The protein or mRNA expression of autophagy-related proteins (Beclin1, microtubule-associated protein light chain 3 (LC3), p62), ER stress marked proteins (CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), caspase-12), oxidative stress-related proteins (p22phox, nuclear factor erythroid2-related factor 2 (Nrf2)) and apoptosis-related proteins (Bax, B cell lymphoma-2 (Bcl-2)) were assayed by western blot or real-time fluorescence quantitative PCR.
The aged ischemic muscles had decreased levels of AdipoR1, peroxisome proliferator activated receptor-γ (PPAR-γ), PPAR-γ co-activator 1α (PGC-1α), phospho-AMP-activated protein kinase α (p-AMPK-α), and B cell lymphoma-2 (Bcl-2) and increased levels of cleaved caspase-8 (C-caspase-8) and gp91phox/p22phox genes or/and proteins, nicotinamide adenine dinucleotide phosphate oxidase activity, superoxide production, and matrix metalloproteinase-2/-9 activity as well as increased numbers of infiltrated macrophages and leucocytes.