Table 2_Oxymatrine-associated protection in an MPTP mouse model is accompanied by increased miR-141-3p and reduced HMGB1.docx

IntroductionOxymatrine (OMT) alleviates damage to dopaminergic (DA) neurons and microglia-mediated neuroinflammation in an MPTP mouse model of parkinsonism by inhibiting the High-mobility group protein B1 (HMGB1) pathway. However, the precise mechanism by which OMT inhibits HMGB1 remains unclear. Although miR-141-3p is downregulated in the peripheral blood serum of Parkinson’s disease (PD) patients, its potential relationship with HMGB1 remains unclear.
MethodsTargetScan software and dual-luciferase reporter gene assays predicted that miR-141-3p binds to the 3’-UTR of HMGB1 mRNA. BV2 cells were transfected with miR-141-3p mimics and stimulated with MPP+in vitro experiments. C57BL/6 J mice received stereotaxic injections of miR-141-3p agomir or miR-141-3p antagomir into the bilateral substantia nigra pars compacta (SNpc). Subsequently, the mice were intraperitoneally injected with MPTP four times within a single day. After miR-141-3p antagomir injection, OMT was administered continuously by injection for 7 days. Behavioral tests were assessed using the rotarod and open field tests. Real-time PCR, western blot, ELISA, and immunofluorescence staining were performed on BV2 cells and SNpc tissues.
ResultsOur study showed an inverse correlation between HMGB1 and miR-141-3p expression in both BV2 microglia exposed to MPP+ and MPTP-treated mice. TargetScan analysis identified complementary binding sites between miR-141-3p and the 3’-UTR of HMGB1 mRNA, which was subsequently confirmed through dual-luciferase reporter assays. Through experiments in BV2 microglia exposed to MPP+ and in MPTP-treated mice, miR-141-3p downregulates HMGB1, reduces pro-inflammatory cytokine readouts, and in vivo is associated with improved rotarod and open-field performance and attenuated Tyrosine Hydroxylase (TH)-positive neuronal loss. OMT increases miR-141-3p in the MPTP model, alongside reduced HMGB1 and inflammatory readouts, and these effects are diminished by miR-141-3p inhibition.
ConclusionmiR-141-3p targets HMGB1 to inhibit microglial reaction and mitigate neuroinflammation both in vivo and vitro experiments, reduce TH-positive neuronal loss in the MPTP model. OMT increases miR-141-3p in the MPTP model, alongside reduced HMGB1 and inflammatory readouts, and these effects are diminished by miR-141-3p inhibition.

引言
氧化苦参碱（Oxymatrine, OMT）可通过抑制高迁移率族蛋白B1（High-mobility group protein B1, HMGB1）通路，减轻MPTP诱导的帕金森病样小鼠模型中多巴胺能（dopaminergic, DA）神经元损伤及小胶质细胞介导的神经炎症。然而，氧化苦参碱抑制HMGB1的具体分子机制仍未阐明。尽管miR-141-3p在帕金森病（Parkinson’s disease, PD）患者外周血清中呈低表达，但其与HMGB1的潜在关联仍有待明确。
方法
TargetScan软件及双荧光素酶报告基因实验预测显示，miR-141-3p可结合至HMGB1 mRNA的3'非翻译区。体外实验中，将miR-141-3p模拟物转染至BV2细胞，并用MPP+进行刺激；体内实验中，向C57BL/6J小鼠双侧黑质致密部（substantia nigra pars compacta, SNpc）立体定向注射miR-141-3p激动剂或拮抗剂。随后，小鼠于单日腹腔注射MPTP共4次。在注射miR-141-3p拮抗剂后，连续腹腔注射氧化苦参碱7天。采用转棒实验与旷场实验评估小鼠行为学变化；分别对BV2细胞及黑质致密部组织开展实时荧光定量PCR、蛋白质印迹法、酶联免疫吸附测定及免疫荧光染色检测。
结果
本研究发现，在MPP+处理的BV2小胶质细胞及MPTP造模小鼠中，HMGB1与miR-141-3p的表达呈负相关。TargetScan分析预测到miR-141-3p与HMGB1 mRNA 3'非翻译区存在互补结合位点，该结果随后通过双荧光素酶报告基因实验得到验证。体外经MPP+处理的BV2小胶质细胞及体内MPTP造模小鼠实验均证实，miR-141-3p可下调HMGB1的表达，减少促炎细胞因子的分泌水平；在体实验中还可改善小鼠的转棒及旷场行为学表现，并减轻酪氨酸羟化酶（Tyrosine Hydroxylase, TH）阳性神经元的丢失。氧化苦参碱可在MPTP模型中上调miR-141-3p的表达，同时降低HMGB1的表达及炎症因子分泌水平，而该效应可因miR-141-3p的抑制而被削弱。
结论
miR-141-3p可通过靶向HMGB1，在体内及体外实验中抑制小胶质细胞活化、减轻神经炎症，并减少MPTP模型中酪氨酸羟化酶阳性神经元的丢失。氧化苦参碱可在MPTP模型中上调miR-141-3p的表达，同时降低HMGB1的表达及炎症因子分泌水平，而该效应可因miR-141-3p的抑制而被削弱。