Heme oxygnease-1 induction by methylene blue protects RAW264.7 cells from hydrogen peroxide-induced injury
Authors: Xue-Qiang Sun
Xiao-Tong Zhang
Jia-Jia Yuan
Chen Wu
Qing-Feng Pang
Jun-Liang Chen
Zhi-Ping Wang
Journal: Biochemical Pharmacology (2018)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: Apoptosis; Heme oxygnease-1; Methylene blue; Mitochondrion; RAW264.7.
Affiliations: Wuxi Medical School, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China., Wuxi Medical School, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Electronic address: ., Department of Anesthesiology, The Wuxi People Hospital, 200 Qingyang Road, Wuxi 214023, China. Electronic address: .

Abstract:  Although methylene blue (MB) has showed strong antioxidant effect, its effect related with heme oxygenase-1 (HO-1) is still unclear. Thus, we investigated the effects of MB on HO-1 protein content and enzyme activity, and its protective effect against hydrogen peroxide (H2O2)-induced oxidative damage in RAW264.7 macrophage. The cell viability and the release of lactate dehydrogenase of RAW264.7 were determined. The mitochondrial functions were valuated through these indexes: content of adenosine triphosphate, superoxide dismutase, concentration of reactive oxygen species and mitochondrial membrane potential. Meanwhile, high content screening tested generation of ROS, MMP and intracellular concentration of calcium ion. qRT-PCR valuated macrophage phenotype markers expression. Lastly, flow cytometry and caspase-3 detection analyzed RAW264.7 apoptosis. Our data showed that (1) Both pretreatment and posttreatment of MB increased HO-1 protein content and enzyme activity; (2) MB rescued cells from H2O2-induced mitochondrial dysfunction; (3) High content screening revealed that MB alleviated the changes including generation of reactive oxygen species, mitochondrial membrane potential and intracellular concentration of calcium ion in H2O2 exposed RAW264.7; (4) MB attenuated H2O2-induced apoptosis; (5) MB pretreatment decreased the expression of M1 macrophage markers (Tnf and Nos2) while increasing the expression of M2 macrophage markers (Mrc1 and Il10); (6) The beneficial effect of MB was abolished by zinc protoporphyrin IX (HO-1 activity inhibitor) or HO-1 siRNA. In summary, MB protects RAW264.7 cells from H2O2-induced injury through up-regulation HO-1. 

Retracted: No

PubPeer comments:
 In Figure 8, two micrographs have been duplicated and presented as something else.  , 
 Dear Tabebuia Rosea: Thanks a lot for your valuable comment. You do help us to discover and identify one big mistake in the published article. We set off to collect all the primary data referring to the original picture. After carefully examining our original data, we found that the errors were caused by our carelessness in exporting the representative confocal images. When we prepared figures, we pasted the same ROS monitoring images image into the two panels rather than pasting the correct images in Figure 8A. The image shown in Figure 8A in MB group is correct. The image shown in Figure 8A in Control group is incorrect. We have now replaced these misplaced panels with correct ones in Fig 8A Control group. Figure 8B shown that the effect of HO-1siRNA on the anti-oxidative role of MB in RAW264.7 cells. There are results of separate experiments in Fig8A and 8B. We synthesize nonspecific siRNA scrambled duplex (NC siRNA) and HO-1siRNA. In principle. NC siRNA cannot decreased H2O2-induced ROS in RAW264.7 cells. Therefore, in NC siRNA+ H2O2 group, fluorescent probe DHE ROS monitoring images maybe be similar to that in Figure 8A H2O2 group. We assure that the data presented now are real and reliable without any subjective processing. We would also like to state that the image substitutions do not change the conclusions of this paper. The authors regret in the original version of the article and will make the corrigendum. We apologize for this mistake due to our carelessness. Thank you again for helping us to find and correct these errors. Sincerely Pang Here are the corrected figures in figure 8  Fig. 8. ZnPP and HO-1siRNA abolished the anti-oxidative role of MB in RAW264.7 cells. (A and B) ROS monitoring images were used the fluorescent probe DHE; (C and D) ROS production detected by the DCFH-DA. (E and F) MMP was measured using the fluorescent dye rhodamine 123. Values represented mean u00b1 SD of five independent experiments. *, significance compared with control group (P 0.05); #, significance compared with H2O2 group (P 0.05); , significance compared with MB + H2O2 group (P 0.05). The another representative picture supply in figure 8A H2O2 group (left) and figure 8B NC siRNA+ H2O2 group (right)   (http: ) ,