Study of mitochondrial energy metabolism in D-galactose-induced cell ageing model
Jianming Cao, Dongjie Hao, Huale Chen, Xiao Yu, Yuanbo Hou, Wei Ye
Published 2017-02-14
Cite as Chin J Geriatr, 2017, 36(2): 199-203. DOI: 10.3760/cma.j.issn.0254-9026.2017.02.019
Abstract
ObjectiveTo investigate the mitochondrial energy metabolism in D-galactose-induced cell ageing model.
MethodsMRC-5 cells were cultivated for 72 hours in a medium containing 55 mmol/L D-galactose.The analysis of cell proliferation capacity by CCK8 method, β-galactosidase staining and detection of p21 protein expression level were performed for identifying cell senescence.The cell oxidation-reduction state was evaluated by an analysis of cellular ROS levels, SOD activity, MDA content and oxidative damage level of mitochondrial DNA(mtDNA). For purpose of detecting mitochondrial function and its impairment, mitochondrial morphology was observed by electron microscope, mitochondrial quantity was analyzed by flow cytometry, mitochondrial membrane potential(ΔΨm)was measured by JC-1 staining, and ATP content was analyzed by HPLC, and mitochondrial oxygen consumption rate was detected by Seahorse cell energy metabolism detection system.
ResultsThe decreased MRC-5 cell proliferation, up-expression of p21 protein, increased β-galactosidase activity were observed in D-Gal-treated cells, which indicated the cell premature senescence.When treated with D-Gal, the significantly increased ROS and MDA level, decreased SOD activity and increased oxidized mtDNA proved that the cells kept higher oxidative stress.D-Gal induced-mitochondrial impairment was evidenced by the dimming of mitochondrial cristae and double membrane structure, decrease of transmembrane potential and ATP synthesis, and decrease of its oxygen consumption rate(OCR).
ConclusionsThe 55 mmol/L D-Gal causes an impairment of mitochondrial structure and a decrease of function of energy metabolism, which is associated with cellular senescence induced by D-Gal.
Key words:
Galactose; Cell aging; Mitochondria; Energy metabolism
Contributor Information
Jianming Cao
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Dongjie Hao
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Huale Chen
Medical Inspection Center, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China
Xiao Yu
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Yuanbo Hou
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China
Wei Ye
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035, China