尽管做出了相当努力，但缺血性卒中（IS）仍然是一个具有挑战性的临床问题。因此，基于其潜在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。从蛙类Rana limnocharis皮肤提取的RNA中克隆了一个编码cDNA的肽，预测并合成了成熟的氨基酸序列。肽的溶血和急性毒性进行了测试。此外，使用大鼠中大脑动脉阻塞/再灌注（MCAO/R）模型和类神经元PC12细胞的缺氧葡萄糖剥夺/再灌注（OGD/R）模型评估了其神经保护特性。使用microRNA（miRNA）测序，实时定量聚合酶链反应，双荧光素酶报告基因实验和西方印迹探索了其潜在分子机制。鉴定了一种新肽（NP1），其氨基酸序列为“FLPAAICLVIKTC”。NP1在体内和体外均未显示出明显的毒性，并能穿过血脑屏障。经腹腔注射NP1（10 nmol/kg）可有效减少MCAO/R模型大鼠的脑梗死体积并减轻神经功能障碍。此外，NP1明显减轻了OGD/R诱导的类神经元PC12细胞存活率下降和凋亡增加的程度。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）水平有效抑制了炎症。此外，NP1上调miR-6328的表达，miR-6328进而下调kappa B激酶β（IKKβ）。IKKβ降低了核因子kappa B p65（NF-κB p65）和NF-κB抑制因子（I-κB）的磷酸化，从而抑制了NF-κB信号通路的激活。新发现的无毒肽NP1（“FLPAAICLVIKTC”）通过miR-6328/IKKβ/NF-κB轴通过减少炎症对脑缺血再灌注损伤产生神经保护作用。我们的发现不仅提供了外源性肽类药物和内源性小分子核酸药物候选者，还提供了用于IS治疗的新药物靶点。该研究强调了肽类在新药物开发，病理机制阐明和新药物靶点发现中的重要性。© 2023作者。

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.© 2023. The Author(s).