The Central Laboratory, Changchun Normal University, Changchun, Jilin, PR China.
College of Life Science, Changchun Normal University, Changchun, Jilin, PR China.
J Ethnopharmacol. 2021 Mar 25;268:113568. doi: 10.1016/j.jep.2020.113568. Epub 2020 Nov 11.
Isolated from Uncaria rhynchophylla (U. rhynchophylla), rhynchophylline (Rhy) has been applied for treating diseases related to central nervous system such as Parkinson's disease. Nevertheless, the molecular mechanism of the neuroprotective effect has not been well interpreted.
To investigate the effects of Rhy on MPTP/MPP + -induced neurotoxicity in C57BL/6 mice or PC12 cells and study the mechanisms involved.
The neuroprotective effect of Rhy on MPTP-induced neurotoxicity was evaluated by spontaneous motor activity test, as well as a test of rota-rod on a rat model of Parkinson's disease. The numbers of TH-positive neurons in the substantia nigra pars compacta (SNpc) was assessed by immunohistological. CCK-8, lactate dehydrogenase (LDH), reactive oxygen species (ROS), the concentration of intracellular calcium ([Ca]i) and flow cytometry analysis were performed to evaluate the pharmacological property of Rhy on 1-methyl-4-phenylpyridinium (MPP) induced neurotoxicity in PC12 cells. Besides, LY294002, a PI3K inhibitor was employed to determine the underlying molecular signaling pathway revealing the effect of Rhy by western-blot analysis.
The results showed that Rhy exhibited a protective effect against the MPTP-induced decrease in tyrosine hydroxylase (TH)-positive fibers in the substantia nigra at 30 mg/kg, demonstrated by the immunohistological and behavioral outcomes. Furthermore, it has been indicated that cell viability was improved and the MPP-induced apoptosis was inhibited after the treatment of Rhy at 20 μM, which were severally analyzed by the CCK-8 and the Annexin V/propidium iodide staining method. In addition, Rhy treatment attenuated MPP-induced up-regulation of LDH, ([Ca]i), and the levels of ROS. Besides, it can be revealed from the Western blot assay that LY294002, as a selective Phosphatidylinositol 3-Kinase (PI3K) inhibitor, effectively inhibited the Akt phosphorylation caused by Rhy, which suggested that Rhy showed its protective property through the activated the PI3K/Akt signaling pathway. Moreover, the Rhy-induced decreases of Bax and caspase-3 as the proapoptotic markers and the increase of Bcl-2 as the antiapoptotic marker, were blocked by LY294002 in the MPP-treated PC12 cells.
Rhy exerts a neuroprotective effect is partly mediated by activating the PI3K/Akt signaling pathway.
从钩藤（U. rhynchophylla）中分离得到的钩藤碱（Rhy）已被用于治疗与中枢神经系统相关的疾病，如帕金森病。然而，其神经保护作用的分子机制尚未得到很好的解释。
研究 Rhy 对 C57BL/6 小鼠或 PC12 细胞中 MPTP/MPP + 诱导的神经毒性的影响，并研究相关机制。
通过自发运动活动试验以及旋转棒试验评估 Rhy 对 MPTP 诱导的帕金森病大鼠模型神经毒性的保护作用。通过免疫组织化学评估黑质致密部（SNpc）中 TH 阳性神经元的数量。通过 CCK-8、乳酸脱氢酶（LDH）、活性氧（ROS）、细胞内钙离子浓度 ([Ca]i) 和流式细胞术分析评估 Rhy 对 1-甲基-4-苯基吡啶（MPP）诱导的 PC12 细胞神经毒性的药理作用。此外，还使用 PI3K 抑制剂 LY294002 通过 Western blot 分析确定潜在的分子信号通路，揭示 Rhy 的作用。
结果表明，Rhy 在 30mg/kg 时表现出对 MPTP 诱导的酪氨酸羟化酶（TH）阳性纤维减少的保护作用，这通过免疫组织化学和行为结果得到证实。此外，研究还表明，在用 20μM Rhy 处理后，细胞活力得到改善，MPP 诱导的细胞凋亡受到抑制，这分别通过 CCK-8 和 Annexin V/碘化丙啶染色法进行了分析。此外，Rhy 处理可减轻 MPP 诱导的 LDH、[Ca]i 和 ROS 水平的上调。此外，Western blot 分析表明，LY294002（一种选择性磷脂酰肌醇 3-激酶（PI3K）抑制剂）可有效抑制 Rhy 引起的 Akt 磷酸化，这表明 Rhy 通过激活 PI3K/Akt 信号通路发挥其保护作用。此外，在 MPP 处理的 PC12 细胞中，LY294002 阻断了 Rhy 诱导的 Bax 和 caspase-3 作为促凋亡标志物的减少以及 Bcl-2 作为抗凋亡标志物的增加。
Rhy 的神经保护作用部分是通过激活 PI3K/Akt 信号通路介导的。
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