基于GRIN2A/PLCB1/PRKCG信号通路探讨白芍总苷减轻马钱子水提物神经损伤的作用机制

Mechanism of Total Glucosides of Paeony in Attenuating Neurotoxicity of Aqueous Extract of Strychni Semen via GRIN2A/PLCB1/PRKCG Signaling Pathway

摘要:

目的 研究白芍总苷（TGP）对马钱子水提物（SA）所致小鼠神经损伤的作用及机制。方法 将32只雄性KM小鼠随机分为正常组、SA组（19.5 mg·kg^-1）、TGP组（225 mg·kg^-1）、SA+TGP组（SA 19.5 mg·kg^-1+TGP 225 mg·kg^-1）。采用旷场实验、平衡木实验观察小鼠行为学变化。尼氏染色观察大脑皮层区尼氏小体的病理变化。通过酶联免疫吸附法（ELISA）检测小鼠脑组织中丙二醛（MDA）、谷氨酸（Glu）和血清中5-羟色胺（5-HT）的含量。运用转录组学测序检测小鼠脑组织的基因表达谱，共同差异表达基因进行基因本体（GO）、京都基因与基因组百科全书（KEGG）富集分析。采用实时荧光定量聚合酶链式反应（Real-time PCR）检测相关靶点mRNA表达水平。结果 与正常组比较，SA组小鼠旷场实验中边上路程和平均速度明显增加，平衡木行走时间明显增加；小鼠皮质神经元轴突消失；脑组织中Glu和MDA含量明显升高（P<0.05，P<0.01），血清中5-HT水平也明显升高（P<0.05）。与SA组比较，SA+TGP组小鼠旷场实验中边上路程和平均速度及平衡木行走时间明显降低（P<0.05，P<0.01）；皮质神经元轴突清晰可见；血清5-HT、Glu和MDA含量降低（P<0.05，P<0.01）。转录组结果发现TGP可调控N-甲基-D-天氡氨酸离子能谷氨酸受体2A（GRIN2A）/磷脂酰肌醇特异性磷酯酶C β₁（PLCB1）/蛋白激酶C-γ（PRKCG）信号通路。与正常组比较，SA可显著降低小鼠脑中GRIN2A、PLCB1、PRKCG mRJA表达（P<0.01），而配伍TGP后，GRIN2A和PRKCG mRNA水平明显升高（P<0.05，P<0.01）。结论 SA可引起小鼠脑内明显的神经毒性，TGP显著减轻SA所诱导的神经损伤，其机制可能与GRIN2A/PLCB1/PRKCG信号通路有关。

Abstract:

Objective To investigate the effect of total glucosides of paeony （TGP） on neurotoxicity induced by aqueous extract of Strychni Semen （SA） in mice and to explore its mechanism.Method Thirty-two male KM mice were randomly divided into normal group，SA group （19.5 mg·kg^-1），TGP group （225 mg·kg^-1），and SA+TGP group （SA 19.5 mg·kg^-1+TGP 225 mg·kg^-1）. The open field test and beam walking test were used to observe the behavioral changes in mice. Pathological changes in the Nissl bodies of the cerebral cortex were assessed through Nissl staining. The levels of malondialdehyde （MDA），glutamate （Glu） in the mouse brain tissue，and serum levels of 5-hydroxytryptamine （5-HT） were detected using enzyme-linked immunosorbent assay （ELISA）. Transcriptome sequencing was employed to analyze gene expression profiles in the brain tissue. Common differentially expressed genes （DEGs） underwent gene ontology （GO） and kyoto encyclopedia of genes and genomes （KEGG） enrichment analyses. The mRNA expression levels of key targets were determined using quantitative real-time polymerase chain reaction （Real-time PCR）.Result Compared with the normal group，the SA group exhibited significant increases in side-to-side distance and average speed in the open field test，as well as increased walking time on the balance beam. The axons of cortical neurons were absent，and the levels of Glu and MDA in the brain tissue were significantly elevated （P<0.05，P<0.01），along with a notable increase in serum 5-HT levels （P<0.05）. In contrast to the SA group，the SA+TGP group significantly reduced the side-to-side distance，average speed，and balance beam walking time （P<0.05 or P<0.01）. The neuronal axons were clearly visible，and levels of 5-HT，Glu，and MDA were decreased （P<0.05，P<0.01）. Transcriptome analysis indicated that TGP could regulate the glutamate receptor，ionotropic，N-methyl-D-aspartate 2a （GRIN2A）/phospholipase C β₁ （PLCB1）/protein kinase C，gamma （PRKCG） signaling pathway. Compared with the normal group，SA significantly decreased the expression of GRIN2A，PLCB1，and PRKCG genes in the mouse brain （P<0.01），while the mRNA levels of GRIN2A and PRKCG significantly increased after TGP administration （P<0.05，P<0.01）.Conclusion SA induces significant neurotoxicity in the mouse brain，and TGP significantly alleviates SA-induced neurological damage，potentially through the GRIN2A/PLCB1/PRKCG signaling pathway.