Title: Effects of rosmarinic acid on oxidative stress injury of H9C2 cardiomyocytes
Abstract: Objective
To investigate the effect of rosmarinic acid (RA) on oxidative stress injury of H9C2 cardiomyocytes.
Methods
The oxidative stress injury model was established by hydrogen peroxide (H2O2) treatment. H9C2 cardiomyocytes were cultured in vitro and randomly divided into control group, H2O2 group and H2O2+RA group. The H2O2 group was treated with 400 μmol/L H2O2 for 4h to establish the oxidative stress injury model; H2O2+RA group was treated with low dose (5μg/ml) , medium dose (10 μg/ml) and high dose (20 μg/ml) of RA for 24 h before adding H2O2. CCK-8 assay was used to detect the survival rate of H9C2 cardiomyocytes; DCFH-DA probe with a microplate reader was used to measure the intracellular reactive oxygen species (ROS) content; specific kits were used to measure the amount of lactate dehydrogenase (LDH) leakage, intracellular malondialdehyde (MDA) content, and activities of catalase (CAT) and glutathione peroxidase (GSH-Px) . Western blotting and real-time fluorescence quantitative PCR were used to detect the levels of reduced coenzyme II oxidase 2 (NOX2) , superoxide dismutase 2 (SOD2) and the expression of apoptosis-related genes Bax, Bcl-2, cleavage-caspase 3 (c-cas 3) and caspase 3 (cas 3) ; TUNEL kit was used to examine the cell apoptosis.
Results
(1) The cell viability of all H2O2-treated group was lower than that of the control group. Lower-dose RA did not, but higher-levels RA (≥30 μg/ml) significantly inhibited the growth of H9C2 cardiomyocytes. (2) Compared with the control group, the cell survival rate in the H2O2 group decreased by 52.9%, and the LDH leakage increased by 73.8%. RA pretreatment increased the cell survival rate and decreased the LDH leakage, and such protective effect increased significantly along with the RA concentration. (3) Compared with the control group, H2O2 group had significantly elevated intracellular oxidation as reflected by a 146.7% increased in MDA level and a 192.2% increase in ROS level. RA pretreatment resulted in reduced ROS production and lower intracellular MDA content, which was more obvious with higher doses of RA. (4) Compared with the control group, apoptosis in the H2O2 group was obvious. RA pretreatment could improve H2O2-induced apoptosis, and the improvement was more prominent with higher doses of RA.
Conclusion
RA exhibits significant protective effect on H9C2 cardiomyocytes under oxidative stress.
Key words:
Myocytes, heart; Oxidative stress; Apoptosis; Rosmarinic acid
Publication Year: 2017
Publication Date: 2017-12-15
Language: en
Type: article
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