Luo Yaowen, Cheng Junkai, Fu Yihao, Zhang Min, Gou Maorong, Li Juan, Li Xiaobing, Bai Jing, Zhou Yuefei, Zhang Lei, Gao Dakuan
Department of Neurosurgery, Xijing Hospital, Air Force Medical University, Changle West Road NO.127, Xi'an, China.
Department of Neurology, Xijing Hospital, Air Force Medical University, Changle West Road 127, Xi'an, China.
Biol Proced Online. 2023 Nov 28;25(1):30. doi: 10.1186/s12575-023-00224-z.
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.
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.
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.
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.
当供应大脑的血管发生阻塞，导致脑缺血时，就会发生缺血性中风（IS）。这种类型的中风约占所有中风的87%。在全球范围内，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-阿洛糖是葡萄糖的一种生物底物，在许多植物中广泛表达。我们之前的研究表明，D-阿洛糖通过减轻神经炎症对急性脑缺血/再灌注（I/R）损伤发挥神经保护作用。在此，我们旨在阐明D-阿洛糖在抑制IS诱导的神经炎症损伤、细胞毒性、神经元凋亡和神经功能缺损方面的有益作用及其在体外和体内的潜在机制。
在体内，通过大脑中动脉闭塞和再灌注（MCAO/R）在C57BL/6 N小鼠中诱导I/R模型，并在再灌注后5分钟内腹腔注射D-阿洛糖。在体外，建立氧糖剥夺和再灌注（OGD/R）的小鼠海马神经元细胞（HT-22）作为IS的细胞模型。测量神经评分、一些细胞因子、大脑和细胞系中的细胞毒性和凋亡。此外，使用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，以揭示分子机制。
D-阿洛糖减轻了OGD/R后HT-22细胞中的细胞毒性，包括细胞活力、乳酸脱氢酶释放和凋亡，这也减轻了I/R小鼠模型中的脑损伤，如病变体积、脑水肿、神经元凋亡和神经功能缺损所示。此外，D-阿洛糖减少了炎症因子如IL-1β、IL-6和TNF-α的释放。此外，通过三维结构预测和免疫共沉淀证实，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。用短发夹RNA沉默Gal-3基因可降低TLR4信号通路的激活，并减轻IS诱导的神经炎症、凋亡和脑损伤。重要的是，Gal-3的缺失增强了D-阿洛糖介导的对I/R诱导的HT-22细胞损伤、炎症损伤和凋亡的保护作用，而选择性激动剂LPS激活TLR4则增加了神经元损伤的程度并消除了D-阿洛糖的保护作用。
总之，D-阿洛糖在体外和体内通过抑制Gal-3/TLR4/PI3K/AKT信号通路在IS后抑制炎症中起关键作用。
