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Ginkgolide B (GB) is a diterpene lactone found in the leaves of the traditional Chinese medicinal plant Ginkgo that has been shown to have various pharmacological effects. However, the anti‑apoptotic properties of GB in cardiovascular disease remain poorly understood. The present study aimed to investigate the effect of GB on hydrogen peroxide‑induced cell injury in cardiac H9c2 cells, and to further clarify its protective mechanism of action. An in vitro hydrogen peroxide‑treated H9c2 cell model was used in order to mimic myocardial ischemia‑reperfusion (I/R) injury. Cell viability was assessed by the Cell Counting Kit‑8 assay. The induction of apoptosis was determined by flow cytometry and staining was performed using Hoechst 33342. In addition, the effect of GB on the expression levels of apoptosis‑associated proteins was evaluated by western blot analysis. The present study demonstrated that GB protected against hydrogen peroxide‑induced cytotoxicity and cell apoptosis in H9c2 cardiac cells. GB upregulated the expression level of the anti‑apoptotic protein Bcl‑2 and downregulated the expression levels of the pro‑apoptotic proteins cleaved caspase‑3 and Bax in hydrogen peroxide‑treated H9c2 cells. The molecular mechanism underlying the anti‑apoptotic effects of GB was subsequently detected. GB pretreatment activated the PI3K/Akt/mTOR signaling pathway and caused an increase in the phosphorylation levels of Akt and mTOR in hydrogen peroxide‑treated H9c2 cells. These results revealed that GB inhibited hydrogen peroxide‑induced apoptosis in H9c2 cells via activation of the PI3K/Akt/mTOR signaling pathway. These findings indicate the potential therapeutic benefits of GB in the treatment of myocardial I/R injury.

Aim: The aim of this study was to characterize the role of Alport syndrome, mental retardation, midface hypoplasia, and elliptocytosis chromosomal region gene 1 (AMMECR1) in human lung cancer cell lines., Materials and Methods: AMMECR1 gene expression was evaluated in four lung cell lines, with A549 then selected for further in-depth examination. To characterize the role of AMMECR1, silencing was achieved utilizing lentivirus-mediated RNA interference, and confirmed by quantitative real-time polymerase chain reaction and western blotting. The impact of AMMECR1 silencing on cellular proliferation was assessed using Celigo-based and MTT assays. Apoptosis was determined using the annexin V-allophycocyanin single staining method. Cell-cycle arrest was assessed by flow cytometry. Finally, colony formation was assessed using Giemsa staining., Results: In A549 cells, AMMECR1 silencing was found to significantly suppress cell proliferation, reduce colony formation, promote apoptosis, and arrest cells in the S and G 2 /M phases., Conclusion: AMMECR1 plays a critical role in cell proliferation, cell-cycle progression, and apoptosis of human lung cancer cells, and may serve as a potential therapeutic target for non-small-cell lung cancer., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Hepatocyte growth factor (HGF) is widely known as a protective factor in ischemic myocardium, however HGF sensitive cellular mechanism remained ill-defined. Autophagy at early stage of hypoxia has been demonstrated to play a role in protecting myocardium both in vivo and vitro. We performed this study to investigate the association between the protective effect of HGF and autophagy., Methods: Ventricular myocytes were isolated from neonatal rat heart (NRVMs). We evaluated cardiomyocytes apoptosis by Hoechst staining and flow cytometry. Autophagy was assessed by transmission electron microscope and mRFP-GFP-LC3 adenovirus infection. Mitochondrial membrane potential was estimated by JC-1 staining. Western blotting and ELISA assay were used to quantify protein concentrations., Results: We found that autophagy in NRVMs increased at early stage after hypoxia and HGF release was consistent with the change of autophagy. Exogenous HGF enhanced autophagy and decreased apoptosis, while neutralizing HGF yielded opposite effects. Besides, inhibition of autophagy increased apoptosis of myocytes. Furthermore, exogenous HGF induced Parkin, the marker of mitochondrial autophagy, indicating increased clearance of injured mitochondria., Conclusions: Our results revealed a potential mechanism in which exogenous HGF prevented NRVMs from apoptosis after hypoxia. Upregulation of Parkin through administration of exogenous HGF may be a potential therapeutic strategy ptotecting myocytes during ischemia., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background/aims: The discovery of c-kit+ cardiac stem cells (CSCs) provided us with new therapeutic targets to repair the damaged heart. However, the precise mechanisms regulating CSC proliferation and differentiation in the aged heart remained elusive. Necroptosis, a type of regulated cell death, has recently been shown to occur following myocardial infarction (MI); however, its effect on c-kit+ CSCs remains unknown. We investigated the effects of hepatocyte growth factor (HGF) and necroptosis on the proliferation and differentiation of endogenous c-kit+ CSCs in aged rat hearts following MI., Methods: The c-kit+ CSCs and HGF/p-Met expression levels in neonatal, adult and aged rats were compared using immunofluorescence and Western blotting. Immediately after MI, adenovirus carrying the HGF gene (Ad-HGF) was injected into the left ventricular wall surrounding the infarct areas of the aged rat heart. The proliferation and differentiation of the endogenous c-kit+ CSCs were studied using immunofluorescence. The signalling pathways were analysed via Western blotting and ELISA., Results: HGF/p-Met expression levels and c-kit+ CSC abundance gradually decreased with age. Ad-HGF promoted c-kit+ CSC differentiation into precursor cells of cardiomyocyte, endothelial and smooth muscle cell lineages and enhanced cardiomyocyte proliferation and angiogenesis in aged rats; these effects were reversed by the inhibition of necroptosis. Ad-HGF administration induced necroptosis by increasing the expression of receptor interacting protein kinase (RIP) 1 and receptor interacting protein kinase (RIP) 3 proteins in the infarcted heart. Moreover, Ad-HGF-induced necroptosis increased high-mobility group box 1 protein (HMGB1) levels and enhanced the abundance of c-kit+ cells in the bone marrow, which may partly account for the beneficial effect of necroptosis on the c-kit+ CSCs., Conclusion: Ad-HGF-induced necroptosis facilitated aged heart repair after MI by promoting c-kit+ CSC proliferation and differentiation. These findings may lead to the development of new methods for the treatment of ischaemic heart disease in aged populations., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Calcium-sensing receptors (CaSR) are G-protein coupled receptors which maintain systemic calcium haemeostasis, participate in hormone secretion, activation of iron channel, cell apoptosis, proliferation and differentiation. Previous studies have show CaSR induce apoptosis in isolated rat adult heart and in normal rat neonatal cardiomyocytes by G-protein-PLC-IP3 signaling transinduction. A few of studies had demonstrated that CaSR induce apoptosis in cultured neonatal rat cardiomyocytes during ischemia/reperfusion. Hepatocyte growth factor (HGF), as a mesenchymally derived heterodimeric glycoprotein, play vital role in mitogenesis, angiogenesis, cellular motility and growth and anti-apoptosis after postinfarction heart failure via activation of transmembrane tyrosine kinase cell surface receptor c-Met. However, little knowledge exists about whether anti-apoptotic role of HGF in preventing cardiomyocytes injury induced by ischemia/reperfusion is associated with downregulation of CaSR expression. We incubated primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2 h, then reincubated them in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, we analyzed the expression of Caspase-3, Bcl-2 and Phosphoinositide 3-kinase (PI3K) by Western blotting. The simulated I/R enhances the expression of CaSR and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSR, further increase the expression of CaSR and Cardiomyocyte apoptosis, along with upregulation of Caspase-3, downregulation of Bcl-2 and inhibiting PI3K phosphorylation. Combination of GdCl3 with LY294002 (a selective PI3K inhibitor) increased Cardiomyocytes apoptosis but did not increased CaSR expression. Treatment of HGF decreased I/R- and GdCl3-induced apoptosis by suppressing Caspase-3 and promoting Bcl-2 and PI3K phosphorylation expression in accordance with downregulation of CaSR expression. HGF exerts protective role in I/R-induced apoptosis at least in part by inhibiting CaSR expression along with promoting Bcl-2, suppressing Caspase-3 expression and stimulating PI3K phosphorylation signaling pathway.

The aim of this present study is to investigate the impacts of combinatorial simvastatin administration and endothelial progenitor cell (EPC) transplantation on therapeutic angiogenesis in an athymic nude mouse model of hind limb ischemia. Athymic nude mice were divided into four groups (n = 10/group): vehicle administration plus PBS injection (control), simvastatin administration plus PBS injection (simvastatin), vehicle administration plus EPC transplantation (EPC), and simvastatin administration plus EPC transplantation (combination). The combination therapy had the greatest laser Doppler blood perfusion imager (LDPI) index and capillary density among the four groups. Importantly, this combination therapy significantly reduced apoptosis of ischemic skeletal muscle cells in part through downregulation of Bax and upregulation of Bcl-2 compared with the other groups. Moreover, the combination therapy exhibited the highest efficacy of increasing the ratio of phospho-Akt to Akt among the four groups. Taken together, the simvastatin and EPC combination therapy promotes powerful angiogenesis in hindlimb ischemia. The combination therapy not only inhibites apoptosis of ischemic skeletal muscle cells partially via downregulation of Bax and upregulation of Bcl-2, but also activates Akt phosphorylation significantly. These efficacies may be mediated by the angiogenic potency of simvastatin, EPCs, and by the beneficial effects of simvastatin on transplanted EPCs as well.

Background. The aim of the present study was to observe the effect of RAGE-HMGB1 signal pathway on remote ischemic postconditioning in mice with myocardial ischemia reperfusion injury. Methods. Mice model of MIRI was established and randomly divided into three groups: control group, ischemia reperfusion group, and remote ischemic postconditioning group. Infarction size was detected by Evans blue and TTC staining. Cardiac function was detected by echocardiography measurement. The protein levels of RAGE, HMGB1, P-AKT, and ERK1/2 were detected by Western blot 120 min following reperfusion. Results. RIPostC could decrease the infarct size and increase LVEF and FS compared with I/R group. Two hours after myocardial ischemia reperfusion, the levels of RAGE and HMGB1 were significantly decreased in RIPostC group compared with those in I/R group. The level of p-AKT was significantly higher in the RIPostC group than in the I/R group. LY294002 significantly attenuated RIPostC-increased levels of Akt phosphorylation. Conclusion. RIPostC may inhibit the expression of RAGE and HMGB1 and activate PI3K/Akt signaling pathway to extenuate ischemic reperfusion injury in mice. It could further suppress the oxidative stress, have antiapoptosis effect, and reduce inflammatory reaction, but this effect has certain timeliness. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Calcium-sensing receptors (CaSRs) are G-protein coupled receptors which maintain systemic calcium homeostasis and participate in hormone secretion, activation of ion channels, cell apoptosis, proliferation, and differentiation. Previous studies have shown that CaSRs induce apoptosis in isolated adult rat heart and in normal neonatal rat cardiomyocytes by G-protein-PLC-IP3 signaling transduction. However, little knowledge is presently available concerning the role of CaSRs in the apoptosis induced by ischemia and reperfusion in neonatal cardiomyocytes. Methods: Primary neonatal rat ventricular cardiomyocytes were incubated in ischemiamimetic solution for 2 h, and then re-incubated in normal culture medium for 24 h to establish a model of simulated ischemia/reperfusion (I/R). Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of CaSRs mRNA was detected by real-time reverse transcription polymerase chain reaction (RT-PCR). In addition, the expressions of caspase-3 and Bcl-2 were analyzed by western blot. Results: The simulated I/R enhanced the expression of CaSRs and cardiomyocyte apoptosis. GdCl3, a specific activator of CaSRs, further increased the expression of CaSRs and cardiomyocyte apoptosis, along with up-regulation of caspase-3 and down-regulation of Bcl-2. Conclusion: CaSRs are associated with I/R injury and apoptosis in neonatal rat ventricular cardiomyocytes via suppressing Bcl-2 and promoting caspase-3 expression. [Copyright &y& Elsevier]

Abstract: Circulating endothelial progenitor cells (EPCs) play a key role in restoring endothelial function and enhancing angiogenesis. However, the effects of low-dose aspirin on circulating EPCs are not well known. We investigated the effects of low-dose aspirin on EPC migration, adhesion, senescence, proliferation, apoptosis and endothelial nitric oxide synthase (eNOS) expression. EPC migration was detected by a modified Boyden chamber assay. EPC adhesion assay was performed by counting adherent cells on fibronectin-coated culture dishes. EPC senescence was assessed by both senescence-associated-β-galactosidase staining and DAPI staining. EPC proliferation was analyzed by MTT assay. EPC apoptosis was evaluated by flow cytometric analysis. eNOS protein expression was measured by Western blotting analysis. Aspirin promoted EPC migratory and adhesive capacity at concentrations between 0.1 and 100μmol/L and prevented senescence at concentrations between 50 and 100μmol/L. Meanwhile, aspirin in a range of these concentrations did not affect EPC proliferation, apoptosis or eNOS expression. Our findings indicate that low-dose aspirin promotes migration and adhesion and delays the onset of senescence of EPCs. [Copyright &y& Elsevier]

Abstract: Iodinated contrast media (CM) can induce apoptosis and necrosis of renal tubular cells. The injuries of endothelial cells induced by CM on the systemic condition have not been fully understood. To assess the toxic effects of non-ionic CM on the glomerular and aortic endothelial cells, iopromide and iodixanol, two kinds of representative non-ionic CM, were used for the in vivo study. Sixty aged rats were respectively received the agents or normal sodium intravascularly. No obvious apoptosis and morphological change was detected in the glomerular and aortic endothelial cells apart from renal tubules after CM administration. However, expressions of the nitric oxide synthase (eNOS) in glomerular endothelium were decreased at 12h after CM injection. Furthermore, plasma creatinine and endothelin-1 were increased and plasma nitric oxide (NO) was decreased significantly after CM administration. However, we failed to observe the significant increase of plasma von Willebrand Factor. These results suggest that non-ionic iodinated CM do not induce apoptosis and necrosis of glomerular and aortic endothelial cells in vivo. Decreased eNOS expression and increased plasma endothelin-1 may be involved in non-ionic iodinated CM-induced endothelial dysfunction and kidney injury. [Copyright &y& Elsevier]