title: Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.
abstract: Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.
authors.affiliation: Post-graduate School, Southern Medical University, Guangzhou, Guangdong, China.|Plastic & Laser Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China.
authors.forename: Baihui
authors.initials: B
authors.lastname: Sun
authors.affiliation: Spine Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Ruoting
authors.initials: R
authors.lastname: Ding
authors.affiliation: Plastic & Laser Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China.
authors.forename: Wenlin
authors.initials: W
authors.lastname: Yu
authors.affiliation: Plastic & Laser Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Post-graduate School, Southern Medical University, Guangzhou, Guangdong, China.
authors.forename: Bulin
authors.initials: B
authors.lastname: Wang
authors.affiliation: Plastic & Laser Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou, Guangdong, China. gzzxwk@163.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Li
mesh_terms: D062107:Advanced Oxidation Protein Products / Q000378:metabolism*; D017209:Apoptosis; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000201:enzymology* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D000074663:NADPH Oxidase 4 / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D062107:Advanced Oxidation Protein Products; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; C498910:NOX4 protein, human; D011065:Poly(ADP-ribose) Polymerases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: AOPPs; Apoptosis; HaCaT cells; MAPK; Oxidative stress