Phytomedicine ( IF 6.7 ) Pub Date : 2023-11-24 , DOI: 10.1016/j.phymed.2023.155238
Junpeng Long 1 , Yang Sun 1 , Shasha Liu 2 , Chen Chen 3 , Qian Yan 1 , Yuting Lin 1 , Zhao Zhang 4 , Shifeng Chu 4 , Yantao Yang 1 , Songwei Yang 1 , Meiyu Lin 1 , Xuan Liu 1 , Jinping Liang 1 , Naihong Chen 5 , Qidi Ai 1
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Background
Ischemic stroke, a severe and life-threatening neurodegenerative condition, currently relies on thrombolytic therapy with limited therapeutic window and potential risks of hemorrhagic transformation. Thus, there is a crucial need to explore novel therapeutic agents for ischemic stroke. Ginsenoside Rg1 (Rg1), a potential neuroprotective agent, exhibits anti-ischemic effects attributed to its anti-inflammatory, anti-oxidant, and anti-apoptotic properties. Nevertheless, the precise underlying mechanism of action remains to be fully elucidated.
Purpose
This study aimed to explore whether Rg1 exerts anti-ischemic stroke effects by inhibiting pyroptotic neuronal cell death through modulation of the chemokine like factor 1 (CKLF1)/ C-C chemokine receptor type 5 (CCR5) axis.
Methods
In this study, the MCAO model was used as an ischemic stroke model, and experimental tests were performed after 6 hours of ischemia. The anti-ischemic effect of Rg1 was examined by TTC staining, nissl-staining and neurobehavioral tests. In the in vitro experiments, PC12 cells were subjected to stimulation with CKLF1′s mimetic peptide C27 to assess the potential of CKLF1 to induce focal neuronal cell death. Additionally, the impact of CKLF1 mimetic peptide C27, antagonistic peptide C19, and CCR5 inhibitor MVC on PC12 cells subjected to oxygen-glucose deprivation (OGD) and subsequently treated with Rg1 was investigated. In vivo, Rg1 treatment was examined by quantitative real-time PCR (qPCR), ELISA, immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), and co-immunoprecipitate (Co-IP) assays to perspective whether Rg1 treatment reduces CKLF1/CCR5 axis-induced pyroptotic neuronal cell death. In addition, to further explore the biological significance of CKLF1 in ischemic stroke, CKLF1−/− rats were used as the observation subjects in this study.
Results
The in vitro results suggested that CKLF1 was able to induce neuronal cells to undergo pyroptosis. In vivo pharmacodynamic results showed that Rg1 treatment was able to significantly improve symptoms in ischemic stroke rats. In addition, Rg1 treatment was able to inhibit the interaction between CKLF1 and CCR5 after ischemic stroke and inhibited CKLF1/CCR5 axis-induced pyroptosis. The results of related experiments in CKLF1−/− rats showed that Rg1 lost its therapeutic effect after CKLF1 knockdown.
Conclusion
Our findings indicate that the activation of the NLRP3 inflammasome is initiated by the CKLF1/CCR5 axis, facilitated through the activation of the NF-κB pathway, ultimately resulting in the pyroptosis of neuronal cells. Conversely, Rg1 demonstrates the capability to mitigate neuronal cell damage following CKLF1-induced effects by suppressing the expression of CKLF1. Thus, CKLF1 represents a crucial target for Rg1 in the context of cerebral ischemia treatment, and it also holds promise as a potential target for drug screening in the management of ischemic stroke.
中文翻译：
人参皂苷 Rg1 通过调节 CKLF1/CCR5 轴诱导的神经元细胞焦亡治疗缺血性中风
背景
缺血性中风是一种严重且危及生命的神经退行性疾病，目前依赖于溶栓治疗，但治疗窗有限且存在出血转化的潜在风险。因此，迫切需要探索缺血性中风的新型治疗剂。人参皂苷 Rg1 (Rg1) 是一种潜在的神经保护剂，由于其抗炎、抗氧化和抗凋亡特性而具有抗缺血作用。然而，确切的潜在作用机制仍有待充分阐明。
目的
本研究旨在探讨Rg1是否通过调节趋化因子样因子1（CKLF1）/ CC趋化因子受体5型（CCR5）轴来抑制焦亡神经元细胞死亡，从而发挥抗缺血性中风作用。
方法
本研究采用MCAO模型作为缺血性脑卒中模型，在缺血6小时后进行实验测试。通过TTC染色、尼氏染色和神经行为测试检查Rg1的抗缺血作用。在体外实验中，用CKLF1的模拟肽C27刺激PC12细胞，以评估CKLF1诱导局灶性神经元细胞死亡的潜力。此外，还研究了 CKLF1 模拟肽 C27、拮抗肽 C19 和 CCR5 抑制剂 MVC 对经历氧糖剥夺 (OGD) 并随后用 Rg1 处理的 PC12 细胞的影响。在体内，通过定量实时 PCR (qPCR)、ELISA、免疫组织化学 (IHC)、免疫荧光 (IF)、蛋白质印迹 (WB) 和免疫共沉淀 (Co-IP) 检测来检查 Rg1 治疗，以了解 Rg1 治疗是否有效减少 CKLF1/CCR5 轴诱导的焦亡神经元细胞死亡。此外，为了进一步探讨CKLF1在缺血性脑卒中中的生物学意义，本研究以CKLF1 −/−大鼠作为观察对象。
结果
体外结果表明CKLF1能够诱导神经元细胞发生焦亡。体内药效学结果表明，Rg1治疗能够显着改善缺血性中风大鼠的症状。此外，Rg1治疗能够抑制缺血性中风后CKLF1和CCR5之间的相互作用，并抑制CKLF1/CCR5轴诱导的细胞焦亡。 CKLF1 −/−大鼠相关实验结果表明，CKLF1敲低后Rg1失去治疗作用。
结论
我们的研究结果表明，NLRP3 炎症小体的激活是由 CKLF1/CCR5 轴启动，并通过 NF-κB 通路的激活促进，最终导致神经元细胞焦亡。相反，Rg1 表现出通过抑制 CKLF1 的表达来减轻 CKLF1 诱导效应后神经元细胞损伤的能力。因此，CKLF1代表了Rg1在脑缺血治疗中的一个关键靶点，并且它也有望成为治疗缺血性中风的药物筛选的潜在靶点。
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