Hydrogen sulfide epigenetically attenuates homocysteine-induced mitochondrial toxicity mediated through NMDA receptor in mouse brain endothelial (bEnd3) cells

Previously we have shown that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulfide (H₂S) has potent anti-inflammatory, anti-oxidative, and anti-apoptotic effects. Therefore, in the present study we examined whether H₂S ameliorates Hcy-induced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100μM Hcy treatment in the presence or absence of 30μM NaHS (donor of H₂S) for 24h. Hcy-activate NMDA receptor and induced mitochondrial toxicity by increased levels of Ca²⁺, NADPH-oxidase-4 (NOX-4) expression, mitochondrial dehydrogenase activity and decreased the level of nitrate, superoxide dismutase (SOD-2) expression, mitochondria membrane potentials, ATP production. To confirm the role of epigenetic, 5'-azacitidine (an epigenetic modulator) treatment was given to the cells. Pretreatment with NaHS (30μM) attenuated the Hcy-induced increased expression of DNMT1, DNMT3a, Ca²⁺, and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also mitigated mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcy-induced LC3-I/II, CSE, Atg3/7, and low p62 expression which confirm its effect on mitophagy. Likewise, NaHS also restored level of eNOS, CD31, VE-cadherin and ET-1 and maintains endothelial function in Hcy treated cells. Molecular inhibition of NMDA receptor by using small interfering RNA showed protective effect whereas inhibition of H₂S production by propargylglycine (PG) (inhibitor of enzyme CSE) showed mitotoxic effect. Taken together, results demonstrate that, administration of H₂S protected the cells from HHcy-induced mitochondrial toxicity and endothelial dysfunction.