Methylene Blue Protects Against Oxidative Damage In RAW264.7 Cells By Enhancing The Expression And Activity Of Heme Oxygenase-1

Methylene blue?MB?is a kind of phenothiazine salts,which is used to treat methemoglobinemia and cyanide poisoning.It has been reported that MB had antioxidant and mitochondrial protective effects.MB could reduce the oxidative damage caused by ischemia and reperfusion.Recently,it has been found that the molecular mechanism of MB mitochondrial protection may be related to the upregulation of Nrf2/ARE genes.As a downstream product of Nrf-2,HO-1 is a key enzyme in cell defense.It plays a key role in the protection of ROS against cell damage and protects against noxious stimuli-induced inflammation and oxidative damage.The purpose of this study was to study the protective effect of MB on oxidative damage induced by hydrogen peroxide?H₂O₂?-induced RAW264.7macrophages and to elucidate the protective mechanism.In this study,an oxidative stress model was established in RAW264.7 cells,and the cells were pretreated and post-treated with different concentrations of MB.The degree of cell damage was detected by MTT and lactate dehydrogenase?LDH?.Reactive oxygen species?ROS?concentration,H₂O₂ activity,superoxide dismutase?SOD?activity and adenosine triphosphate?ATP?were used to detected oxidative damage degree.Meanwhile,High Content Screening was employed to test mitochondrial membrane potential?MMP?and intracellular calcium concentration.Finally,apoptosis of RAW264.7 cells was detected by flow cytometry and protein assay of caspase 3 by western-blot.The results showed that both 0.1 and 1?M concentrations of MB pretreatment and posttreatment could protect the oxidative damage induced by H₂O₂ in RAW264.7 cells,especially the pretreatment of MB at a concentration of0.1?M which had an effective reduction of cell damage,enhanced mitochondrial function,and decreased apoptosis.In order to elucidate wether HO-1 participate in the protective role of MB on H₂O₂-induced RAW264.7 cellular injury,the expression and activity of HO-1 protein was examined.The results showed that both the pretreatment and post-treatment at concentrations of 0.1 and 1?M MB were increased HO-1 protein expression in H₂O₂-treated RAW264.7cells.Then,HO-1 protein expression and enzyme activity were inhibited by HO-1 siRNA and the HO-1 activity inhibitor zinc protoporphyrin when MB administration.The results showed that HO-1 siRNA or zinc protoporphyrin abolished the beneficial effects of MB on H₂O₂-induced cell injury,oxidative stress,and mitochondrial damage.It can be seen from the results that the protective effect of MB on H₂O₂-induced cell injury was at least partly dependent on HO-1.In conclusion,MB protects H₂O₂-induced oxidative damage of RAW264.7 cells by up-regulating HO-1 protein expression and enzyme activity.