背景:缺血性卒中是一种常见且常见的临床疾病。近年来研究表明鞘脂在缺血性卒中的病理过程中起重要作用。PI3K-Akt是脑缺血损伤的经典保护性信号通路。作用于S1P受体后,S1P可激活下游PI3K/Akt信号通路,发挥抗脑缺血作用。补阳还五汤(BHD)是一种用于治疗缺血性中风的中药配方。然而,根据S1P/S1PR1/PI3K/Akt信号通路,BHD对缺血性卒中的作用机制尚不清楚.
目的:本研究基于S1P/S1PR1/PI3K/Akt信号通路,从多角度探讨BHD对缺血性脑卒中的作用机制。
方法:采用真空冷冻干燥法制备BHD冻干品,其化学成分通过UPLC-Q-TOF/MS测定。采用缝合闭塞法建立小鼠永久性大脑中动脉闭塞(pMCAO)模型。雄性KM小鼠随机分为7组:假手术组,模型组,FTY720(阳性对照)组,BHD组,BHD+W146(选择性S1PR1抑制剂)组,SEW2871(选择性S1PR1激动剂)组,和Calycosin组。每组连续给药14天,1日进行改良神经严重度评分(mNSS)和脑梗死体积评估,第四,Seven,第14天SphK1、SphK2、S1PR1、PI3K、Akt,和P-Akt蛋白在前额叶,海马体,分别通过Westernblot和免疫组织化学(IHC)实验对纹状体和纹状体进行定量。采用qRT-PCR方法评估上述组织中SphK1、SphK2和S1PR1mRNA的表达。
结果:BHD和Calycosin均有效改善mNSS评分,梗死体积较小。前额叶的SphK1水平,海马体,BHD组小鼠纹状体明显降低,和SphK2、PI3K、海马和纹状体的p-Akt明显高于模型组。BHD显著降低了SphK1mRNA在前额叶的表达,海马体,和纹状体,显著上调SphK2mRNA和S1PR1mRNA表达。此外,SphK1蛋白在前额叶的表达水平,海马体,BHD组纹状体明显低于模型组,和SphK2、S1PR1、PI3K、Akt,p-Akt蛋白表达水平明显升高。此外,SEW2871可以增加S1PR1和Akt的表达,并上调SphK2和S1PR1mRNA表达。BHD+W146减弱了BHD对S1P/S1PR1/PI3K/Akt信号通路相关蛋白和mRNA表达的影响。
结论:BHD和Calycosin显着改善了pMCAO小鼠的神经功能缺损症状,减少脑梗塞体积,上调SphK2和S1PR1mRNA水平,增强型SphK2、S1PR1、PI3K、Akt,前额叶p-Akt蛋白表达,海马和纹状体,并下调SphK1mRNA和蛋白表达,这可能有助于阐明BHD通过S1P/S1PR1/PI3K/Akt信号通路保护脑缺血损伤的机制。
BACKGROUND: Ischemic stroke is a common and frequent clinical disease. Recent studies have demonstrated that sphingolipid plays an important role in the pathological process of ischemic stroke. PI3K-Akt is a classic protective signaling pathway of cerebral ischemic injury. After acting on the S1P receptor, S1P can activate the downstream PI3K/Akt signaling pathway and play an anti-cerebral ischemia role. Buyang Huanwu Decoction (BHD) is a traditional Chinese medicine formula used to treat ischemic stroke. However, the mechanisms of BHD on ischemic stroke remain unclear based on S1P/S1PR1/PI3K/Akt signaling pathway.
OBJECTIVE: The present
study is intended to investigate the action mechanism of BHD on ischemic stroke based on the S1P/S1PR1/PI3K/Akt signaling pathway from multiple perspectives.
METHODS: The BHD lyophilized product was prepared by vacuum freeze-drying method, of which the chemical composition was determined by UPLC-Q-TOF/MS. The mouse permanent middle cerebral artery occlusion (pMCAO) model was established by the suture-occluded method. Male KM mice were randomly divided into seven groups: sham group, model group, FTY720 (positive control) group, BHD group, BHD + W146 (selective S1PR1 inhibitor) group, SEW2871 (selective S1PR1 agonist) group, and Calycosin group. Each group was administered continuously for 14 days and evaluated with modified neurological severity score (mNSS) and cerebral infarct volume on the 1st, 4th, 7th, and 14th days. The SphK1, SphK2, S1PR1, PI3K, Akt, and p-Akt protein in the prefrontal lobe, hippocampus, and striatum was quantified by Western blot and immunohistochemical (IHC) experiment respectively. The qRT-PCR method was employed to evaluate SphK1, SphK2, and S1PR1 mRNA expression in the above tissue.
RESULTS: BHD and Calycosin both effectively improved mNSS scores with smaller infarct volumes. The SphK1 level in the prefrontal lobe, hippocampus, and striatum of mice in the BHD group was significantly lower, and SphK2, PI3K, and p-Akt in the hippocampus and striatum were significantly higher than those in the model group. BHD significantly decreased SphK1 mRNA expression in the prefrontal lobe, hippocampus, and striatum, and significantly up-regulated SphK2 mRNA and S1PR1 mRNA expression. Additionally, SphK1 protein expression levels of the prefrontal lobe, hippocampus, and striatum in the BHD group was significantly lower than model group, and SphK2, S1PR1, PI3K, Akt, and p-Akt protein expressions levels were increased obviously. Furthermore, SEW2871 can increase S1PR1 and Akt expression, and up-regulate SphK2 and S1PR1 mRNA expression. The effect of BHD on the expression of S1P/S1PR1/PI3K/Akt signaling pathway-related proteins and mRNA were weakened by BHD + W146.
CONCLUSIONS: BHD and Calycosin significantly improved the symptoms of neurological deficits in pMCAO mice, reduced the cerebral infarction volume, up-regulated SphK2 and S1PR1 mRNA levels, enhanced SphK2, S1PR1, PI3K, Akt, p-Akt protein expression of the prefrontal lobe, hippocampus and striatum, and down-regulated SphK1 mRNA and protein expression, which may be helpful to clarify the mechanism of BHD through S1P/S1PR1/PI3K/Akt signaling pathway to protect against cerebral ischemic injury.