背景:Isoorientin(ISO),黄酮C-糖苷,是糖原合酶激酶3β(GSK3β)底物竞争性抑制剂。ISO在治疗阿尔茨海默病(AD)方面具有潜力。GSK3β的过度活化可导致引起神经元损伤的神经炎症。小胶质细胞,作为中枢神经系统的固有免疫细胞,介导神经炎症。这里,我们研究了ISO对小胶质细胞活化以减轻神经炎症的影响.
方法:观察ISO对脂多糖(LPS)刺激小鼠小胶质细胞BV2或SIM-A9细胞的作用。氯化锂(LiCl)是作为GSK3β抑制剂的阳性对照。通过ELISA和Griess测定法分析TNF-α和NO的释放,而COX-2、Iba-1、BDNF的表达,GSK3β,NF-κBp65,IκB,通过蛋白质印迹法检测Nrf2和HO-1。在SIM-A9细胞和分化的SH-SY5Y人神经母细胞瘤细胞共培养模型中,用MTS试验评价ISO对小胶质细胞介导的神经元损伤的影响。
结果:ISO显著抑制TNF-α的产生(p<0.01),LPS诱导NO(p<0.001)、COX-2(p<0.01)和Iba-1(p<0.05)的表达,增加BDNF。在共培养中,SH-SY5Y的细胞活力被LPS抑制,这是由ISO预处理防止。ISO增加了p-GSK3β(Ser9)的表达,细胞质中的IκB和HO-1,与LPS组相比,细胞核中NF-κBp65降低,Nrf2升高。
结论:ISO通过调节GSK3β减弱小胶质细胞的活化,NF-κB和Nrf2/HO-1信号通路发挥神经保护作用。
Background: Isoorientin (ISO), a flavone C-glycoside, is a glycogen synthase kinase 3β (GSK3β) substrate-competitive inhibitor. ISO has potential in treatment of Alzheimer\'s disease (AD). An excessive activation of GSK3β can lead to neuroinflammation causing neuronal damage. Microglia cells, as resident immune cells of the central nervous system, mediate neuroinflammation. Here, we studied the effects of ISO on microglial activation to alleviate neuroinflammation.
Methods: Effects of ISO were observed upon the stimulation of mouse microglia BV2 or SIM-A9 cells by lipopolysaccharide (LPS). Lithium chloride (LiCl) was the positive control as a GSK3β inhibitor. The release of TNF-α and NO were analyzed by ELISA and Griess assays, while expressions of COX-2, Iba-1, BDNF, GSK3β, NF-κB p65, IκB, Nrf2 and HO-1 were detected by Western blotting. In the co-culture model of SIM-A9 cells and differentiated SH-SY5Y human neuroblastoma cells, effects of ISO on microglia-mediated neuronal damage were evaluated with the MTS assay.
Results: ISO significantly inhibited the production of TNF-α (p < 0.01), NO (p < 0.001) and the expression of COX-2 (p < 0.01) and Iba-1 (p < 0.05) induced by LPS, and increased BDNF. The cell viability of SH-SY5Y was inhibited by LPS in the co-culture, which was prevented by ISO pretreatment. ISO increased the expression of p-GSK3β (Ser9), IκB and HO-1 in the cytoplasm, decreased NF-κB p65 and increased Nrf2 in the nucleus compared with the LPS group.
Conclusion: ISO attenuated the activation of microglia through regulating the GSK3β, NF-κB and Nrf2/HO-1 signaling pathways to exert neuroprotection.