Protective effect of α-mangostin derivatives on hypoxia/reoxygenation-induced apoptosis in H9C2 cells and their mechanism

Abstract Cardiomyopathy and liver damage due to iron-overload are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes and hepatic cells, and that TPO may exert protective effect on apoptosis of cardiomyocytes (Circulation, 2006). In this study, we demonstrated firstly that iron induced apoptosis in cardiomyocytes. Using H9C2 cells, we have shown that iron reduced cell viability in a dose-dependent manner (0.003–3 mM) (n=6). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.3 mM, 72 hrs) (n=6). The expression of active caspase-3 was significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (5, 10, 20, 50, 100 ng/mL, 72 hrs). The cell viability was significantly increased with the treatment of TPO at 50 ng/mL and 100 ng/mL (n=4). Dot-plot analysis of annexin V/PI staining demonstrated that TPO (50 ng/mL) significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis of H9C2 cells also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6). The population of phospho-Akt and Erk1/2 were also significantly increased after treatment by TPO (P<0.05, n=4). Human liver cell line MIHA was also used as a cell model. We showed that iron-overload reduced cell viability in a dose-dependent manner (0.0375–0.6 mM) (n=7). By annexin V and PI staining, apoptotic cells were found to be significantly increased after iron treatment (0.15–0.6 mM) for 72 hrs (n=7). The expression of active caspase-3 was also significantly increased in iron-treated cells (n=5). We also found that TPO exerted proliferation effect on MIHA cell by activation of phospho-Akt. However, MIHA cells were cultured in the presence of iron (0.3 mM) with TPO (50 ng/mL, 72 hrs). The cell viability was not significantly increased with the treatment of TPO (n=5). Dot-plot analysis of annexin V/PI staining did not demonstrated that TPO reduced the population of apoptotic cells induced by iron-overload (n=5). Also, incubation with TPO did not decrease the iron-induced caspase-3 expression in these cells (n=5). Our findings suggest that iron-overload induces apoptosis in cardiomyocytes and hepatocytes via mitochondrial/caspase-3 pathways and that TPO might exert a protective effect on iron-overload induced apoptosis via the activation of Akt and Erk1/2 pathways in cardiomyocytes.

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CCL6 aggravates hypoxia-reoxygenation-induced apoptosis in H9c2 cells through enhancing the expression of IGF2-AS

Journal of Cardiovascular Pharmacology ◽

10.1097/fjc.0000000000000905 ◽

2020 ◽

Vol Publish Ahead of Print ◽

Author(s):

Xiufen Li ◽

Bilali Aishan ◽

Yan Yang ◽

Yang Xie ◽

Dilimulati Dilimulati ◽

...

Keyword(s):

H9c2 Cells ◽

Induced Apoptosis ◽

Hypoxia Reoxygenation

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Globular Adiponectin, Acting via AdipoR1/APPL1, Protects H9c2 Cells from Hypoxia/Reoxygenation-Induced Apoptosis

PLoS ONE ◽

10.1371/journal.pone.0019143 ◽

2011 ◽

Vol 6 (4) ◽

pp. e19143 ◽

Cited By ~ 45

Author(s):

Min Park ◽

ByungSoo Youn ◽

Xi-long Zheng ◽

Donghai Wu ◽

Aimin Xu ◽

...

Keyword(s):

H9c2 Cells ◽

Globular Adiponectin ◽

Induced Apoptosis ◽

Hypoxia Reoxygenation

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Abstract 248: Stabilization of cIAP2 during Postconditioning protects Endothelial Cells from Reperfusion Injury

Circulation ◽

10.1161/circ.116.suppl_16.ii_29-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Krishnaveni Gadiraju ◽

Frauke V Haertel ◽

Hans M Piper ◽

Thomas Noll

Keyword(s):

Endothelial Cells ◽

Protective Effect ◽

Reperfusion Injury ◽

Intermittent Hypoxia ◽

Umbilical Vein ◽

Fold Increase ◽

Protective Mechanism ◽

Second Phase ◽

Induced Apoptosis ◽

Hypoxia Reoxygenation

Hypoxia-reperfusion causes a perturbance in the complex equilibrium of pro and anti apoptotic mechanisms which ultimately leads to apoptosis in the reperfused myocardium. Postconditioning (intermittent hypoxia at the onset of reperfusion) is a proven strategy to reduce reperfusion injury, however, the mechanisms are largely unknown in endothelial cells. Here we analyze the effect of postconditioning in endothelial cells and hypothesize that the ‘inhibitors of apoptosis proteins’ (IAPs), known as antiapoptotic mediators, are key elements of this protective mechanism. Methods and Results: Exposure of human umbilical vein endothelial cells to severe hypoxia (Po 2 < 2 mmHg) for 2 hrs causes a 2.1±0.5-fold increase in caspase 3 activation (western blot analysis; P<0.05, n=3, for all further parameters) and a 2.3±04-fold increase in apoptosis (annexin V staining) after 24 hrs of reoxygenation. cIAP2 but not cIAP1 is rapidly increased during hypoxia in a biphasic manner. Transcription inhibitor, Actinomycin D (20μg/ml) reveals that the 2.5-fold increase within 5 min of hypoxia (first phase) was independent of transcription, but the 3.1-fold increase after 60 min (second phase) was induced by gene transcription. cIAP2 levels dropped down to basal value with the onset of reperfusion. Importantly, cIAP2 levels could be maintained by postconditioning (3 short periods of intermittent hypoxia, 5 minutes each separated by a 5 minute reoxygenation) which abolished hypoxia-reoxygenation-induced apoptosis. Down regulation of IAP2 by siRNA strategy enhanced hypoxia-reoxygenation-induced apoptosis and diminished the protective effect of postconditioning. Conclusions: The present study shows for the first time that postconditioning can protect endothelial cells against hypoxia-reoxygenation induced apoptosis. This protective effect is conferred by the cIAP2, which is stabilized during hypoxia and could be maintained at an elevated level by postconditioning.

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Mongolian Medicinal Formula Anshen-Buxin-Liuwei Pill Alleviate Cardiomyocyte Hypoxia/Reoxygenation Injury via Mitochondrion Pathway

10.21203/rs.3.rs-839312/v1 ◽

2021 ◽

Author(s):

Yu-jia Huang ◽

Hai-ying Tong ◽

Xian-ju Huang ◽

Xin-Cai Xiao ◽

Yue Dong ◽

...

Keyword(s):

Protective Effect ◽

Cell Viability ◽

Cell Apoptosis ◽

Therapeutic Drug ◽

Mrna Levels ◽

Dependent Manner ◽

H9c2 Cells ◽

Mitochondrial Energy Metabolism ◽

H9c2 Cardiomyocytes ◽

Hypoxia Reoxygenation

Abstract Anshen Buxin Liuwei pill (ABLP), a Mongolian medicinal formula, composed of the six medicinal materials of Mongolian medicine Bos taurus domesticus Gmelin, Choerospondias axillaris (Roxb. ) Burtt et Hill, Myristica fragrans Houtt., Eugenia caryophµllata Thunb., Aucklandia lappa Decne., Liqui dambar formosana Hance, is considered to have a therapeutic effect on the symptoms such as coronary heart disease, angina pectoris, arrhythmia, depression and irritability, palpitation, and shortness of breath. Therefore, the present study was employed a network pharmacology approach to identify the potentially active ingredients and to evaluate the protective effect of ABLP on hypoxia/reoxygenation (HR)-induced H9c2 cardiomyocytes, and its influence on cell viability, apoptosis, oxidative stress. H9c2 cardiomyocytes were used to construct a HR injury model. CCK-8 assay and AnnexinV-FITC cell apoptosis assays were used for cell viability and cell apoptosis determination. The levels of LDH, SOD, MDA, CAT, CK, GSH-Px, Na+-K+-ATPase, and Ca2+-ATPase in the cells were determined to assess the effect of ABLP. the mRNA levels of Sirtuin3 (Sirt3) and Cytochrome C (Cytc) in H9c2 cells were determined by quantitative real-time PCR. The finding of this study indicates that HR treatment cells began to shrink from the spindle in an irregular shape with some floated in the medium, well by increasing the therapeutic dose of ABLP (5 µg/mL, 25 µg/mL, and 50 µg/mL), the cells gradually reconverted in a concentration-dependent manner. The release of CK in HR-treated cells was significantly increased, indicating that ABLP exerts a protective effect in H9c2 cells against HR injury and can improve the mitochondrial energy metabolism and mitochondrial function integrity. The present study scrutinized the cardio-protective effect of ABLP against the HR-induced H9c2 cells injury through antioxidant and mitochondrion pathways. ABLP could be a promising therapeutic drug for the treatment of myocardial ischemic cardiovascular disease. The results will provide reasonable information for clinical use of the ABLP.

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TRPV1 Activation Exacerbates Hypoxia/Reoxygenation-Induced Apoptosis in H9C2 Cells via Calcium Overload and Mitochondrial Dysfunction

International Journal of Molecular Sciences ◽

10.3390/ijms151018362 ◽

2014 ◽

Vol 15 (10) ◽

pp. 18362-18380 ◽

Cited By ~ 32

Author(s):

Zewei Sun ◽

Jie Han ◽

Wenting Zhao ◽

Yuanyuan Zhang ◽

Shuai Wang ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Calcium Overload ◽

H9c2 Cells ◽

Induced Apoptosis ◽

Hypoxia Reoxygenation

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Thrombopoietin Protects Cardiomyocytes from Iron-Overload Induced Oxidative Stress and Mitochondrial Injury

Cellular Physiology and Biochemistry ◽

10.1159/000430173 ◽

2015 ◽

Vol 36 (5) ◽

pp. 2063-2071 ◽

Cited By ~ 11

Author(s):

Shing Chan ◽

Godfrey Chifung Chan ◽

Jieyu Ye ◽

Qizhou Lian ◽

Jianliang Chen ◽

...

Keyword(s):

Oxidative Stress ◽

Membrane Potential ◽

Iron Overload ◽

Protective Effect ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Lipid Hydroperoxides ◽

Ros Production ◽

H9c2 Cells ◽

Induced Apoptosis

Background/Aims: Thalassaemia accompanied with iron-overload is common in Hong Kong. Iron-overload induced cardiomyopathy is the commonest cause of morbidity and mortality in patients with β-thalassaemia. Chronic iron-overload due to blood transfusion can cause cardiac failure. Decreased antioxidant defence and increased ROS production may lead to oxidative stress and cell injury. Iron-overload may lead to heart tissue damage through lipid peroxidation in response to oxidative stress, and a great diversity of toxic aldehydes are formed when lipid hydroperoxides break down in heart and plasma. Methods: Iron entry into embryonic heart H9C2 cells was determined by calcein assay using a fluorometer. Reactive oxygen species (ROS) production in cells treated with FeCl3 or thrombopoietin (TPO) was monitored by using the fluorescent probe H2DCFDA. Changes in mitochondrial membrane potential of H9C2 cells were quantified by using flow cytometry. Results: We demonstrated that iron induced oxidative stress and apoptosis in cardiomyocytes, and that iron increased ROS production and reduced cell viability in a dose-dependent manner. Iron treatment increased the proportion of cells with JC-1 monomers, indicating a trend of drop in the mitochondrial membrane potential. TPO exerted a cardio-protective effect on iron-induced apoptosis. Conclusions: These findings suggest that iron-overload leads to the generation of ROS and further induces apoptosis in cardiomyocytes via mitochondrial pathways. TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and suppressing the mitochondrial pathways in cardiomyocytes.

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Protective Effect of Two Alkaloids from Hippophae rhamnoides Linn. against Doxorubicin-Induced Toxicity in H9c2 Cardiomyoblasts

Molecules ◽

10.3390/molecules26071946 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1946

Author(s):

Wenna Zhou ◽

Jian Ouyang ◽

Na Hu ◽

Gang Li ◽

Honglun Wang

Keyword(s):

Protein Expression ◽

Protective Effect ◽

Caspase 3 ◽

Cardiac Cells ◽

Potential Candidate ◽

H9c2 Cells ◽

Cardiac Muscle Cells ◽

Anti Cancer ◽

H9c2 Cardiomyoblasts ◽

Induced Apoptosis

Background: Doxorubicin (Dox) is one of the most frequently prescribed anti-cancer drugs. However, clinical application with Dox is limited due to its potentially fatal cumulative cardiotoxicity. N-p-coumaroyl-4-aminobutan-1-ol (alk-A), an organic amide alkaloid and hippophamide (alk-B), a rare pyridoindole alkaloid were successfully obtained by purification and separation of seabuckthorn seed residue in our previous research. This study was undertaken to investigate the protective effect of alk-A and alk-B against Dox-induced embryonic rat cardiac cells (H9c2 cells) apoptosis. Methods: H9c2 cells were treated with Dox (2.5 µM) in the presence of alk-A and alk-B (10, 20, and 40 µM) and incubated for 24 h. Results: It was shown that pretreatment of the H9c2 cells with alk-A and alk-B significantly reduced Dox-induced apoptosis. Alk-A and alk-B both inhibited reactive oxygen species (ROS) production and suppressed cleaved-caspase-3 protein expression and the activation of JNK (Jun N-terminal kinases), as well as increasing ATP levels, favoring mitochondrial mitofusin protein expression, and relieving damage to mitochondrial DNA. Conclusions: These results suggest that alk-A and alk-B can inhibit Dox-induced apoptosis in H9C2 cardiac muscle cells via inhibition of cell apoptosis and improvement of mitochondrial function, while alk-B showed more protection. Alk-B could be a potential candidate agent for protecting against cardiotoxicity in Dox-exposed patients.

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