Department of Anatomy and Histology and Embryology, Faculty of Basic Medical Science, Kunming Medical University, Kunming, 650500, Yunnan, China.
Department of Orthopedics, 920th Hospital of Joint Logistics Support Force of PLA, Kunming, 650032, Yunnan, China.
J Neuroinflammation. 2023 Feb 28;20(1):53. doi: 10.1186/s12974-023-02739-4.
Despite considerable efforts, ischemic stroke (IS) remains a challenging clinical problem. Therefore, the discovery of effective therapeutic and targeted drugs based on the underlying molecular mechanism is crucial for effective IS treatment.
A cDNA-encoding peptide was cloned from RNA extracted from Rana limnocharis skin, and the mature amino acid sequence was predicted and synthesized. Hemolysis and acute toxicity of the peptide were tested. Furthermore, its neuroprotective properties were evaluated using a middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats and an oxygen-glucose deprivation/reperfusion (OGD/R) model in neuron-like PC12 cells. The underlying molecular mechanisms were explored using microRNA (miRNA) sequencing, quantitative real-time polymerase chain reaction, dual-luciferase reporter gene assay, and western blotting.
A new peptide (NP1) with an amino acid sequence of 'FLPAAICLVIKTC' was identified. NP1 showed no obvious toxicities in vivo and in vitro and was able to cross the blood-brain barrier. Intraperitoneal administration of NP1 (10 nmol/kg) effectively reduced the volume of cerebral infarction and relieved neurological dysfunction in MCAO/R model rats. Moreover, NP1 significantly alleviated the decrease in viability and increase in apoptosis of neuron-like PC12 cells induced by OGD/R. NP1 effectively suppressed inflammation by reducing interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels in vitro and in vivo. Furthermore, NP1 up-regulated the expression of miR-6328, which, in turn, down-regulated kappa B kinase β (IKKβ). IKKβ reduced the phosphorylation of nuclear factor-kappa B p65 (NF-κB p65) and inhibitor of NF-κB (I-κB), thereby inhibiting activation of the NF-κB pathway.
The newly discovered non-toxic peptide NP1 ('FLPAAICLVIKTC') exerted neuroprotective effects on cerebral ischemia-reperfusion injury by reducing inflammation via the miR-6328/IKKβ/NF-κB axis. Our findings not only provide an exogenous peptide drug candidate and endogenous small nucleic acid drug candidate but also a new drug target for the treatment of IS. This study highlights the importance of peptides in the development of new drugs, elucidation of pathological mechanisms, and discovery of new drug targets.
尽管已经做出了相当大的努力，但是缺血性脑卒中（IS）仍然是一个具有挑战性的临床问题。因此，基于潜在的分子机制发现有效的治疗和靶向药物对于有效的 IS 治疗至关重要。
从中华大蟾蜍皮肤中提取的 RNA 中克隆出一个 cDNA 编码肽，预测并合成成熟的氨基酸序列。测试肽的溶血和急性毒性。此外，还在大鼠大脑中动脉闭塞/再灌注（MCAO/R）模型和神经元样 PC12 细胞的氧葡萄糖剥夺/再灌注（OGD/R）模型中评估其神经保护作用。使用 microRNA（miRNA）测序、定量实时聚合酶链反应、双荧光素酶报告基因检测和 Western blot 探索潜在的分子机制。
鉴定出一种新的肽（NP1），其氨基酸序列为“FLPAAICLVIKTC”。NP1 在体内和体外均无明显毒性，并且能够穿过血脑屏障。腹腔内给予 NP1（10 nmol/kg）可有效减小 MCAO/R 模型大鼠脑梗死体积并改善神经功能障碍。此外，NP1 可显著减轻 OGD/R 诱导的神经元样 PC12 细胞活力下降和凋亡增加。NP1 通过降低体外和体内白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）水平有效抑制炎症。此外，NP1 上调了 miR-6328 的表达，进而下调了κB 激酶β（IKKβ）。IKKβ 降低了核因子-κB p65（NF-κB p65）和 NF-κB 抑制剂（I-κB）的磷酸化，从而抑制了 NF-κB 通路的激活。
新发现的无毒肽 NP1（“FLPAAICLVIKTC”）通过 miR-6328/IKKβ/NF-κB 轴减少炎症发挥对脑缺血再灌注损伤的神经保护作用。我们的研究结果不仅提供了一种外源性肽类药物候选物和内源性小核酸药物候选物，还为 IS 的治疗提供了一个新的药物靶点。本研究强调了肽在新药开发、病理机制阐明和新药物靶点发现中的重要性。
