亚甲基蓝减轻创伤后应激障碍模型小鼠的神经元焦亡

Methylene blue alleviate neuronal pyroptosis in animal models of post-traumatic stress disorder

摘要:

【摘要】目的 研究亚甲基蓝（MB）对创伤后应激综合症模型（PTSD）小鼠的神经炎症和神经元焦亡的影响及其分子机制。 方法 60只C57BL/6小鼠被随机分为对照组（Control组）、模型组（PTSD组）、低剂量治疗组（MB 2mg 组）及中剂量组（MB 10mg 组）4组。PTSD 组采用条件性足部电击( CF) 和单次-持续应激( SPS) 来制备 PTSD 动物模型。通过旷场实验和高架十字迷宫实验来检测小鼠模型焦虑和抑郁反应。通过胡须刺激测试和条件性恐惧测试检测模型小鼠的恐惧等行为学。利用 Western Blot 检测NLRP3、Caspase1、和GSDMD的蛋白水平表达。利用免疫组织化学染色检测 GSDMD的表达。利用ELISA试剂盒检测IL-1β和IL-18等。 结果 与Con组比较，PTSD组小鼠表现出明显的焦虑、抑郁和恐惧等行为学改变；GSDMD的阳性细胞数和蛋白水平显著升高（P<0.05），且NLRP3和Cleaved-Caspase1的表达也显著增加；同时IL-1β和IL-18等炎性因子释放显著增加，导致神经元焦亡增加。无论低剂量或中剂量MB治疗后，小鼠的应激样症状均获得改善，焦亡通路的相关分子也表达下降（P<0.05），神经元焦亡明显减少；2mg与10mg MB在治疗效果上无明显差异（P>0.05）。 结论 中、低剂量的MB均显著改善PTSD小鼠的行为学异常，其机制可能是通过抑制NLRP3炎性小体激活，下调GSDMD表达，抑制神经炎症反应进而减轻神经细胞焦亡。

Abstract:

【Abstract】Objective To investigate the effect of methylene blue (MB) on inflammatory response and neuronal pyroptosis in a post-traumatic stress disorder (PTSD) mouse model and its molecular mechanisms. Methods Sixty C57BL/6 mice were randomly divided into four groups: control group (Con group), model group (PTSD group), low-dose treatment group (MB 2 mg group), and medium-dose treatment group (MB 10 mg group). The PTSD group was subjected to conditioned foot shock (CF) and single prolonged stress (SPS) to establish the PTSD animal model. The open field test (OFT) and elevated plus maze (EPM) test were used to assess anxiety- and depression-like behaviors in the model mice. Whisker stimulation test and conditioned fear test were employed to evaluate fear-related behaviors. Western blot was used to measure the protein expression levels of NLRP3, Caspase1, and GSDMD. Immunohistochemical staining was performed to detect GSDMD expression. ELISA kits were used to measure the levels of inflammatory cytokines such as IL-1β and IL-18. Results Compared with the Con group, the PTSD group exhibited significant anxiety-, depression-, and fear-like behavioral changes. The number of GSDMD-positive cells and its protein expression level were significantly increased (P < 0.05), along with elevated expression of NLRP3 and cleaved Caspase1. Meanwhile, the release of inflammatory cytokines such as IL-1β and IL-18 was significantly increased, leading to enhanced neuronal pyroptosis. After treatment with either low-dose or medium-dose MB, the stress-like symptoms in mice were alleviated, and the expression of pyroptosis-related molecules was reduced (P < 0.05), with a significant decrease in neuronal pyroptosis. No significant difference was observed between the therapeutic effects of 2 mg and 10 mg MB (P > 0.05). Conclusion Both medium- and low-dose MB significantly improved behavioral abnormalities in PTSD mice, and the mechanism may be through down-regulating NLRP3 and GSDMD, as well as inhibiting neuroinflammatory response and pyroptosis.