Probenecid and N-Acetylcysteine Prevent Loss of Intracellular Glutathione and Inhibit Neuronal Death after Mechanical Stretch Injury In Vitro
Probenecid and N-Acetylcysteine Prevent Loss of Intracellular Glutathione and Inhibit Neuronal Death after Mechanical Stretch Injury In Vitro
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DOI：
10.1089/neu.2015.4342
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发表时间：
2016-10-15
影响因子：
4.2
通讯作者：
Clark, Robert S. B.
中科院分区：
文献类型：
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作者：
Du, Lina;Empey, Philip E.;Clark, Robert S. B.
Probenecid和n -乙酰半胱氨酸（NAC）可以通过两种不同的生化途径维持细胞内重要抗氧化剂谷胱甘肽（GSH）的水平。Probenecid抑制转运体介导的谷胱甘肽外排，NAC作为谷胱甘肽合成的半胱氨酸供体。我们假设probenecid和NAC单独使用可以维持细胞内GSH浓度，抑制外伤性拉伸损伤后的神经元死亡，并且两种药物联合使用会产生叠加效应。用probenecid （100M）和NAC （50M）单独或联合（Pro-NAC）处理性别分离大鼠皮层原代神经元，然后进行机械拉伸（10s（-1）应变率，50%膜变形）。24h时，probenecid和NAC均抑制外伤诱导的XY-和xx -神经元细胞内GSH消耗、乳酸脱氢酶（LDH）释放和碘化丙啶（PI）摄取。在维持细胞内谷胱甘肽和以PI摄取评估的神经元死亡方面，联合Pro-NAC治疗优于probenecid或NAC单独治疗。有趣的是，机械损伤后24小时caspase 3活性在XX-神经元中更为突出，并且在XX-神经元中观察到治疗效果（probenecid、NAC和Pro-NAC），而在xy -神经元中则没有；然而，xy神经元最终比XX神经元更容易受到机械拉伸引起的损伤，LDH释放和PI摄取检测到更多的神经元死亡证明了这一点。此外，在HT22海马细胞拉伸损伤后，二氯荧光素检测到NAC和probenecid均能有效降低氧化应激。这些体外数据支持在体内创伤性神经元损伤模型中进一步测试这种药物组合。
Probenecid and N-acetylcysteine (NAC) can preserve intracellular levels of the vital antioxidant glutathione (GSH) via two distinct biochemical pathways. Probenecid inhibits transporter-mediated GSH efflux and NAC serves as a cysteine donor for GSH synthesis. We hypothesized that probenecid and NAC alone would maintain intracellular GSH concentrations and inhibit neuronal death after traumatic stretch injury, and that the drugs in combination would produce additive effects. Sex-segregated rat primary cortical neurons were treated with probenecid (100M) and NAC (50M), alone and in combination (Pro-NAC), then subjected to mechanical stretch (10s(-1) strain rate, 50% membrane deformation). At 24h, both probenecid and NAC inhibited trauma-induced intracellular GSH depletion, lactate dehydrogenase (LDH) release, and propidium iodide (PI) uptake in both XY- and XX-neurons. Combined Pro-NAC treatment was superior to probenecid or NAC alone in maintenance of intracellular GSH and neuronal death assessed by PI uptake. Interestingly, caspase 3 activity 24h after mechanical trauma was more prominent in XX-neurons, and treatment effects (probenecid, NAC, and Pro-NAC) were observed in XX- but not XY-neurons; however, XY-neurons were ultimately more vulnerable to mechanical stretch-induced injury than their XX counterparts, as was evidenced by more neuronal death detected by LDH release and PI uptake. In addition, after stretch injury in HT22 hippocampal cells, both NAC and probenecid were highly effective at reducing oxidative stress detected by dichlorofluorescein fluorescence. These in vitro data support further testing of this drug combination in models of traumatic neuronal injury in vivo.