PQQ 通过 gsk-3/Akt 信号通路减轻 d- 半乳糖所致小鼠认知功能障碍
PQQ ameliorates D-galactose induced cognitive impairments by reducing glutamate neurotoxicity via the GSK-3β/Akt signaling pathway in mouse
Oxidative stress is known to be associated with various age-related diseases. D-galactose (D-gal) has been considered a senescent model which induces oxidative stress response resulting in memory dysfunction. Pyrroloquinoline quinone (PQQ) is a redox cofactor which is found in various foods. In our previous study, we found that PQQ may be converted into a derivative by binding with amino acid, which is beneficial to several pathological processes. In this study, we found a beneficial glutamate mixture which may diminish neurotoxicity by oxidative stress in D-gal induced mouse. Our results showed that PQQ may influence the generation of proinflammatory mediators, including cytokines and prostaglandins during aging process. D-gal-induced mouse showed increased MDA and ROS levels, and decreased T-AOC activities in the hippocampus, these changes were reversed by PQQ supplementation. Furthermore, PQQ statistically enhanced Superoxide Dismutase SOD2 mRNA expression. PQQ could ameliorate the memory deficits and neurotoxicity induced by D-gal via binding with excess glutamate, which provide a link between glutamate-mediated neurotoxicity, inflammation and oxidative stress. In addition, PQQ reduced the up-regulated expression of p-Akt by D-gal and maintained the activity of GSK-3β, resulting in a down-regulation of p-Tau level in hippocampus. PQQ modulated memory ability partly via Akt/GSK-3β pathway.
氧化应激与各种与年龄相关的疾病有关。D- 半乳糖(D-gal)被认为是一种衰老模型，它能诱导氧化应激反应导致记忆功能障碍。吡咯并喹啉醌是一种存在于各种食物中的氧化还原辅助因子。在我们以前的研究中，我们发现 PQQ 可以通过与氨基酸结合转化为一个衍生物，这有利于一些病理过程。在这项研究中，我们发现了一种有益的谷氨酸混合物，它可以减少氧化应激对 d- 半乳糖诱导的小鼠的神经毒性。我们的结果表明，PQQ 可能影响老化过程中前炎症介质的产生，包括细胞因子和前列腺素。D- 半乳糖诱导小鼠海马组织 MDA 和 ROS 水平升高，T-AOC 活性降低，PQQ 可逆转这些变化。此外，PQQ 显著增强了超氧化物歧化酶 SOD2 mRNA 的表达。PQQ 通过与过量谷氨酸结合，改善 d- 半乳糖所致的记忆缺陷和神经毒性，这为谷氨酸介导的神经毒性、炎症和氧化应激提供了一个联系。此外，PQQ 抑制 d- 半乳糖上调 p-Akt 的表达，维持 gsk-3的活性，导致海马 p-Tau 水平下调。PQQ 部分通过 Akt/GSK-3通路调节记忆能力。
Aging is a complicated multifactorial process that results in a gradual slow decline in physiological function and the ability of an organism to survive. Oxidative damage has been implicated to be a major factor in the decline in physiologic function that occurs during the aging process1,2. Several studies have suggested that accumulation of reactive oxygen species (ROS) and elevated oxidative stress are associated with neuronal dysfunction in various age-related neurodegenerative disorders3,4,5. Mitochondria have been revealed as an important link between the age-related oxidative damage and the alterations in physiologic function associated with aging6,7,8. Furthermore, studies reported that oxidative damage to mitochondria would increase the generation of ROS, and lead to impaired cognition6,9,10,11. Oxidative damage stimulates the generation of free radicals and release high level of glutamate that may aggravate damage to brain cells12,13. Glutamate is the critical excitatory neurotransmitter in the central nervous system. However, excessive glutamate stimulation can produce ROS, which will contribute to neuronal damage14,15,16,17. In addition, the cumulate of Glutamate and continuous increase of ROS trigger neuroinflammatory pathways, increasing the chemokines, cytokines, and prostaglandins18,19,20,21,22,23. Moreover, reports demonstrated that glutamate decreased the expression of p-GSK-3β, which would increase GSK-3β activity during glutamate-induced neurotoxicity24. Pharmacological treatments, which inhibit GSK-3β, have been reported to reduce cognitive impairment in AD mice25.
衰老是一个复杂的多因素过程，导致生理功能和生物体生存能力的逐渐下降。氧化损伤被认为是老化过程中生理功能下降的一个主要因素。一些研究表明，在各种与年龄相关的神经退行性紊乱中，活性氧类和氧化应激的积累与神经功能障碍有关。线粒体被揭示为与年龄相关的氧化损伤和与年龄相关的生理功能改变之间的重要联系。此外，研究报告称，线粒体的氧化损伤会增加 ROS 的产生，并导致认知障碍6,9,10,11。氧化损伤刺激自由基的产生和释放高水平的谷氨酸盐，可能加重对脑细胞的损伤12,13。谷氨酸是中枢神经系统的重要兴奋性神经递质。然而，过度的谷氨酸刺激可以产生活性氧，这将有助于神经损伤14,15,16,17。此外，谷氨酸的累积和活性氧的持续升高可触发神经炎症通路，增加趋化因子、细胞因子和前列腺素18、19、20、21、22、23。此外，研究表明谷氨酸降低谷氨酸诱导的神经毒性24小时内 p-gsk-3的表达，增加 gsk-3的活性。药物治疗，抑制 gsk-3，已报告减少认知障碍的 AD mice25。
D-galactose (D-gal) has been considered an artificial aging model which induces oxidative stress and inflammatory response resulting in memory and synaptic dysfunction26,27,28. Chronic systemic administration of D-gal in rodents has been extensively used as an animal model for brain aging in various anti-aging studies29,30,31. It has been reported that animals receiving chronic successive administration of D-gal (50–500 mg/kg) for 4–8 weeks experienced cognitive and memory dysfunction24,25,30. It has been reported that D-gal induced behavioral and neurochemical changes that could mimic many characteristics of the natural process of brain aging26,32,33. This model exhibits accelerated aging in tissues especially brain34,35,36,37,38. The brain is particularly vulnerable to oxidative damage because of its high oxygen demand, high unsaturated lipid content, and relative deficiency in anti-oxidative defense mechanisms. Thus, initial studies suggested that antioxidant therapy is crucial to prevent aging or age-related neurodegenerative disorders39,40.
D- 半乳糖(D-gal)被认为是一种人工衰老模型，它能诱导氧化应激和炎症反应，导致记忆和突触功能障碍26,27,28。慢性系统给予 d- 半乳糖作为脑衰老的动物模型，在各种抗衰老研究中得到了广泛的应用。据报道，长期连续给予 d- 半乳糖(50-500mg/kg)4-8周的动物会出现认知和记忆功能障碍24,25,30。据报道，d- 半乳糖诱导的行为和神经化学改变可以模拟大脑活动自然过程的许多特征。该模型显示了组织特别是脑34、35、36、37、38的衰老加速作用。由于大脑需氧量高，不饱和脂肪含量高，抗氧化防御机制相对缺乏，因此特别容易受到氧化损伤。因此，最初的研究表明，抗氧化剂治疗对预防衰老或年龄相关的神经退行性疾病至关重要。
Pyrroloquinoline quinone (PQQ) is a redox cofactor. It has been found in various foods, including vegetable and animal tissues41,42,43. PQQ not only serves to mediate redox reactions in the mitochondrial respiratory chain, but also plays a potential role of scavenging ROS and attenuating oxidative stress in mitochondria44. Accumulating evidence shows that PQQ can protect neurons against glutamate-induced damage by scavenging ROS17.
吡咯并喹啉醌是一种氧化还原辅助因子。在各种食物，包括蔬菜和动物组织中都发现了这种物质。PQQ 不仅在线粒体呼吸链中介导氧化还原反应，而且在线粒体44中发挥清除活性氧和衰减氧化应激的潜在作用。越来越多的证据表明，PQQ 通过清除 ROS17对谷氨酸诱导的神经元损伤有保护作用。
In the present study, we investigated that the protective effects of PQQ against D-gal induced oxidative stress, cognitive impairment, glutamate level and inflammation factors in mouse brain. We further explored the link between oxidative stress, neurotoxicity, neuroinflammation, and its potential contribution in aging related disease.
在本研究中，我们研究了 PQQ 对 d- 半乳糖诱导的氧化应激、认知功能障碍、谷氨酸水平和炎症因子的保护作用。我们进一步探讨了氧化应激、神经毒性、神经炎症及其在衰老相关疾病中的潜在作用之间的联系。
Effects of PQQ on cognitive impairment induced by D-gal
PQQ 对 d- 半乳糖所致认知功能障碍的影响
The spatial working memory using spontaneous alteration behavior percentage (%) was analyzed using Y-maze task. Our results indicated that D-gal had a lower percentage of spontaneous alteration than control group, indicating less working memory (P < 0.05). However, the spontaneous alteration was significantly increased in mice who received PQQ either alone or in combination with D-gal (P < 0.05), indicating that PQQ improved memory in D-gal treated mice (Fig. 1).
采用 y 迷宫任务分析空间工作记忆的自发改变行为百分比(%)。结果表明，d- 半乳糖自发改变率低于对照组，表明工作记忆减退(p < 0.05)。而 PQQ 单独或与 D-gal 联合使用组小鼠的自发改变明显增加(p < 0.05) ，表明 PQQ 改善了 D-gal 小鼠的记忆力(图1)。
Passive avoidance test
The results showed that PQQ significantly ameliorated cognitive ability of D-gal-treated mice for Passive avoidance test (Fig. 2). The model group mice had more error times and shorter latency for retention than those of control group mice (*P < 0.05, **P < 0.01 relative to control group, respectively). PQQ contributed to decreased number of mistakes (#P < 0.05, ##P < 0.01 relative to D-gal group, respectively) during the training session both alone and combining with D-gal. In the retention session, PQQ treatment with D-gal group mice showed longer latency and less mistakes compared with the model group mice (##P < 0.01 relative to D-gal group).
结果表明，PQQ 能显著改善 d- 半乳糖处理小鼠被动回避试验的认知能力(图2)。与对照组相比，模型组小鼠出错次数明显增多，记忆潜伏期明显缩短(* p < 0.05，* * p < 0.01)。PQQ 有助于减少单独训练和与 D-gal 联合训练时的错误次数(# p < 0.05，# p < 0.01)。在记忆保持阶段，dgal 组小鼠 PQQ 治疗的潜伏期较模型组小鼠延长，错误发生率较低(# # p < 0.01，dgal 组小鼠相对)。
PQQ ameliorated D-gal-induced oxidative stress and restored T-AOC activity in D-gal-induced aging mice
PQQ 改善 d- 半乳糖诱导的小鼠氧化应激，恢复 d- 半乳糖诱导的小鼠 T-AOC 活性
Treatment with PQQ reduced the hippocampal MDA level, and thus reduced oxidative stress (Fig. 3A). In addition, the ROS levels were significantly higher in the hippocampal (p < 0.01) of D-gal-induced mice compared to the control mice. Treatment with PQQ inhibited the increase of levels of ROS in hippocampal (p < 0.01) (Fig. 3B). T-AOC activities were significantly lower in the hippocampal (p < 0.05) of D-gal-induced mice compared with normal control mice. Treatment with PQQ significantly ameliorated the reduction in hippocampal T-AOC activity (p < 0.05) (Fig. 3C). In addition, PQQ alone had no effect on MDA, ROS level or T-AOC activity in normal mice.
PQQ 治疗可降低海马 MDA 水平，从而降低氧化应激。此外，d- 半乳糖诱导小鼠海马内活性氧水平显著高于对照组(p < 0.01)。PQQ 治疗可抑制海马 ROS 的升高(p < 0.01)(图3B)。D- 半乳糖诱导小鼠海马 T-AOC 活性明显低于正常对照小鼠(p < 0.05)。PQQ 治疗可显著改善海马 T-AOC 活性的降低(p < 0.05)。此外，PQQ 单独使用对正常小鼠的 MDA、 ROS 水平和 T-AOC 活性均无影响。
Effect of PQQ on SOD expression in D-gal-induced mice
PQQ 对 d- 半乳糖诱导小鼠 SOD 表达的影响
Semiquantitative RT-PCR (Fig. 4A, Supplementary Info 2) was carried out to determine the levels of SOD1 and SOD2 expression. The mean levels of SOD1 mRNA and, to a greater extent, SOD2 mRNA were lower in D-gal mice than in the control group (P < 0.01) (Fig. 4). PQQ combine with D-gal treatment did not alter mRNA expression of SOD1 compared to the D-gal group, but statistically enhanced SOD2 expression (P < 0.01).
半定量 RT-PCR (图4A，补充信息2)检测 SOD1和 SOD2的表达水平。D- 半乳糖组小鼠 SOD1 mRNA 和 SOD2 mRNA 的平均水平较对照组明显降低(p < 0.01)(图4)。PQQ 联合 d- 半乳糖治疗组与 d- 半乳糖治疗组相比，未改变 SOD1的 mRNA 表达，但显著增强了 SOD2的表达(p < 0.01)。
Effect of PQQ on inflammatory factors include IL-2, IFN-γ levels and the production of Prostaglandin E2 (PGE2) in D-gal-induced mice
PQQ 对 d- 半乳糖诱导的小鼠炎症因子的影响包括 IL-2、 ifn- 水平和前列腺素E2的产生
The effects of PQQ on IL-2 and IFN-γ levels in serum were detected by ELISA (Fig. 5A,B). D-gal treatment significantly increased IL-2 and IFN-γ levels 1.8- and 2.4-times (P < 0.01), respectively. Combining D-gal treatment with PQQ significantly decreased IL-2 from 2.63 ± 0.08 pg/ml in the D-gal group to 1.26 ± 0.12 pg/ml in the D-gal + PQQ group (P < 0.01). IFN-γ secretion was decreased from 42.35 ± 2.12 pg/ml in the D-gal group to 11.26 ± 1.18 pg/ml in the D-gal + PQQ group (P < 0.01). Figure 5C showed the effect of PQQ on the production of PGE2 in the hippocampus of D-gal-induced mice. As compared to control group, D-gal significantly increased the production of PGE2 to 6.41 times (P < 0.01). Treating the mice with PQQ significantly depressed the increase of production of PGE2 (p < 0.05), as compared to the D-gal-treated group. The production of PGE2 level in hippocampus were lower in PQQ treated mice than D-gal treated group (p < 0.01). These data indicate that PQQ reduced the enhanced IL-2 and IFN-γ caused by D-gal and regulated the production of PGE2.
ELISA 法检测 PQQ 对血清 IL-2和 ifn- 水平的影响(图5A，b)。D- 半乳糖治疗组 IL-2和 ifn- 水平分别提高1.8倍和2.4倍(p < 0.01)。D-gal 联合 PQQ 治疗组 IL-2水平由 D-gal 组的2.63 ± 0.08 pg/ml 降至 D-gal + PQQ 组的1.26 ± 0.12 pg/ml (p < 0.01)。D- 半乳糖组 ifn- 分泌量由42.35 ± 2.12 pg/ml 降至11.26 ± 1.18 pg/ml (p < 0.01)。图5C 显示 PQQ 对 d- 半乳糖诱导小鼠海马产生 PGE2的影响。与对照组相比，d- 半乳糖能显著提高前列腺素 e2的产量，提高幅度为对照组的6.41倍(p < 0.01)。与 d- 半乳糖处理组相比，PQQ 处理组小鼠前列腺素 e2产生明显减少(p < 0.05)。PQQ 组小鼠海马 PGE2含量明显低于 d- 半乳糖组(p < 0.01)。提示 PQQ 可降低 d- 半乳糖所致 IL-2和 ifn- 的增强，并可调节 PGE2的产生。
Effects of PQQ on glutamate content in D-gal-induced mice
PQQ 对 d- 半乳糖诱导小鼠谷氨酸含量的影响
Increased glutamate concentration in hippocampus were found in D-gal induced mice compared to control groups (25.14% vs p < 0.05). Only PQQ treated, the glutamate levels were not significant compared to control mouse. Glutamate level in hippocampus was lower in PQQ treated mouse than D-gal treated mouse (27.86% vs p < 0.05). The increased glutamate level in D-gal treatment were ameliorated significantly by PQQ treatment in hippocampus (23.32% vs p < 0.05) (Fig. 6A). Additionally, there was no significant difference in cortex and cerebellumin between these groups (Data not shown).
D- 半乳糖诱导小鼠海马内谷氨酸浓度较对照组升高(25.14% vs p < 0.05)。只有 PQQ 处理后，谷氨酸水平与对照组相比无显著性差异。PQQ 组小鼠海马谷氨酸含量低于 d- 半乳糖组(27.86% vs p < 0.05)。在 d- 半乳糖处理中，PQQ 处理可显著改善海马区谷氨酸水平(23.32% vs p < 0.05)(图6A)。此外，这些组之间在大脑皮层和小脑蛋白方面没有显著差异(数据未显示)。
Furthermore, in molecular masses spectrum (Fig. 6B), the ions at m/z 414.19 ([M + H]+), 415.2([M + 2 H]+) was found in brain tissue of PQQ treatment group. We infer that the production has a molecular mass of 413 Da. These results are agreement with the previously reporters that PQQ can easily react with amino acids to form stable imidazolopyrroloquinoline29,30. Accordingly, the production with 413 Da molecular weight was tentatively identified as imidazolopyrroloquinoline PQQ-Glu (Fig. 6B). Given the evidence that imidazolopyrroloquinoline have redox and antioxidant activity as well as growth-promoting properties, a clear demonstration that complexes PQQ binding with amino acids exist in tissues has considerable importance. Further study on the beneficial effects of PQQ-Glu in neural function is necessary.
PQQ 治疗组脑组织中 m/z414.19([ m + h ] +)、415.2([ m + 2h ] +)离子的分子质量谱(图6B)。我们推断该产物的分子量为413da。结果表明，PQQ 能与氨基酸反应生成稳定的咪唑吡咯喹啉29、30。据此，初步鉴定其分子量为413da 的产物为咪唑并吡咯喹啉 PQQ-Glu (图6B)。由于咪唑并吡咯喹啉具有氧化还原和抗氧化活性以及促生长的特性，显然证明 PQQ 与氨基酸的结合在组织中存在具有相当重要的意义。进一步研究 PQQ-Glu 对神经功能的有益作用是必要的。
Effect of PQQ on expressions of Akt/GSK3β signaling markers
PQQ 对 akt/gsk3信号标记表达的影响
Western blot analysis (Fig. 7A, Supplementary Info 1) was used to assess the effect of PQQ on the Akt/GSK3β pathway in hippocampus subjected to D-gal. The results show that D-gal had no significant effect on the overall expression of Tau in hippocampus (p > 0.05). But mice treated with D-gal exhibited significant stimulation in the expression of p-Tau in hippocampus (p < 0.01), whereas the expressions of p-Tau in hippocampus were significantly attenuated by co-treatment with PQQ (p < 0.05, Fig. 7D). Although PQQ failed to modulate the expression of Akt in hippocampus, it was able to reduce the p-Akt expression in D-gal treated groups so as to decrease the ratio of p-Akt/Akt from 1.05 to 0.77 (p < 0.05, Fig. 7C). Likewise, PQQ was able to reduce the ratio of p-GSK-3β/GSK-3β from 1.11 to 0.91, which was beneficial to regulate the expression of GSK-3β during D-gal-induced aging (p < 0.05, Fig. 7A,B). However, there were no significant differences in cortex and cerebellumin in these groups (Data not shown).
应用免疫印迹分析(图7A，补充信息1)评价 PQQ 对 d- 半乳糖致海马 akt/gsk3通路的影响。结果表明，d- 半乳糖对海马 Tau 蛋白的整体表达无明显影响(p > 0.05)。Dgal 处理组小鼠海马 p-Tau 蛋白表达明显增强(p < 0.01) ，PQQ 处理组小鼠海马 p-Tau 蛋白表达明显减弱(p < 0.05，图7D)。PQQ 虽然不能调节海马中 Akt 的表达，但可以降低 D-gal 治疗组 p-Akt 的表达，使 p-Akt/Akt 的比值从1.05降至0.77(p < 0.05，图7C)。PQQ 可使 p-gsk-3/gsk-3的比值从1.11降低到0.91，有利于调节 d- 半乳糖诱导衰老过程中 gsk-3的表达(p < 0.05，图7A，b)。然而，在这些组中，大脑皮层和小脑蛋白没有显著差异(数据未显示)。
The present study aimed to investigate the protective effect of PQQ on cognitive impairment in the aging mouse induced by D-gal and the possible underlying mechanisms.
本研究旨在探讨 PQQ 对 d- 半乳糖致衰老小鼠认知功能障碍的保护作用及其可能的机制。
The chronic administration of D-gal induces changes that resemble natural aging in animals, such as cognitive dysfunction, oxidative stress and neurodegradation. D-gal induced senescence acceleration has been accepted as a rodent model for use in research on aging studies45,46. D-gal can cause the accumulation of ROS in vivo, ultimately result in oxidative stress37. In addition, oxidative stress contributes to a chronic inflammatory process during aging and causes age-associated diseases22,47. It is well known that glutamate is the critical excitatory neurotransmitter in the central nervous system. Oxidative stress increases the generation of ROS and release excess glutamate48,49. Excess glutamate stimulation is a common pathway in brain injuries and degenerative diseases, and ROS production is usually considered to be involved in glutamate stimulation50,51. Furthermore, oxidative injury, excitotoxicity and inflammatory seem to be three important factors involved in process of aging.
长期服用 d- 半乳糖会引起类似于动物自然衰老的变化，如认知功能障碍、氧化应激和神经退化。D- 半乳糖诱导衰老加速作为一种啮齿动物模型已被接受用于衰老研究。D- 半乳糖可引起体内活性氧的积累，最终导致氧化应力。此外，氧化应激在衰老过程中促成慢性炎症过程，并引起年龄相关疾病22,47。众所周知，谷氨酸是中枢神经系统的关键兴奋性神经递质。氧化应激增加活性氧的产生并释放过量的谷氨酸。过量的谷氨酸刺激是脑损伤和退行性疾病的常见途径，而活性氧的产生通常被认为与谷氨酸的刺激有关。此外，氧化损伤、兴奋毒性和炎症反应似乎是参与衰老过程的三个重要因素。
Some studies had already confirmed that PQQ prevents oxidative damage in the brain and reduces the cognitive deficit caused by oxidative stress in rats during aging52. In our previous study, we found that PQQ may be converted into a derivative by binding with amino acid, which is beneficial to several pathological processes53,54. However, which derivative promotes antioxidant in aging brain and the underlying mechanism is yet unclear. For the first time, we found a beneficial glutamate mixture which may diminish neurotoxicity by oxidative stress. As well, we will discuss the relevant aspects concerning possible neuroprotection of PQQ in the oxidative stress and neuroinflammatory changes observed in aging.
一些研究已经证实，PQQ 可以防止大脑中的氧化性损伤，并减少老年时期氧化应激对大鼠造成的认知缺陷。在我们以前的研究中，我们发现 PQQ 可以通过与氨基酸结合转化为衍生物，这对一些病理过程有益。然而，哪种衍生物促进抗氧化剂在老化的大脑和潜在的机制尚不清楚。第一次，我们发现了一种有益的谷氨酸混合物，它可以减少氧化应激的神经毒性。同时，我们将讨论 PQQ 在氧化应激中可能的神经保护作用的相关方面以及在衰老过程中观察到的神经炎症改变。
The evidence showed that PQQ treatment can affect learning ability and memory function during oxidative stress in rats55. It is well known that PQQ is a well-characterized, free and redox cycling planar orthoquinone. Similar to other polyphenolic biofactors, there is strong evidence PQQ may play an important role in pathways to antioxidant potential56. Our results showed D-gal seriously impaired the learning and memory abilities. We found that this behavioral impairment was reversed when D-gal treated mice were simultaneously administered PQQ for the duration of the treatment (Fig. 1). The results are supported by previous studies, using MK-801 treatment in mice54 and hypoxia treatment in rats55, which proved that PQQ would prevent the cognitive deficit resulting from the oxidative stress.
证据显示 PQQ 治疗可以影响55只大鼠在氧化应激一周的学习能力和记忆功能。众所周知，PQQ 是一个良好的特点，自由和氧化还原循环平面原醌。与其他多酚类生物因子一样，有强有力的证据表明 PQQ 可能在通往抗氧化剂的途径中发挥重要作用。结果显示 d- 半乳糖严重损害学习记忆能力。我们发现，当 d- 半乳糖处理的小鼠在治疗期间同时给予 PQQ 时，这种行为障碍被逆转(图1)。这一结果得到了先前研究的支持，使用 MK-801治疗 mice54和缺氧治疗大鼠55，这证明 PQQ 可以防止由氧化应激引起的认知缺陷。
We also observed that oxidative damage occurred in the D-gal induced aging mouse brain, and this damage was reversed by PQQ intervention. The consequences of oxidative stress can be measured by markers of damage including ROS, MDA and T-AOC. It has been suggested that accumulation of ROS induces severe damage to neuronal system. An increase in MDA is another important marker for lipid peroxidation37, which is a well-known indicator of oxidative damage of membranes under conditions of oxidative stress. In addition, total antioxidant capacity (T-AOC), as a non-enzymatic antioxidant, also indirectly reflects the level of oxidative stress57. According to our researches, PQQ decreased MDA (Fig. 3A) levels, and reduced ROS in D-gal aging model (Fig. 3B). Furthermore, in our T-AOC test, PQQ could renew the activity of T-AOC in the brain of D-gal-induced aging mice (Fig. 3C).
我们还观察到 d- 半乳糖诱导的衰老小鼠大脑发生了氧化损伤，PQQ 干预可逆转这种损伤。氧化应激的后果可以通过损伤指标来衡量，包括活性氧、丙二醛和总抗氧化能力。有研究表明，活性氧的积累会对神经系统造成严重损伤。MDA 的增加是脂质过氧化的另一个重要标志，脂质过氧化是氧化应激条件下细胞膜氧化损伤的一个著名指标。此外，总抗氧化能力(T-AOC)作为一种非酶抗氧化剂，也间接反映了氧化应力水平。根据我们的研究，PQQ 降低了 d- 半乳糖衰老模型的 MDA (图3A)水平，降低了 ROS (图3B)。此外，在我们的 T-AOC 实验中，PQQ 可以更新 d- 半乳糖诱导衰老小鼠脑内 T-AOC 的活性(图3C)。
Apart from amelioration of MDA and ROS, PQQ also increased the activity of SOD, a major mitochondrial antioxidant enzyme that converts toxic superoxide to hydrogen peroxide. SOD is the first and the most important line of antioxidant enzyme defense against ROS protecting cells and tissues58. SOD2 null mice have been known to have short survival. B-Sod2−/− showed brain lipid peroxidation significantly increased, causing severe growth retardation59. Further more, mitochondrial SOD2 is thought to play an important role in cellular defense against oxidative damage by ROS60.
除了改善丙二醛和活性氧，PQQ 还增加了 SOD 的活性，这是一种主要的线粒体抗氧化酶，能将有毒的超氧化物转化为过氧化氢。超氧化物歧化酶(SOD)是抗氧化酶防御活性氧保护细胞和组织的第一道也是最重要的一道防线。众所周知，SOD2缺失小鼠的存活时间很短。B 型 sod2-/-表明大脑脂质过氧化明显增加，导致严重的生长迟缓59。此外，线粒体 SOD2被认为在 ROS60抗氧化损伤的细胞防御中起重要作用。
In this study, PQQ statistically enhanced SOD2 mRNA expression compared to the D-gal group (Fig. 4). Our findings provide direct supportive experimental evidence supporting that PQQ might have anti-aging effects due to its antioxidant role.
在这项研究中，PQQ 与 D-gal 组相比，统计学上增强了 SOD2 mRNA 的表达(图4)。我们的研究结果提供了直接的支持性实验证据，支持 PQQ 由于其抗氧化作用可能具有抗衰老作用。
In addition, the development of neuroinflammation has also been related to cognitive changes in aging related diseases61,62,63. Neuroinflammation in aging is characterized by the accumulation of many cytokines, in which the most obvious change is IL-2 production64. IL-2 and IFN-γ are produced by activated T cells, and aging disorders have been related to these cytokines exceedingly. Furthermore, chronic hypobaric hypoxia prompts the release of cytokines/chemokines viz. IFN-γ and IL-2 activating the signal transduction pathway related to oxidative stress65. In this study, the production of important cytokines such as IL-2 and IFN-γ was also investigated. D-gal increased the levels of the pro-inflammatory cytokines IL-2 and IFN-γ. PQQ decreased cytokines IL-2 (52.1%), and IFN-γ (73.4%) from D-gal treatment mice (Fig. 5). The results are agreed with the report that PQQ deprivation bring about decline immune function66. This study indicated that PQQ markedly reduce the inflammation in D-gal-treated brain, and thus protect the nervous system against oxidative damage.
此外，神经炎症的发展也与衰老相关疾病的认知改变有关。衰老过程中的神经炎症拥有属性是多种细胞因子的积累，其中最明显的变化是 IL-2的产生64。IL-2和 ifn- 是由活化的 t 细胞产生的，衰老障碍与这些细胞因子关系密切。此外，慢性低压缺氧促进细胞因子/趋化因子的释放。Ifn- 和 IL-2激活与氧化应激相关的信号转导通路65。在这项研究中，重要的细胞因子，如 IL-2和 ifn- 的产生也进行了调查。D- 半乳糖增加促炎细胞因子 IL-2和 ifn- 的水平。PQQ 使 d- 半乳糖处理小鼠的细胞因子 IL-2(52.1%)和 ifn- (73.4%)降低(图5)。结果与 PQQ 剥夺导致免疫功能下降的报道一致。本研究表明，PQQ 能明显减轻 d- 半乳糖处理后脑组织的炎症反应，从而保护神经系统免受氧化损伤。
More over, prostaglandins are major components of the neuroinflammatory process. Prostaglandin E2 (PGE2), a product of oxidative stress, is one of the most reliable biomarkers of lipid peroxidation in the human body, and of aging related diseases67,68.
It has been reported that prostaglandin levels are higher in the brains of AD patients than in control brains69,70. Our results showed that hippocampus PGE2 was significantly increased in D-gal treated mouse. Treatment with PQQ suppressed hippocampus level of PGE2. As the levels of pro-inflammatory prostaglandins increase, the capability of clearance must also be improved to maintain low level prostaglandin and prevent additional inflammation. Our results suggest that PQQ may influence the generation of proinflammatory mediators, including cytokines and prostaglandins during in aging process.
据报道，AD 患者大脑中的前列腺素水平高于对照组大脑中的69,70。结果表明，d- 半乳糖处理小鼠海马 PGE2明显升高。PQQ 抑制前列腺素 e2水平的治疗作用。随着前列腺素水平的增加，清除能力也必须提高，以维持低水平的前列腺素和防止额外的炎症。提示 PQQ 在衰老过程中可能影响前炎症介质的产生，包括细胞因子和前列腺素。
Glutamate is the main excitatory neurotransmitter in the central nervous system (CNS) and the toxicity of glutamate has been shown to induce neuronal cell death through oxidative stress71. In mammalian brain, Glu is by far the most prevalent neurotransmitter, which is excitatory working on over 90% of the synapses in the brain72. It is considered to be the major mediator of excitatory signals and is probably involved in cognitive domain of the central nervous system73. Glutamatergic neurotransmission has already been adopted as a promising target for neuro disorder drug development. Furthermore, Liang et al. reported that reduction of the SOD2 level increased the levels of glutamate at the synapse and led to pathological conditions due to the downregulation of glutamate transporter 1 that clear glutamate from synapse74. In the brains of B-Sod2−/−, the potently downregulated expression of GLT-1 could induce a resultant increase in the glutamate concentration at synapse, leading to excitotoxicity. Therefore, it is of critical importance that the extracellular glutamate concentration must be kept enough low due to the toxicity of glutamate in high concentrations. It should be revealed that the distribution of glutamate in brain is a dynamic equilibrium with a rapid turnover in normal condition. Extracellular glutamate levels are maintained through a balance between release and uptake in the normal brain function. Abnormal glutamate uptake has been shown to be involved in the pathogenesis of neurological disorders where glutamate neurotoxicity plays a major role. Glutamate uptake is one of the main mechanisms responsible for keeping glutamate concentration at low level to resistant glutamate toxicity in normal intact brain tissue for the long-term75. However, there is no match to the capabilities of glutamate uptake in abnormal condition. The previous research was focused on the ability of glutamate transporter function for promoting protective effects against glutamate excitotoxicity to neurons. It has been proved that glutamate bound to various proteins is likely to reduce the concentration of free glutamate in glutamate transport cycles76. Inspired by the successful studies of binding with proteins, it seemed tempting to look for effective compounds binding to glutamate for the normal brain function. It is well known that PQQ has special chemical structure and characteristics and easy to combine with different substances42. Our previous studies found that PQQ was easily converted into derivatives with neurotransmitters amino acid which have beneficial capabilities to several physiological processes53,77. The concentrations of amino acid in the central nervous system tissue vary according to the physiological state. From Fig. 6A, it can be seen that the concentrations of glutamate in D-gal-induced mice were significantly higher than those in control group. D-gal is a reducing sugar having affinity for free amines of amino acid in proteins which lead to accumulation of advanced glycation end products, subsequently lead to elevated oxidative stress78. Because of the competitive interaction with free amines of amino acid, the potential efficacy of PQQ in inhibiting the production of glycation end products by binding with amino acid, and prevented the oxidative stress. Moreover, the “bingding” reduced excitability toxicity by decreasing the content of glutamate. From Fig. 6B, new production was found and identified in the brain of PQQ treated mouse compared with untreated ones by UPLC/MS. The results indicated that the complex may be beneficial for anti-aging effects of PQQ, which were related to the action of mechanism. PQQ inhibits excitotoxicity by which it may produce complex via binding with free glutamate in the brain, providing new insights into future research of central nervous system and drug discovery.
谷氨酸是中枢神经系统(CNS)的主要兴奋性神经递质，谷氨酸的毒性作用是通过氧化应激引起神经细胞死亡。在哺乳动物大脑中，Glu 是目前最普遍的神经递质，它对大脑90% 以上的突触起兴奋作用。它被认为是兴奋性信号的主要介质，可能参与中枢神经系统的认知功能。谷氨酸能神经传导已经被用作神经疾病药物开发的一个有前途的靶点。此外，Liang 等人还报道了 SOD2水平的降低增加了突触的谷氨酸水平，并导致了病理状态，这是由于谷氨酸转运体1的下调使突触74中的谷氨酸得以清除。在 B-Sod2-/-脑中，GLT-1的强烈下调可引起突触谷氨酸浓度的升高，从而导致兴奋毒性。因此，由于高浓度谷氨酸的毒性作用，细胞外谷氨酸浓度必须保持在足够低的水平。应该揭示的是，谷氨酸在大脑中的分布是一个动态平衡，在正常情况下周转迅速。细胞外的谷氨酸水平通过正常的大脑功能释放和摄取之间的平衡来维持。谷氨酸摄取异常已被证明参与了神经系统疾病的发病机制，其中谷氨酸神经毒性起主要作用。谷氨酸摄取是维持谷氨酸浓度在低水平的主要机制之一，以抵抗正常完整脑组织长期存在的谷氨酸毒性。然而，在异常情况下，这些细胞的谷氨酸摄取能力并不匹配。前人的研究主要集中在谷氨酸转运体对神经元兴奋毒性的保护作用。结果表明，谷氨酸与多种蛋白质结合可能降低游离谷氨酸在谷氨酸转运周期中的浓度。受到与蛋白质结合的成功研究的启发，寻找与谷氨酸结合的有效化合物来维持大脑的正常功能似乎很有诱惑力。PQQ 具有特殊的化学结构和性质，易与不同的物质结合42。我们以前的研究发现，PQQ 很容易转化为具有神经递质的氨基酸衍生物，这些衍生物对多个生理过程具有有益的作用。中枢神经系统组织中氨基酸的浓度随着生理状态的不同而不同。从图6A 可以看出 d- 半乳糖诱导小鼠的谷氨酸浓度明显高于对照组。半乳糖是一种还原糖，与蛋白质中氨基酸的游离胺有亲和力，可导致晚期糖基化终产物的积累，进而导致氧化应力升高。由于 PQQ 与氨基酸游离胺的竞争性相互作用，PQQ 通过与氨基酸结合抑制糖基化终产物的生成，并阻止氧化应激。此外，“拼食”通过降低谷氨酸含量而降低兴奋性毒性。在图6B 中，用 UPLC/MS 技术在 PQQ 处理的小鼠脑内发现了新的产物，并与未处理的小鼠进行了比较。结果表明，该复合物可能有利于 PQQ 的抗衰老作用，其作用机制可能与 PQQ 的抗衰老作用有关。PQQ 通过与脑内游离谷氨酸结合，抑制兴奋毒性，为今后中枢神经系统研究和药物开发提供了新的思路。
Glycogen Synthase Kinase-3β (GSK-3β) is a protein kinase involved in memory formation which impairs memory through excessive phosphorylation of substrates, such as tau protein79,80. GSK-3β has been revealed to provide an opportunistic target for learning and memory related disorders in aging process81. Several studies have demonstrated the link between GSK-3β and the neuropathology of AD82,83. Sintoni also reported that GSK-3β dysregulation in the hippocampus play a key role in memory impaired rat models84. In addition, the regulation of transcription process by GSK-3β is an important survival pathway against oxidative stress85. Pharmacological treatments, which inhibit GSK-3β, have been reported to repair cognitive impairment in AD mice25. GSK-3β acts as a downstream regulator, and its activity is mainly regulated by phosphorylation or dephosphorylation via signaling pathways86. In addition, decreased the expression of p-GSK-3β was also reported during glutamate-induced neurotoxicity24. It has been documented that protein kinase B (Akt) is a major kinases to inactivate GSK-3βthrough phosphorylation87. The high enzymatic activity is inhibited upon phosphorylation on residue ser9 of GSK-3β by Akt88. Therefore, drugs that are able to down-regulate the activity of GSK-3β hold high promise of therapeutic effects for aging-related diseases.
糖原合成酶激酶 -3(gsk-3)是一种参与记忆形成的蛋白激酶，通过过度磷酸化底物(如 tau 蛋白79,80)损害记忆。Gsk-3已被发现为老化过程81中的学习和记忆相关疾病提供了一个机会靶点。一些研究已经证实了 gsk-3与 AD82,83的神经病理学之间的联系。辛托尼还报告说，海马中的 gsk-3失调在记忆受损的大鼠模型中起着关键作用。另外，gsk-3对转录过程的调控是抗氧化应激的重要生存途径。药物治疗可以抑制 gsk-3，对 AD 患者认知功能损害有修复作用。Gsk-3作为下游调控因子，其活性主要通过信号途径86磷酸化或去磷酸化来调控。另外，在谷氨酸诱导的神经毒性过程中，p-gsk-3的表达也有所下降。蛋白激酶 b (Akt)是通过磷酸化作用使 gsk-3失活的主要激酶。Akt88对 gsk-3残基 ser9的磷酸化可抑制高酶活性。因此，能够下调 gsk-3活性的药物对衰老相关疾病具有很高的治疗效果。
Obviously, PQQ has been proved to attenuate oxidative stress in mitochondria44,89, and antagonize glutamate-induced neuronal injury both in vitro and in vivo17,90,91. It has been observed that phosphorylated Akt was regulated by PQQ treatment in glutamate-injured hippocampal neurons17. The molecular mechanisms underlying these effects have not been fully elucidated. Although it was well proposed that PQQ might have antioxidant role, the direct supportive experimental evidences linking the mechanism of action with anti-aging have rarely been reported so far. The biological effects of PQQ have been intensively studied, but it is not clear whether the neuroprotective effects of PQQ against oxidative stress is mediated through reducing glutamate and suppressing neuroinflammatory mediators by Akt/GSK-3β signal pathway interruption.
显然，PQQ 已被证明能减弱线粒体 a44,89中的氧化应激，并在体外和 vivo17,90,91中拮抗谷氨酸诱导的神经元损伤。观察到 PQQ 对谷氨酸损伤海马神经元磷酸化 Akt 的调节作用。这些作用的分子机制尚未完全阐明。虽然 PQQ 可能具有抗氧化作用，但直接支持 PQQ 抗衰老作用机制的实验证据至今鲜见报道。PQQ 的生物学效应已经得到了广泛的研究，但是 PQQ 对氧化应激的神经保护作用是否通过 Akt/GSK-3信号通路阻断降低谷氨酸和抑制神经炎症介质而介导尚不清楚。
In this experiment, we found D-gal up-regulated the expression of p-AKT in the hippocampus, the active form of AKT, leading to decrease the activity of GSK-3β. Fortunately, the expression of p-Akt was markedly reduced and maintained the activity of GSK-3β by PQQ, resulting in an down-regulation of p-Tau level in hippocampus. Moreover, our present study indicated that PQQ prevents cognitive impairment of D-gal induced mouse. Interestingly, PQQ reduced the phosphorylation of Tau and maintained the activity of GSK-3β, which was essential for memory and learning in aging process. In addition, the inhibition of MDA activity and ROS production might be involved in the anti-oxidative stress effect of PQQ. Its mechanism may be related to the down-regulation the expression of SOD2. The decline of SOD2 activity can lead to the disruption of oxidative stress balance, which damages mitochondrial function and causes aging related diseases. This effect is likely related to the reduction of oxidative stress via the Akt/GSK-3β pathway. Therefore we inferred that PQQ modulated memory ability partly via Akt/GSK-3β pathway. Our results showed that antioxidant capability of PQQ might be mediated by the inactivation of GSK-3β in an increase of p-GSK-3β. Furthermore, for the first time, we revealed that PQQ could ameliorate the memory deficits and neurotoxicity induced by D-gal via binding with excess glutamate, inhibiting oxidative stress, and eliminating inflammation through GSK-3β/Akt signaling pathway. This is in agreement with the evidence that PQQ protected cultured hippocampal neurons against glutamate excitotoxicity90,91
在本实验中，我们发现 d- 半乳糖上调海马 p-AKT 的表达，导致 gsk-3的活性下降。幸运的是，PQQ 明显降低了 p-Akt 的表达，并维持了 gsk-3的活性，导致海马 p-Tau 水平下调。此外，本研究还表明 PQQ 对 d- 半乳糖所致小鼠认知功能障碍具有保护作用。有趣的是，PQQ 可以减少 Tau 蛋白的磷酸化，维持 gsk-3的活性，而 gsk-3在衰老过程中对记忆和学习是必不可少的。此外，抑制丙二醛(MDA)活性和活性氧(ROS)产生可能参与 PQQ 的抗氧化应激作用。其机制可能与下调 SOD2的表达有关。2活性的下降会导致氧化应激平衡的破坏，从而损害线粒体功能并引起与衰老相关的疾病。这种效应可能与通过 Akt/GSK-3途径减少氧化应激有关。因此，我们推测 PQQ 调制记忆能力部分是通过 Akt/GSK-3通路实现的。我们的结果表明，PQQ 的抗氧化能力可能是通过 gsk-3的失活增加 p-gsk-3而介导的。此外，我们首次发现 PQQ 通过与过量谷氨酸结合，抑制氧化应激，通过 gsk-3/Akt 信号通路消除炎症反应，从而改善 d- 半乳糖所致的记忆缺陷和神经毒性。这与 PQQ 保护培养海马神经元免受谷氨酸兴奋毒性90,91的证据一致
In conclusion, our findings demonstrate that PQQ effectively improves D-gal-induced cognitive dysfunction provide a link between Glu-mediated neurotoxicity, inflammation and oxidative stress. Taken together, our results suggest that PQQ is beneficial in preventing cognitive deficits during aging process. More work remains to be done before we elucidate these mechanisms.
总之，我们的研究结果表明，PQQ 有效地改善 d- 半乳糖诱导的认知功能障碍提供了谷氨酸介导的神经毒性、炎症和氧化应激之间的联系。综上所述，我们的研究结果表明 PQQ 在预防老化过程中的认知缺陷方面是有益的。在阐明这些机制之前，还有更多的工作要做。
Materials and Methods
Reagents and drugs
D-gal (Sigma-Aldrich, USA) and PQQ (Shanghai Med. Co., China) solutions were freshly prepared in sterile water. MDA (catalog no. S0131) were purchased from Beyotime Institute of Biotechnology (China). T-AOC (catalog no. S0116, Beyotime Institute of Biotechnology, China). IL-2 and IFN-γ production were measured using ELISA Ready-SET-Go! ® kits (eBioscience, USA). PGE2 were tested using PGE2 EIA kit (Cayman Chemical Co., Ann Arbor, MI, USA). Total RNA Extraction Reagent was purchased from Beyozol (China); High-Capacity cDNA Reverse Transcription Kits were purchased from Applied Biosystems (China). Semiquantitative RT-PCR was performed using primers designed by and obtained from Sangon Biotech, LTD (China). Glu were purchased from ACROS (Belgium, USA). Methanol was of HPLC grade and obtained from CNW Technologies GmbH (Hanau, Germany).
D-gal (Sigma-Aldrich，USA)和 PQQ (Shanghai Med)。在无菌水中新鲜制备的溶液。MDA (目录号。S0131)购自碧昂斯生物技术研究所(中国)。目录编号。中国碧昂斯生物技术研究所 S0116)。用 ELISA Ready-SET-Go 法测定 IL-2和 ifn- 的产生！工具箱(美国 eBioscience)。采用 PGE2环境影响评价试剂盒(安阿伯 Cayman Chemical co. ，MI，USA)测定 PGE2。总 RNA 提取试剂购自 Beyozol，大容量 cDNA 逆转录试剂盒购自美国 Applied Biosystems 公司。利用中国三光生物技术有限公司自行设计和获得的引物进行半定量 RT-PCR。Glu 是从 ACROS (比利时，美国)购买的。甲醇采用高效液相色谱法，从哈瑙 CNW 技术有限公司获得。
Animals and treatment
ICR mice were supplied from Cavens Lab Animal Co. (Changzhou, China). The mice were aged 3 months old, weighed 32 ± 2 g. Animals were housed at a temperature of 25 ± 1 °C and relative humidity of 55%–60% with a controlled light-dark cycle. The mice had free access to food and water. The mice were divided into four groups of 10 male mice at random. Two groups of mice received daily subcutaneous injection of D-gal at dose of 150 mg·kg−1 · d−1 for 6 weeks, and the third group of mice served as normal control was injected with saline (0.9% NaCl) only. Meanwhile, one group of D-gal-treated mice received PQQ at a dose of 100 μg · kg−1 · d−1. At the same time, the other group of D-gal-treated mice and the control group of mice were given distilled water, and the fourth group received PQQ (100 μg·kg−1 · d−1) only. The dosages and pretreatment duration for the compounds were used based on the previous studies56,57,58. All experiments were approved by the Animal Care and Ethics Committee of Jiangsu Institute of Nuclear Medicine and carried out according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
ICR 小鼠由卡文斯实验室动物公司(常州)提供。这些小鼠3个月大，体重32 ± 2 g，温度为25 ± 1 ° c，相对湿度为55%-60% 。老鼠可以自由地获得食物和水。将小鼠随机分为4组，每组10只雄性小鼠。两组小鼠每日给予150mg/kg-1 d-1剂量的 d- 半乳糖，连续6周，第三组作为正常对照，仅给予生理盐水(0.9% NaCl)。同时，一组 d- 半乳糖处理小鼠给予 PQQ，剂量为100g kg-1d-1。另一组给予 d- 半乳糖处理的小鼠和对照组给予蒸馏水，第四组只给予 PQQ (100gkg-1d-1)。在前人研究的基础上，对化合物的用量和预处理时间进行了研究。所有的实验都得到了江苏核医学研究所动物护理和伦理委员会的批准，并且是根据美国国立卫生研究院实验室动物护理和使用指南进行的。
Y-maze constructed from black painted wood with three arms of 50 cm long, 20 cm high, and 10 cm wide at the bottom and at the top was used for behavioral analysis. The mice were placed at the center and were allowed to move freely for three 8-min sessions. The test was performed in a sound-isolated and dark room. The arm entries series was noted for observation. Spontaneous alteration was defined as the successive entry of the mice into the three arms in overlapping triplet sets. Alteration behavior (%) was calculated as [successive triplet sets(entries into three different arms consecutively)/total number of arms entries −2] × 10059.
Y 型迷宫由三条长50厘米、高20厘米、底部和顶部宽10厘米的黑色漆木构成，用于行为分析。老鼠被放置在中心，并被允许自由移动三个8分钟的会议。实验在隔音暗室中进行。手臂入口系列注意观察。自发性改变被定义为老鼠以重叠的三连体组连续进入三只手臂。改变行为(%)计算为[连续三个三连音组(连续三个不同的臂)/臂入口总数 -2]10059。
Passive avoidance test
The passive avoidance apparatus contains two chambers (20 × 25 × 30 cm each) of equal size, one is an illuminated by a 4 W fluorescent lamp and other is a dark one. Both of these compartments are linked with a door which allows mice to cross freely from one compartment to another. These compartments contain a grid floor that consists of rods having a distance of 0.5 cm between the rods. Initially the animals were trained by placing them in the illuminated compartment, and an electric shock (40 V, 0.5 A, 1 s) was delivered to their paws via the rods of the grid floor, causing them to move into the dark chamber. Mice were then immediately removed and returned to their home cages. During the retention trial that occurred 24 h later, mice were placed in the illuminated chamber, and the latency to enter the dark chamber was recorded (i.e., step-through latency). In this session, the number of repeated step-down in 300 s was counted as errors.
这种被动回避装置包含两个相同大小的腔室(每个202530厘米) ，一个被4w 的荧光灯照亮，另一个是黑暗的腔室。这两个隔间都与一扇门相连，这扇门可以让老鼠自由地从一个隔间穿过到另一个隔间。这些隔间包含一个由杆组成的网格层，杆之间的距离为0.5厘米。起初，这些动物被放置在照明的隔间里进行训练，然后通过栅格地板的杆子向它们的爪子传递电击(40伏，0.5 a，1秒) ，使它们移动到黑暗的房间里。然后，老鼠被立即移走并放回它们的笼子里。在24小时后进行的保留试验中，将小鼠放在发光的腔室中，记录进入暗腔的潜伏期(即逐级潜伏期)。在这次会议中，300秒内重复减速的次数被计算为错误。
Measurement of ROS, MDA levels, and total antioxidant capacity (T-AOC)
MDA, ROS, and T-AOC, were determined by using commercially available kits. ROS was measured as described previously with some modification, based on oxidation of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) to 2,7-dichlorodihydrofluorescein60. The data are expressed as picomole DCF formed per minute per milligram protein. The thiobarbituric acid reaction method was used to determine MDA content. MDA reacts with thiobarbituric acid to form a stable chromophoric molecule that can be measured at the wavelength of 532 nm61. MDA content was expressed as nanomoles per milligram of tissue protein. The Ferric reducing power (FRAP) test method was used to quantify the T-AOC of the organs62. After mixing the FRAP reagent with the organ sample, the absorbance was determined every 1 min for a total of 10 min. Fe (II) standard solution was tested in parallel.
用市售试剂盒测定丙二醛(MDA)、活性氧(ROS)和总抗氧化能力(T-AOC)。以2,7- 二氯二氢荧光素二乙酸酯(DCFH-DA)氧化为2,7- 二氯二氢荧光素60为基础，经过一些修饰，测定了活性氧。这些数据表示为每毫克蛋白质每分钟形成的微微粒 DCF。采用硫代巴比妥酸反应法测定丙二醛含量。丙二醛与硫代巴比妥酸反应生成稳定的发色分子，可在532 nm61波长下测定。MDA 含量以每毫克组织蛋白质的纳米微粒表达。采用铁还原力(FRAP)试验方法，测定了有机物62。将 FRAP 试剂与器官样品混合后，每隔1分钟测定一次吸光度，共10分钟。并对 Fe (II)标准溶液进行了平行测定。
RNA extraction and RT-PCR analysis
RNA 提取及 RT-PCR 分析
T RNA was extracted from brain using the kit as directed by the manufacturer’s protocol. Using the High-Capacity cDNA Reverse Transcription Kits, 1.5 μg of RNA was transcribed into cDNA according to the manufacturer’s instructions, and cDNA samples were stored at −70 °C. Semiquantitative RT-PCR was performed using the primers obtained from Sangon Biotech, LTD (China). The sequences of the primers are designed as follows: SOD1(352 bp), Forward 5′-ACCATCCACTTCGAGCAG-3′, Reverse 5′-TTTCTTCATTTCCACCTTTG-3′; SOD2(396 bp), Forward, 5′-GCACCACAGCAAGCACC-3′, Reverse, 5′-CCCAGCAGCGGAATAAG-3′; β-actin(384 bp), Forward, 5′-GGGAAATCGTGCGTGACAT-3′, Reverse, 5′-CAGGAGGAGCAATGATCTT-3′.
根据制造商的协议，t RNA 是通过试剂盒从大脑中提取的。利用高容量 cDNA 反转录试剂盒，按制造商指示将1.5 g RNA 转录成 cDNA，并将 cDNA 样品保存在 -70 ° c。利用从中国桑贡生物技术有限公司获得的引物进行半定量 RT-PCR。设计的引物序列如下: SOD1(352bp) ，正向5′-acccatccagccag-3′ ，逆向5′-tttctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctccaccacc-3′ ，正向5′-gccacc-3′ ，反向5′-cccagcagc-3′ ;-actin (384bp) ，正向5′-ggggggggggcgat-3′ ，反向5′-gagcagatcgat-3′。
ELISA analysis for IL-2, IFN-γ and PGE2
IL-2、 ifn- 和前列腺素 e2的 ELISA 检测
IL-2 and IFN-γ production were measured using ELISA Ready-SET-Go! ® kits with sensitivity of 2 and 4 pg/ml, respectively as described by the manufacturer. The absorbance of the reaction was measured at 450 nm with subtraction of background at 570 nm using a microplate reader. PGE2 concentrations were measured using a commercially available PGE2 EIA kit according to manufacturer’s instructions as the previous described31.
用 ELISA Ready-SET-Go 法测定 IL-2和 ifn- 的产生！按制造商所述，其敏感度分别为2及4皮克/毫升。反应的吸光度在450nm 处测量，背景色在570nm 处减去。根据制造商的说明书，使用市售的 PGE2 EIA 试剂盒测定了前列腺素 e2的浓度。
Western blot analysis
The Western blot assay was performed as previously described24,88. Briefly, the hippocampus was homogenized in lysis buffer (Beyotime Biotechnology, Haimen, China) and centrifuged at 12,000 rpm for 10 min at 4 °C. The concentration of hippocampal protein was measured using BCA assay kit (Sangon, Shanghai, China). The dilution of primary antibodies as follows: β-actin (1:4000, Abcam, UK); GSK-3β; p-GSK-3β; AKT; p-AKT; Tau, p-Tau (1:2000, Cell Signaling Technology, USA). Each membrane was rinsed three times for 15 min and incubated with the secondary antibodies (1:8000, Abcam, UK). β-actin was used as the loading reference for data analysis.
免疫印迹实验按之前描述的24,88。简单地说，海马匀浆在裂解缓冲液中(海门 Beyotime 生物技术公司) ，在12,000转/分钟的速度下离心，在4 ° c 下10分钟。应用 BCA 检测试剂盒测定海马蛋白质浓度。初级抗体的稀释度如下:-actin (1:4000，Abcam，UK) ; gsk-3; p-gsk-3; AKT; p-AKT; Tau，p-Tau (1:2000，Cell signal Technology，USA)。每层膜清洗3次，持续15分钟，并与次级抗体共同孵育(英国 Abcam，1:8000)。采用肌动蛋白作为载荷参考，进行数据分析。
Measurement of glutamate
The levels of glutamate in brain tissues were analyzed using HPLC system as the our confirmed method. Furthermore, a related marker for the protection of glutamate neurotoxicity in hippocampus has been explored by UPLC/MS. The brain tissues were homogenized in ice-cold 0.5 M formic acid (5 mL/g) and the mixtures were centrifuged 15,000 g for 30 min at 4 °C. The supernatant was collected and run through solid-phase extraction columns (Styre Screen® H2P) with the aid of vacuum. Extraction cartridges were pretreated by rinsing with 1 ml of ethanol, followed by 1 ml deionized water contain 0.1% trifluoroacetic acid (TFA). After the samples were loaded onto columns, the columns were washed with 1 ml deionized water, followed by 2 ml of ethanol/o.1%TFA (60/40) in water. Eluants were evaporated to dryness by vacuum freeze-drying and waiting for analysis. The content of glutamate were determinated using UPLC-MS49. Molecular masses were carried out on a Waters Acquity ultraperformance liquid chromatography system (Waters, MA, USA) with a SQ Detector 2 mass spectrometer equipped with diode-array detector. Data were collected and processed with Waters MassLynx. A Waters BEH C18 column (2.1 × 100 mm, 1.7 um) was applied for reverse phase chromatographic separation. The optimized MS parameterswere set as follows: Desolvation temperature 400 °C, desolvation gas flow 600 L/h, source temperature 110 °C, cone gas flow 50 L/h, capillary voltage 3000 V, cone voltage 30 V. The initial mobilephase composition was held at 5/95 ACN/water contain 0.1% TFA (v/v) for 5 min followed by a linear gradient to 75:25 ACN:water contain 0.1% TFA (v/v) at 25 min.
采用高效液相色谱法测定脑组织中谷氨酸含量。在此基础上，利用超高效液相色谱/质谱联用技术研究了谷氨酸对海马神经毒性的保护作用。冰冻0.5 m 甲酸(5ml/g) ，离心15000g，在4 ° c 条件下离心30min。收集上清液，用固相萃取塔(Styre Screen H2P) ，真空辅助萃取。萃取筒用1毫升乙醇冲洗预处理，其次是1毫升去离子水含有0.1% 的三氟乙酸。样品装入柱后，用1ml 去离子水洗涤柱，然后用2ml 乙醇/0.1% TFA (60/40)水洗涤柱。洗脱液通过真空冷冻干燥蒸发干燥等待分析。采用超高效液相色谱法测定谷氨酸的含量。采用二极管阵列检测器，在美国马萨诸塞州沃特斯超高效液相色谱系统上进行了分子质量的测定。数据由 Waters MassLynx 收集和处理。采用 Waters BEH C18色谱柱(2.1100 mm，1.7 um)进行反相色谱分离。优化后的 MS 参数为: 去溶温度400 ° c，去溶气流600 L/h，源温度110 ° c，锥气流50 L/h，毛细管电压3000 v，锥电压30 v。初始活性相组成保持在含0.1% TFA (v/v)的活性炭纳米粒子5/95/水中5分钟，然后呈线性梯度变化到75:25: 水含0.1% TFA (v/v) ，25分钟。
All statistical tests were performed with the SPSS v16.0 statistical software. One way analysis of variance was applied using Tukey’s posthoc comparisons. The data were expressed as mean ± SD of triplicate experiments. P-values below 0.05 were considered statistically significant.
用 SPSS v16.0统计软件进行统计学检验。使用 Tukey 的方差分析比较法进行了单向实验。数据表示为三次实验的平均标准偏差。P 值低于0.05被认为具有统计学意义。