Investigation Of The Role of Sphingosine-1-Phosphate 1 (S1P1) Receptors in The Vascular Relaxant Effect of İcariin
Project no. 75582 · Status: Kapanmış (Yayın bekleniyor) · 2022
Summary
<p>Sphingosine-1-phosphate(S1P) is a bioactive sphingolipid metabolite produced by sphingosine kinases(SK). S1P is important in regulating vascular development/stability, angiogenesis and vascular tone, and disorders in this signaling pathway are reported in the pathology of atherosclerosis, diabetes, and hypertension. S1P has five different receptors, called sphingosine-1-phosphate 1-5 receptors(S1PR1-5). S1P is formed by SK enzymes; It shows its effects on vasodilation and vascular permeability through S1PR1 and S1PR3. Icariin(ICA), which has smooth muscle relaxant, neuroprotective, cardiovascular disease protective, and anti-inflammatory effects; shows its smooth muscle relaxant and antiatherosclerotic effects through activation of the PI3K/Akt-eNOS-NO pathway, similar to S1P. Since ICA and S1PR1/S1PR3 agonists share common mechanisms of action related to inflammation such as myocardial ischemia/reperfusion injury and Parkinson-Alzheimer, the question is "Could Icariin exert its vascular relaxant effect through S1PR1 and/or S1PR3?". The question aimed to investigate, for the first time, the contribution of S1PR1 and S1PR3 to the relaxant effect of icariin on the aorta through biochemical/pharmacological studies. To prove that ICA relaxations are endothelium-dependent and/or NO-mediated, responses were obtained in the presence of endothelium-stripped aortas and nitric oxide synthase inhibitor L-NAME(100 µM, 30 min.) incubation. The relationship of ICA with the sphingosine pathway was examined through functional studies using enzyme and receptor inhibitors on the pathway. The relationship of ICA with the ceramidase enzyme, the ceramidase enzyme inhibitor D-erythro-MAPP(MAPP; 3-10 µM, 30 min.), the relationship with the sphingosine kinase enzyme, the sphingosine kinase enzyme inhibitor N,N-Dimethylsphingosine(DMS; 10-30 µM, 30min.), its relationship with S1PR1 was investigated through the S1PR1 receptor antagonist W146(1-3 µM, 30min.) and its relationship with S1PR3 was investigated through the S1PR3 receptor antagonist CAY10444(10-30 µM, 30min.). Cumulative ICA(3x10−7M -10−4 M) relaxation responses were obtained from aortic tissues after pre-contraction with phenylephrine(10-7 M) in the presence/absence of inhibitors. EC50 value of ICA(3 µM) was used as the incubation concentration in aortic tissue culture (24 hours, 37 °C, 5% CO2 incubator), SK-1, S1PR1, and S1PR3 expression levels were investigated by western blot. Nitric oxide(NO) levels were measured spectrophotometrically using the Griess reaction by the kit protocol. According to our results, ICA relaxations are endothelium-dependent/NO-mediated. While relaxations decreased statistically significantly in the presence of DMS and W146, they did not change in the presence of MAPP and CAY10444. While SK-1 and S1PR1 protein expression levels increased, S1PR3 expression levels did not change. While ICA increased total nitrate/nitrite levels, this increase was reversed in the presence of inhibitors (W146, DMS, L-NAME). It has been shown that ICA has a relaxant effect in the rat aorta through the SK enzymes responsible for S1P production and/or S1PR1. In NO-related pathologies, it is important to determine the importance of the signaling pathways responsible for NO production and the agent chosen for treatment accordingly. The use of ICA in the treatment of vascular diseases such as hypertension and atherosclerosis, especially those associated with endothelial dysfunction in cases where the sphingosine signaling pathway is impaired and NO deficiency associated with this pathway, may be of clinical importance as it has the potential to be a new and effective drug candidate.</p>