Objective: To investigate the effect of 8-week dihydromyricetin (DHM) treatment on motor ability of mice with MPTP/probenecid-induced Parkinson's disease (PD) and explore the molecular mechanism.
Methods: Sixty C57BL/6 mice were randomized into the control group, PD model group, PD+DHM group and PD+NEC-1 group (n=15). In the latter 3 groups, the mice were treated with 25 mg·kg-1·d-1 MPTP and 250 mg·kg-1·d-1 probenecid twice a week for 5 weeks to establish PD models; DHM (100 mg·kg-1·d-1) was administered 5 times a week via gavage for 8 weeks and NEC-1 (6.25 mg·kg-1·d-1, twice a week) via intraperitoneal injection for 5 weeks. The changes in motor function of the mice were assessed, and the expressions of TH, GFAP and Iba-1 in the substantia nigra were detected with immunofluorescence assay; serum levels of IL-1β and LDH were detected using ELISA. The mRNA expressions of TNF-α and IL-6 were determined with RT-PCR, and the expressions of TH and proteins associated with pyroptosis, neuroinflammation, necroptosis and autophagy in the striatum were detected using Western blotting. MPP +-activated Bv-2 cells were treated with different concentrations of DHM or 3-MA, and the expressions of proteins associated with autophagy and NLRP3 were detected using Western blotting; PI staining was used to detect cell necroptosis.
Results: The PD mouse models showed significantly reduced TH-positive cells and TH protein expression (P < 0.001). DHM obviously ameliorated motor deficits and TH loss in PD mice, increased TH expression (P=0.0023), decreased α-syn levels (P < 0.001), lowered the protein expressions of GFAP (P=0.045) and Iba-1 (P < 0.001) and the mRNA and protein levels of TNF-α (P=0.0015) and IL-6 (P < 0.001), and increased IL-4 level (P < 0.001). The 8-week DHM treatment significantly suppressed pyroptosis and necroptosis and activated autophagy in the striatum of the PD mice. In MPP +-induced Bv-2 cells, DHM treatment effectively reversed autophagy impairment and inhibited NLRP3 and TNF-α, IL-6 and IL-1β release, and the anti--inflammatory effects of DHM was obviously blunted by 3-MA.
Conclusion: DHM can improve motor function of PD mice probably by activating autophagy to inhibit pyroptosis and necroptosis and reduce neuroinflammation.
目的: 探讨8周的双氢杨梅素（DHM）对1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶（MPTP）/丙磺舒（probenecid）（MPTP/p）诱导慢性帕金森病（PD）小鼠运动功能障碍的影响及其分子机制。
方法: C57BL/6小鼠腹腔注射MPTP/p构建PD模型，将60只健康雄性小鼠随机分为4组（n=15）：对照组（Control）、模型组（PD）、DHM干预组（PD+DHM）、阳性对照NEC-1抑制剂组（PD+NEC-1）。除对照组外，其余小鼠（n=45）腹腔注射MPTP（25 mg·kg-1·d-1）联合Probenecid（250 mg·kg-1·d-1），每周2次，干预5周构建慢性PD模型。造模结束后，DHM干预组进行8周DHM灌胃处理（100 mg·kg-1·d-1，5 d/周），阳性对照组进行5周NEC-1腹腔注射（6.25 mg·kg-1·d-1，2 d/周）。随机选取小鼠进行运动协调性测试（n=10）；免疫荧光染色观察黑质内TH、GFAP、Iba-1的表达（n=3）；ELISA检测小鼠血清中IL-1β；试剂盒测定纹状体内LDH酶活性；RT-PCR测定TNF-α及IL-6的mRNA水平；Western blot检测小鼠纹状体中TH和病理蛋白α-syn、神经炎症、细胞焦亡、坏死性凋亡及自噬相关蛋白的表达。1-甲基-4-苯基吡啶离子（MPP+）激活Bv-2小胶质细胞，采用不同浓度的DHM（3.12、6.25、12.5 μg/mL）及自噬抑制剂3-MA进行干预；Western blot检测干预后Bv-2细胞自噬及NLRP3炎症小体相关蛋白的表达水平；PI染色检测细胞的死亡情况。
结果: 与对照组相比，PD组小鼠存在运动功能障碍，且TH的阳性细胞数及蛋白水平显著下降（P < 0.001），而DHM干预显著改善了小鼠的运动协调能力，增加TH（P=0.0023），降低α-syn的表达（P < 0.001）。8周的DHM干预降低了PD小鼠纹状体中胶质细胞异常活化，下调GFAP（P=0.045）和Iba-1（P < 0.001）的表达；降低TNF-α（P=0.0015）、IL-6（P < 0.001）的mRNA和蛋白水平，上调IL-4（P < 0.001），减轻神经炎症反应；DHM通过下调Caspase1的活性、NLRP3炎症小体、GSDMD-N、IL-1β、IL-18的表达水平（P < 0.001）抑制细胞焦亡；降低LDH、p-RIPK1、RIPK1、p-RIPK3、RIPK3、MLKL的水平（P < 0.001），上调Caspase8（P=0.8944），改善坏死性凋亡；上调Beclin1和ULK1（P < 0.001），下调p62和LC3 Ⅰ（P < 0.001），激活自噬。体外实验结果表明，不同浓度的DHM干预（3.25、6.5、12.5 μg/mL）均可通过改善MPP+诱导的Bv-2细胞自噬受损，抑制NLRP3炎症小体的激活减轻炎症，而抑制自噬钝化了DHM的抗炎功效。
结论: DHM能改善慢性MPTP/p诱导PD小鼠的运动协调能力障碍，其机制可能是通过激活自噬抑制神经胶质细胞活化引起的神经炎症反应、细胞焦亡以及坏死性凋亡。
Keywords: MPTP; autophagy; dihydromyricetin; necroptosis; neuroinflammation; probenecid; pyroptosis.