, 50 mM Tris, pH 7.five, two mM EDTA and 1 Triton X-100) supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany), and adjusted to a protein concentration of 10 mg/ml. Carbonylated proteins have been biotinylated by incubating with biotin-hydrazide (Sigma) of 5 mM within the dark for two hr at area temperature, and stabilized by further incubating with NaBH4 of 50 mM for 1 hr. Aliquots on the samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membrane was blocked in 1 bovine serum albumin solution for 1 h and incubated inside a diluted avidin-biotin horseradish peroxidase option (ready making use of the VECTASTAIN Elite ABC Kit) for 30 min with gentle agitation.