State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College and Beijing Key Laboratory of Drug Target and Screening Research, Beijing 100050, China.
College of Pharmacy, Harbin University of Commerce, Harbin 150076, China.
Oxid Med Cell Longev. 2020 Feb 27;2020:6431459. doi: 10.1155/2020/6431459. eCollection 2020.
Oxidative stress has been recognized as the contributor to diabetic peripheral neuropathy (DPN). Antioxidant strategies have been most widely explored; nevertheless, whether antioxidants alone prevent DPN still remains inconclusive. In the present study, we established an DPN cell model for drug screening using Schwann RSC96 cells under high glucose (HG) stimulation, and we found that salvianolic acid A (SalA) mitigated HG-induced injury evidenced by cell viability and myelination. Mechanistically, SalA exhibited strong antioxidative effects by inhibiting 1,1-diphenyl-2-picrylhydrazyl (DPPH) and reducing reactive oxygen species (ROS), malondialdehyde (MDA), and oxidized glutathione (GSSG) content, as well as upregulating antioxidative enzyme mRNA expression. In addition, SalA significantly extenuated neuroinflammation with downregulated inflammatory factor mRNA expression. Furthermore, SalA improved the mitochondrial function of HG-injured Schwann cells by scavenging mitochondrial ROS, decreasing mitochondrial membrane potential (MMP), and enhancing ATP production, as well as upregulating oxidative phosphorylation gene expression. More importantly, we identified nuclear factor-E2-related factor 2 (Nrf2) as the upstream regulator which mediated protective effects of SalA on DPN. SalA directly bound to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) and thus disrupted the interaction of Nrf2 and Keap1 predicted by LibDock of Discovery Studio. Additionally, SalA significantly inhibited Nrf2 promoter activity and downregulated Nrf2 mRNA expression but without affecting Nrf2 protein expression. Interestingly, SalA upregulated the nuclear Nrf2 expression and promoted Nrf2 nuclear translocation by high content screening assay, which was confirmed to be involved in its antiglucotoxicity effect by the knockdown of Nrf2 in RSC96 cells. In KK-Ay mice, we demonstrated that SalA could effectively improve the abnormal glucose and lipid metabolism and significantly protect against DPN by increasing the mechanical withdrawal threshold and sciatic nerve conduction velocity and restoring the ultrastructural impairment of the injured sciatic nerve induced by diabetes. Hence, SalA protected against DPN by antioxidative stress, attenuating neuroinflammation, and improving mitochondrial function via Nrf2. SalA may be prospective therapeutics for treating DPN.
氧化应激被认为是导致糖尿病周围神经病变（DPN）的原因。抗氧化策略是最广泛探索的方法；然而，单独使用抗氧化剂是否能预防 DPN 仍然没有定论。在本研究中，我们使用高糖（HG）刺激下的施万细胞（RSC96 细胞）建立了 DPN 细胞模型，用于药物筛选，结果发现丹酚酸 A（SalA）通过提高细胞活力和髓鞘形成来减轻 HG 诱导的损伤。在机制上，SalA 通过抑制 1,1-二苯基-2-苦基肼（DPPH）和减少活性氧（ROS）、丙二醛（MDA）和氧化型谷胱甘肽（GSSG）的含量，以及上调抗氧化酶 mRNA 表达，表现出很强的抗氧化作用。此外，SalA 还通过下调炎症因子 mRNA 表达显著减轻神经炎症。此外，SalA 通过清除线粒体 ROS、降低线粒体膜电位（MMP）和增强 ATP 产生，以及上调氧化磷酸化基因表达，改善 HG 损伤施万细胞的线粒体功能。更重要的是，我们确定核因子 E2 相关因子 2（Nrf2）是介导 SalA 对 DPN 保护作用的上游调节剂。SalA 直接与 Kelch 样 ECH 相关蛋白 1（Keap1）的 Kelch 结构域结合，从而破坏了 Discovery Studio 的 LibDock 预测的 Nrf2 和 Keap1 的相互作用。此外，SalA 显著抑制 Nrf2 启动子活性并下调 Nrf2 mRNA 表达，但不影响 Nrf2 蛋白表达。有趣的是，通过高内涵筛选试验，SalA 上调了核 Nrf2 表达并促进了 Nrf2 的核转位，这通过 RSC96 细胞中的 Nrf2 敲低得到证实，该作用涉及到其抗糖毒性作用。在 KK-Ay 小鼠中，我们证明 SalA 可以通过增加机械退缩阈值和坐骨神经传导速度，以及恢复糖尿病引起的损伤坐骨神经的超微结构损伤，有效改善异常血糖和脂质代谢，显著预防 DPN。因此，SalA 通过抗氧化应激、减轻神经炎症和改善线粒体功能来保护 DPN，这是通过 Nrf2 实现的。SalA 可能是治疗 DPN 的有前途的治疗方法。
