Low Concentration Ethanol Prevents Oxidative Stress-Induced Cardiac Cells Injury Under Hyperglycemic Conditions by Inhibiting the SAPK/JNK Signaling Pathway and ROS Generation

Research Article

Gerontol Geriatr Res. 2021; 7(4): 1062.

Low Concentration Ethanol Prevents Oxidative Stress- Induced Cardiac Cells Injury Under HyperglycemicConditions by Inhibiting the SAPK/JNK Signaling Pathway and ROS Generation

Tian Y1, Tian W3*, Bai Y2, Zhang A4, Zhu L4, Zhang X4 and Xu J2*

1School of Basic Medical Sciences, North China University of Science and Technology, The Hebei Key Laboratory for Chronic Diseases, Tangshan, Hebei Province, China

2School of Public Health, North China University of Science and Technology, Tangshan, Hebei Province, China

3Integrated Testing Centre, North China University of Science and Technology, Tangshan, Hebei Province, China

4Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin 300010, China

*Corresponding author: Wei Tian, North China University of Science and Technology, 21 Bohai Avenue, Caofeidian District, Tangshan, Hebei, 063000, China

Jingman Xu, North China University of Science and Technology, 21 Bohai Avenue, Caofeidian District, Tangshan, Hebei, 063000, China

Received: July 31, 2021; Accepted: August 25, 2021; Published: September 01, 2021

Abstract

The purpose of this study was to determine the effects and mechanisms of ethanol on oxidative stress-induced cardiac H9c2 cells mitochondrial injury under hyperglycemic conditions. Under hyperglycemic conditions, ethanol pretreatment (10-100 μM) prevented H2O2-induced mitochondria swelling, as well as decreased cell viability and Respiratory Control Ratio (RCR) in the H9c2 cells. It also prevented TMRE fluorescence intensity loss and DCF fluorescence intensity increase under hyperglycemic conditions. These effects of ethanol were reversed by the SAPK/JNK agonist, anisomycin. Finally, treatment of H9c2 cells with 33mM glucose significantly enhanced Akt and ERK phosphorylation, which was not affected by ethanol. However, ethanol decreased the phosphorylation of SAPK/JNK under hyperglycemic conditions. Collectively, these findings indicate that under hyperglycemic conditions, that ethanol prevents oxidative stress-induced mitochondrial injury in cardiac H9c2 cells by preventing ROS generation via inhibiting the SAPK/JNK signaling pathway.

Keywords: Ethanol; Hyperglycemia; Oxidative stress injury; SAPK/JNK

Abbreviations

Δψm: Mitochondrial Membrane Potential; OCR: Oxygen Consumption Rate; RCR: Respiratory Control Ratio; SAPK/JNK: Stress-Activated Protein Kinase/c-Jun N-terminal Kinase

Introduction

Although many studies suggest that alcohol consumption is closely associated with cardiovascular disease in diabetic populations [1,2], several studies have demonstrated that moderate consumption of alcohol has a direct cardioprotective effect on myocardium in various experimental models [3-5]. The beneficial effect of ethanol on ischemic heart disease was proposed to be attributable to its positive effects on antioxidant capacity [6], lipid profile (Lindberg and Amsterdam, 2008) and the coagulation system [7]. Since several signaling pathways have been proposed to be essential for ethanolinduced cardioprotection against oxidative injury in nondiabetic people [4,7,8], these pathways may be also involved in ethanolinduced cardioprotective effects under diabetic conditions.

The mitochondrial Permeability Transition Pore (mPTP) opening has been proposed as a critical determinant of myocardial oxidative stress [9-11]. Zhou et al. [4] reported that ethanol at low doses can prevent oxidant-induced mPTP opening by activating Phosphatidyl Inositol 3-Kinase (PI3K)/v-akt-murinet hymoma viral onco-gene homolog (Akt) signaling pathway. Although the signal factors such as Akt\and extracellular signal-regulated kinase (ERK) have been proposed to be linked to the inhibition of the mPTP opening in nondiabetic models, the exact signaling mechanism by which cardioprotective interventions prevent mitochondrial oxidant injury in diabetic patients remains unclear [12,13].

In the current study, we hypothesized that ethanol prevents oxidative stress induced-mPTP opening in cardiac H9c2 cells cultured in high glucose conditions. We also sought to define the signaling mechanism by which ethanol prevents the mitochondrial oxidative stress injury.

Materials and Methods

Cell culture

The rat heart tissue-derived H9c2 cardiac myoblast cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100U penicillin/streptomycin at 37oC in a humidified 5% CO2-95% air atmosphere.

Chemicals and antibodies

Ethanol was purchased from Sigma Chemical (St. Louis, MO); SAPK/JNK agonist anisomycin was purchased from LC Laboratories (LC, USA); Tetramethylrhodamine ethyl ester (TMRE) was from Molecular Probes (Eugene, OR, USA); 2’,7’-dichlorofluorescein diacetate (H2DCF-DA) was from Beyotime (Shanghai, China); and all antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Cell viability assays

For cell viability assays, cells were plated in 96-well dishes (10000 cells/well) and grown over 24h. Then cells were treated with different doses of glucose. Cell viability was assessed using the CCK-8 assay (Beyotime, Shanghai, China) according to the manufacturer’s instruction.

Confocal imaging of mitochondrial membrane potential (Δψm)

Δψm was measured by loading cardiomyocytes with Tetramethylrhodamine Ethyl Ester (TMRE). TMRE is a cell permeable, cationic, nontoxic, fluorescent dye that specifically stains live mitochondria. TMRE is accumulated specifically by the mitochondria in proportion to membrane potential. A number of studies have measured Δψm by imaging cardiac cells loaded with TMRE [4,14]. Briefly, cardiac cells cultured in a specific temperaturecontrolled culture dish (120,000 cells/dish) were incubated with TMRE (100nM) in a standard Tyrode solution containing (in mM) NaCl 140, KCl 6, MgCl2 1, CaCl2 1, HEPES 5 and glucose 5.8 (pH 7.4) for 10min. Cells were then mounted on the stage of an Olympus FV 1000 laser scanning confocal microscope. The red fluorescence was excited with a 543-nm line of argon-krypton laser line and imaged through a 560-nm-long path filter. Temperature was maintained at 37oC.

Confocal imaging of intracellular ROS

H9c2 cells (120,000 cells/dish) were plated in specific temperaturecontrolled culture dishes and grown over 24h. Then cells were treated with different doses of glucose. Cells were incubated with 20μM H2DCF-DA in a standard Tyrode solution containing (in mM) NaCl 140, KCl 6, MgCl2 1, CaCl2 1, HEPES 5 and glucose 5.8 (pH 7.4) for 20 min. After washing with phosphate buffered saline (PBS), the cells were incubated in new standard Tyrode solution for ROS measurements. Cells were then mounted on the stage of an Olympus FV 1000 laser scanning confocal microscope. The DCF fluorescence was excited at 480nm and collected at 530nm. Temperature was maintained at 37oC throughout the experiment.

Western blot analysis

Equal amounts of protein lysates (40μg) were loaded and electrophoresed on SDS-polyacrylamide gel and transferred to a PVDF membrane. Membranes were probed with primary antibodies that recognize phosphorylation of Akt (Ser473), ERK (Thr202/Tyr204) and SAPK/JNK (Thr183/Tyr185). Each primary antibody binding was detected with a secondary antibody and visualized by the Enhanced Chemiluminescence (ECL) method. Equal loading of samples were confirmed by reprobing membranes with anti-Tubulin antibody. The images were recorded on a computer and were quantified by Image J.

Experimental protocols

To determine the effect of hyperglycemia on cardiac H9c2 cells, cells were cultured with glucose (5.5, 22, 33 mM) DMEM from 12 to 48 h separately. To examine the effect of ethanol on cardiac H9c2 cells under oxidative stress and hyperglycemic conditions, cells were cultured with high glucose (33mM) for 48h before exposure to H2O2 (550μM) for 20min. Ethanol (1-1000 μM) was given 1 h before exposure to H2O2. Anisomycin (4μM) was given 10min before exposure to ethanol. To examine the effect of ethanol on Akt (or ERK, SAPK/JNK) phosphorylation, cells were exposed to ethanol for 1h.

Statistical analysis

Data were collected from repeated experiments and are presented as mean ± SD. One-way ANOVA and the Student’s t test were used for statistical analysis. Differences were considered to be significant at P <0.05.

Results

Hyperglycemia increases cardiac H9c2 cells mitochondrial injury

Hyperglycemia is a major risk factor for cardiovascular disease. To determine the effects of high doses of glucose on H9c2 cells, we first examined the change of cell viability. As shown in Figure 1, compared to the cells cultured in 5.5mM glucose, treatment of cells with 33mM glucose for 48h decreased the cell viability markedly, implying that hyperglycemia increases H9c2 cells injury. In addition, the Δψm and cell viability were tested to evaluate the oxidative stress injury of cells cultured in different glucose conditions. Compared to the control group, the Δψm and cell viability decreased in the H2O2 group, implying that oxidative stress increases mitochondrial injury in different glucose conditions. The data suggests that oxidative stress-induced cell injury is increased in a glucose dependent manner (Figure 2A-2D).