技术领域technical field
本发明涉及药物技术领域，具体来说是一种用于防治血管性痴呆的磷酸二酯酶9A抑制剂的用途。The invention relates to the technical field of medicine, in particular to the use of a phosphodiesterase 9A inhibitor for preventing and treating vascular dementia.
背景技术Background technique
血管性痴呆(Vascular Dementia，VaD)是由流向大脑的血流量减少而引起的一种影响认知能力的进行性疾病,其约占所有痴呆患者的17-20％，成为继阿尔茨海默病(AD)的第二大主要形式的痴呆症，并且在老年人群中普遍存在。临床表现为认知功能障碍、神经功能缺失等。Vascular dementia (Vascular Dementia, VaD) is a progressive disease that affects cognitive ability caused by reduced blood flow to the brain. It accounts for about 17-20% of all dementia patients. (AD) is the second leading form of dementia and is prevalent in the elderly population. Clinical manifestations include cognitive impairment and neurological deficits.
脑血管疾病的异质性使得阐明神经病理学机制和血管性痴呆机制具有挑战性。脑血流的充足供应对于维持大脑结构和功能的完整性具有重要的意义，脑血管的细微病变都可能对认知功能造成长远的不可逆的损伤。目前公认的由心脏疾病或者颈动脉狭窄或堵塞导致的脑血流灌注低于某个阈值时可以导致认知功能障碍。Marshall等在文章中提出脑血流降低40％～50％可导致不可逆的大脑功能抑制和认知功能障碍。而各种细胞信号转导和调节机制，包括细胞凋亡、自噬，氧化应激和炎症，由于它们参与脑缺血而与VaD相关。小胶质细胞，星形胶质细胞以及一些受神经损伤的神经元和少突胶质细胞在经危险性刺激后被激活时可以分泌炎症介质和其它标志物。一些数据报道，TNF-α、IL-1β和IL-6等促炎细胞因子在血管性痴呆进程中发挥关键作用。The heterogeneity of cerebrovascular disease makes elucidation of neuropathological mechanisms and mechanisms of vascular dementia challenging. Sufficient supply of cerebral blood flow is of great significance to maintain the integrity of brain structure and function. Even minor lesions of cerebral blood vessels may cause long-term irreversible damage to cognitive function. It is currently recognized that cerebral perfusion below a certain threshold, caused by heart disease or carotid artery stenosis or blockage, can lead to cognitive impairment. In their article, Marshall et al. proposed that a 40% to 50% reduction in cerebral blood flow can lead to irreversible brain function inhibition and cognitive dysfunction. Various cellular signaling and regulatory mechanisms, including apoptosis, autophagy, oxidative stress, and inflammation, are associated with VaD due to their involvement in cerebral ischemia. Microglia, astrocytes, and some nerve-damaged neurons and oligodendrocytes secrete inflammatory mediators and other markers when activated after dangerous stimuli. Some data report that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 play critical roles in the progression of vascular dementia.
迄今为止，VaD治疗还没有特效药，当前的治疗手段主要是控制VaD导致的认知障碍的发展进程，但都缺乏显著疗效。目前临床上应用的胆碱酯酶抑制剂如石杉碱甲、多奈哌齐等以及NMDA受体阻断剂美金刚(Memantine)以及其他一些脑循环促进剂尼莫地平、施普善等均不能有效地改善或逆转VaD患者的症状和体征。随着VaD患者的病情加剧，生活往往无法自理，长期需要家人或专职医疗人员的护理，给家庭和社会带来严重的精神损失和经济压力。而且目前尚无FDA批准用于治疗VaD的有效药物。因此寻找新靶点、新机制的有效药物成为迫切需求。So far, there is no specific drug for VaD treatment. The current treatment methods are mainly to control the development process of cognitive impairment caused by VaD, but they all lack significant efficacy. Currently clinically used cholinesterase inhibitors such as Huperzine A, Donepezil, etc., as well as NMDA receptor blocker Memantine and other cerebral circulation promoters such as Nimodipine and Sprusan, cannot effectively improve or Reversal of signs and symptoms in VaD patients. As the condition of VaD patients aggravates, they are often unable to take care of themselves and require long-term care from family members or professional medical personnel, which brings serious mental loss and economic pressure to the family and society. And there is currently no FDA-approved effective drug for the treatment of VaD. Therefore, the search for effective drugs with new targets and new mechanisms has become an urgent need.
LW33是PDE9A的强效选择性抑制剂(IC50为5.0nM，对其他PDE亚型的选择性大于50倍)，是基于PDE9A晶体结构设计并合成的全新结构高效价N-取代吡唑并[3,4-d]嘧啶酮类优选化合物。分子式C18H20CIN5O，分子量357.8。物化特性分析显示，白色至淡黄色粉末，易溶于甲醇、乙醇、丙酮，难溶于水、氯仿、乙酸乙酯等弱极性有机溶剂；药效及初步安全性分析显示，IC50＝5.0nM，小鼠急毒LD50接近1.5g/kg；药代动力学分析显示，大鼠单次灌胃5mg/kg后，Tmax＝2.0h，Cmax＝372.54ng/L，AUC(0-t)＝1640.87μg/L*hr，t1/2＝1.46h，口服生物利用度F＝57％。LW33 is a potent and selective inhibitor of PDE9A (IC50 of 5.0 nM, greater than 50-fold selectivity for other PDE isoforms), a novel structure designed and synthesized based on the crystal structure of PDE9A with high valency N-substituted pyrazolo[ 3,4-d]pyrimidinones are preferred compounds. Molecular formula C18 H20 CIN5 O, molecular weight 357.8. Analysis of physicochemical properties shows that it is white to light yellow powder, easily soluble in methanol, ethanol, acetone, and insoluble in weakly polar organic solvents such as water, chloroform and ethyl acetate; efficacy and preliminary safety analysis show that IC50 =5.0 nM, the acute toxicity LD50 of mice was close to 1.5g/kg; pharmacokinetic analysis showed that after a single gavage of 5mg/kg in rats, Tmax =2.0h,Cmax =372.54ng/L, AUC(0- t) =1640.87 μg/L*hr, t1/2 =1.46h, oral bioavailability F=57%.
该化合物与PDE9A复合物晶体结构显示，其与辉瑞化合物有类似的作用机制。前期药理研究表明，在动物体内东莨菪碱模型中，1.0mg/kg LW33对东莨菪碱致小鼠记忆障碍有显著性记忆改善作用，其药效与阳性对照物石杉碱甲一致。Aβ1-40脑内注射大鼠模型中，LW33低(0.35mg/kg)、中(0.7mg/kg)、高剂量(1.4mg/kg)干预后，能改善模型大鼠的认知功能，缩短潜伏期、增加经过平台次数、延长在目标象限停留时间。海马区cGMP水平显著上升。The crystal structure of this compound in complex with PDE9A shows that it has a similar mechanism of action to the Pfizer compound. Preliminary pharmacological studies have shown that in the scopolamine model in animals, 1.0mg/kg LW33 has a significant memory improvement effect on scopolamine-induced memory impairment in mice, and its efficacy is consistent with that of the positive control, Huperzine A. In the rat model of intracerebral injection of Aβ1-40 , LW33 low (0.35mg/kg), medium (0.7mg/kg) and high dose (1.4mg/kg) interventions can improve the cognitive function of the model rats. Shorten the latency period, increase the number of passes over the platform, and prolong the dwell time in the target quadrant. The level of cGMP in the hippocampus was significantly increased.
此外，LW33还可以诱导海马脑片早期LTP及后期LTP的形成，以及早期LTP和后期LTP的融合，而AChEI多奈哌齐仅能诱导早期LTP形成，与文献报道PDE9抑制剂BAY73-6691的研究结果一致，提示PDE9A抑制剂较AChEI能更有效持久地增强学习记忆。机制研究发现，LW33主要通过cGMP/PKG/CREB信号通路产生抗神经保护作用。但是，截止到现在，尚未见LW33作为治疗血管性痴呆的应用。In addition, LW33 can also induce the formation of early LTP and late LTP, as well as the fusion of early LTP and late LTP in hippocampal slices, while AChEI donepezil can only induce early LTP formation, which is consistent with the research results reported in the literature on PDE9 inhibitor BAY73-6691. It is suggested that PDE9A inhibitor can enhance learning and memory more effectively and lastingly than AChEI. Mechanistic studies have found that LW33 mainly produces anti-neuroprotective effects through the cGMP/PKG/CREB signaling pathway. However, up to now, there is no application of LW33 for the treatment of vascular dementia.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题是：提供LW33在制备预防和/或治疗血管性痴呆疾病药物中的用途，黄芪甲苷通过下调LPS诱导的BV2炎症模型中IL-1β、IL-6和TNF-α的mRNA水平，改善UCCAo模型小鼠和模型大鼠的学习记忆功能障碍，缩短潜伏期、增加经过平台次数、延长在目标象限停留时间。同时可降低模型大鼠血清中MDA含量、升高SOD活性，并降低血清中IL-1β、IL-6的含量及改善血管性痴呆模型小鼠和大鼠脑组织皮层区和海马区的神经元坏死等病理状态，具有防治血管性痴呆的新用途。The technical problem to be solved by the present invention is: to provide the use of LW33 in the preparation of drugs for preventing and/or treating vascular dementia, and astragaloside IV can reduce IL-1β, IL-6 and TNF-α in the BV2 inflammation model induced by LPS. The mRNA level of UCCAo improved the learning and memory dysfunction of UCCAo model mice and model rats, shortened the latency period, increased the number of passing platforms, and prolonged the residence time in the target quadrant. At the same time, it can reduce the content of MDA in the serum of the model rats, increase the activity of SOD, and reduce the content of IL-1β and IL-6 in the serum, and improve the neurons in the cortex and hippocampus of the vascular dementia model mice and rats. Necrosis and other pathological states, and has a new use in preventing and treating vascular dementia.
为实现上述目的，本发明提供了一种一种由下述化合物(Ⅰ)表示的磷酸二酯酶9A抑制剂LW33，或其药学上可接受的盐或含有它们任何一种的药物组合物在制备用于预防或/和治疗血管性痴呆疾病药物的用途。In order to achieve the above object, the present invention provides a phosphodiesterase 9A inhibitor LW33 represented by the following compound (I), or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them. Use in the preparation of a medicament for preventing or/and treating vascular dementia.
进一步地，所述LW33在制备预防和/或治疗血管性痴呆疾病药物中缓解LPS诱导的BV2炎症反应的作用浓度为0.1～1μM。Further, the concentration of LW33 for relieving LPS-induced BV2 inflammatory response in the preparation of a drug for preventing and/or treating vascular dementia is 0.1-1 μM.
进一步地，所述LW33在用于治疗血管性痴呆时降低炎症因子TNF-α、IL1-β和IL-6的mRNA表达的浓度为0.1～1μM。Further, when the LW33 is used to treat vascular dementia, the concentration of reducing the mRNA expression of inflammatory factors TNF-α, IL1-β and IL-6 is 0.1-1 μM.
进一步地，本发明还提供了LW33或其药学上可接受盐或含有它们任何一种的药物组合物在制备影响单侧颈总动脉结扎(UCCAo)模型小鼠脑组织病理结构药物中的用途。Further, the present invention also provides the use of LW33 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in preparing a medicament affecting the pathological structure of brain tissue in unilateral common carotid artery ligation (UCCAo) model mice.
进一步地，本发明还提供了LW33或其药学上可接受盐或含有它们任何一种的药物组合物在制备改善单侧颈总动脉结扎(UCCAo)模型小鼠学习记忆功能障碍药物的用途。Further, the present invention also provides the use of LW33 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in preparing a drug for improving learning and memory dysfunction in unilateral common carotid artery ligation (UCCAo) model mice.
进一步地，所述化合物或其药学上可接受盐或含有它们任何一种的药物组合物的使用剂量为10～20mg/kg。Further, the used dose of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them is 10-20 mg/kg.
本发明还提供了LW33或其药学上可接受盐或含有它们任何一种的药物组合物在制备改善血管性痴呆模型大鼠学习记忆功能障碍的用途。The present invention also provides the use of LW33 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in the preparation of improving the learning and memory dysfunction of vascular dementia model rats.
进一步地，所述化合物或其药学上可接受盐或含有它们任何一种的药物组合物的使用剂量为1～9mg/kg。Further, the used dose of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them is 1-9 mg/kg.
本发明还提供了一种LW33或其药学上可接受盐或含有它们任何一种的药物组合物在制备用于降低血管性痴呆模型大鼠血清中MDA含量、升高血清中SOD含量的药物中的用途。The present invention also provides a LW33 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in the preparation of a medicine for reducing the MDA content in the serum of vascular dementia model rats and increasing the SOD content in the serum the use of.
本发明还提供了LW33或其药学上可接受盐或含有它们任何一种的药物组合物在制备降低血管性痴呆模型大鼠血清中IL-1β、IL-6药物中的用途。The present invention also provides the use of LW33 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in preparing a medicine for reducing IL-1β and IL-6 in the serum of vascular dementia model rats.
本发明还提供了LW33或其药学上可接受盐或含有它们任何一种的药物组合物在制备影响血管性痴呆模型大鼠脑组织病理结构药物中的用途。The present invention also provides the use of LW33 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them in preparing a medicine affecting the pathological structure of brain tissue in vascular dementia model rats.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.
图1为LW33对小鼠小胶质细胞BV2增殖的细胞毒作用；Figure 1 shows the cytotoxic effect of LW33 on the proliferation of mouse microglia BV2;
图2为LW33对LPS诱导的BV2细胞增殖的影响；Figure 2 shows the effect of LW33 on LPS-induced BV2 cell proliferation;
图3a为LW33对LPS诱导BV2炎症模型的炎症因子IL1-β的mRNA水平的影响；Figure 3a shows the effect of LW33 on the mRNA level of the inflammatory factor IL1-β in the LPS-induced BV2 inflammation model;
图3b为LW33对LPS诱导BV2炎症模型的炎症因子IL-6的mRNA水平的影响；Figure 3b shows the effect of LW33 on the mRNA level of the inflammatory factor IL-6 in the LPS-induced BV2 inflammation model;
图3c为LW33对LPS诱导BV2炎症模型的炎症因子TNF-α的mRNA水平的影响；Figure 3c shows the effect of LW33 on the mRNA level of the inflammatory factor TNF-α in the LPS-induced BV2 inflammation model;
图4为LW33治疗21天对UCCAo模型小鼠脑组织海马CA1区神经元的影响(HE染色)；Figure 4 shows the effect of LW33 treatment for 21 days on neurons in the hippocampal CA1 region of the brain tissue of UCCAo model mice (HE staining);
图5为LW33治疗14天对模型大鼠脑组织海马CA1区神经元的影响(HE染色)；Figure 5 shows the effect of LW33 treatment for 14 days on neurons in the hippocampal CA1 area of model rat brain tissue (HE staining);
图6为LW33治疗14天对模型大鼠脑组织皮层区神经元的影响(HE染色)；Figure 6 shows the effect of LW33 treatment on neurons in the cortical area of model rat brain tissue for 14 days (HE staining);
图7为LW33的化学结构式。Figure 7 is the chemical structural formula of LW33.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
实施例1、LW33对BV2细胞活力的影响Example 1. The effect of LW33 on the viability of BV2 cells
(1)实验材料：BV2(小鼠小胶质细胞)，购于中国科学院细胞库；DMEM培养基(美国Gibco)；胎牛血清(美国Gibco)；CCK8(日本同仁化学研究所)；Bay73-6691(1-(2-氯苯基)-6-[(2R)-3,3,3-三氟-2-甲基丙基]-1,5-二氢-4H-吡唑并[3,4-d]嘧啶-4-酮,拜耳)；PF-04447943(6-[(3S，4S)-4-甲基-1-(嘧啶-2-基甲基)吡咯烷-3-基]-1-(四氢-2H-吡喃-4-基)-1,5-二氢-4H-吡唑并[3,4-d]嘧啶-4-酮，美国MCE)。(1) Experimental materials: BV2 (mouse microglia), purchased from the Cell Bank of the Chinese Academy of Sciences; DMEM medium (Gibco, USA); fetal bovine serum (Gibco, USA); CCK8 (Tongjin Institute of Chemistry, Japan); Bay73- 6691 (1-(2-Chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3 ,4-d]pyrimidin-4-one, Bayer); PF-04447943 (6-[(3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl] -1-(Tetrahydro-2H-pyran-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one, MCE, USA).
(2)实验方法：5.0×103的BV2细胞加入到96孔板的板孔中，培养24h。吸出原培养液，加入含有不同浓度(0.1μM、0.3μM、1μM)的LW33的完全培养液，以加入不含LW33的完全培养液作为正常对照。继续培养24h，向每孔中加入10μL CCK8，将培养板在孵育箱内培养2h，用酶标仪在450nm处检测各孔吸光度值，检测细胞活力。(2) Experimental method: 5.0×103 BV2 cells were added to the wells of a 96-well plate and cultured for 24 hours. The original culture solution was aspirated, and the complete culture solution containing different concentrations (0.1 μM, 0.3 μM, 1 μM) of LW33 was added, and the complete culture solution without LW33 was added as a normal control. Continue to culture for 24 hours, add 10 μL of CCK8 to each well, culture the plate in an incubator for 2 hours, and use a microplate reader to detect the absorbance value of each well at 450 nm to detect cell viability.
(3)实验结果：由图1结果表明，LW33在0.1～1μM范围内，对细胞活力没有明显的改变，无明显细胞毒性作用。(3) Experimental results: As shown in Figure 1, LW33 in the range of 0.1-1 μM has no obvious change in cell viability and no obvious cytotoxic effect.
实施例2、LW33对LPS诱导的BV2细胞增殖的影响Example 2. The effect of LW33 on LPS-induced BV2 cell proliferation
(1)实验材料(1) Experimental materials
小鼠小胶质细胞BV2(中国科学院细胞库)，DMEM培养基(美国Gibco)、胎牛血清(美国Gibco)、CCK8(日本同仁)、LPS(Sigma)Mouse microglia BV2 (Cell Bank of Chinese Academy of Sciences), DMEM medium (Gibco, USA), fetal bovine serum (Gibco, USA), CCK8 (Japanese colleagues), LPS (Sigma)
(2)实验方法(2) Experimental method
5.0×103的BV2细胞加入到96孔板的板孔中，培养24小时。将BV2细胞分为：5.0×103 BV2 cells were added to wells of a 96-well plate and cultured for 24 hours. BV2 cells are divided into:
对照组：LPS处理组(1μg/mL作用时间24h)；Control group: LPS treatment group (1μg/mL for 24h);
LPS处理组(1μg/mL作用时间24h)+LW33 0.1μM；LPS treatment group (1μg/mL for 24h)+LW33 0.1μM;
LPS处理组(1μg/mL作用时间24h)+LW33 0.3μM；LPS treatment group (1μg/mL for 24h)+LW33 0.3μM;
LPS处理组(1μg/mL作用时间24h)+LW33 1μM；LPS treatment group (1μg/mL for 24h)+LW33 1μM;
LPS处理组(1μg/mL作用时间24h)+Bay73-6691 3μM；LPS treatment group (1μg/mL for 24h)+Bay73-6691 3μM;
LPS处理组(1μg/mL作用时间24h)+PF-04447943 3μM；LPS treatment group (1μg/mL for 24h)+PF-04447943 3μM;
24h后CCK8检测细胞增殖情况。以LPS未刺激组作为对照，分析各组BV2细胞增殖率的差异。Cell proliferation was detected by CCK8 after 24h. Taking the LPS-unstimulated group as a control, the differences in the proliferation rate of BV2 cells in each group were analyzed.
(3)实验结果：详见图2。与LPS未刺激组相比，LPS刺激BV2(小鼠小胶质细胞)24h后，细胞活力下降；而LW33在0.1μM～0.3μM浓度范围内可以升高BV2活力，具有保护作用。(3) Experimental results: see Figure 2 for details. Compared with the LPS-unstimulated group, the cell viability decreased after LPS-stimulated BV2 (mouse microglia) for 24 h; while LW33 in the concentration range of 0.1μM～0.3μM could increase the BV2 activity and had a protective effect.
实施例3、LW33对LPS诱导的BV2炎症因子mRNA水平的影响Example 3. The effect of LW33 on LPS-induced BV2 inflammatory factor mRNA levels
(1)材料与方法：HLF细胞购于中山大学实验动物中心；(1) Materials and methods: HLF cells were purchased from the Experimental Animal Center of Sun Yat-sen University;
BV2购于中国科学院细胞库，DMEM培养基(美国Gibco)、胎牛血清(美国Gibco)、0.25％胰酶(美国Gibco)、双抗(美国Gibco)、LPS(Sigma)、DMSO(MPBIO)、IL-1、IL-1β、TNF-α引物(上海生工)、Thermo Revert Aid Kit试剂盒(Thermo Fisher Scientific)BV2 was purchased from the Cell Bank of the Chinese Academy of Sciences, DMEM medium (Gibco, USA), fetal bovine serum (Gibco, USA), 0.25% trypsin (Gibco, USA), double antibody (Gibco, USA), LPS (Sigma), DMSO (MPBIO), IL-1, IL-1β, TNF-α primers (Shanghai Shenggong), Thermo Revert Aid Kit (Thermo Fisher Scientific)
(2)试验方法(2) Test method
将细胞接种于60mm中皿，胞培养箱中培养。当细胞生长融合至50～60％，分别用0.1、0.3、1μM LW33以及3μM Bay73-6691、PF-04447943对BV2细胞进行预处理1h，随后再加入1μg/mL的LPS进行刺激，作用24h后提取细胞总RNA进行实时荧光定量PCR试验。测得浓度后，取1μg总RNA按照Thermo Revert Aid Kit试剂盒说明书逆转为cDNA，Q-PCR步骤参照TOYOBO SYBR Premix EX TaqTM试剂盒说明书推荐用量进行优化，按95℃for 60s，95℃for10s，60℃for 30s进行扩增40个循坏。以β-action为内参，根据2-ΔΔCt法计算各基因的相对表达量，即表达量的变化倍数。The cells were seeded in a 60mm medium dish and cultured in a cell culture incubator. When the cell growth and confluence reached 50-60%, the BV2 cells were pretreated with 0.1, 0.3, 1 μM LW33 and 3 μM Bay73-6691 and PF-04447943 for 1 h, and then 1 μg/mL LPS was added for stimulation, and the cells were extracted after 24 h. The total cellular RNA was subjected to real-time fluorescence quantitative PCR assay. After measuring the concentration, take 1 μg of total RNA and reverse it to cDNA according to the instructions of the Thermo Revert Aid Kit. The Q-PCR step is optimized according to the recommended dosage of the TOYOBO SYBR Premix EX TaqTM kit. Amplification was performed for 40 cycles at 60°C for 30s. With β-action as the internal reference, the relative expression of each gene, that is, the fold change of the expression, was calculated according to the 2-ΔΔCt method .
(3)实验结果：详见图3a、3b、3c。在0.1～1μM范围内，LW33呈剂量依赖性下调IL-1、IL-1β、TNF-α的mRNA水平，与模型组相比，有统计学差异。与模型组相比，LW33在0.1～1μM范围内，能缓解LPS诱导的炎症反应。(3) Experimental results: see Figures 3a, 3b, and 3c for details. In the range of 0.1-1 μM, LW33 down-regulated the mRNA levels of IL-1, IL-1β and TNF-α in a dose-dependent manner, and compared with the model group, there was a statistical difference. Compared with the model group, LW33 in the range of 0.1-1 μM could alleviate the inflammatory response induced by LPS.
实施例4、LW33对单侧颈总动脉结扎(UCCAo)模型小鼠的影响Example 4. Effect of LW33 on unilateral common carotid artery ligation (UCCAo) model mice
有研究表明，单侧CCA(UCCAo)的永久性闭塞模型显示CBF(脑血流量)，WM(白质)病变和记忆障碍延迟的慢性轻度减少。此外，促炎细胞因子的水平增加，而抗炎细胞因子的水平在脑中降低。这种小鼠模型是研究炎症反应，WM损伤和记忆障碍之间关联的有力工具，这可能有助于阐明VaD的病理机制，特别是SIVD(皮质下缺血性血管性痴呆)。Studies have shown that the permanent occlusion model of unilateral CCA (UCCAo) shows chronic mild reductions in CBF (cerebral blood flow), WM (white matter) lesions and delayed memory impairment. In addition, levels of pro-inflammatory cytokines increased, while levels of anti-inflammatory cytokines decreased in the brain. This mouse model is a powerful tool to study the association between inflammatory response, WM impairment and memory impairment, which may help to elucidate the pathological mechanism of VaD, especially SIVD (subcortical ischemic vascular dementia).
(1)实验材料(1) Experimental materials
实验动物：SPF级KM小鼠40只，8～9周龄，体重28～40g，雄性，购自于中山大学实验动物中心(许可证号：SYXK(粤)2016-0112)，环境温度:20～25℃，湿度50～70％，自由摄食饮水。Experimental animals: 40 SPF grade KM mice, 8-9 weeks old, weighing 28-40 g, male, purchased from the Experimental Animal Center of Sun Yat-sen University (license number: SYXK (Guangdong) 2016-0112), ambient temperature: 20 ~ 25 ℃, humidity 50 ~ 70%, free access to food and water.
LW33(中山大学药学院)、多奈哌齐(卫材药业有限公司)羧甲基纤维素钠CMCNa(天津致远化学试剂有限公司)LW33 (School of Pharmacy, Sun Yat-Sen University), Donepezil (Eisai Pharmaceutical Co., Ltd.) Sodium Carboxymethyl Cellulose CMCNa (Tianjin Zhiyuan Chemical Reagent Co., Ltd.)
(2)实验方法(2) Experimental method
动物分组：SPF级KM小鼠40只，按体重随机分为5组，分别是假手术组，模型组，LW33低、高剂量组(10、20mg/kg)、多奈哌齐组，每组8只。Animal grouping: 40 SPF grade KM mice were randomly divided into 5 groups according to body weight, namely the sham operation group, the model group, the LW33 low and high dose groups (10, 20 mg/kg), and the donepezil group, with 8 mice in each group.
模型制备：除假手术组外，其他各组小鼠在1周的适应期后，给小鼠施用0.45％戊巴比妥钠用于诱导麻醉。通过中线颈部切口，从邻近的迷走神经分离右颈总动脉(CCA)，并用6-0丝线缝合(n＝32)。假手术组(n＝8)，对小鼠进行相同的外科手术而不进行颈动脉结扎。手术后，将小鼠放置笼子中，并给予食物和水。Model preparation: Except for the sham-operated group, mice in other groups were administered 0.45% pentobarbital sodium for inducing anesthesia after a 1-week adaptation period. The right common carotid artery (CCA) was isolated from the adjacent vagus nerve through a midline neck incision and sutured with 6-0 silk suture (n=32). Sham-operated group (n=8), mice were subjected to the same surgery without carotid artery ligation. After surgery, mice were placed in cages and given food and water.
给药方案：假手术组灌胃给予同等量溶媒0.5％CMCNa，LW33低(10mg/kg)、LW33高剂量组(20mg/kg)以及多奈哌齐组(2.1mg/kg)给予相应受试药物。造模后次日开始给药，每天给药1次，连续给药21天。末次给药1h后再测相关指标。Dosing scheme: The sham operation group was given the same amount of vehicle 0.5% CMCNa by gavage, and the LW33 low (10mg/kg), LW33 high dose group (20mg/kg) and donepezil group (2.1mg/kg) were given the corresponding test drugs. The drug was administered the next day after modeling, once a day, for 21 consecutive days. Relevant indicators were measured 1 hour after the last administration.
检测指标：每天观察各组小鼠一般状态、给药前和给药结束后分别称量体重，给药第21天为实验终点，末次给药1h后进行Morris水迷宫试验，测定完毕后假手术或UCCAO手术后30天，用戊巴比妥钠深度麻醉小鼠，并用0.01MPBS经心脏灌注，然后用含有4％多聚甲醛的0.1M磷酸盐缓冲液(PB，pH7.4)的固定剂灌注。脑组织固定24h后做HE染色。Detection indicators: The general state of the mice in each group was observed every day, and the body weights were weighed before and after the administration. The 21st day of administration was the end point of the experiment. Morris water maze test was performed 1 hour after the last administration, and sham operation was performed after the measurement. Or 30 days after UCCAO surgery, mice were deeply anesthetized with sodium pentobarbital and transcardially perfused with 0.01M PBS, followed by a fixative containing 4% paraformaldehyde in 0.1M phosphate buffered saline (PB, pH 7.4) perfusion. The brain tissue was fixed for 24h for HE staining.
(3)实验结果(3) Experimental results
1)小鼠一般状态及体重1) General state and body weight of mice
与假手术组比较，造模各组动物活动减少，毛发欠光滑，其它外观体征、粪便性状等未见明显差异。与模型对照组比较，LW33组及多奈哌齐组动物外观体征、行为活动、粪便性状等未见明显差异。由表1可见，与正常对照组比较,造模组动物体重没有明显改变，未见统计学差异(P>0.05)。与模型组比较，LW33组小鼠体重未见统计学差异(P>0.05)，但是0周时，多奈哌齐组小鼠体重有所减轻，有统计学差异(P<0.05)。Compared with the sham operation group, the animals in each model group had less activity, less smooth hair, and no significant difference in other appearance signs and feces. Compared with the model control group, the LW33 group and the donepezil group showed no significant difference in appearance signs, behavioral activities, and fecal traits. It can be seen from Table 1 that compared with the normal control group, the body weight of the animals in the modeling group did not change significantly, and there was no statistical difference (P>0.05). Compared with the model group, there was no statistical difference in the body weight of the mice in the LW33 group (P>0.05), but at 0 weeks, the body weight of the mice in the donepezil group decreased, and there was a statistical difference (P<0.05).
表1 LW33对UCCAo小鼠体重的影响(g，Means±SD，n＝8)Table 1 The effect of LW33 on the body weight of UCCAo mice (g, Means±SD, n=8)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
2)LW33对UCCAo小鼠空间探索和学习记忆能力的影响2) Effects of LW33 on spatial exploration and learning and memory abilities of UCCAo mice
与假手术组比较，模型组小鼠逃避潜伏期显著升高，在定位航行的第一天和第四天，有统计学差异(P<0.05)，提示单侧结扎CCA可导致小鼠学习记忆障碍。与模型组比较，LW33给药组和多奈哌齐组组可降低小鼠逃避潜伏期，但是没有统计学上的差异(P>0.05)。与假手术组比较，模型组小鼠穿越平台次数和在目标象限的停留时间明显减少(P＜0.01)，提示单侧结扎CCA造模3周可导致小鼠空间探索能力减弱。与模型组比较，LW33高剂量组和多奈哌齐组可以改善模型小鼠的空间探索障碍(P<0.05)。见表2、表3：Compared with the sham operation group, the escape latency of mice in the model group was significantly increased, and there was a statistical difference on the first and fourth days of positioning navigation (P<0.05), suggesting that unilateral ligation of CCA can lead to learning and memory impairment in mice . Compared with the model group, the LW33 administration group and the donepezil group could reduce the escape latency of mice, but there was no statistical difference (P>0.05). Compared with the sham-operated group, the number of times of crossing the platform and the stay time in the target quadrant of the model group were significantly reduced (P<0.01), suggesting that unilateral ligation of CCA for 3 weeks could lead to weakened spatial exploration ability of mice. Compared with the model group, the LW33 high-dose group and the donepezil group could improve the spatial exploration disorder of the model mice (P<0.05). See Table 2 and Table 3:
表2 LW33对UCCAo小鼠逃避潜伏期的影响(Means±SD，n＝8)Table 2 The effect of LW33 on escape latency in UCCAo mice (Means±SD, n=8)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
表3 LW33对UCCAo小鼠空间探索的影响(Means±SD，n＝8)Table 3 Effects of LW33 on spatial exploration in UCCAo mice (Means±SD, n=8)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
3)LW33对UCCAo小鼠脑组织HE染色的影响3) The effect of LW33 on HE staining of brain tissue of UCCAo mice
如图4的HE染色结果所示：假手术小鼠海马CA1区正常神经元数量较多，细胞核呈圆形，核仁明显且胞浆着色均匀，包膜完整。模型组小鼠海马CA1区正常神经元明显减少，细胞核固缩，神经元细胞周围疏松，间隙变大。As shown in the HE staining results in Figure 4: the number of normal neurons in the hippocampal CA1 area of sham-operated mice is large, the nucleus is round, the nucleolus is obvious, the cytoplasm is uniformly colored, and the envelope is complete. The normal neurons in the hippocampal CA1 area of the mice in the model group were significantly reduced, the nuclei were pyknotic, the surrounding cells of the neurons were loose, and the gap became larger.
由实验结果可知，PDE9A抑制剂LW33在高剂量(20mg/kg)下可以改善UCCAo模型小鼠的学习记忆功能障碍，其作用与阳性药多奈哌齐效果相当。LW33高剂量组和多奈哌齐组能改善小鼠海马CA1区神经元损伤，且神经元数量较模型组有所增加。The experimental results show that the PDE9A inhibitor LW33 can improve the learning and memory dysfunction of UCCAo model mice at a high dose (20 mg/kg), and its effect is equivalent to that of the positive drug donepezil. LW33 high-dose group and donepezil group can improve the damage of neurons in the CA1 region of the hippocampus of mice, and the number of neurons is increased compared with the model group.
实施例5、LW33对致血管性痴呆模型大鼠的影响Example 5. Effect of LW33 on vascular dementia model rats
使用缺血性中风的啮齿动物模型可能有助于阐明造成人类认知障碍的病变类型。大鼠血管内大脑中动脉(MCA)闭塞被认为是人脑缺血的方便可靠的模型。在大鼠血管内MCA闭塞模型中可以诱导感觉运动和认知功能障碍，但是由血管内MCA闭塞引起的感觉运动缺陷可能随时间而改善，研究数据表明，在大鼠中，该模型可导致认知障碍的进行性过程。这与VaD的临床进展一致。到目前为止，使用该模型的实验研究已经证明了原发性缺血区域中脑缺血损伤和AD型神经病理学的直接相互作用。此外，与认知功能的进行性衰退相一致，在大鼠血管内MCA闭塞模型中已经证实了在原发性缺血区域，海马远端的远端区域的延迟神经变性。因此该模型可用于研究VaD。并提供对VaD的一些病理生理学机制的深入了解。The use of rodent models of ischemic stroke may help to elucidate the types of lesions that contribute to cognitive impairment in humans. Rat intravascular middle cerebral artery (MCA) occlusion is considered a convenient and reliable model of human cerebral ischemia. Sensorimotor and cognitive dysfunction can be induced in a rat model of intravascular MCA occlusion, but sensorimotor deficits caused by intravascular MCA occlusion may improve over time, and data suggest that in rats, this model can lead to cognitive impairment. The progressive process of cognitive impairment. This is consistent with the clinical progression of VaD. To date, experimental studies using this model have demonstrated a direct interaction of cerebral ischemic injury and AD-type neuropathology in primary ischemic regions. In addition, consistent with the progressive decline of cognitive function, delayed neurodegeneration in the region of primary ischemia, distal to the hippocampus, has been demonstrated in the rat intravascular MCA occlusion model. Therefore this model can be used to study VaD. And provide insight into some of the pathophysiological mechanisms of VaD.
(1)实验材料(1) Experimental materials
实验动物：SPF级SD大鼠72只，雄性，体重200～240g，购自于中山大学实验动物中心(许可证号：SCXK(粤)2016-0029)，环境温度:20～25℃，湿度50～70％，自由摄食饮水。Experimental animals: 72 SPF SD rats, male, weighing 200-240 g, purchased from the Experimental Animal Center of Sun Yat-sen University (license number: SCXK (Guangdong) 2016-0029), ambient temperature: 20-25 °C, humidity 50 ~70%, free access to food and water.
LW33(中山大学药学院)、尼莫地平片(广东华南药业集团有限公司)、羧甲基纤维素钠(天津致远化学试剂有限公司)、多聚甲醛(国药集团化学试剂有限公司)、大鼠SOD试剂盒(南京建成生物工程研究所)、大鼠MDA试剂盒(南京建成生物工程研究所)、大鼠IL-6试剂盒(武汉华美生物工程有限公司)、大鼠TNF-α试剂盒(武汉华美生物工程有限公司)、大鼠IL-1β试剂盒(武汉华美生物工程有限公司)。LW33 (School of Pharmacy, Sun Yat-sen University), Nimodipine Tablets (Guangdong Huanan Pharmaceutical Group Co., Ltd.), Sodium Carboxymethyl Cellulose (Tianjin Zhiyuan Chemical Reagent Co., Ltd.), Paraformaldehyde (Sinopharm Group Chemical Reagent Co., Ltd.), Rat SOD Kit (Nanjing Jiancheng Bioengineering Institute), Rat MDA Kit (Nanjing Jiancheng Bioengineering Institute), Rat IL-6 Kit (Wuhan Huamei Bioengineering Co., Ltd.), Rat TNF-α Kit (Wuhan Huamei Biological Engineering Co., Ltd.), rat IL-1β kit (Wuhan Huamei Biological Engineering Co., Ltd.).
(2)实验方法(2) Experimental method
动物分组：SPF级雄性Sprague Dawley大鼠72只，按体重随机分为6组，分别是假手术组、模型组、LW33低剂量组(1mg/kg)、LW33中剂量组(3mg/kg)、LW33高剂量组(9mg/kg)、尼莫地平组(9.3mg/kg)。每组12只。Animal grouping: 72 SPF grade male Sprague Dawley rats were randomly divided into 6 groups according to body weight, namely sham operation group, model group, LW33 low dose group (1mg/kg), LW33 medium dose group (3mg/kg), LW33 high dose group (9mg/kg), nimodipine group (9.3mg/kg). 12 per group.
模型制备：除假手术组外，其他各组大鼠根据Nagasawa等方法改良的线栓法制作模型。以2％戊巴比妥钠腹腔注射(40mg/kg)麻醉，仰卧固定于手术板上。右颈部备皮、清洗，纵向剪开约1cm小口，分离CCA、ICA及ECA并挂线备用，结扎ECA与CCA,用动脉夹夹闭ICA远心端后,迅速于ECA与ICA分叉处作一切口,从切口处插入一端加热成光滑球形尼龙线(直径为0.25mm,距球端2cm处作标记)。线插入ICA后,于入口处稍稍结扎尼龙线与入口处ICA段,然后松开夹闭ICA的动脉夹,继续插入尼龙线至稍有阻力后略回撤,至线插入深度为(18.5±0.5)mm左右,实现大脑中动脉阻塞导致脑缺血。再次结扎入口处,尼龙线外留约1cm,缝合皮肤。缺血1h后轻轻提拉所留线头至有阻力,实现大脑中动脉再灌注,则造模完成。假手术组只结扎ECA与ICA。Model preparation: Except for the sham-operated group, the models of the other groups were made according to the suture method modified by Nagasawa et al. Anesthetized with 2% sodium pentobarbital intraperitoneal injection (40mg/kg), supine and fixed on the operation board. The skin of the right neck was prepared and cleaned, and a small incision of about 1 cm was cut longitudinally. The CCA, ICA and ECA were separated and hung up for use. The ECA and CCA were ligated. Make an incision, insert one end from the incision and heat it into a smooth spherical nylon thread (0.25mm in diameter, marked at 2cm from the ball end). After the thread is inserted into the ICA, the nylon thread and the ICA segment at the entrance are ligated slightly, then the artery clip that clamps the ICA is released, and the nylon thread is continued to be inserted until there is a little resistance and then retracted slightly until the thread insertion depth is (18.5±0.5 ) mm to achieve cerebral ischemia caused by middle cerebral artery occlusion. The entrance was ligated again, leaving about 1cm outside the nylon thread, and the skin was sutured. After 1 h of ischemia, the remaining thread was gently pulled until there was resistance to achieve reperfusion of the middle cerebral artery, and the modeling was completed. In the sham operation group, only ECA and ICA were ligated.
给药方案：假手术组灌胃给予同等量溶媒(0.5％CMCNa)，LW33低剂量组(1mg/kg)、LW33中剂量组(3mg/kg)、LW33高剂量组(9mg/kg)、尼莫地平组(9.3mg/kg)给予相应受试药物。造模后次日开始给药，每天给药1次，连续给药14天。末次给药1h后再测相关指标。Dosing scheme: the sham operation group was given the same amount of vehicle (0.5% CMCNa) by gavage, LW33 low dose group (1 mg/kg), LW33 medium dose group (3 mg/kg), LW33 high dose group (9 mg/kg), Modipine group (9.3 mg/kg) was given the corresponding test drug. The drug was administered the next day after modeling, once a day, for 14 consecutive days. Relevant indicators were measured 1 hour after the last administration.
检测指标：每天观察各组大鼠一般状态，给药第14天为实验终点，末次给药1h后依次进行被动回避(PAT)试验、Morris水迷宫试验，测定完毕后取大鼠腹腔静脉血置于含有EDTA的抗凝采血管中，3000rpm 4℃离心10min，吸取上清测定SOD、MDA、IL-1β、IL-6含量，取血后剪开颈胸部皮肤，暴露气管，迅速解剖胸腔，暴露心、肺，并用0.01M PBS经心脏灌注，然后用含有4％多聚甲醛的0.1M磷酸盐缓冲液(PB，pH7.4)的固定剂灌注。脑组织固定24h后做HE染色。Detection indicators: The general state of the rats in each group was observed every day, and the 14th day of administration was the end point of the experiment. Passive avoidance (PAT) test and Morris water maze test were performed in sequence 1 h after the last administration. In an anticoagulant blood collection tube containing EDTA, centrifuge at 3000rpm for 10min at 4°C, aspirate the supernatant to measure the content of SOD, MDA, IL-1β, and IL-6. After blood collection, cut the skin of the neck and chest to expose the trachea. Hearts, lungs, and transcardially perfused with 0.01M PBS, followed by a fixative containing 4% paraformaldehyde in 0.1M phosphate buffered saline (PB, pH 7.4). The brain tissue was fixed for 24h for HE staining.
(3)实验结果(3) Experimental results
1)大鼠一般状态与假手术组比较，造模各组动物活动减少，毛发欠光滑，其它外观体征、粪便性状等未见明显差异。与模型对照组比较，各给药组组及动物外观体征、行为活动、粪便性状等未见明显差异。1) Compared with the sham operation group, the general state of the rats in each model group decreased in activity, the hair was not smooth, and there was no significant difference in other appearance signs and fecal properties. Compared with the model control group, there were no significant differences in the appearance signs, behavioral activities, and fecal properties of the administration groups and animals.
2)LW33对模型大鼠在获得被动回避反应过程中记忆巩固的影响2) The effect of LW33 on memory consolidation in the process of acquiring passive avoidance response in model rats
术后给予LW33治疗2周后，在PAT测试期，与假手术相比，模型组大鼠进入暗箱的逃避潜伏期明显缩短(P<0.01)，错误次数增多(P<0.05)，提示可诱导SD大鼠获得性记忆巩固障碍；与模型组大鼠相比，LW33高剂量以及尼莫地平组可以延长大鼠进入暗箱的逃避潜伏期(P<0.01)且减少错误次数(P<0.01)。After 2 weeks of LW33 treatment, in the PAT test period, compared with the sham operation, the escape latency of rats in the model group into the dark box was significantly shortened (P<0.01), and the number of errors was increased (P<0.05), suggesting that SD can be induced Rat acquired memory consolidation disorder; compared with the model group, the high dose of LW33 and the nimodipine group could prolong the escape latency (P<0.01) and reduce the number of mistakes (P<0.01) in the dark box.
表4.LW33对模型大鼠在训练期和测试期中潜伏期的影响(g，Means±SD，n＝12)Table 4. The effect of LW33 on the latency of model rats during training and testing (g, Means±SD, n=12)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
3)LW33对模型大鼠在Morris水迷宫试验中长时程记忆和空间探索能力的影响3) Effects of LW33 on long-term memory and spatial exploration ability of model rats in Morris water maze test
在适应性训练中，与假手术组比较，各组大鼠游泳速度没有明显差异；假手术组大鼠学习记忆能力最好，找到平台的时间(潜伏期)随着训练时间而缩短；模型组大鼠学习记忆能力最差，潜伏期并没有随着训练天数的增加而有明显改变，提示可诱导大鼠产生认知功能障碍；与模型组相比，LW33高剂量组与阳性药尼莫地平组作用相当，特别是在第五天与模型组的潜伏期存在统计学差异(P<0.05)。见表5、表6：In the adaptive training, compared with the sham operation group, there was no significant difference in the swimming speed of the rats in each group; the sham operation group had the best learning and memory ability, and the time to find the platform (latency) shortened with the training time; the model group had the best learning and memory ability. The learning and memory ability of the rats was the worst, and the latency did not change significantly with the increase of training days, suggesting that cognitive dysfunction could be induced in rats; quite, especially on the fifth day, there was a statistical difference between the incubation period and the model group (P<0.05). See Table 5 and Table 6:
表5.LW33对模型大鼠逃避潜伏期的影响(g，Means±SD，n＝12)Table 5. Effects of LW33 on escape latency in model rats (g, Means±SD, n=12)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
表6.LW33对模型大鼠空间探索能力的影响(g，Means±SD，n＝12)Table 6. Effects of LW33 on the spatial exploration ability of model rats (g, Means±SD, n=12)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
4)LW33对模型大鼠血清SOD、MDA含量的影响4) Effects of LW33 on serum SOD and MDA contents in model rats
与假手术组比较，模型组大鼠SOD含量显著下降(P＜0.01)，说明可导致大鼠血清SOD含量明显下降。与模型组比较，LW33高剂量组大鼠SOD含量升高有统计学差异(P＜0.01)；与假手术组比较，模型组大鼠血清MDA含量显著增加(P＜0.01)，说明可导致大鼠血清MDA水平明显升高。与模型组比较，LW33高剂量组以及尼莫地平组可降低模型大鼠血清MDA水平(P＜0.01)；见表7Compared with the sham operation group, the SOD content of the rats in the model group decreased significantly (P<0.01), indicating that the serum SOD content of the rats was significantly decreased. Compared with the model group, the SOD content in the high-dose LW33 group was significantly increased (P<0.01); compared with the sham-operated group, the serum MDA content in the model group was significantly increased (P<0.01), indicating that it can lead to a significant increase in serum MDA content (P<0.01). The serum MDA level of the mice was significantly increased. Compared with the model group, the LW33 high-dose group and the nimodipine group could reduce the serum MDA level of the model rats (P<0.01); see Table 7
表7 LW33对模型大鼠血清SOD、MDA含量的影响(Means±SD，n＝6)Table 7 Effects of LW33 on serum SOD and MDA contents in model rats (Means±SD, n=6)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
5)LW33对模型大鼠血清IL-1β、IL-6含量的影响5) The effect of LW33 on serum IL-1β and IL-6 levels in model rats
与假手术组比较，模型组大鼠IL-1β、IL-6含量升高(P＜0.01)，说明可导致大鼠血清IL-1β、IL-6含量明显升高。与模型组比较，LW33高剂量组大鼠IL-1β、IL-6含量降低，分别有统计学差异(P＜0.01，P＜0.05)；见表8Compared with the sham operation group, the levels of IL-1β and IL-6 in the model group were increased (P<0.01), indicating that the levels of IL-1β and IL-6 in the serum of the rats were significantly increased. Compared with the model group, the levels of IL-1β and IL-6 in the LW33 high-dose group were decreased, and there were statistical differences (P<0.01, P<0.05); see Table 8
表8 LW33对模型大鼠血清IL-1β、IL-6含量的影响(Means±SD，n＝10)Table 8 Effects of LW33 on serum IL-1β and IL-6 levels in model rats (Means±SD, n=10)
与模型对照组比较，*为P<0.05，**为P<0.01。Compared with the model control group, * means P<0.05, ** means P<0.01.
6)LW33对模型大鼠脑组织HE染色的影响6) The effect of LW33 on HE staining of model rat brain tissue
如图5、图6的HE染色结果所示：假手术组大鼠脑组织切片HE染色光镜下可见海马CA1区以及皮层区神经元细胞数目较多，排列整齐且形态结构完整。核仁明显且胞浆着色均匀，包膜完整。而模型组大鼠海马CA1区以及皮层区正常神经元细胞明显减少，神经元椎体细胞排列稀疏散乱，且皮层区出现明显的神经元坏死和空泡状。LW33高剂量组和多奈哌齐组大鼠海马CA1区及皮层区神经元损伤有所改善，正常神经元数量较模型组有所增加。As shown in the HE staining results shown in Figure 5 and Figure 6: the brain tissue sections of the sham-operated group were stained with HE staining under light microscope. The number of neurons in the hippocampal CA1 area and the cortical area were large, neatly arranged and morphologically complete. The nucleolus was obvious and the cytoplasm was uniformly colored, and the envelope was intact. In the model group, normal neurons in the CA1 area of the hippocampus and cortex were significantly reduced, the neurons in the pyramidal cells were sparsely arranged, and the cortical area had obvious neuronal necrosis and vacuoles. The damage of neurons in the hippocampal CA1 area and cortical area of the rats in the LW33 high-dose group and donepezil group was improved, and the number of normal neurons increased compared with the model group.
结论：在PAT(被动回避测试)试验中，高剂量(9mg/kg)的LW33及尼莫地平(9.3mg/kg)可以改善被动回避任务中大鼠的记忆巩固；在Morris水迷宫试验中，随着训练时间的增加，高剂量的LW33及尼莫地平可以提高模型大鼠的潜伏期、增加穿越目标象限平台的次数以及目标象限停留时间，对模型大鼠的学习记忆和空间探索障碍有所改善。同时，高剂量的LW33及尼莫地平也可以升高血清中SOD活性并降低MDA、IL-1β、IL-6含量，这提示LW33可能具有抗氧化应激、抗炎等作用。Conclusion: In the PAT (passive avoidance test) test, high doses (9mg/kg) of LW33 and nimodipine (9.3mg/kg) can improve the memory consolidation of rats in the passive avoidance task; in the Morris water maze test, With the increase of training time, high doses of LW33 and nimodipine can increase the latency of model rats, increase the number of crossing the target quadrant platform and the dwell time in the target quadrant, and improve the learning and memory and spatial exploration obstacles of model rats. . At the same time, high doses of LW33 and nimodipine can also increase the activity of SOD in serum and reduce the content of MDA, IL-1β, and IL-6, suggesting that LW33 may have anti-oxidative stress and anti-inflammatory effects.
本技术领域技术人员可以理解，除非另外定义，这里使用的所有术语(包括技术术语和科学术语)具有与本发明所属领域中的普通技术人员的一般理解相同的意义。还应该理解的是，诸如通用字典中定义的那些术语应该被理解为具有与现有技术的上下文中的意义一致的意义，并且除非像这里一样定义，不会用理想化或过于正式的含义来解释。It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and, unless defined as herein, are not to be taken in an idealized or overly formal sense. explain.
最后所应说明的是：以上实施例仅用以说明而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应该理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围。Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified. Or equivalents can be substituted without departing from the spirit and scope of the present invention.
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| CN201910551371.1ACN110339197A (en) | 2019-06-24 | 2019-06-24 | Application of a phosphodiesterase 9A inhibitor for preventing and treating vascular dementia |
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| CN201910551371.1ACN110339197A (en) | 2019-06-24 | 2019-06-24 | Application of a phosphodiesterase 9A inhibitor for preventing and treating vascular dementia |
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