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张艳菊, 潘婷婷, 杨辉, 张林.愈痫灵含药血清抑制小胶质细胞Panx1/P2X7R-NLRP3炎性活化及神经元保护效应研究[J].湖南中医药大学学报英文版,2025,45(12):2280-2287.[Click to copy
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| 愈痫灵含药血清抑制小胶质细胞Panx1/P2X7R-NLRP3炎性活化及神经元保护效应研究 |
| 张艳菊,潘婷婷,杨辉,张林 |
| (贵州中医药大学研究生学院, 贵州 贵阳 550025;贵州中医药大学第二附属医院, 贵州 贵阳 550003) |
| 摘要: |
| 目的 研究愈痫灵含药血清对脂多糖（LPS）诱导下BV-2小胶质细胞神经炎症的调控作用及神经炎症导致的神经元细胞损伤的保护作用。方法 建立LPS诱导的BV-2小胶质细胞神经炎症模型，设置正常对照组、模型组、空白血清组、愈痫灵含药血清（5%、10%、20%）组。采用CCK-8法检测细胞活力；采用Western blot分析泛连接蛋白1（Panx1）、嘌呤能离子通道型受体7（P2X7R）、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、凋亡相关斑点样蛋白（ASC）、半胱天冬酶-1（Caspase-1）、离子钙结合衔接分子1（Iba-1）蛋白表达水平；采用免疫荧光观察Panx1、P2X7R、NLRP3、Iba-1蛋白的表达；采用ELISA检测细胞上清液中白细胞介素-1（IL-1β）、白细胞介素-18（IL-18）、肿瘤坏死因子-α（TNF-α）含量。随后收集BV-2小胶质细胞培养上清液，将其加入到纯净的HT22海马神经元细胞中共同孵育（即条件培养基处理），观察愈痫灵含药血清对HT22神经元的保护作用。采用CCK-8 法检测HT22细胞存活率。结果 与正常对照组比较，模型组Panx1、P2X7R、NLRP3、ASC、Caspase-1、Iba-1蛋白表达较高（P＜0.01），IL-1β、IL-18和TNF-α含量升高（P＜0.01）；海马神经元细胞活力降低（P＜0.01）。与模型组比较，20%愈痫灵含药血清组Panx1、P2X7R、NLRP3、ASC、Caspase-1、Iba-1蛋白表达降低（P＜0.01），IL-1β、IL-18和TNF-α含量降低（P＜0.01）；海马神经元细胞活力增加（P＜0.01）。结论 愈痫灵含药血清可减轻LPS诱导下BV-2小胶质细胞神经炎症，进而减轻其对HT22神经元的损伤作用。 |
| 关键词: 癫痫 愈痫灵含药血清 脂多糖 BV-2细胞 HT22神经元 |
| DOI：10.3969/j.issn.1674-070X.2025.12.006 |
| Received:July 21, 2025 |
| 基金项目:国家自然科学基金项目（82060863）；贵州省教育厅青年科技拔尖人才项目（黔教技〔2024〕331号）；贵州省中医药、民族医药科学技术研究专项课题（QZYY-2024-068）；贵州中医药大学校级课题（贵中医科院内〔2019〕20）；贵州中医药大学第二附属医院院内项目（GZEYK〔2020〕11）。 |
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| Inhibitory effects of Yuxianling medicated serum on microglial Panx1/P2X7R-NLRP3 inflammatory activation and its neuroprotective effects |
| ZHANG Yanju, PAN Tingting, YANG Hui, ZHANG Lin |
| (Graduate School, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou 550025, China;The Second Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou 550003, China) |
| Abstract: |
| Objective To investigate the regulatory effects of Yuxianling medicated serum on neuroinflammation in lipopolys-accharide (LPS)-induced BV-2 microglial cells and its protective role against neuronal damage resulting from neuroinflammation. Methods A neuroinflammation model was established using LPS-induced BV-2 microglial cells, with experimental groups including a normal control group, a model group, a blank serum group, and Yuxianling medicated serum groups (5%, 10%, and 20%). Cell viability was assessed using the cell counting kit-8 (CCK-8) assay. The protein expression levels of Pannexin 1 (Panx1), Purinergic P2X Receptor 7 (P2X7R), NOD-like receptor family pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a card (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1), and ionized calcium-binding adapter molecule 1 (Iba-1) were analyzed by Western blot. The expression of Panx1, P2X7R, NLRP3, and Iba-1 proteins were observed using immunofluo-rescence. The concentrations of interleukin-1β (IL-1β), IL-18, and tumour necrosis factor-α (TNF-α) in the cell culture supernatant were measured by ELISA. Subsequently, the culture supernatant from BV-2 microglial cells was collected and added to pure HT22 hippocampal neuronal cells for co-incubation (i.e., conditioned medium treatment) to investigate the protective effects of Yuxianling medicated serum on HT22 neurons. The survival rate of HT22 cells was determined using the CCK-8 assay. Results Compared to the normal control group, the model group exhibited higher protein expression levels of Panx1, P2X7R, NLRP3, ASC, Caspase-1, and Iba-1 (P<0.01); elevated cytokine expressions of IL-1β, IL-18, and TNF-α (P<0.01); reduced hippocampal neuronal cell viability (P<0.01). In comparison with the model group, the 20% concentration Yuxianling medicated serum group showed decreased protein expressions of Panx1, P2X7R, NLRP3, ASC, Caspase-1, and Iba-1 (P<0.01); reduced expression levels of IL-1β, IL-18, and TNF-α (P<0.01); and increased viability of hippocampal neuronal cells (P<0.01). Conclusion Yuxianling medicated serum can alleviate neuroinflammation in LPS-induced BV-2 microglial cells, thereby reducing their damaging effects on HT22 neurons. |
| Key words: epilepsy Yuxianling medicated serum lipopolysaccharide BV-2 cell HT22 neuron |
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