槲皮素对NRTI类药物AZT引起的神经炎症的影响及机制研究

发布时间：2018-05-27 18:44

本文选题：HIV + NRTI　；参考：《浙江理工大学》2017年硕士论文

【摘要】：核苷类逆转录酶抑制剂(NRTI)是一类重要的抑制人类免疫缺陷病毒(HIV)的抗病毒药物,在高效抗逆转录病毒治疗(HAART)中发挥关键作用,而AZT是HAART中应用最广泛的药物之一,被推荐为抗逆转录病毒药物(ARVs)的首选药物。虽然可以有效抑制HIV,但长期的HAART会导致HIV相关神经障碍(HAND)。有报道称HAART导致的不良反应很大程度与神经炎症有关。因此,目前急需寻找新的治疗策略作为HAART的辅助疗法来降低其引起的神经炎症。据报道黄酮类化合物槲皮素有很好的抗炎效果,能够减轻神经炎症的症状,但是,槲皮素对NRTI引起的神经炎症的影响及其机制尚未见报道,因此,本课题在这方面进行了系统的探索研究。首先进行体外实验:通过Western blotting检测不同浓度AZT(15,25,50μM)作用BV2细胞24 h后炎症因子的表达情况,以及不同浓度槲皮素(10,20,30μM)预处理后,再以50μM AZT作用BV2细胞24 h后的炎症因子表达情况。结果发现AZT药物显著增加BV2细胞炎症因子(IL-1β,IL-6)释放,与AZT组相比,浓度为15,25,50μM的槲皮素对BV2细胞中AZT诱发的IL-1β的抑制率分别是32.07±6.59%,27.75±6.92%,35.44±17.28%;对BV2细胞中IL-6的抑制率分别是29.80±1.49%,66.11±5.65%,68.63±14.27%。因此,槲皮素可有效降低AZT引起的炎症因子释放。体外实验的探究初步证实槲皮素对NRTI药物AZT导致的炎症因子释放具有显著的抑制效果。其次进行体内实验:以100mg/kg/day槲皮素和100mg/kg/dayAZT处理小鼠,8天后收集小鼠中枢神经组织(CNS:皮层,海马,脊髓)。通过Western blotting检测小鼠中枢神经组织(CNS)中炎症因子(IL-1β,IL-6)的表达。结果发现槲皮素对小鼠中枢神经系统(CNS:皮层,海马,脊髓)中AZT诱发的IL-1β的抑制率分别为35.02±3.61%,16.59±5.58%,20.78±6.15%;槲皮素对小鼠中枢神经系统(CNS:皮层,海马,脊髓)中AZT诱发的IL-6的抑制率分别为39.43±14.21%,20.87±7.62%,17.67±1.17%。因此,槲皮素可以显著降低小鼠CNS中AZT导致的炎症因子上调。说明槲皮素可以有效抑制小鼠CNS中NRTI诱发的神经炎症。神经炎症以胶质细胞活化为标志,分别以GFAP和CD11b来标记星形胶质细胞和小胶质细胞,通过Western blotting和免疫组织化学染色的方法,进一步探究槲皮素对NRTI诱发的神经炎症的效果。结果发现槲皮素可以有效抑制小鼠CNS中AZT引起的星型胶质细胞活化,以及脊髓和海马组织中部分小胶质细胞的活化。说明槲皮素主要是通过抑制星型胶质细胞活化来抑制NRTI导致的神经炎症,而对小胶质细胞的活化有部分抑制。从而进一步证明了槲皮素对NRTI导致的神经炎症有抑制作用。上述实验初步证实槲皮素可以抑制NRTI导致的神经炎症,但其作用机制仍不太清楚。而Wnt5a信号通路与很多炎性疾病相关,并且最近很多研究发现Wnt5a信号通路参与神经炎症调节,因此我们猜测Wnt5a信号与槲皮素对NRTI导致的神经炎症的抑制作用有关。因此,我们进行体内实验和体外实验通过Western blotting方法检测Wnt5a表达水平的变化。结果发现AZT处理组Wnt5a显著上调,而槲皮素可有效抑制Wnt5a的上调,说明Wnt5a参与调节槲皮素抑制NRTI药物AZT导致的神经炎症。因此,通过本课题的系统性研究证明槲皮素可以有效抑制胶质细胞活化进而降低NRTI导致的神经炎症,并且Wnt5a细胞信号通路参与调节槲皮素对NRTI药物AZT诱发的神经炎症过程。
[Abstract]:Nucleoside reverse transcriptase inhibitor (NRTI) is an important class of antiviral drugs that inhibit human immunodeficiency virus (HIV) and plays a key role in high performance antiretroviral therapy (HAART). AZT is one of the most widely used drugs in HAART and is recommended as the first drug of the anti retroviral drug (ARVs). Although it can be effectively suppressed, it can be effectively suppressed. HIV, but long-term HAART can lead to HIV related neurological disorders (HAND). It is reported that the adverse reactions caused by HAART are largely associated with neuroinflammation. Therefore, it is urgent to find a new treatment strategy as a adjuvant therapy for HAART to reduce the neuroinflammation caused by it. It is reported that the flavonoid compound quercetin has good anti-inflammatory effects. It can alleviate the symptoms of neuro inflammation, but the effect of quercetin on NRTI induced neuroinflammation and its mechanism have not yet been reported. Therefore, this topic has been systematically explored in this field. First of all, in vitro experiment: the expression of inflammatory factors after 24 h of BV2 cells with different concentrations of AZT (15,25,50 uh M) was detected by Western blotting As well as the expression of inflammatory factors after the pretreatment of quercetin (10,20,30 mu M) at different concentrations (10,20,30 mu M), and after 24 h of BV2 cells with 50 micron AZT, it was found that AZT drugs significantly increased the release of BV2 cell inflammatory factors (IL-1 beta, IL-6), and the inhibitory rate of quercetin, which was the concentration of 15,25,50 micron, was compared with AZT group. It is 32.07 + 6.59%, 27.75 + 6.92%, 35.44 + 17.28%, and the inhibition rate of IL-6 in BV2 cells is 29.80 + 1.49%, 66.11 + 5.65%, 68.63 + 14.27%., so quercetin can effectively reduce the release of inflammatory factors caused by AZT. In vitro experiments have preliminarily confirmed that quercetin has a significant inhibitory effect on the release of inflammatory factors caused by NRTI drug AZT. Second, in vivo experiment: the mice were treated with 100mg/kg/day quercetin and 100mg/kg/dayAZT, and the mice central nervous tissue (CNS: cortex, hippocampus, spinal cord) were collected 8 days later. The expression of inflammatory factors (IL-1 beta, IL-6) in the central nervous tissue (CNS) of mice was detected by Western blotting. The results showed that quercetin was used in the central nervous system of mice (CNS: cortex, CNS: cortex,). The inhibitory rates of AZT induced IL-1 beta in the hippocampus and spinal cord were 35.02 + 3.61%, 16.59 + 5.58%, 20.78 + 6.15%, respectively. The inhibition rate of AZT induced IL-6 in the central nervous system (CNS: cortex, hippocampus, and spinal cord) of mice was 39.43 + 14.21%, 20.87 + 7.62%, 17.67 + 1.17%., respectively. Quercetin could significantly reduce AZT induced inflammation in CNS in mice. It is suggested that quercetin can effectively inhibit NRTI induced neuroinflammation in CNS of mice. Neuroglia is marked by glial cell activation, and astrocytes and microglia are marked with GFAP and CD11b respectively. The NRTI induced God by quercetin is further explored by Western blotting and immunohistochemical staining. The results showed that quercetin could effectively inhibit the activation of astrocytes induced by AZT in CNS and the activation of some microglia in the spinal and hippocampal tissues. It shows that quercetin mainly inhibits the activation of astrocytes by inhibiting the activation of NRTI, and has a part of the activation of microglia. The inhibitory effect of quercetin on NRTI induced neuroinflammation was further demonstrated. The above experiments preliminarily confirmed that quercetin inhibited NRTI induced neuroinflammation, but its mechanism was still unclear. The Wnt5a signaling pathway was associated with many inflammatory diseases, and the most recent studies found that Wnt5a signaling pathways involved nerves. We speculated that the Wnt5a signal and quercetin were associated with the inhibitory effect of quercetin on NRTI induced neuroinflammation. Therefore, in our in vivo and in vitro experiments, the expression of Wnt5a was detected by the Western blotting method. The results showed that the Wnt5a in the AZT treatment group was significantly adjusted, and quercetin could effectively inhibit the up regulation of Wnt5a. Wnt5a is involved in regulating quercetin to inhibit the neuroinflammation caused by the NRTI drug AZT. Therefore, the systematic study of this topic has demonstrated that quercetin can effectively inhibit glial activation and reduce the neuroinflammation caused by NRTI, and the Wnt5a cell signaling pathway participates in the regulation of quercetin on the process of neuroinflammation induced by NRTI drug AZT.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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