核苷类似物中枢神经细胞线粒体毒性及机制研究

As a staple of combined antiretroviral therapy, nucleoside analogue (NA) plays a crucial role in anti-HIV treatment. Due to the potential viral reservoir, most HIV-1 infected patients must administer long-term combined antiretroviral therapy including two nucleoside analogues, resulting in drug toxicity. As the substrate of DNA polymerase and viral reverse transcriptase, phosphorylated NA anti-HIV by competitive inhibition of viral reverse transcription. Simultaneously, phosphorylated NA is also the substrate of mitochondral DNA (mtDNA) polymerase gama and induces mitochondral damage, resulting in lactic acidosis, hepatic steatosis, peripheral neuropathy, etc. However, the central nervous toxicity of NA is unclear. Our previous studies found that NA might in vitro induce neuronal apoptosis and inhibit neurite outgrowth; Long-term oral administration of NAs could cause mtDNA deletion in mouse cortical neurons; AIDS patients with combined antiretroviral therapy had increased superoxide ions in cerebrospinal fluid and oxidative damage and mtDNA damage (deletion and mutation) in cerebral cortical autopsy. Therefore, in this research project, we intend to in vivo and in vitro detect central nervous mitochondral toxicity and analyze its potential mechanism by primary neuronal cultures and NA mouse models, and a series of molecular biology and cell biology and neurophysiological experiments, for the purpose of supportion reasonable choice of anti-HIV therapy regimen and reductiion drug nerve toxicity.

以核苷类似物（NA）为主要成分的联合抗反转录病毒治疗是目前抗HIV主要治疗措施。由于病毒潜藏库的存在，多数病人需要长期大剂量服用两种以上核苷类似物，易于产生药物毒副反应。NA通过磷酸化成为DNA聚合酶和病毒逆转录酶底物，抑制病毒逆转录酶而发挥抗病毒作用。NA也是线粒体DNA（mtDNA）聚合酶pol γ的底物，可导致线粒体损伤，产生乳酸性酸中毒、肝脂肪变性、周围神经病变等，但NA中枢神经毒性尚不清楚。我们前期研究发现NA可体外诱导小鼠神经元细胞凋亡，抑制神经突起形成；长期喂服NA可致小鼠皮层神经元mtDNA缺失；包含NA的联合抗病毒治疗的艾滋病人脑脊液超氧离子增加，尸检大脑皮层组织标本存在氧化损伤，mtDNA缺失和突变。因此，本研究中我们拟通过原代神经细胞培养和NA小鼠模型体内和体外研究NA中枢神经线粒体毒性及其损伤机制，为合理选择抗HIV治疗方案，减少药物神经毒副反应提供参考。

以核苷类似物（NA）为骨架的联合抗反转录病毒治疗（cART）是目前抗HIV主要治疗措施。NA也是线粒体DNA（mtDNA）聚合酶polγ的底物，长期服用可导致线粒体损伤，产生乳酸性酸中毒、肝脂肪变性、周围神经病变等毒副反应，但NA中枢神经毒性尚不清楚。我们前期研究发现NA可体外诱导小鼠神经元细胞凋亡，抑制神经突起形成；长期喂服NA可致小鼠皮层神经元mtDNA缺失；包含NA的联合抗病毒治疗的艾滋病人脑脊液超氧离子增加，尸检大脑皮层组织标本存在氧化损伤，mtDNA缺失和突变。本研究通过小鼠大脑皮层原代神经细胞培养和NA小鼠模型体外和体内研究NA中枢神经线粒体毒性及其损伤机制。研究发现NA神经毒性呈时间和浓度依赖性特点。NA神经元细胞mtDNA缺失以mtDNA主环损伤为主，小环损伤不明显。神经胶质细胞mtDNA缺失不明显；mtDNA损伤也可表现为线粒体DNA突变，且为基因转换形式为主的基因多态性变化。高剂量NA可通过线粒体机制诱导神经细胞凋亡，低剂量NA则诱导神经细胞自噬。低剂量NA诱导神经细胞自噬调控与非p53基因介导的DRAM调控有关。p53基因缺失小鼠进一步验证非p53基因介导的DRAM调控在NA诱导神经自噬中的作用；认知功能测试24周核苷类似物模型小鼠多数认知功能受累不明显（仅AZT组小鼠认知功能有影响）。本课题研究结果提示一定浓度的核苷类似物存在中枢神经元细胞线粒体损伤，且表现为mtDNA主环为主的mtDNA缺失，突变以及多态性变化等，伴有神经元细胞凋亡或自噬变化，而自噬调控与非p53依赖的DRAM调控有关。这种损伤可能与认知功能障碍有关。相关研究还需要进一步深入和拓展。cART使得HIV/AIDS病人寿命延长，核苷类似物的神经线粒体毒性将会同其它药物相关并发症一样会逐步显现，相关研究结果在加强艾滋病人健康管理和改善预后方面具有非常重要的意义。