Liu Yang, Chang Dennis, Liu Tianqing, Zhou Xian
NICM Health Research Institute, Western Sydney University, Westmead, NSW, Australia.
School of Science, Western Sydney University, Penrith, NSW, Australia.
Front Pharmacol. 2023 Feb 15;14:1135934. doi: 10.3389/fphar.2023.1135934. eCollection 2023.
Neuroinflammation is an important pathological event contributing to the onset and progression of neurodegenerative diseases. The hyperactivation of microglia triggers the release of excessive proinflammatory mediators that lead to the leaky blood-brain barrier and impaired neuronal survival. Andrographolide (AN), baicalein (BA) and 6-shogaol (6-SG) possess anti-neuroinflammatory properties through diverse mechanisms of action. The present study aims to investigate the effects of the pair-combinations of these bioactive compounds in attenuating neuroinflammation. A tri-culture model with microglial N11 cells, microvascular endothelial MVEC(B3) cells, and neuroblastoma N2A cells was established in a transwell system. AN, BA and 6-SG used alone (25 µM) or in pair-wised combinations (12.5 + 12.5 µM) were subjected to the tri-culture system. Upon the stimulation of lipopolysaccharides (LPS) at 1 μg/mL, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) levels were determined by ELISA assays. Immunofluorescence staining was applied to investigate the nuclear translocation of nuclear factor kappa B p65 (NF-κB p65) on N11 cells, expressions of protein zonula occludens-1 (ZO-1) on MVEC cells and phosphorylated tau (p-tau) on N2A cells, respectively. The endothelial barrier permeability of MVEC cells was assessed by the Evans blue dye, and the resistance from the endothelial barrier was measured by transepithelial/endothelial electrical resistance (TEER) value. Neuronal survival of N2A cells was determined by Alamar blue and MTT assays. Combinations of AN-SG and BA-SG synergistically lowered the TNF and IL-6 levels in LPS-induced N11 cells. Remarkably, the combined anti-neuroinflammatory effects of AN-SG and BA-SG remained significantly greater compared to their individual components at the same concentration level. The molecular mechanism of the attenuated neuroinflammation was likely to be mediated by downregulation of NF-κB p65 translocation ( < 0.0001 vs. LPS stimulation) in N11 cells. In the MVEC cells, both AN-SG and BA-SG restored TEER values, ZO-1 expression and reduced permeability. Furthermore, AN-SG and BA-SG significantly improved neuronal survival and reduced expressions of p-tau on N2A cells. The AN-SG and BA-SG combinations showed greater anti-neuroinflammatory potential than those used alone in mono- and tri-cultured N11 cells, thereby further protecting endothelial tight junction and neuronal survival. Taken together, AN-SG and BA-SG may provide improved anti-neuroinflammatory and neuroprotective activities.
神经炎症是导致神经退行性疾病发生和发展的重要病理事件。小胶质细胞的过度激活会触发过量促炎介质的释放，进而导致血脑屏障渗漏和神经元存活受损。穿心莲内酯（AN）、黄芩素（BA）和6-姜辣素（6-SG）通过多种作用机制具有抗神经炎症特性。本研究旨在探讨这些生物活性化合物的组合在减轻神经炎症方面的作用。在Transwell系统中建立了包含小胶质细胞N11、微血管内皮细胞MVEC(B3)和成神经细胞瘤N2A细胞的共培养模型。单独使用（25 μM）或两两组合使用（12.5 + 12.5 μM）的AN、BA和6-SG被应用于该共培养系统。在用1 μg/mL脂多糖（LPS）刺激后，通过ELISA测定肿瘤坏死因子-α（TNF-α）和白细胞介素6（IL-6）水平。采用免疫荧光染色分别研究N11细胞中核因子κB p65（NF-κB p65）的核转位、MVEC细胞中紧密连接蛋白1（ZO-1）的表达以及N2A细胞中磷酸化tau蛋白（p-tau）的表达。用伊文思蓝染料评估MVEC细胞的内皮屏障通透性，通过跨上皮/内皮电阻（TEER）值测量内皮屏障的电阻。通过alamar蓝和MTT测定法确定N2A细胞的神经元存活率。AN-SG和BA-SG组合协同降低了LPS诱导的N11细胞中TNF和IL-6水平。值得注意的是，在相同浓度水平下，AN-SG和BA-SG的联合抗神经炎症作用仍显著大于其各自成分。减轻神经炎症的分子机制可能是通过下调N11细胞中NF-κB p65的转位（与LPS刺激相比，P < 0.0001）来介导的。在MVEC细胞中，AN-SG和BA-SG均恢复了TEER值、ZO-1表达并降低了通透性。此外，AN-SG和BA-SG显著提高了神经元存活率并降低了N2A细胞上p-tau的表达。与单独用于单培养和共培养的N11细胞相比，AN-SG和BA-SG组合显示出更大的抗神经炎症潜力，从而进一步保护内皮紧密连接和神经元存活。综上所述，AN-SG和BA-SG可能具有更好的抗神经炎症和神经保护活性。
