College of Life and Health Sciences, Northeastern University, Shenyang, China; Key Laboratory of Data Analytics and Optimization for Smart Industry, Northeastern University, Ministry of Education, Shenyang, China.
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China.
Phytomedicine. 2020 Dec;79:153353. doi: 10.1016/j.phymed.2020.153353. Epub 2020 Sep 22.
Increasing evidence has shown that microglia-induced neuroinflammation is involved in the pathogenesis of ischemic stroke. Stepharine, one of the alkaloids extracted from Stephania japonica (Thunb.) Miers, exhibited strong inhibitory effect on microglial overactivation. However, it is not known whether it has the potential to prevent ischemic stroke.
The neuroprotective and anti-neuroinflammatory effects of stepharine were investigated in vivo and in vitro, using a rat model of middle cerebral artery occlusion (MCAO) and lipopolysaccharide (LPS)-stimulated BV-2 cells, respectively.
In vivo, stepharine (500 μg/kg) suppressed neurological deficits scores, brain water content and cerebral infarct volume induced by MCAO. Moreover, stepharine (500 μg/kg) inhibited NeuN cells loss and Iba-1 cells increase in the MCAO ischemic cortex. In vitro, stepharine (10, 30 μM) substantially inhibited nitric oxide release as well as the mRNA and protein expression of pro-inflammatory mediators [inducible nitric oxide synthase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-1β] in LPS-activated BV-2 cells. LPS-induced increase of TLR4 expression, IκBα phosphorylation, and NF-κB p65 nuclear translocation was inhibited by stepharine (10, 30 μM). Molecular docking analysis showed that stepharine directly interacted with TLR4. SPR assay further confirmed that stepharine could bind to the TLR4/MD2 complex. Meanwhile, stepharine exhibited neuroprotective effects on SH-SY5Y cells cultured with LPS-treated conditioned medium.
Our study demonstrated for the first time that stepharine improved the outcomes in MCAO rats, reduced neuronal loss, and suppressed microglial overactivation via the inhibition of TLR4/NF-κB pathway. These results suggest that stepharine might be a potential therapeutic agent for the treatment of ischemic stroke.
越来越多的证据表明，小胶质细胞诱导的神经炎症参与了缺血性中风的发病机制。从Stephania japonica（Thunb.）Miers 中提取的生物碱之一Stepharine 对小胶质细胞过度激活表现出强烈的抑制作用。然而，目前尚不清楚它是否有预防缺血性中风的潜力。
使用大脑中动脉闭塞（MCAO）大鼠模型和脂多糖（LPS）刺激的 BV-2 细胞，分别在体内和体外研究了 Stepharine 的神经保护和抗炎作用。
体内，Stepharine（500μg/kg）抑制 MCAO 引起的神经功能缺损评分、脑水含量和脑梗死体积。此外，Stepharine（500μg/kg）抑制 MCAO 缺血皮质中 NeuN 细胞丢失和 Iba-1 细胞增加。体外，Stepharine（10、30μM）显著抑制一氧化氮释放以及 LPS 激活的 BV-2 细胞中促炎介质[诱导型一氧化氮合酶、白细胞介素（IL）-6、肿瘤坏死因子（TNF）-α、IL-1β]的 mRNA 和蛋白表达。Stepharine（10、30μM）抑制 LPS 诱导的 TLR4 表达、IκBα磷酸化和 NF-κB p65核易位增加。分子对接分析表明 Stepharine 直接与 TLR4 相互作用。SPR 分析进一步证实 Stepharine 可与 TLR4/MD2 复合物结合。同时，Stepharine 对 LPS 处理的条件培养基培养的 SH-SY5Y 细胞表现出神经保护作用。
我们的研究首次表明，Stepharine 通过抑制 TLR4/NF-κB 通路改善 MCAO 大鼠的预后，减少神经元丢失，并抑制小胶质细胞过度激活。这些结果表明 Stepharine 可能是治疗缺血性中风的潜在治疗剂。
