目的 研究哈巴俄苷对神经炎症诱导神经元损伤的作用。方法 采用MTT检测血管紧张素II（angiotensin II，Ang II）和哈巴俄苷对小鼠小胶质BV2细胞活性的影响；观察BV2细胞的形态变化；检测细胞上清液中炎症因子水平；采用免疫荧光检测核因子-κB p65（nuclear factor-κB p65，NF-κB p65）核易位以及CD86、髓系细胞触发受体2（triggering receptor expressed on myeloid cells 2，TREM2）的表达；采用Western blotting检测Toll样受体4（Toll like receptor 4，TLR4）、髓样分化因子88（molecule myeloid differentiation factor 88，MyD88）、诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）蛋白表达。分别使用脂多糖（lipopolysaccharide，LPS）和TLR4抑制剂（TAK-242）进一步验证哈巴俄苷的作用机制。建立BV2-HT22细胞共培养模型，检测线粒体膜电位、细胞凋亡情况以及凋亡相关蛋白表达，研究哈巴俄苷对神经炎症引起的神经元损伤的影响。结果 Ang II显著上调BV2细胞TLR4、MyD88、IL-1β、TNF-α和iNOS蛋白表达（P＜0.01、0.001），促进NF-κB p65入核（P＜0.001），调节小胶质细胞表面炎症标志物CD86和TREM2的表达（P＜0.05）。哈巴俄苷和TAK-242可逆转Ang II或LPS诱导的BV2细胞的作用（P＜0.05、0.01、0.001）。此外，哈巴俄苷可显著下调BV2-HT22共培养模型HT22细胞中凋亡相关蛋白的表达（P＜0.01、0.001），抑制HT22细胞凋亡（P＜0.05、0.001）。结论 哈巴俄苷可能通过抑制Ang II诱导的BV2细胞TLR4/MyD88/ NF-κB通路的激活来减轻炎症反应，进而缓解炎症引起的神经元细胞的凋亡。

Objective To study the effect of harpagoside on neuron injury induced by neuroinflammation. Methods The effects of angiotensin II (Ang II) and harpagoside on viability of mouse microglia BV2 cells were detected by MTT assay; The morphological changes of BV2 cells was observed; The levels of inflammatory factors in cell supernatant were detected; The nuclear translocation of nuclear factor-κB p65 (NF-κB p65) and expressions of CD86 and triggering receptor expressed on myeloid cells 2 (TREM2) in myeloid cells were detected by immunofluorescence; Toll like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88), inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) protein expressions were detected by Western blotting. Lipopolysaccharide (LPS) and TLR4 inhibitor (TAK-242) were used to further verify the mechanism of harpagoside. The co-culture model of BV2-HT22 cells was established, mitochondrial membrane potential, apoptosis and expressions of apoptosis-related proteins were detected to study the effect of harpagoside on neuronal damage caused by neuroinflammation. Results Ang II significantly increased the protein expressions of TLR4, MyD88, IL-1β, TNF-α and iNOS in BV2 cells (P < 0.01, 0.001), promoted the nucleation of NF-κB p65 (P < 0.001), and regulated the expressions of inflammatory markers CD86 and TREM2 on the surface of microglia (P < 0.05). Harpagoside and TAK-242 could reverse the effects of Ang II or LPS on BV2 cells (P < 0.05, 0.01, 0.001). In addition, harpagoside could significantly down-regulate the expressions of apoptosis-related proteins in HT22 cells of BV2-HT22 co-culture model (P < 0.01, 0.001), and inhibit the apoptosis of HT22 cells (P < 0.05, 0.001). Conclusion Harpagoside may alleviate the inflammatory response by inhibiting the activation of TLR4/MyD88/ NF-κB pathway in BV2 cells induced by Ang II, thus alleviating the apoptosis of neurons caused by inflammation.

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国家重点研发计划项目（2022YFC3501403）；江苏省自然科学基金资助项目（BK20191506）；深圳市“医疗卫生三名工程”项目（SZZYSM202111011）