Betulinic acid derivatives can protect human Muller cells from glutamate-induced oxidative stress

Muller cells are the predominant retinal glial cells. One of the key roles of Muller cells is in the uptake of the neurotransmitter glutamate and in its conversion to glutamine. Muller cell dysfunction due to oxidative stress elicited by high glutamate concentrations can lead to toxicity, which promote the pathogenesis of retinal diseases like diabetic retinopathy and glaucoma. This study investigated the anti-oxidant activity and mechanisms of betulinic acid (BA) and its derivatives in human Muller cells. Human MIO-M1 Muller cells were pre-treated in the presence or absence of BA, BE as well as their derivatives (named H3-H20) followed by incubation with glutamate. Cell viability was evaluated with the MTT and calcein-AM assays. Reactive oxygen species (ROS) production in MIO-Ml cells was measured using CM-H(2)DCFDA and flow cytometry. The activation of cellular apoptosis and necrosis was analyzed with annexin V/PI staining and flow cytometry. The modulation of signaling pathways involved in glutamate-mediated cytotoxicity and ROS production was evaluated by immunoblotting. The BA derivatives H3, H5 and H7 exhibited minimal cytotoxicity and significant anti-oxidant activity. These compounds significantly suppressed ROS production and attenuated cellular necrosis elicited by glutamate-induced oxidative stress. The protective effects of H3, H5 and H7 in MIO-M1 cells were associated with the attenuation of Akt, Erk, and JNK signaling. The BA analogues H3, H5 and H7 are protective against glutamate-induced oxidative stress in human Muller cells, and elicit their actions by modulation of the Erk, Akt and JNK signaling pathways. These agents are potential candidate molecules for the prevention or treatment of human retinal diseases.