背景:当供应大脑的血管阻塞时,就会发生缺血性中风(IS),导致脑缺血。这种类型的中风占所有中风的大约87%。全球范围内,IS导致高死亡率和不良预后,并与神经炎症和神经元凋亡有关。D-阿洛糖是葡萄糖的生物底物,其在许多植物中广泛表达。我们先前的研究表明,D-allose通过减少神经炎症对急性脑缺血/再灌注(I/R)损伤具有神经保护作用。这里,我们旨在阐明D-Allose在抑制IS诱导的神经炎症损伤中的有益作用,细胞毒性,体外和体内神经元凋亡和神经功能缺损及其潜在机制。
方法:体内,通过C57BL/6N小鼠大脑中动脉阻塞再灌注(MCAO/R)诱导I/R模型,再灌注后5min内给予D-阿洛糖腹腔注射。体外,建立小鼠海马神经元细胞(HT-22)氧糖剥夺再灌注(OGD/R)作为IS细胞模型。神经学评分,一些细胞因子,测量脑和细胞系中的细胞毒性和凋亡。此外,Gal-3短发夹RNA,慢病毒和腺相关病毒在体外和体内调节神经元中Gal-3的表达以揭示其分子机制。
结果:D-allose减轻了细胞毒性,包括细胞活力,LDH释放和凋亡,在OGD/R后的HT-22细胞中,这也减轻了脑损伤,如病变体积所示,脑水肿,神经元凋亡,和神经功能缺损,在I/R的小鼠模型中此外,D-Allose降低了炎症因子的释放,如IL-1β,IL-6和TNF-α。此外,在野生型小鼠和HT-22细胞中,I/R增加了Gal-3的表达,并且该因子进一步与TLR4结合,如三维结构预测和Co-IP所证实的。用shRNA沉默Gal-3基因可降低TLR4信号的激活并减轻IS诱导的神经炎症,细胞凋亡和脑损伤。重要的是,Gal-3的缺失增强了D-allose介导的抗I/R诱导的HT-22细胞损伤的保护作用,炎性损伤和细胞凋亡,而选择性激动剂LPS对TLR4的激活增加了神经元损伤的程度,并消除了D-allose的保护作用。
结论:总之,D-阿洛糖通过在体外和体内抑制Gal-3/TLR4/PI3K/AKT信号通路在抑制IS后炎症中起着至关重要的作用。
BACKGROUND: Ischemic stroke (IS) occurs when a blood vessel supplying the brain becomes obstructed, resulting in cerebral ischemia. This type of stroke accounts for approximately 87% of all strokes. Globally, IS leads to high mortality and poor prognosis and is associated with neuroinflammation and neuronal apoptosis. D-allose is a bio-substrate of glucose that is widely expressed in many plants. Our previous study showed that D-allose exerted neuroprotective effects against acute cerebral ischemic/reperfusion (I/R) injury by reducing neuroinflammation. Here, we aimed to clarify the beneficial effects D-allose in suppressing IS-induced neuroinflammation damage, cytotoxicity, neuronal apoptosis and neurological deficits and the underlying mechanism in vitro and in vivo.
METHODS: In vivo, an I/R model was induced by middle cerebral artery occlusion and reperfusion (MCAO/R) in C57BL/6 N mice, and D-allose was given by intraperitoneal injection within 5 min after reperfusion. In vitro, mouse hippocampal neuronal cells (HT-22) with oxygen-glucose deprivation and reperfusion (OGD/R) were established as a cell model of IS. Neurological scores, some cytokines, cytotoxicity and apoptosis in the brain and cell lines were measured. Moreover, Gal-3 short hairpin RNAs, lentiviruses and adeno-associated viruses were used to modulate Gal-3 expression in neurons in vitro and in vivo to reveal the molecular mechanism.
RESULTS: D-allose alleviated cytotoxicity, including cell viability, LDH release and apoptosis, in HT-22 cells after OGD/R, which also alleviated brain injury, as indicated by lesion volume, brain edema, neuronal apoptosis, and neurological functional deficits, in a mouse model of I/R. Moreover, D-allose decreased the release of inflammatory factors, such as IL-1β, IL-6 and TNF-α. Furthermore, the expression of Gal-3 was increased by I/R in wild-type mice and HT-22 cells, and this factor further bound to TLR4, as confirmed by three-dimensional structure prediction and Co-IP. Silencing the Gal-3 gene with shRNAs decreased the activation of TLR4 signaling and alleviated IS-induced neuroinflammation, apoptosis and brain injury. Importantly, the loss of Gal-3 enhanced the D-allose-mediated protection against I/R-induced HT-22 cell injury, inflammatory insults and apoptosis, whereas activation of TLR4 by the selective agonist LPS increased the degree of neuronal injury and abolished the protective effects of D-allose.
CONCLUSIONS: In summary, D-allose plays a crucial role in inhibiting inflammation after IS by suppressing Gal-3/TLR4/PI3K/AKT signaling pathway in vitro and in vivo.