High-Throughput Screening Assay Identifies Berberine and Mubritinib as Neuroprotection Drugs for Spinal Cord Injury via Blood-Spinal Cord Barrier Protection
High-Throughput Screening Assay Identifies Berberine and Mubritinib as Neuroprotection Drugs for Spinal Cord Injury via Blood-Spinal Cord Barrier Protection
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DOI：
10.1007/s13311-022-01310-y
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发表时间：
2022-09
影响因子：
5.7
通讯作者：
Yuki Suzuki;Shinsuke Nakagawa;Takeshi Endo;Akihito Sotome;Rufei Yuan;T. Asano;Satoko Otsuguro;K. Maenaka;N. Iwasaki;K. Kadoya
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作者：
Yuki Suzuki;Shinsuke Nakagawa;Takeshi Endo;Akihito Sotome;Rufei Yuan;T. Asano;Satoko Otsuguro;K. Maenaka;N. Iwasaki;K. Kadoya
由于血脑脊髓屏障（BBSCB）的破坏导致许多中枢神经系统（CNS）疾病，因此预防BBSCB的破坏一直是主要的治疗目标，特别是对于脊髓损伤（SCI）。然而，保护BBSCB功能的有效药物尚未开发。本研究的目的是：1）开发一种高通量筛选试验（HTSA）来鉴定保护BBSCB功能的候选药物，2）用新开发的HTSA从现有药物中鉴定候选药物，3）检查候选药物对SCI的治疗效果。我们的HTSA包括用候选药物引发的永生化人脑内皮细胞培养物，用H2 O2应激，并评估其活力。基于刃天青的测定与0.45 mM H2 O2的组合被认为是可靠的HTSA。对1，570种现有药物的筛查确定了90种药物为热门药物。通过重复性测试、排除不适合临床转化的药物以及剂量依赖性测试的组合，小檗碱、莫布替尼和吡格列酮被确定为候选药物。体外BBSCB功能测试显示，小檗碱和mubritinib，但不是吡格列酮，保护BBSCB从氧-葡萄糖剥夺和复氧应激。此外，这两种药物使小鼠颈脊髓损伤后1天BBSCB的分解最小化。此外，小檗碱和mubritinib减少神经元丢失和改善SCI后8周的步态表现。总的来说，目前的研究建立了一个有用的HTSA，以确定潜在的神经保护药物，通过维持BBSCB功能，并证明了SCI后的神经保护作用的小檗碱和mubritinib。
Because the breakdown of the blood–brain spinal cord barrier (BBSCB) worsens many central nervous system (CNS) diseases, prevention of BBSCB breakdown has been a major therapeutic target, especially for spinal cord injury (SCI). However, effective drugs that protect BBSCB function have yet to be developed. The purpose of the current study was 1) to develop a high-throughput screening assay (HTSA) to identify candidate drugs to protect BBSCB function, 2) to identify candidate drugs from existing drugs with newly developed HTSA, and 3) to examine the therapeutic effects of candidate drugs on SCI. Our HTSA included a culture of immortalized human brain endothelial cells primed with candidate drugs, stress with H2O2, and evaluation of their viability. A combination of the resazurin-based assay with 0.45 mM H2O2qualified as a reliable HTSA. Screening of 1,570 existing drugs identified 90 drugs as hit drugs. Through a combination of reproducibility tests, exclusion of drugs inappropriate for clinical translation, and dose dependency tests, berberine, mubritinib, and pioglitazone were identified as a candidate. An in vitro BBSCB functional test revealed that berberine and mubritinib, but not pioglitazone, protected BBSCB from oxygen–glucose deprivation and reoxygenation stress. Additionally, these two drugs minimized BBSCB breakdown 1 day after cervical SCI in mice. Furthermore, berberine and mubritinib reduced neuronal loss and improved gait performance 8 weeks after SCI. Collectively, the current study established a useful HTSA to identify potential neuroprotective drugs by maintaining BBSCB function and demonstrated the neuroprotective effect of berberine and mubritinib after SCI.