6-Gingerol Pretreatment Alleviates Hypoxia/Reoxygenation-induced H9C2 Cardiomyocyte Injury by Inhibiting Oxidative Stress and Inflammation

QIN Qiu-yu, LÜ Xiang-wei, WU Qi, LU Pan, ZHAO Wei-kun

Abstract

To observe the effect of 6-gingerol (6-G) pretreatment on hypoxia/reoxygenation (H/R) induced injury in H9C2 myocardial cell and investigate its related mechanism.  Methods  The H/R in vitro model of cardiomyocytes was prepared by conventional methods. In detail, H9C2 cells were added with the nitrogen-saturated hypoxic liquid, and placed in an incubator, mixed with gas (1% O2, 5% CO2, 94% N2) applying for 15 min. After culturing for 3 h, the cells were taken out and placed in an incubator (37℃, 5% CO2) for 1 h. Before establishing the cell model, the cells were pretreated with 6-G, and the cell viability was measured by MTT method to observe the protective effect of different concentrations of 6-G on H/R-induced cell damage. The 6-G mass concentration for pretreatment that led to the highest cell viability was used for follow-up experiments. DCFH-DA fluorescent probe was used to detect the effect of 6-G pretreatment on H9C2 oxidative stress level, and the intracellular oxidative stress was observed with fluorescence microscope and flow cytometry. Western blot method was used to detect the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in H/R-induced cell inflammatory responses.  Results  Compared with the H/R group, the cell viability of the 6-G+H/R group began to increase when the concentration of 6-G promoted to50 μg/mL. The cell viability was the highest after pretreated with 200 μg/mL 6-G. Therefore, 200 μg/mL was considered as the best 6-G intervention concentration for subsequent experiment. The content of reactive oxygen species (ROS) in the 200 μg/mL 6-G group had no significant changes compared with the control group (P>0.05), and the ROS fluorescence peak did not migrate significantly. However the ROS content in the H/R group increased significantly compared with the control (P<0.05), and the ROS fluorescence peak shifted to the right. Compared with the H/R group, the ROS content of the 6-G+H/R group decreased (P<0.05), and the ROS fluorescence peak shifted to the left. Compared with the control group, the expressions of TNF-α, IL-6, IL-1β in the 6-G group had no significant changes (P>0.05); the expressions of TNF-α, IL-6, IL-1β in the H/R group increased (P<0.05). Compared with H/R group, the expressions of TNF-α, IL-6 and IL-1β in 6-G+H/R group decreased (P<0.05).  Conclusion  6-G pretreatment can alleviate H/R-induced H9C2 myocardial injury, which may be related to the inhibition of oxidative stress and inflammatory responses.

 

Keywords: 6-Gingerol, Hypoxia/reperfusion, Oxidative stress, Inflammation, H9C2 myocardial cell

 

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