脑多肽口服液通过改善脂质代谢异常发挥对缺血性脑卒中的治疗作用

Cerebroprotein hydrolysate oral liquid alleviates ischemic stroke through improving lipid metabolism abnormalities

脑多肽口服液(cerebroprotein hydrolysate oral liquid,COL)是一种由氨基酸和多肽组成的神经保护制剂,对多种中枢系统疾病具有保护作用。然而,口服COL对缺血性脑卒中(ischemic stroke,IS)的治疗作用和潜在机制仍需探索。本研究旨在探讨COL对IS体内外模型的治疗效果及其潜在的作用机制。通过大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)手术建立IS大鼠模型,采用氯化三苯基四氮唑(triphenyltetrazolium chloride,TTC)染色、行为学评分、伊文思蓝渗漏实验、酶联免疫吸附实验(enzyme-linked immunosorbent assay,ELISA)和免疫荧光染色等考察COL对IS大鼠脑梗死面积、神经功能、血脑屏障(blood brain barrier,BBB)和神经炎症的影响;通过糖氧剥夺/复氧复糖(oxygen-glucose deprivation/reperfusion,OGD/R)在hCMEC/D3细胞上建立IS的体外模型,采用cell counting kit-8法、活性氧(reactive oxygen species,ROS)检测、划痕实验及Transwell实验等考察COL对细胞活力、氧化应激和迁移能力的影响;采用脂质组学技术、实时定量PCR和Western blot实验等考察COL对IS大鼠脑内脂质代谢的调控及作用机制。所有动物实验均经中国药科大学实验动物伦理委员会批准(批准号:2022-02-23)。研究结果显示,口服COL可以显著减少IS大鼠的脑梗死面积,改善神经功能缺陷,降低炎症因子水平并减轻BBB的破坏。OGD/R造模导致hCMEC/D3细胞的活力显著下降、细胞内ROS水平升高且细胞的迁移能力减弱,而给予COL可有效改善OGD/R对hCMEC/D3细胞造成的损伤。此外,脑缺血的发生导致大鼠脑内出现显著的脂质代谢紊乱,其中甘油三酯(triglyceride,TAG)和神经酰胺(ceramide,Cer)类脂质在IS大鼠脑内显著蓄积。COL可以显著逆转IS大鼠脑内的脂质代谢紊乱,并可通过上调酸性神经酰胺酶即N-酰基鞘氨醇酰胺水解酶1(N-acylsphingosine amidohydrolase 1,ASAH1)的水平,降低细胞毒性脂质Cer的含量。综上,COL被证明是一种有效且具有良好前景的IS治疗候选药物,可通过调节脂质代谢异常减轻缺血性脑损伤。

Cerebroprotein hydrolysate oral liquid(COL)is a neuroprotective preparation composed of various amino acids and peptides,which has beneficial effects on diverse central system diseases.However,the therapeutic effect and potential mechanism of oral COL on ischemic stroke(IS)still need to be explored.This study aims to investigate the therapeutic effects and underlying mechanisms of COL on IS in vivo and in vitro.An IS rat model was established through middle cerebral artery occlusion(MCAO)surgery,and triphenyltetrazolium chloride(TTC)staining,behavioral scoring,Evans blue leakage,enzyme-linked immunosorbent assay(ELISA)and immunofluorescence staining were performed to investigate the effects of COL on cerebral infarct size,neurological function,blood-brain barrier(BBB)and neuroinflammation in IS rats.An in vitro model of IS was established on hCMEC/D3 cells through oxygen-glucose deprivation/reperfusion(OGD/R),and cell counting kit-8assay,reactive oxygen species(ROS)detection,scratch test and Transwell test were conducted to examine the influence of COL on cell viability,oxidative stress and migration ability.Lipidomics technology,real-time quantitative PCR and Western blot experiments were used to investigate the regulation and mechanism of COL on lipid metabolism in the brain of IS rats.All the animal experiments were approved by Ethical Committee of Animal Experiments of China Pharmaceutical University(No.2022-02-23).Our results showed that intragastric administration of COL could significantly reduce the area of cerebral infarction,improve neurological deficits,lower the levels of inflammatory factors,and protect against the blood-brain barrier damage of rats with IS.OGD/R modeling resulted in a significant decrease in the viability,an elevation in intracellular ROS levels,and a weakened cell migration ability of hCMEC/D3 cells.COL treatment could effectively protect hCMEC/D3 cells from damage caused by OGD/R.More importantly,cerebral ischemia led to significant lipid metabolism disorders in the brain of rats,and significant accumulation of intracerebral triglycerides(TAG)and ceramides(Cer)was observed in IS rats.COL was proved to significantly reverse lipid metabolism disorders in the brain of IS rats and reduce the content of cytotoxic lipid Cer by upregulating the intracerebral levels of an acid ceramidase called N-acylsphingosine amidehydrolase 1(ASAH1).In summary,COL was proved to be a promising and effective candidate for IS treatment,which could alleviate cerebral ischemic injury by regulating abnormal lipid metabolism.