title: Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy.
abstract: Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl(2) to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl(2) induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl(2)-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl(2)-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.
authors.affiliation: College of Life Science, Sichuan University, Chengdu 610064, PR China.
authors.forename: Xin-Xin
authors.initials: XX
authors.lastname: Tong
authors.forename: Dan
authors.initials: D
authors.lastname: Wu
authors.forename: Xue
authors.initials: X
authors.lastname: Wang
authors.forename: Hua-Li
authors.initials: HL
authors.lastname: Chen
authors.forename: Jia-Xiang
authors.initials: JX
authors.lastname: Chen
authors.forename: Xiao-Xiao
authors.initials: XX
authors.lastname: Wang
authors.forename: Xu-Lei
authors.initials: XL
authors.lastname: Wang
authors.forename: Lu
authors.initials: L
authors.lastname: Gan
authors.forename: Zhi-Yun
authors.initials: ZY
authors.lastname: Guo
authors.forename: Gui-Xiu
authors.initials: GX
authors.lastname: Shi
authors.forename: Yi-Zheng
authors.initials: YZ
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D002374:Catalase / Q000378:metabolism; D015687:Cell Hypoxia / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003035:Cobalt; D054439:Ghrelin / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000074624:NADPH Oxidase 1; D018384:Oxidative Stress / Q000187:drug effects*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D054439:Ghrelin; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D003035:Cobalt; D002374:Catalase; D013482:Superoxide Dismutase; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614712:NOX1 protein, rat; D055372:AMP-Activated Protein Kinases; C018021:cobaltous chloride