Carnosine protects brain microvascular endothelial cells against rotenone‐induced oxidative stress injury through histamine H1 and H2 receptors in vitro

Abstract

Although it is believed that carnosine has protective effects on various cell types, its effect on microvascular endothelial cells has not been well defined. In the present study, we investigated the protective effects of carnosine in microvascular endothelial cells using an in vitro rotenone-induced oxidative stress model. Mouse brain microvascular endothelial cells were exposed to 1μmol/L rotenone for 18h. In some experiments, carnosine (100nmol/L-1mmol/L) was added 30min prior to rotenone exposure. When used, histamine receptor antagonists (100nmol/L-10μmol/L) were added 15min before carnosine treatment. After rotenone exposure, apoptosis of microvascular cells was analysed by Hoechst 33342 staining, whereas mitochondrial membrane potential was assessed by JC-1 staining. Intracellular carnosine and histamine levels were determined using HPLC or ultra-HPLC. Over the range 1μmol/L-1mmol/L, carnosine concentration-dependently decreased the number of apoptotic cells after 18h exposure to rotenone. This effect was reversed by the histamine H1 receptor antagonists pyrilamine and diphenhydramine (1 and 10μmol/L) and the H2 receptor antagonists cimetidine (100nmol/L-10μmol/L) and zolatidine (10μmol/L). α-Fluoromethylhistidine (100μmol/L), a selective and irreversible inhibitor of histidine decarboxylase, also significantly inhibited the protective effects of carnosine. At 0.1mmol/L, carnosine restored the decrease in mitochondrial membrane potential after 6h exposure to 1μmol/L rotenone and this effect was also reversed by the H1 and H2 receptor antagonists. Moreover, intracellular carnosine levels increased as early as 1h after carnosine treatment, whereas intracellular histamine levels increased 18h after carnosine treatment. The results of the present study indicate that carnosine protects brain microvascular endothelial cells against rotenone-induced oxidative stress injury via histamine H1 and H2 receptors. The findings suggest that carnosine may be beneficial in the treatment of microvascular endothelial dysfunction induced by oxidative stress.