亚甲蓝对创伤后应激障碍模型小鼠应激和神经元焦亡的作用机制研究

Study on the mechanisms by which methylene blue affects stress responses and neuronal pyroptosis in a mouse model of post-traumatic stress disorder

摘要:

目的 研究亚甲蓝（MB）对创伤后应激障碍（PTSD）模型小鼠的神经炎症和神经元焦亡的影响及其可能分子机制。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 2 mg组）及中剂量治疗组（MB 10 mg组）4组。PTSD组采用单次长时间应激／电击（SPSS）在小鼠中诱导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。利用蛋白质印迹法检测核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3（NLRP3）、裂解型胱天蛋白酶-1（Cleaved-Caspase-1）和N端加斯德敏D蛋白质（N-GSDMD）的蛋白质表达水平。利用免疫荧光技术检测N-GSDMD和NLRP3的表达。利用酶联免疫吸附试验检测白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）等炎症因子的水平。结果 与Control组比较，PTSD组小鼠表现出明显的僵直和恐惧等行为学改变；N-GSDMD的阳性细胞数和蛋白质水平升高（P<0.05），且NLRP3和Cleaved-Caspase-1、N-GSDMD的表达也增加；同时IL-1β和IL-18等炎症因子释放增加，神经元焦亡增加。无论低剂量还是中剂量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表达水平也下降（P<0.05），神经元焦亡减少；2 mg与10 mg MB在治疗效果上差异有统计学意义（P<0.05）。结论 低、中剂量的MB均改善PTSD小鼠的行为学异常，其机制可能是通过抑制NLRP3炎症小体激活，下调N-GSDMD表达，抑制神经炎症反应进而减轻神经细胞焦亡。

Abstract:

Objective To investigate the effects of methylene blue （MB） on neuroinflammation and neuronal pyroptosis in a post-traumatic stress disorder （PTSD） mouse model and its possible molecular mechanisms.Methods Sixty C57BL/6 mice were randomly divided into four groups： control group， model group （PTSD group）， low-dose treatment group （MB 2 mg group）， and medium-dose treatment group （MB 10 mg group）. PTSD was induced using single prolonged stress/shock （SPSS） in mice， that is， SPS with additional foot shock stimulation. The open field test and elevated plus maze test were used to assess anxiety- and depression-like behaviors in the model mice. Western blotting was used to measure the protein expression levels of nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 （NLRP3）， Cleaved-Caspase-1， and N-terminal gasdermin D （N-GSDMD）. Immunohistochemical staining was performed to detect the expression of N-GSDMD and NLRP3. Enzyme-linked immunosorbent assay kits were used to measure the levels of inflammatory cytokines such as IL-1β and IL-18.Results Compared with the control group， the PTSD group exhibited obvious anxiety-， depression-， and fear-like behavioral changes； the number of N-GSDMD-positive cells and the expression of N-GSDMD were increased （P<0.05）， the expression of NLRP3 and Cleaved-Caspase-1 and N-GSDMD was elevated， the release of inflammatory cytokines such as IL-1β and IL-18 was increased， and neuronal pyroptosis was enhanced. After treatment with either low-dose or medium-dose MB， the stress-like symptoms in mice were alleviated， the expression of pyroptosis pathway-related molecules was reduced （P<0.05）， and neuronal pyroptosis was decreased. There was significant difference in therapeutic effect between 2 mg and 10 mg MB （P<0.05）.Conclusions Both medium- and low-dose MB improve behavioral abnormalities in PTSD mice， and the underlying mechanisms may involve inhibiting NLRP3 inflammasome activation， down-regulating N-GSDMD expression， and inhibiting the neuroinflammatory response， thereby alleviating neuronal pyroptosis.