The cytotoxicity and protective effects of Astragalus membranaceus extracts and butylated hydroxyanisole on hydroxyl radical-induced apoptosis in fish erythrocytes

Erythrocytes play an essential role in transporting O2and CO2for respiration in fish. However,erythrocytes continuously suffer from reactive oxygen species(ROS)-induced oxidative stress and apoptosis. Thus, it is essential to expand our knowledge of how to protect erythrocytes against ROS-induced oxidative stress and apoptosis in fish. In this study, we explored the cytotoxicity and the effects of butylated hydroxyanisole(BHA), ethyl ether extracts, ethyl acetate extracts, acetone extracts(AE), ethanol extracts, and aqueous extracts of Astragalus membranaceus(EAm) on hydroxyl radical(·OH)-induced apoptosis in carp erythrocytes. The rat hepatocytes and carp erythrocytes were incubated with different concentrations of BHA or EAm(0.125 to 1 mg/mL). The toxicity in rat hepatocytes and carp erythrocytes was then measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay and a haemolysis assay,respectively. The carp erythrocytes were treated with BHA or EAm in the presence of 40 μmol/L FeSO4 and20 μmol/L H2O2at 37℃, except for the control group. Oxidative stress and apoptosis parameters in the carp erythrocytes were then evaluated using the commercial kit. The results indicated that at high concentrations, BHA and EAm could induce toxicity in rat hepatocytes and fish erythrocytes. However, BHA was more toxic than EAm at the same concentrations. Moreover, the toxicity order of BHA and EAm in the fish erythrocytes approximately agreed with that for the rat hepatocytes. Butylated hydroxyanisole and EAm suppressed the ·OH-induced phosphatidylserine exposure and DNA fragmentation(the biomarkers of apoptosis) by decreasing the generation of ROS, inhibiting the oxidation of cellular components, and restoring the activities of antioxidants in carp erythrocytes. Of all of the examined EAm, the AE showed the strongest effects. The effects of AE on superoxide anion, H2O2, met-haemoglobin and reduced glutathione levels, as well as glutathione reductase activity and apoptosis were equivalent to or stronger than those of BHA. These results revealed that the AE of Astragalus membranaceus could be used as a potential natural antioxidant or apoptosis inhibitor in fish erythrocytes.