Supplementary Material for: Isotetrandrine Reduces Astrocyte Cytotoxicity in Neuromyelitis Optica by Blocking the Binding of NMO-IgG to Aquaporin 4

<b><i>Objective:</i></b> Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease that affects the optic nerves and spinal cord with no cure and no FDA-approved therapy. Research over the last decade revealed that the binding of NMO-IgG to the water channel protein astrocyte aquaporin 4 (AQP4) might be the primary cause of NMO pathogenesis. The purpose of this study was to identify potential blockers of NMO-IgG and AQP4 binding. <b><i>Methods:</i></b> We developed a two-step screening platform consisting of a reporter cell-based high-throughput screen assay and a cell viability-based assay. Purified NMO-IgG from NMO patient serum and transfected Chinese hamster lung fibroblast V79 cells stably expressing human M23-AQP4 were used for primary screening of 40,000 small molecule fractions from 500 traditional Chinese herbs. <b><i>Results:</i></b> Thirty-six positive fractions were identified, of which 3 active fractions (at 50 μg/ml) were found to be from the same Chinese traditional herb <i>Mahonia japonica </i>(Thunb.). A bioactivity-guided method based on a primary screening assay for blocking activity led to the isolation of an active single natural compound, isotetrandrine, from the 3 fractions. Our immunofluorescence staining results showed that isotetrandrine can block NMO-IgG binding to AQP4 without affecting the expression and function of AQP4. It can also inhibit NMO-IgG binding to astrocyte AQP4 in NMO patient sera and block NMO-IgG-dependent complement-mediated cytotoxicity with the IC₅₀ at ∼3 μM. <b><i>Conclusions:</i></b> The present study developed a cell-based high-throughput screen to identify small molecule inhibitors for NMO-IgG and AQP4 binding, and suggests a potential therapeutic value of isotetrandrine in NMO.

**<i>研究目的：</i>** 视神经脊髓炎（Neuromyelitis optica, NMO）是一种累及视神经与脊髓的重症神经脱髓鞘自身免疫性疾病，目前尚无治愈方案，亦无经美国食品药品监督管理局（Food and Drug Administration, FDA）批准的治疗手段。近十年的研究显示，NMO-IgG与星形胶质细胞水通道蛋白4（aquaporin 4, AQP4）的结合可能是NMO发病的核心致病机制，本研究旨在筛选可阻断NMO-IgG与AQP4结合的潜在抑制剂。
**<i>实验方法：</i>** 本研究构建了两步法筛选平台，包含基于报告基因细胞的高通量筛选实验与基于细胞活力的验证实验。实验使用从NMO患者血清中纯化得到的NMO-IgG，以及稳定表达人源M23-AQP4的转染中国仓鼠肺成纤维细胞V79，对500种中药材的40000个小分子组分进行初筛。
**<i>实验结果：</i>** 本次初筛共获得36个阳性组分，其中3个活性组分（作用浓度为50 μg/ml）来源于同一种中药材——日本十大功劳（<i>Mahonia japonica</i> (Thunb.)）。研究人员基于初筛的阻断活性，采用生物活性导向分离策略，从这3个活性组分中分离得到单一活性天然化合物异粉防己碱（isotetrandrine）。免疫荧光染色实验结果表明，异粉防己碱可阻断NMO-IgG与AQP4的结合，且不影响AQP4的表达与功能；此外，该化合物还可抑制NMO患者血清中NMO-IgG与星形胶质细胞AQP4的结合，并能以约3 μM的半最大效应浓度（IC₅₀）阻断NMO-IgG依赖的补体介导细胞毒性。
**<i>研究结论：</i>** 本研究成功构建了基于细胞的高通量筛选体系，用于筛选可阻断NMO-IgG与AQP4结合的小分子抑制剂，研究结果提示异粉防己碱在NMO治疗中具有潜在的临床应用价值。