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.
Cerebral stroke is an acute cerebrovascular disease that includes hemorrhagic and ischemic stroke (IS). IS, which accounts for ~70% of all strokes, is caused by stenosis or occlusion of arteries supplying blood and oxygen to the brain. Stroke is one of the leading causes of death globally. Hypoxia and cerebral ischemia can lead to severe clinical symptoms, such as hemiplegia, hemianopia, aphasia, sensory disturbance, and decrease in consciousness. These symptoms are caused by a series of complex pathological changes, including brain oxidative stress, neuroexcitotoxicity, and neuroinflammation. Inflammation can have a considerable impact on prognosis after cerebral ischemia reperfusion. With the increase in reactive oxygen species (ROS) and microglial activation in cerebrovascular disease, adhesion molecules are induced and enter the microvessels, leading to occlusion. Inflammatory cells also release a variety of toxic substances, such as matrix metalloproteinases and nitric oxide, which can exacerbate neuronal death and disrupt the extracellular matrix and blood–brain barrier, leading to death or severe and lasting neurological damage. Therefore, inhibition of inflammation is crucial for the effective treatment of IS.
The occurrence of inflammation is affected by many molecules and signaling pathways, including mitogen-activated protein kinase (MAPK), nuclear transcription factor-κB (NF-κB), and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt). Among them, NF-κB is a key nuclear transcription factor, and activation of the NF-κB pathway is closely related to the occurrence of neuroinflammation. Ischemia–reperfusion (I/R) injury activates the NF-κB pathway, which releases NF-κB from the cytoplasmic NF-κB/I-κB complex to undergo nuclear translocation, thereby initiating the transcription of target genes, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Thus, the NF-κB signaling pathway is an important target for studying neuroinflammation in cerebral I/R.
Regulation of the NF-κB signaling pathway is affected by many factors. In recent years, the regulation of microRNAs (miRNAs) involved in the NF-κB signaling pathway has attracted increasing attention. MiRNAs are a class of non-coding RNAs consisting of 20–25 nucleotides, which can inhibit the translation of mRNAs by binding to their 3′ untranslated region (UTR). MiRNAs regulate various pathophysiological processes in the development of IS, such as neuronal death, oxidative stress, and inflammation. For example, miR-21 reduces neuronal apoptosis during cerebral ischemia by targeting the Fas ligand, miR-148a-3p modulates Rock2 expression to alter oxygen–glucose deprivation/reperfusion (OGD/R)-induced oxidative stress, and miR-421-3p inhibits activation of the NF-κB pathway and reduces inflammation after cerebral ischemia. Thus, miRNAs may be important biomarkers for IS prognosis and the discovery of novel miRNAs may provide new therapeutic targets for IS therapy and prognosis.
The high rates of IS-related disability and mortality place a severe burden on patients, their families, and society. However, effective treatments and drugs are currently lacking. Recombinant tissue plasminogen activator (rtPA) thrombolysis remains the only treatment approved by the US Food and Drug Administration (FDA). While rtPA thrombolysis can break down blood clots, it cannot rescue neuronal damage caused by hypoxia and must be administered within a demanding time window (4.5 h). Furthermore, reperfusion after thrombolysis can cause severe secondary damage to brain tissue and the cerebrovascular system, aggravating the inflammatory response and risk of hemorrhagic transformation (HT), disability, and mortality. Thus, the discovery of alternative drugs and new drug targets for cerebral I/R injury is critical.
In recent years, neuroprotective agents derived from plants such as scutellarin and gastrodin have been reported. However, these are far from enough for the needs of the disease. Animal-derived act
