title: Geniposide Prevents Hypoxia/Reoxygenation-Induced Apoptosis in H9c2 Cells: Improvement of Mitochondrial Dysfunction and Activation of GLP-1R and the PI3K/AKT Signaling Pathway.
abstract: OBJECTIVE
  Myocardial ischemia/reperfusion injury is a major cause of morbidity and mortality associated with coronary heart disease. Many studies have demonstrated that natural products are promising chemotherapeutic drugs counteracting the loss of cardiomyocytes. Thus, the purpose of the present study was to investigate the effects of geniposide, a traditional Chinese herb extract from Gardenia jasminoides J. Ellis, on cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) in H9c2 cells, and their underlying mechanisms.
  
  
  METHODS
  Cell viability and apoptosis ratio were assessed using the cell counting kit-8 assay and Annexin V/propidium iodide (PI) staining. The concentrations of lactate dehydrogenase (LDH), intracellular total superoxide dismutase (T-SOD), and malondialdehyde (MDA) were detected by microplate reader. The production of reactive oxygen species/reactive nitrogen species (ROS/RNS), the level of mitochondrial calcium, and mitochondrial membrane potential depolarization were measured by confocal laser scanning microscopy. Mitochondrial morphology was visualized using transmission electron microscopy. The expressions of Bcl-2 mRNA and Caspase-3 mRNA were measured by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of cleaved caspase-3, Bcl-2, Bax, AKT, p-AKTserine473, cytochrome-c were detected by western bloting.
  
  
  RESULTS
  Geniposide pretreatment increased cell viability, decreased LDH levels in the supernatant, and inhibited cardiomyocyte apoptosis caused by H/R. Furthermore, geniposide reversed mitochondrial dysfunction by decreasing oxidative stress products (ROS/RNS and MDA), increasing anti-oxidative enzyme (T-SOD) level, improving mitochondrial morphology, attenuating mitochondrial calcium overload and blunting depolarization of mitochondrial membrane. Moreover, geniposide pretreatment increased Bcl-2 level and decreased Bax level, thus enhancing the Bcl-2/Bax ratio. Consistent with the above result, Bcl-2 mRNA expression was upregulated and caspase-3 mRNA expression was downregulated by geniposide. In addition, geniposide decreased the protein expression of cleaved caspase-3 and cytochrome-c and increased the level p-AKTserine473. The protective effects of geniposide were partially reversed by glucagon-like pepitide-1 receptor antagonist exendin-(9-39) and the phosphatidylinositol 3 kinase (PI3K) inhibitor LY294002.
  
  
  CONCLUSIONS
  Our results suggest that geniposide pretreatment inhibits H/R-induced myocardial apoptosis by reversing mitochondrial dysfunction, an effect in part due to activation of GLP-1R and PI3K/AKT signaling pathway.
authors.affiliation: Department of Cardiology, First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing, China.
authors.forename: You-Qin
authors.initials: YQ
authors.lastname: Jiang
authors.forename: Guang-Lei
authors.initials: GL
authors.lastname: Chang
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Dong-Ying
authors.initials: DY
authors.lastname: Zhang
authors.forename: Li
authors.initials: L
authors.lastname: Cao
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004365:Drugs, Chinese Herbal / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D000067757:Glucagon-Like Peptide-1 Receptor / Q000378:metabolism*; D039823:Iridoids / Q000494:pharmacology*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008929:Mitochondria, Heart / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D010100:Oxygen / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D004365:Drugs, Chinese Herbal; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D039823:Iridoids; D019253:Proto-Oncogene Proteins c-bcl-2; C007835:geniposide; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3; D010100:Oxygen