A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine

描述（由申请人提供）：HIV-1感染的主要并发症是HIV-1相关痴呆（HAD）的发展。HAD背后的机制尚不清楚。一些研究表明HIV-1包膜糖蛋白（gp120）和转调控蛋白（Tat）在HAD的发展中起作用。特别是，这些蛋白通过穿越血脑屏障、改变血脑屏障转运蛋白和破坏血脑屏障来影响血脑屏障的完整性。我们的研究表明，这些蛋白在体外血脑屏障模型RBE4细胞中也能诱导氧化应激。我们通过测量选定的氧化应激参数，确定gp120和Tat诱导RBE4细胞氧化应激。随后，负责维持细胞氧化平衡的主要细胞内硫醇谷胱甘肽（GSH）水平显著下降，表明细胞处于氧化应激状态。抗氧化剂在氧化应激相关疾病中越来越受欢迎，并有望成为治疗药物。我们还发现n -乙酰半胱氨酸酰胺（NACA），一种新的巯基抗氧化剂，显著增加gp120和暴露于tat的RBE4细胞的GSH水平。此外，许多艾滋病/艾滋病毒阳性患者使用成瘾药物，包括甲基苯丙胺（METH）。由于甲基苯丙胺也会引起氧化应激，滥用药物的患者可能面临协同效应和增加损害的风险。我们的初步结果表明，METH确实与gp120和Tat协同作用，诱导RBE4细胞的氧化应激。由于RBE4细胞的工作是开放的批评，讨论在“具体目标”部分，我们计划使用一个更合适的体外血脑屏障模型。人脑微血管内皮细胞（HBMVECs）将用于评估甲基安非他明和两种HIV蛋白在诱导氧化应激中的协同作用。过表达gp120或Tat的转基因小鼠将作为我们的体内模型。血脑屏障模型（hbmvec和转基因动物分离的脑微血管内皮细胞）的通透性实验也将作为功能终点进行。因此，我们建议确定强抗氧化剂NACA是否能保护血脑屏障免受gp120、Tat或METH（单独和/或联合）的伤害。尽管开发和使用了有效的抗逆转录病毒疗法，HIV-1相关痴呆（HAD）仍然存在。一些研究表明HIV-1包膜糖蛋白（gp120）和转调控蛋白（Tat）在HAD的发展中起作用。一种被提出的机制是HIV-1诱导的神经毒性可能是由于HIV-1蛋白（gp120和Tat）产生活性氧（ROS）的增加。此外，许多艾滋病/艾滋病毒阳性患者使用甲基苯丙胺（冰毒）等成瘾药物。在这些成瘾性药物中，甲基苯丙胺尤其能诱导各种细胞中的自由基。如果发现HIV感染的神经发病机制是由甲基苯丙胺和gp120和/或Tat的氧化损伤显著增加引起的，则应将抗氧化剂纳入治疗以预防HIV诱导的痴呆。谷胱甘肽（GSH）是负责清除活性氧（ROS）和维持组织氧化平衡的主要细胞内硫醇。半胱氨酸和谷胱甘肽传递化合物保护细胞免受自由基损伤。例如，n -乙酰半胱氨酸（NAC）通过半胱氨酸去乙酰化间接补充谷胱甘肽，防止氧化应激。然而，NAC的口服生物利用度非常低（30%），只有静脉给药时其疗效才显著。NAC在体内不能提供显著的抗氧化作用可能源于其低溶解度和组织分布。这种无法提供抗氧化防御可能是由于NAC的带负电荷的羧基。最近，合成了一种酰胺形式的NAC，我们的实验室在各种氧化应激模型中对这种新化合物进行了测试。由于NACA中的羧基被中和，NACA比NAC亲脂性更强，可以很容易地穿过细胞膜。因此，我们假设成瘾性药物，如甲基安非他明，增强了gp120和Tat在血脑屏障（BBB）诱导的氧化应激，而NACA阻断氧化应激并保护血脑屏障。

DESCRIPTION (provided by applicant): A major complication of HIV-1 infection is the development of HIV-1-associated dementia (HAD). The mechanisms behind HAD are not yet known. Certain studies have indicated that the HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) play a role in the development of HAD. In particular, these proteins affect the integrity of the blood- brain barrier (BBB) by crossing the BBB, altering BBB transporters, and disrupting the BBB. Our studies show that these proteins also induce oxidative stress in RBE4 cells, an in vitro BBB model. We determined that gp120 and Tat induced oxidative stress in RBE4 cells by measuring selected oxidative stress parameters. Subsequently, the levels of glutathione (GSH), the principal intracellular thiol responsible for maintaining the oxidative balance in cells, significantly decreased, indicating that the cells were undergoing oxidative stress. Antioxidants are becoming increasingly popular in oxidative stress-related disorders and hold promise as therapeutic agents. We have also found that N-acetylcysteine amide (NACA), a new thiol antioxidant, significantly increased the levels of GSH in gp120 and Tat-exposed RBE4 cells. Furthermore, many AIDS/HIV-positive patients use addictive drugs, including methamphetamines (METH). Since METH induces oxidative stress as well, drug abusing patients might be at risk of a synergistic effect and increased damage. Our preliminary results showed that METH does indeed work synergistically with gp120 and Tat to induce oxidative stress in RBE4 cells. Since work with RBE4 cells is open to the criticism, discussed under the "Specific Aims" section, we plan to use a more appropriate in vitro BBB model. Human brain microvascular endothelial cells (HBMVECs) will be used to evaluate the synergistic effect of METH and the two HIV proteins in inducing oxidative stress. Transgenic mice overexpressing gp120 or Tat will serve as our in vivo model. Permeability experiments in BBB models (both in HBMVECs and isolated brain microvascular endothelial cells from the transgenic animals) will also be performed to serve as functional endpoints. Therefore, we propose to determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH (alone and/or in combination).Despite the development and use of effective antiretroviral therapy, HIV-1 associated dementia (HAD) still persists. Certain studies have indicated that the HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) play a role in the development of HAD. One proposed mechanism is that HIV-1 induced neurotoxicity may be due to an increased production of reactive oxygen species (ROS) by HIV-1 proteins (gp120 and Tat). Furthermore, many AIDs/HIV-positive patients use addictive drugs such as methamphetamine (METH). Among these addictive drugs, METH, in particular, induces free radicals in various cells. If neuropathogenesis of HIV infection is found to be caused by a significant increase in oxidative damage by METH and gp120 and/or Tat, antioxidants should be included in the treatment to prevent HIV-induced dementia. Glutathione (GSH) is the principal intracellular thiol responsible for scavenging reactive oxygen species (ROS) and maintaining the oxidative balance in tissues. Cysteine and glutathione delivery compounds protect cells from free radical damage. For example, N-acetylcysteine (NAC) indirectly replenishes GSH through deacetylation to cysteine and prevents oxidative stress. However, NAC has a very low oral bioavailability (30%) and its efficacy is only significant when given intravenously. NAC's failure to provide significant antioxidant effects in vivo might originate from its low solubility and tissue distribution. This inability to provide antioxidant defense may result from NAC's negatively charged carboxyl group. Recently, an amide form of NAC has been synthesized and our laboratory has tested this new compound in various oxidative stress models. Because the carboxylic group in NACA is neutralized, NACA is more lipophilic than NAC and can easily cross the cell membranes. Therefore, we hypothesize that addictive drugs, as exemplified by METH, potentiate the oxidative stress induced by gp120 and Tat at the blood brain barrier (BBB) and that NACA blocks oxidative stress and protects the BBB.