Effect of methylene blue analogues on glutamate-induced HT22 neuron cell injury and its mechanism
CHEN Ziwei1, CHEN Qiuhe2, CAI Yu1, HOU Shanshan1, LIU Fuhe1.
1.Pharmaceutical Department, Zhejiang pharmaceutical college, Ningbo 315000, China; 2.School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
CHEN Ziwei,CHEN Qiuhe,CAI Yu, et al. Effect of methylene blue analogues on glutamate-induced HT22 neuron cell injury and its mechanism[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2022, 52(12): 956-964,971.
Abstract：Objective: To investigate the effect of methylene blue (MB) analogues (2-Chlorophenothiazine,Neutral red, Azure A and Azure B) on glutamate-induced HT22 neuronal cell injury and its mechanism. Methods:The toxicity effect of 2-Chlorophenothiazine (2-C), Azure A (A-A), Azure B (A-B) and Neutral red (NR) on HT22 neuronal cells were detected by MTT. The protect effect of methylene blue analogues against glutamateinduced cell death was detected by MTT and LDH release assay; ROS production was measured by H2DCF-DA staining; siRNA was used to download the expression of MEF2D. Western blot was used to detect the protein levels of MEF2D, ND6, Akt and GSK-3β. Results: LD50 of compound was calculated according to the results of MTT experiment,: MB (50.66 μmol/L), 2-C (175.90 μmol/L), A-A (152.40 μmol/L), A-B (140.40 μmol/L), NR (73.19 μmol/L). Except for the NR, the other compounds had significant protective effect on glutamate-induced oxidative damage of HT22 cells. The EC50 value of each compound was calculated as follows: MB (42.13 nmol/L),2-C (34.32 nmol/L), A-A (48.96 nmol/L), A-B (44.42 nmol/L). MB, 2-C, A-B and A-A significantly reduced glutamate-induced ROS generation in HT22 cells detected by H2DCF-DA staining at 100 nmol/L. However, NR had no obvious effect on glutamate-induced ROS generation in HT22 cells. MB, 2-C, A-B and A-A elevated the protein level of MEF2D (P<0.05). To investigate the role of MEF2D in the neuroprotective effect of MB analogues against glutamate-induced cell death, we knocked down MEF2D by siRNA in HT22 cells (P<0.05). MTT results showed that the knockdown of MEF2D abolished part of neuroprotective effect of MB, 2-C, A-B and A-A against glutamate in HT22 cells. Further studies on the mechanism showed that 2-C, A-B, A-A and MB could activate Akt, increase GSK-3β phosphorylation (P<0.05) and inactivate GSK-3β, which may be related to the activation of MEF2D and ND6. Conclusion: Compared with MB, 2-C has lower cytotoxicity, and stronger damage activity to the anti-glutamate-induced cells.Compounds with phenothiazine structure such as 2-C, A-B,A-A and MB have excellent antioxidant activity. The Akt/GSK-3β/MEF2D/ND6 pathway may be related to its antioxidant activity.
