Aqueous extract of Cortex Dictamni protects H9c2 cardiomyocytes from hypoxia/reoxygenation-induced oxidative stress and apoptosis by PI3K/Akt signaling pathway.

Ischemia-reperfusion injury is the major manifestation of ischemic heart disease, which facilitates cardiac arrhythmias, heart failure and death. Oxidative stress and apoptosis have been involved in the pathogenesis of myocardial ischemia-reperfusion injury. Modern pharmacological studies have indicated that the extracts and active compounds of Cortex Dictamni exhibit antioxidative and cardiovascular protective activities. This study was designed to investigate the protective effect of aqueous extract of Cortex Dictamni (CDAE) on regulating hypoxia/reoxygenation (H/R)-induced cardiomyocytes oxidative stress and apoptosis. H9c2 cardiomyocytes pretreatmented with CDAE for 24h were exposed to hypoxia/reoxygenation. Cell survival was measured by methyl thiazolyl tetrazolium (MTT) assay, and by the detections of lactate dehydrogenase (LDH) activity and cardiac troponin I (cTn-I) content in cultured supernatant. Cell apoptosis was measured by Hoechst 33342/propidium iodide (PI) staining, Annexin-V/PI staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Intracellular reactive oxygen species (ROS) production, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were measured to examine antioxidant activity. Mitochondrial membrane potential and release of cytochrome c were measured to examine mitochondrial changes. The expressions of anti-oxidant, pro-apoptosis and anti-apoptosis proteins were measured by performing western blotting assay. Inhibitor LY294002 was used to confirm the regulation effect of CDAE on PI3K/Akt signaling pathway. CDAE pretreatment prevents H/R-induced cardiomyocytes oxidative stress and apoptosis through activation of PI3K/Akt signaling pathway.

Li L ，Zhou Y ，Li Y ，Wang L ，Sun L ，Zhou L ，Arai H ，Qi Y ，Xu Y ... -  《-》

Geniposide Prevents Hypoxia/Reoxygenation-Induced Apoptosis in H9c2 Cells: Improvement of Mitochondrial Dysfunction and Activation of GLP-1R and the PI3K/AKT Signaling Pathway.

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. 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. 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. 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.

Jiang YQ ，Chang GL ，Wang Y ，Zhang DY ，Cao L ，Liu J ... -  《-》

Shikonin protects H9C2 cardiomyocytes against hypoxia/reoxygenation injury through activation of PI3K/Akt signaling pathway.

Myocardial ischemic/reperfusion (I/R) injury often leads to irreversible myocardial cell death and even heart failure, with limited therapeutic possibilities. In the present study, we evaluated the protective effects of shikonin (SHK) against hypoxia/reoxygenation (H/R)-induced cardiomyocyte damage and explored the underlying mechanisms. H9C2 cardiomyocytes were pretreated with different doses of SHK prior to H/R exposure. We observed that SHK pretreatment significantly increased cell viability, attenuated LDH release, and suppressed cardiomyocyte apoptosis induced by H/R exposure. SHK pretreatment also restored the loss of mitochondrial membrane potential (MMP) and cytochrome c release. In addition, SHK significantly enhanced the phosphorylation of Akt and GSK-3β in H/R-treated H9C2 cells. These protective effects of SHK were partially reversed by LY294002, a specific PI3K/Akt inhibitor. Therefore, our findings suggested that SHK might be a promising agent for myocardial I/R injury, and PI3K/Akt signaling plays a crucial role during this process.

Wang S ，Zhu Y ，Qiu R 《-》

25-Hydroxyl-protopanaxatriol protects against H(2)O(2)-induced H9c2 cardiomyocytes injury via PI3K/Akt pathway and apoptotic protein down-regulation.

Oxidative stress injury and apoptosis are the main mechanisms of myocardial ischemia-reperfusion injury (MI/RI). Compounds with anti-oxidant properties can treat MI/RI. Therefore, identification of natural antioxidants such as herbs or botanical drugs is an ideal strategy to develop safe and effective anti-MI/RI drugs. Cardioprotective effects of Ginseng are well documented and are attributable to its anti-oxidant, anti-inflammatory, anti-tumorigenic, anti-arrhythmic, anti-ischemic properties. Ginseng monomer 20®-dammarane -3 beta,6 alpha,12 beta,20,25-pentol(25-hydroxyl-Protopanaxatriol,25-OH-PPT), a novel compound, which belongs to panaxatriol category, is extracted from the leaves and stem of ginseng. It was first investigated for its anti-tumorigenic properties. In this study, we explored whether 25-OH-PTT plays a role in antioxidant stress injury and anti-apoptosis in cardiomyocytes. We also explored the mechanisms in order to provide a theoretical basis to develop 25-OH-PPT as a new drug for treatment of MI/RI. First, we used H2O2 to induce H9c2 cardiomyocytes in vitro resulting in an oxidative stress injury model and pretreated with 25-OH-PPT. Secondly, we examined the viability of H9c2 cells by MTT assay, the reactive oxygen species (ROS) content and mitochondrial membrane potential by flow cytometry as well as cell apoptosis by flow cytometry Annexin-FITC/PI and Hoechst 33258 staining. Furthermore, we pretreated H9c2 cells with PI3K inhibitor, LY294002, and observed the above phenomenon. Lastly, we examined the expressions of proteins related to the PI3K/Akt signaling pathway and the apoptotic proteins. We found that 25-OH-PPT can protect H9c2 cells against H2O2-induced injury, decrease apoptosis of H9c2 cells and ROS generation, and increase the mitochondrial membrane potential. It can also upregulate the protein expressions of p-Akt, p-eNOS, and Bcl-2 and down-regulate apoptotic proteins Bax and Caspase-3. Our results indicate that 25-OH-PPT inhibits H2O2-induced H9c2 cardiomyocytes injury through PI3K/Akt pathway. It may become a potential safe and effective traditional Chinese medicine monomer for treatment of MI/RI.

Wang Z ，Su G ，Zhang Z ，Dong H ，Wang Y ，Zhao H ，Zhao Y ，Sun Q ... -  《-》

Troxerutin attenuates oxygen‑glucose deprivation and reoxygenation‑induced oxidative stress and inflammation by enhancing the PI3K/AKT/HIF‑1α signaling pathway in H9C2 cardiomyocytes.

Yu ZP ，Yu HQ ，Li J ，Li C ，Hua X ，Sheng XS ... -  《-》