scholarly journals Dexmedetomidine Postconditioning Alleviates Hypoxia/Reoxygenation Injury in Senescent Myocardial Cells by Regulating lncRNA H19 and m6A Modification

2020 ◽  
Vol 2020 ◽  
pp. 1-13 ◽  
Author(s):  
Xuan Zhang ◽  
Qiang Fu ◽  
Longhe Xu ◽  
Yitian Yang ◽  
Weixing Zhao ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Myocardial Cell ◽  
Rna Modification ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Myocardial Cells ◽  
Apoptosis Protein ◽  
Hypoxia Reoxygenation

H19, a long noncoding RNA (lncRNA), reportedly protects myocardial cells (H9c2 cell line) against hypoxia-reoxygenation- (H/R-) induced injury. Dexmedetomidine (Dex) has an important myocardial protective effect, although its function and mechanism in cardiac ischemia/reperfusion (I/R) injury, especially for senile patients, requires further study. RNA N6-methyladenosine (m6A) is the most abundant endogenous RNA modification. However, the effect of Dex postconditioning on RNA m6A modification has rarely been reported. The aim of this study was to evaluate roles of H19 and m6A modification in Dex postconditioning of aged cardiomyocytes. Hydrogen peroxide (H2O2) was used to induce senescence of H9c2 cells. After 6 h of hypoxia, H9c2 cells were exposed to different concentrations of dexmedetomidine (0, 500 nM, 1 μM, and 2 μM) for 6 h. After knockdown or overexpression of H19 and its downstream gene miR-29b-3p and cellular inhibitor of apoptosis protein 1 (cIAP1), Dex postconditioning experiments were performed to examine effects on myocardial cell injury. Global m6A levels after H/R with or without Dex postconditioning were measured with a colorimetric m6A RNA Methylation Quantification Kit. The mechanism by which RNA m6A methylation regulated genes mediating H19 expression was verified by m6A RNA immunoprecipitation (MeRIP), and the function of Dex postconditioning of aged cardiomyocytes was investigated. Dex postconditioning protected against H/R-induced injury of aged myocardial cells through H19/miR-29b-3p/cIAP1, increased methylation of RNA m6A elicited by H/R, and attenuated H/R-induced injury by suppressing expression of the RNA m6A demethylase gene alkB homolog 5 (ALKBH5). In addition, AKLBH5 regulated the expression of H19, and Dex postconditioning attenuated H/R-induced injury via ALKBH5 in aged cardiomyocytes.

scholarly journals Anshen Buxin Liuwei Pill, a Mongolian Medicinal Formula, Could Protect H2O2-Induced H9c2 Myocardial Cell Injury by Suppressing Apoptosis, Calcium Channel Activation, and Oxidative Stress

2022 ◽  
Vol 2022 ◽  
pp. 1-11
Author(s):  
Yue Dong ◽  
Hai-Ying Tong ◽  
Xian-Ju Huang ◽  
Ghulam Murtaza ◽  
Yu-Jia Huang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Injury ◽  
Cardiac Injury ◽  
Myocardial Cell ◽  
H9c2 Cell ◽  
Mrna Levels ◽  
H9c2 Cells ◽  
Therapeutic Benefits ◽  
Myocardial Cell Injury ◽  
And Oxidative Stress

Background. Anshen Buxin Liuwei pill (ABLP) is a Mongolian medicinal formula which has a therapeutic effect on the symptoms such as coronary heart disease, angina pectoris, arrhythmia, depression and irritability, palpitation, and short breath. However, its bioactivity against cardiac injury remains unclear. Methods. The protective effect of ABLP was evaluated using H9c2 cells. Cell viability, intracellular Ca2+, reactive oxidative indices, and mitochondrial membrane potential (∆ψ) were assessed, respectively. The mRNA levels of Ca2+ channel-related genes (DHPR, RyR2, and SCN5A) and oxidative stress-related genes (Keap1, Nrf2, and HO-1) were measured by RT-PCR. Results. 0.5–50 μg/mL ABLP could significantly decrease H2O2-induced cell injury by suppressing cell necrosis/apoptosis and excess oxidative stress, ameliorating the collapse of ∆ψ, and reducing intracellular Ca2+ concentration. Furthermore, 0.5–50 μg/mL ABLP reversed H2O2-induced imbalance in the mRNA levels of DHPR, RyR2, SCN5A, Keap1, Nrf2, and HO-1 gene in H9c2 cells, which further illustrate the mechanism. Conclusion. ABLP provided protective and therapeutic benefits against H2O2-induced H9c2 cell injury, indicating that this formula can effectively treat coronary disease. In addition, the present study also provides an in-depth understanding of the pharmacological functions of ABLP, which may lead to further successful applications of Mongolian medicine.

LncRNA SNHG12 downregulates RAGE to attenuate hypoxia-reoxygenation-induced apoptosis in H9c2 cells

2021 ◽  
Vol 85 (4) ◽  
pp. 866-873
Author(s):  
Ping Lu ◽  
Shihui Xiao ◽  
Shaoze Chen ◽  
Youlin Fu ◽  
Peng Zhang ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Ischemia Reperfusion ◽  
H9c2 Cells ◽  
Tnf Α ◽  
Glycation End Products ◽  
Induced Apoptosis ◽  
High Level ◽  
Injury Causes ◽  
Hypoxia Reoxygenation

ABSTRACT Ischemia-reperfusion (I/R) injury causes cardiac dysfunction through several mechanisms including the irregular expression of some long noncoding RNA. However, the role of SNHG12 in myocardial I/R injury remains unclear. Here, we found the increase of the SNHG12 level in hypoxia-reoxygenation (H/R)-injured-H9c2 cells. SNHG12 silencing enhanced the apoptosis of H/R-injured H9c2 cells, while SNHG12 overexpression relieved the cardiomyocyte apoptosis induced by H/R stimulation. Additionally, the suppression of SNHG12 significantly boosted the H/R-induced expression and the production of TNF-α, IL-6, and IL-1β, as well as the activation of NF-κB, which were fully reversed after overexpression of SNHG12. Mechanistically, SNHG12 adversely regulated the production of receptor for advanced glycation end products (RAGE) in H/R-stimulated H9c2 cells. Antibody blocking of RAGE alleviated the apoptosis of H/R-injured H9c2 cells. Collectively, we have determined a valuable mechanism by which the high level of SNHG12 contributes to H9c2 cells against H/R injury through the reduction of RAGE expression.

scholarly journals Wenxin Granule Ameliorates Hypoxia/Reoxygenation-Induced Oxidative Stress in Mitochondria via the PKC-δ/NOX2/ROS Pathway in H9c2 Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Qihui Jin ◽  
Yanhong Jiang ◽  
Lizhong Fu ◽  
Yanqiu Zheng ◽  
Yuxia Ding ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Injury ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion ◽  
Control Group ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Cytochrome C Release ◽  
Mitochondrial Ros ◽  
Hypoxia Reoxygenation

Myocardial infarction and following reperfusion therapy-induced myocardial ischemia/reperfusion (I/R) injury have been recognized as an important subject of cardiovascular disease with high mortality. As the antiarrhythmic agent, Wenxin Granule (WXG) is widely used to arrhythmia and heart failure. In our pilot study, we found the antioxidative potential of WXG in the treatment of myocardial I/R. This study is aimed at investigating whether WXG could treat cardiomyocyte hypoxia/reoxygenation (H/R) injury by inhibiting oxidative stress in mitochondria. The H9c2 cardiomyocyte cell line was subject to H/R stimuli to mimic I/R injury in vitro. WXG was added to the culture medium 24 h before H/R exposing as pretreatment. Protein kinase C-δ (PKC-δ) inhibitor rottlerin or PKC-δ lentivirus vectors were conducted on H9c2 cells to downregulate or overexpress PKC-δ protein. Then, the cell viability, oxidative stress levels, intracellular and mitochondrial ROS levels, mitochondrial function, and apoptosis index were analyzed. In addition, PKC-δ protein expression in each group was verified by western blot analysis. Compared with the control group, the PKC-δ protein level was significantly increased in the H/R group, which was remarkably improved by WXG or rottlerin. PKC-δ lentivirus vector-mediated PKC-δ overexpression was not reduced by WXG. WXG significantly improved H/R-induced cell injury, lower levels of SOD and GSH/GSSG ratio, higher levels of MDA, intracellular and mitochondrial ROS content, mitochondrial membrane potential and ATP loss, mitochondrial permeability transition pore opening, NOX2 activation, cytochrome C release, Bax/Bcl-2 ratio and cleaved caspase-3 increasing, and cell apoptosis. Similar findings were obtained from rottlerin treatment. However, the protective effects of WXG were abolished by PKC-δ overexpression, indicating that PKC-δ was a potential target of WXG treatment. Our findings demonstrated a novel mechanism by which WXG attenuated oxidative stress and mitochondrial dysfunction of H9c2 cells induced by H/R stimulation via inhibitory regulation of PKC-δ/NOX2/ROS signaling.

scholarly journals Lipopolysaccharide (LPS) Aggravates High Glucose- and Hypoxia/Reoxygenation-Induced Injury through Activating ROS-Dependent NLRP3 Inflammasome-Mediated Pyroptosis in H9C2 Cardiomyocytes

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Zhen Qiu ◽  
Yuhong He ◽  
Hao Ming ◽  
Shaoqing Lei ◽  
Yan Leng ◽  
...  
Keyword(s):  
Cell Viability ◽  
Nlrp3 Inflammasome ◽  
High Glucose ◽  
Cell Injury ◽  
H9c2 Cell ◽  
Ros Production ◽  
H9c2 Cells ◽  
Caspase 1 ◽  
H9c2 Cardiomyocytes ◽  
Hypoxia Reoxygenation

Diabetes aggravates myocardial ischemia-reperfusion (I/R) injury because of the combination effects of changes in glucose and lipid energy metabolism, oxidative stress, and systemic inflammatory response. Studies have indicated that myocardial I/R may coincide and interact with sepsis and inflammation. However, the role of LPS in hypoxia/reoxygenation (H/R) injury in cardiomyocytes under high glucose conditions is still unclear. Our objective was to examine whether lipopolysaccharide (LPS) could aggravate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by upregulating ROS production to activate NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. H9C2 cardiomyocytes were exposed to HG (30 mM) condition with or without LPS, along with caspase-1 inhibitor (Ac-YVAD-CMK), inflammasome inhibitor (BAY11-7082), ROS scavenger N-acetylcysteine (NAC), or not for 24 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). The cell viability, lactate dehydrogenase (LDH) release, caspase-1 activity, and intracellular ROS production were detected by using assay kits. The incidence of pyroptosis was detected by calcein-AM/propidium iodide (PI) double staining kit. The concentrations of IL-1β and IL-18 in the supernatants were assessed by ELISA. The mRNA levels of NLRP3, ASC, and caspase-1 were detected by qRT-PCR. The protein levels of NF-κB p65, NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18 were detected by western blot. The results indicated that pretreatment LPS with 1 μg/ml not 0.1 μg/ml could efficiently aggravate HG and H/R injury by activating NLRP3 inflammasome to mediate pyroptosis in H9C2 cells, as evidenced by increased LDH release and decreased cell viability in the cells, and increased expression of NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18. Meanwhile, Ac-YVAD-CMK, BAY11-7082, or NAC attenuated HG- and H/R-induced H9C2 cell injury with LPS stimulated by reversing the activation of NLRP3 inflammasome-mediated pyroptosis. In conclusion, LPS could increase the sensitivity of H9C2 cells to HG and H/R and aggravated HG- and H/R-induced H9C2 cell injury by promoting ROS production to induce NLRP3 inflammasome-mediated pyroptosis.

scholarly journals Melatonin Protects Cardiomyocytes from Oxygen Glucose Deprivation And Reperfusion-Induced Injury by Inhibiting Rac1/JNK/Foxo3a/Bim Signaling Pathway

2021 ◽  
Author(s):  
Yulin Wang ◽  
Ying Jian ◽  
Xiaofu Zhang ◽  
Bin Ni ◽  
Mingwei Wang ◽  
...  
Keyword(s):  
Protective Effect ◽  
Signaling Pathway ◽  
Cell Injury ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
H9c2 Cell ◽  
Protective Mechanism ◽  
H9c2 Cells

Abstract Melatonin has been shown to exert protective effect during myocardial ischemia/reperfusion (I/R). However, the underlying mechanism is not completely understood. Using the oxygen-glucose deprivation and reperfusion (OGD/R) model of H9c2 cells in vitro, we found that melatonin alleviated OGD/R-induced H9c2 cell injury via inhibiting Foxo3a/Bim signaling pathway. Inhibition of Rac1 activation contributed to the protective effect of melatonin against OGD/R injury in H9c2 cells. Additionally, melatonin inhibited OGD/R-activated Foxo3a/Bim signaling pathway through inactivation of Rac1. Furthermore, JNK inactivation was responsible for Rac1 inhibition-mediated inactivation of Foxo3a/Bim signaling pathway and decreased cell injury in melatonin-treated H9c2 cells. Taken together, these findings identified a Rac1/JNK/Foxo3a/Bim signaling pathway in melatonin-induced protective effect against OGD/R injury in H9c2 cells. This study provided a novel insight into the protective mechanism of melatonin against myocardial I/R injury.

scholarly journals Inhibition of DNMT-1 alleviates ferroptosis through NCOA4 mediated ferritinophagy during diabetes myocardial ischemia/reperfusion injury

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Wenyuan Li ◽  
Wei Li ◽  
Yao Wang ◽  
Yan Leng ◽  
Zhongyuan Xia
Keyword(s):  
High Glucose ◽  
Myocardial Injury ◽  
Cell Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
H9c2 Cell ◽  
Nuclear Receptor Coactivator ◽  
Myocardial Ischemia Reperfusion Injury ◽  
Myocardial Ischemia Reperfusion ◽  
Hypoxia Reoxygenation

AbstractThe purpose of this study was to investigate whether inhibition of DNA (cytosine-5)-methyltransferase 1 (DNMT-1) alleviated ferroptosis through nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy during diabetes myocardial (DM) ischemia/reperfusion (I/R) injury (IRI). Rat DM + sham (DS), I/R, and DM + I/R (DIR), H9c2 cell high glucose (HG), hypoxia reoxygenation (H/R), and high-glucose hypoxia reoxygenation (HH/R) models were established. DNMT-1 inhibitor 5-Aza-2’-deoxycytidine (5-aza-CdR) was administered to rat and cell models. The protein level of DNMT-1, NCOA4, FTH, GPX4, Beclin-1, and P62 was detected by western blotting. Compared with normal sham (NS) group, myocardial tissue was injured in DS and I/R models. The level of DNMT-1, NCOA4, and ferroptosis was increased. Moreover, the cell injury was more serious in rat DIR or HH/R model. 5-Aza-CdR could reduce NCOA4-mediated ferritinophagy and myocardial injury in DIR and HH/R models. Moreover, the siRNA for NCOA4 could also reduce the level of ferritinophagy and cell injury in HH/R model. 5-Aza-CdR enhanced the protective effect for NCOA4-siRNA in the process of cell injury. Inhibition of DNMT-1 could reduce ferroptosis during DIR, which the NCOA4-mediated ferritinophagy might be regulated.

scholarly journals Propofol postconditioning ameliorates hypoxia/reoxygenation induced H9c2 cell apoptosis and autophagy via upregulating forkhead transcription factors under hyperglycemia

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Rong-Hui Han ◽  
He-Meng Huang ◽  
Hong Han ◽  
Hao Chen ◽  
Fei Zeng ◽  
...  
Keyword(s):  
Transcription Factors ◽  
High Glucose ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Underlying Mechanism ◽  
Cardiac Cells ◽  
H9c2 Cell ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Hypoxia Reoxygenation

Abstract Background Administration of propofol, an intravenous anesthetic with antioxidant property, immediately at the onset of post-ischemic reperfusion (propofol postconditioning, P-PostC) has been shown to confer cardioprotection against ischemia–reperfusion injury, while the underlying mechanism remains incompletely understood. The FoxO transcription factors are reported to play critical roles in activating cardiomyocyte survival signaling throughout the process of cellular injuries induced by oxidative stress and are also involved in hypoxic postconditioning mediated neuroprotection, however, the role of FoxO in postconditioning mediated protection in the heart and in particular in high glucose condition is unknown. Methods Rat heart-derived H9c2 cells were exposed to high glucose (HG) for 48 h (h), then subjected to hypoxia/reoxygenation (H/R, composed of 8 h of hypoxia followed by 12 h of reoxygenation) in the absence or presence of postconditioning with various concentrations of propofol (P-PostC) at the onset of reoxygenation. After having identified the optical concentration of propofol, H9c2 cells were subjected to H/R and P-PostC in the absence or presence of FoxO1 or FoxO3a gene silencing to explore their roles in P-PostC mediated protection against apoptotic and autophagic cell deaths under hyperglycemia. Results The results showed that HG with or without H/R decreased cell viability, increased lactate dehydrogenase (LDH) leakage and the production of reactive oxygen species (ROS) in H9c2 cells, all of which were significantly reversed by propofol (P-PostC), especially at the concentration of 25 µmol/L (P25) (all P < 0.05, NC vs. HG; HG vs. HG + HR; HG + HR + P12.5 or HG + HR + P25 or HG + HR + P50 vs. HG + HR). Moreover, we found that propofol (P25) decreased H9c2 cells apoptosis and autophagy that were concomitant with increased FoxO1 and FoxO3a expression (all P < 0.05, HG + HR + P25 vs. HG + HR). The protective effects of propofol (P25) against H/R injury were reversed by silencing FoxO1 or FoxO3a (all P < 0.05, HG + HR + P25 vs. HG + HR + P25 + siRNA-1 or HG + HR + P25 + siRNA-5). Conclusion It is concluded that propofol postconditioning attenuated H9c2 cardiac cells apoptosis and autophagy induced by H/R injury through upregulating FoxO1 and FoxO3a under hyperglycemia.

scholarly journals The Exosomal lncRNA KLF3-AS1 From Ischemic Cardiomyocytes Mediates IGF-1 Secretion by MSCs to Rescue Myocardial Ischemia-Reperfusion Injury

2021 ◽  
Vol 8 ◽  
Author(s):  
Gecai Chen ◽  
Aihuan Yue ◽  
Meixiang Wang ◽  
Zhongbao Ruan ◽  
Li Zhu
Keyword(s):  
Myocardial Ischemia ◽  
Myocardial Injury ◽  
Cell Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Myocardial Ischemia Reperfusion

The purpose of the study was to explore the mechanism by which myocardial ischemia-reperfusion (I/R) injury-induced exosomes modulate mesenchymal stem cells (MSCs) to regulate myocardial injury. In this study, we established an I/R injury model in vivo and a hypoxia-reoxygenation (H/R) model in vitro. Then, exosomes isolated from H/R-exposed H9c2 cells were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot analysis. CCK-8 assays and flow cytometry were performed to assess cell injury. ELISA was applied to determine the level of insulin-like growth factor 1 (IGF-1). Echocardiography was used to assess cardiac function in vivo. HE staining and TUNEL assays were conducted to analyze myocardial injury in vivo. In the present study, H/R-exposed H9c2 cells induced IGF-1 secretion from MSCs to inhibit cell myocardial injury. Moreover, exosomes derived from H/R-exposed H9c2 cells were introduced to MSCs to increase IGF-1 levels. The lncRNA KLF3-AS1 was dramatically upregulated in exosomes derived from H/R-treated H9c2 cells. Functional experiments showed that the exosomal lncRNA KLF3-AS1 promoted IGF-1 secretion from MSCs and increased H9c2 cell viability. In addition, miR-23c contains potential binding sites for both KLF3-AS1 and STAT5B, and miR-23c directly bound to the 3'-UTRs of KLF3-AS1 and STAT5B. Furthermore, the lncRNA KLF3-AS1 promoted IGF-1 secretion from MSCs and rescued myocardial cell injury in vivo and in vitro by upregulating STAT5B expression. The lncRNA KLF3-AS1 may serve as a new direction for the treatment of myocardial I/R injury.

Isosteviol protects H9c2 cells against hypoxia-reoxygenation by activating ERK1/2

2021 ◽  
Vol 21 ◽  
Author(s):  
Khaja Shameem Mohammed Abdul ◽  
Neha Faiz ◽  
Aleksandar Jovanović ◽  
Wen Tan
Keyword(s):  
Ischemia Reperfusion ◽  
Underlying Mechanism ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Compound A ◽  
Protein Levels ◽  
Candidate Drug ◽  
Myocardial Ischemia Reperfusion ◽  
Hypoxia Reoxygenation ◽  
In Cells

Aims: In the present study, we have investigated cardioprotective properties of Isosteviol (STV) under conditions of hypoxia-reoxygenation and elucidated underlying mechanism. Background: In our previous studies, we have determined that STV exhibits neuro- and cardio-protective properties. However, the mechanism underlying STV-induced cardioprotection has not been yet fully understood. Methods: All experiments were performed on rat heart embryonic H9c2 cell line. To induce hypoxia-reoxygenation cells were exposed to 1% oxygen (in no glucose and no sodium pyruvate DMEM) following by reoxygenation (using fully supplemented MEM). Cells viability was tested by MTT assay and protein levels were compared by Western blotting. Conclusions: Taken all together, our finding demonstrate that 1) STV protects H9c2 cells against hypoxia-reoxygenation and that 2) this effect is mediated via ERK1/2. The property of STV that selectively activates ERK1/2 in cells exposed to stress, but not in cells under non-stress conditions makes this compound a promising candidate-drug for a therapy against myocardial ischemia-reperfusion in clinical practice.

scholarly journals MiR-21 mediates the protection of kaempferol against hypoxia/reoxygenation-induced cardiomyocyte injury via promoting Notch1/PTEN/AKT signaling pathway

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0241007
Author(s):  
Jinxi Huang ◽  
Zhenhui Qi
Keyword(s):  
Signaling Pathway ◽  
Cell Injury ◽  
Akt Signaling ◽  
Flavonoid Compound ◽  
H9c2 Cell ◽  
H9c2 Cells ◽  
Akt Signaling Pathway ◽  
Down Regulation ◽  
Apoptotic Protein ◽  
Hypoxia Reoxygenation

Kaempferol, a natural flavonoid compound, possesses potent myocardial protective property in ischemia/reperfusion (I/R), but the underlying mechanism is not well understood. The present study was aimed to explore whether miR-21 contributes to the cardioprotective effect of kaempferol on hypoxia/reoxygenation (H/R)-induced H9c2 cell injury via regulating Notch/phosphatase and tensin homologue (PTEN)/Akt signaling pathway. Results revealed that kaempferol obviously attenuates H/R-induced the damages of H9c2 cells as evidence by the up-regulation of cell viability, the down-regulation of lactate dehydrogenase (LDH) activity, the reduction of apoptosis rate and pro-apoptotic protein (Bax) expression, and the increases of anti-apoptotic protein (Bcl-2) expression. In addition, kaempferol enhanced miR-21 level in H9c2 cells exposed to H/R, and inhibition of miR-21 induced by transfection with miR-21 inhibitor significantly blocked the protection of kaempferol against H/R-induced H9c2 cell injury. Furthermore, kaempferol eliminated H/R-induced oxidative stress and inflammatory response as illustrated by the decreases in reactive oxygen species generation and malondialdehyde content, the increases in antioxidant enzyme superoxide dismutase and glutathione peroxidase activities, the decreases in pro-inflammatory cytokines interleukin (IL)-1β, IL-8 and tumor necrosis factor-alpha levels, and an increase in anti-inflammatory cytokine IL-10 level, while these effects of kaempferol were all reversed by miR-21 inhibitor. Moreover, results elicited that kaempferol remarkably blocks H/R-induced the down-regulation of Notch1 expression, the up-regulation of PTEN expression, and the reduction of P-Akt/Akt, indicating that kaempferol promotes Notch1/PTEN/AKT signaling pathway, and knockdown of Notch1/PTEN/AKT signaling pathway induced by Notch1 siRNA also abolished the protection of kaempferol against H/R-induced the damage of H9c2 cells. Notably, miR-21 inhibitor alleviated the promotion of kaempferol on Notch/PTEN/Akt signaling pathways in H9c2 cells exposed to H/R. Taken together, these above findings suggested thatmiR-21 mediates the protection of kaempferol against H/R-induced H9c2 cell injuryvia promoting Notch/PTEN/Akt signaling pathway.

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