亚甲基蓝通过减轻帕金森病模型小鼠多巴胺能神经元焦亡以改善其运动功能障碍的实验研究

Methylene blue alleviates dopaminergic neuronal pyroptosis to improve motor dysfunction in Parkinson's disease mouse models

目的探讨亚甲基蓝对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱导的帕金森病(PD)模型小鼠运动功能障碍的影响及其分子机制。方法将40只健康雄性C57BL/6小鼠按随机数字表法分为对照组、模型组、低剂量治疗组及中剂量治疗组,每组10只。其中,模型组小鼠连续7 d腹腔注射MPTP 25 mg/(kg·d)构建PD模型;低剂量治疗组及中剂量治疗组分别于造模前连续3 d腹腔注射亚甲基蓝2 mg/(kg·d)或10 mg/(kg·d)预处理,然后连续7 d腹腔注射MPTP 25 mg/(kg·d)+亚甲基蓝2 mg/(kg·d)或10 mg/(kg·d),期间注射亚甲基蓝与MPTP时间间隔12 h。造模结束后第8天,对各组小鼠进行旷场实验、爬杆实验和转棒实验以评估其自发运动情况、协调性、耐力及运动能力等。随后处死小鼠并取脑组织,采用免疫荧光染色观察各组小鼠黑质中酪氨酸羟化酶(TH)的表达情况,采用Western blotting实验检测各组小鼠黑质及纹状体中TH、α-突触核蛋白及NOD样受体热蛋白结构域相关蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、活化半胱氨酸蛋白酶-1(cleaved-Caspase-1)和Gasdermin D(GSDMD)的表达情况,采用ELISA法检测各组小鼠黑质及纹状体中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β和IL-18的含量。结果与对照组相比,模型组小鼠的转棒停留时间明显缩短、爬杆下降时间明显延长、旷场箱内移动总路程明显减少,黑质中TH阳性细胞数明显减少,以及黑质及纹状体中TH蛋白水平明显降低,NLRP3、ASC、cleaved-Caspase-1及GSDMD、GSDMD-N蛋白水平明显升高,TNF-α、IL-1β和IL-18含量明显增加,差异均有统计学意义(P<0.05)。与模型组相比,低剂量治疗组及中剂量治疗组小鼠的转棒停留时间明显延长、爬杆下降时间明显缩短、旷场箱内移动总路程明显增多,黑质中TH阳性细胞数明显增多,以及黑质及纹状体中TH蛋白水平明显升高,NLRP3、ASC、cleaved-Caspase-1蛋白水平明显降低,TNF-α、IL-1β和IL-18含量明显减少,差异均有统计学意义(P<0.05)。低剂量治疗组与中剂量治疗组小鼠间上述指标的差异均无统计学意义(P>0.05)。结论低中剂量亚甲基蓝能改善PD模型小鼠的运动功能障碍,其机制与下调黑质及纹状体中NLRP3炎性小体表达、抑制神经炎症反应,从而减少多巴胺能神经元焦亡有关。

Objective To investigate the effect of methylene blue(MB)on motor dysfunction and its mechanism in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson's disease(PD)mouse models.Methods Forty healthy male C57BL/6 mice were randomly divided into 4 groups:control group,model group,low-dose treatment group and medium-dose treatment group(n=10);PD mouse models were established by intraperitoneal injection of 25 mg/kg/d MPTP for a consecutive 7 d;low-dose treatment group and medium-dose treatment group were pretreated intraperitoneally with MB 2 mg/kg/d or MB 10 mg/kg/d for a consecutive 3 d,respectively;and then,MPTP 25 mg/kg/d+MB 2 mg/kg/d or MPTP 25 mg/kg/d+MB 10 mg/kg/d were injected intraperitoneally into the low-dose treatment group or medium-dose treatment group for a consecutive 7 d(MPTP and MB were given at 12 h of interval).Eight d after modeling,open field experiment,pole climbing experiment and rod rotating experiment were carried out to evaluate the spontaneous movement,coordination,endurance and motor ability.And then,the mice were sacrificed;immunofluorescent staining was used to observe tyrosine hydroxylase(TH)expression in the substantia nigra;Western blotting was used to detect the expressions of TH,α-synuclein,nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3(NLRP3),apoptosis-associated speck-like protein containing a CARD(ASC),cleaved-Caspase-1 and Gasdermin D(GSDMD)in the striatum and substantia nigra of mice.Contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-18 in the substantia nigra and striatum of mice were detected by ELISA.Results Compared with the control group,the model group had shortened residence time in rod rotating,prolonged descent time in rod climbing,reduced total movement distance in open field,decreased number of TH-positive cells in the substania nigra,decreased TH protein levels in the substania nigra and striatum,and increased NLRP3,ASC,cleaved-Caspase-1,GSDMD and GSDMD-N protein levels in the substania nigra and striatum,and increased TNF-α,IL-1βand IL-18 contents in the substania nigra and striatum,with significant differences(P<0.05).Compared with the model group,low-dose treatment group and medium-dose treatment group had prolonged residence time in rod rotating,shortened descent time in rod climbing,increased total movement distance in open field,increased number of TH-positive cells in the substania nigra,and increased TH protein levels in the substania nigra and striatum,decreased NLRP3,ASC,and cleaved-Caspase-1 levels in the substania nigra and striatum,and decreased TNF-α,IL-1βand IL-18 contents in the substania nigra and striatum,with significant differences(P<0.05).No statistical differences in the above indexes were noted between the low-dose treatment group and medium-dose treatment group(P>0.05).Conclusion Low-/medium-dose MB can ameliorate motor dysfunction in PD mouse models,whose mechanism may be related to downregulate NLRP3 inflammasome and inhibit neuroinflammatory response to reduce dopaminergic neuron pyroptosis.