Toro Carlos A, Johnson Kaitlin, Hansen Jens, Siddiq Mustafa M, Vásquez Walter, Zhao Wei, Graham Zachary A, Sáez Juan C, Iyengar Ravi, Cardozo Christopher P
bioRxiv. 2023 Feb 15:2023.02.15.528337. doi: 10.1101/2023.02.15.528337.
Membrane channels such as connexins (Cx), pannexins (Panx) and P2X receptors (P2X R) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing (of the spinal cord revealed that boldine modulated a large number of genes involved in neurotransmission in in spinal cord tissue just below the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.
连接蛋白（Cx）、泛连接蛋白（Panx）和P2X受体（P2XR）等膜通道对钙离子以及ATP和谷氨酸等其他小分子具有通透性。通过这些通道释放ATP和谷氨酸是驱动组织对脊髓损伤（SCI）等创伤作出反应的关键机制。从智利波尔多树中分离出的生物碱波丹宁，可阻断Cx半通道（HC）和Panx。为了测试波丹宁是否能改善脊髓损伤后的功能，将波丹宁或赋形剂用于治疗中度挫伤性脊髓损伤的小鼠。根据巴索小鼠评分量表和水平梯级行走测试，波丹宁可使白质保留更多，并提高运动功能。波丹宁治疗减少了活化小胶质细胞标志物（Iba1）和星形胶质细胞标志物（GFAP）的免疫染色，同时增加了轴突生长和神经可塑性标志物（GAP-43）的免疫染色。细胞培养研究表明，波丹宁可阻断培养的星形胶质细胞中的胶质HC，特别是Cx26和Cx30，并阻断通过活化的P2XR的钙内流。RT-qPCR研究表明，波丹宁治疗可降低趋化因子Ccl2、细胞因子IL-6和小胶质细胞基因CD68的表达，同时增加神经传递基因Snap25、Grin2b和Gap-43的表达。脊髓的批量RNA测序显示，在脊髓损伤后14天，波丹宁调节了损伤震中下方脊髓组织中大量参与神经传递的基因。损伤后28天，波丹宁调节的基因数量要少得多。这些结果表明，波丹宁治疗可减轻损伤并保留组织以提高运动功能。
