小胶质细胞激活是甲氨蝶呤(MTX)诱导的神经毒性的基础;然而,确切机制尚不清楚.本研究评估了芹菜素(Api)的潜在影响,一种神经保护类黄酮,通过靶向miR-15a/Rho相关蛋白激酶-1(ROCK-1)/细胞外信号调节激酶1/2(ERK1/2)途径,在MTX诱导的大鼠小胶质细胞激活方面的神经毒性。雄性SpragueDawley大鼠随机分为4组:正常对照组(每天腹腔注射生理盐水,第8天和第15天静脉注射);Api对照(20mg/kg,p.o.)每日30天;单独使用MTX(75mg/kg,i.v.)在第8天和第15天,然后四次i.p.注射亚叶酸(LCV):18小时后6mg/kg,然后MTX后每8小时三次剂量(3mg/kg);和Api共治疗(20mg/kg/天,p.o.)在整个模型中持续30天,与第3组一样,给予MTX和LCV。MTX给药升高海马离子化钙结合衔接蛋白-1(Iba-1)免疫染色,表明小胶质细胞激活。这伴有神经炎症,氧化应激,海马白细胞介素-1β升高表现为细胞凋亡增强,丙二醛,和caspase-3,并降低谷胱甘肽水平。同时,减少miR-15a表达,其靶ROCK-1过表达,下游ERK1/2和cAMP反应元件结合蛋白(CREB)磷酸化减少,观察到海马脑源性神经营养因子(BDNF)水平降低。Api通过逆转生化来减轻MTX诱导的神经毒性,组织病理学,以及通过新颖的物体识别和莫里斯水迷宫测试测试的行为紊乱。最后,Api减轻MTX诱导的神经炎症,氧化应激,通过调节miR-15a/ROCK-1/ERK1/2/CREB/BDNF途径抑制小胶质细胞活化,促进细胞凋亡和认知功能。显示甲氨蝶呤和芹菜素共同治疗在MTX诱导的神经毒性模型中的作用的图形摘要。在左边,甲氨蝶呤(MTX)给大鼠导致海马miR-15a下调,这触发了其靶ROCK-1的表达增强,从而抑制了下游的ERK1/2/CREB/BDNF途径,激发小胶质细胞活化的状态,神经炎症,氧化应激,和凋亡。另一方面,芹菜素(Api)共同治疗恢复的miR-15a,抑制ROCK-1表达,并激活ERK1/2/CREB/BDNF通路,导致海马小胶质细胞激活减少,神经炎症,和细胞凋亡,恢复氧化还原平衡,随着MTX治疗大鼠记忆和认知功能的改善。
Microglial activation underpins the methotrexate (MTX)-induced neurotoxicity; however, the precise mechanism remains unclear. This study appraised the potential impact of apigenin (Api), a neuroprotective flavonoid, in MTX-induced neurotoxicity in rats in terms of microglial activation through targeting the miR-15a/Rho-associated protein kinase-1 (ROCK-1)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Male Sprague Dawley rats were randomly divided into 4 groups: Normal control (saline i.p. daily and i.v. on days 8 and 15); Api control (20 mg/kg, p.o.) daily for 30 days; MTX-alone (75 mg/kg, i.v.) on days 8 and 15, then four i.p. injections of leucovorin (LCV): 6 mg/kg after 18 h, then three doses (3 mg/kg) every 8 h post-MTX; and Api co-treated (20 mg/kg/day, p.o.) throughout the model for 30 days, with administration of MTX and LCV as in group 3. MTX administration elevated hippocampal ionized calcium-binding adaptor protein-1 (Iba-1) immunostaining, indicating microglial activation. This was accompanied by neuroinflammation, oxidative stress, and enhanced apoptosis manifested by elevated hippocampal interleukin-1β, malondialdehyde, and caspase-3, and decreased reduced glutathione levels. Concurrently, abated miR-15a expression, overexpression of its target ROCK-1, diminished downstream ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation, and decreased hippocampal brain-derived neurotrophic factor (BDNF) levels were observed. Api mitigated the MTX-induced neurotoxicity by reversing the biochemical, histopathological, and behavioral derangements tested by novel object recognition and Morris water maze tests. Conclusively, Api lessens MTX-induced neuroinflammation, oxidative stress, and apoptosis and boosts cognitive function through inhibiting microglial activation via modulating the miR-15a/ROCK-1/ERK1/2/CREB/BDNF pathway. Graphical abstract showing the effects of methotrexate and apigenin co-treatment in MTX-induced neurotoxicity model. On the left, methotrexate (MTX) administration to rats resulted in hippocampal miR-15a downregulation, which triggered an enhanced expression of its target ROCK-1, consequently inhibiting the downstream ERK1/2/CREB/BDNF pathway, instigating a state of microglial activation, neuroinflammation, oxidative stress, and apoptosis. On the other hand, apigenin (Api) co-treatment restored miR-15a, inhibited ROCK-1 expression, and activated the ERK1/2/CREB/BDNF pathway, leading to diminished hippocampal microglial activation, neuroinflammation, and apoptosis, and restoration of the redox balance, along with improvement in memory and cognitive function of the MTX-treated rats.