CN113181190A - 脂氧素a4在制备治疗年龄相关性黄斑变性的药物中的应用 - Google Patents
脂氧素a4在制备治疗年龄相关性黄斑变性的药物中的应用 Download PDFInfo
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Abstract
本发明公开了脂氧素A4在制备治疗年龄相关性黄斑变性的药物中的应用,属于医药技术领域。本发明首次将内源性脂质介质引入年龄相关性黄斑变性的治疗领域,通过动物实验、细胞模型以及LXA4抑制RPE细胞光损伤的作用机制研究,发现了LXA4通过抑制RPE细胞光损伤干预年龄相关性黄斑变性,将为年龄相关性黄斑变性的诊断和治疗提供科学依据,不仅为年龄相关性黄斑变性的治疗提供新的治疗药物,同时也为改善和恢复视力的新疗法提供了可能的靶点。
Description
技术领域
本发明涉及脂氧素A4在制备治疗年龄相关性黄斑变性的药物中的应用,属于医药技术领域。
背景技术
AMD(age-related macular degeneration,年龄相关性黄斑变性)是导致老年人视力损伤的主要原因。目前,中国约有湿性(进展性)AMD患者260万人,在美国约有175万AMD患者。由于人口老龄化,AMD患者数量将在未来5年快速增长。在美国,每年用于治疗AMD的成本约300亿美元,而且预计该成本还将逐年上升。我国作为一个人口大国,每年约有4万人因AMD而失明,因病致盲或因病致贫的不在少数。目前AMD缺乏早期治疗的方法,仍有大量的早期AMD患者进展为湿性AMD,甚至是失明。显而易见,AMD是一个重大的公共卫生问题,且迫切需要探索预防和治疗的方法。尽管抗VEGF(vascular endothelial growth factor,血管内皮生长因子)药物为治疗进展性AMD提供了新的治疗途径,但由于其价格昂贵且复发率高,因此抗VEGF药物的治疗仍不是治疗AMD的最佳方法。由于AMD发病机制复杂,尤其缺乏对其早期病变的病理生理的认识,因而探究AMD早期治疗的方法已成为现阶段研究的热点和难点。
在关于AMD发病机制的众多学说中,光损伤学说和氧化应激学说是被最为广泛接受的。RPE(Retinal pigment epithelium,视网膜色素上皮)细胞是视网膜组织的重要结构,RPE细胞的损伤被认为是AMD发生的始动因素。光损伤是RPE细胞损伤的主要原因,尽管RPE细胞的光损伤机制尚未完全明了,但是,越来越多的研究证实抑制RPE光损伤将对视网膜功能产生显著的保护作用。
LXA4(脂氧素A4,lipoxin A4)是重要的抗炎抗氧化物质,其既可以通过内源性产生,亦可以通过阿司匹林诱导产生,已有研究证实LXA4可在体内多个器官中发挥抗氧化作用,然而,目前关于LXA4在眼部的作用研究甚少,尚未有文献报道其对RPE细胞的抗氧化损伤作用。本研究拟通过细胞和动物实验,验证LXA4是否对RPE细胞的光损伤有保护作用,并探索其可能的作用机制,为治疗AMD提供新的思路。
发明内容
为了实现上述发明目的,本发明采用以下技术手段:
本发明的第一个目的是提供脂氧素A4在制备治疗黄斑变性的药物中的应用。
在本发明的一种实施方式中,所述黄斑变性为年龄相关性黄斑变性。
本发明的第二个目的是提供脂氧素A4在制备抑制RPE细胞损伤或凋亡的药物中的应用。
本发明的第三个目的是提供一种治疗年龄相关性黄斑变性的药物组合物,所述药物包括脂氧素A4,以及任选的药学可接受的载体和/或辅料。
在本发明的一种实施方式中,药物组合物可呈选自如下的剂型:溶液、悬液、乳剂、丸剂、片剂、胶嚢、粉末、控制释放或持续释放制剂。
本发明的第四个目的是提供一种抑制RPE细胞损伤或凋亡的药物组合物,所述药物包括脂氧素A4,以及任选的药学可接受的载体和/或辅料。
在本发明的一种实施方式中,药物组合物可呈选自如下的剂型:溶液、悬液、乳剂、丸剂、片剂、胶嚢、粉末、控制释放或持续释放制剂。本发明药物组合物可根据本领域公知的方法制备,用于此目的时,如果需要,可将本发明化合物与一种或多种固体或液体药物赋形剂和/或辅剂结合,制成可作为人药使用的适当的施用形式或剂量形式。
经过验证可知,脂氧素A4通过激活Nrf2/ARE通路,促进Nrf2的核转位,增加下游抗氧化因子的表达,发挥抗氧化作用,从而减少RPE细胞损伤和凋亡。LXA4可改善光照或碘酸钠诱导的视网膜结构破坏,并抑制RPE细胞损伤从而干预AMD发生发展,具有潜在的临床应用前景。
本发明首次将内源性脂质介质引入年龄相关性黄斑变性的治疗领域,LXA4干预年龄相关性黄斑变性的研究,将为年龄相关性黄斑变性的诊断和治疗提供科学依据,为进一步开发脂氧素A4治疗年龄相关性黄斑变性提供新思路。
有益效果:
1、LXA4在治疗年龄相关性黄斑变性中有很好的应用前景,增加研究的力度有可转化为眼用制剂的可能。
2、对LXA4治疗年龄相关性黄斑变性的研究,不仅为年龄相关性黄斑变性的治疗提供新的治疗药物,同时也为改善和恢复视力的新疗法提供了可能的靶点。
附图说明
图1为本发明实施例的实验设计路线。
图2为LXA4逆转蓝光辐射形成的视网膜及RPE脉络膜-巩膜复合体的形态改变。其中:A为彩色眼底图像和OCT图像;B为小鼠视网膜石蜡包埋组织H&E染色照片;C为图B中的ONL, INL, IPL层厚度统计图;D为图B中的视网膜总厚度统计图,n=6。
图3为LXA4阻止蓝光辐射形成的RPE层损伤。其中:A为小鼠视网膜组织切片亚甲蓝染色;B为小鼠视网膜组织脉络膜铺片;C为根据B图统计单位面积RPE细胞数量,n=6。
图4为蓝光辐射诱导RPE细胞氧化损伤。其中:A为A2E负载的RPE细胞在蓝光暴露后不同时间点DHE染色的代表性图像;B为蓝光照射后DHE阳性细胞的定量分析;C为CCK8法检测蓝光照射后不同时间的细胞活力,n=6;D为免疫荧光法检测RPE细胞中ZO-1的表达;E为每个区域ZO-1阳性细胞的定量,n=6。
图5为LXA4减轻蓝光辐射形成的RPE细胞氧化损伤。其中:A为流式细胞术检测蓝光照射后RPE细胞凋亡;B为各组RPE细胞凋亡率统计图,n=4;C为蓝光暴露后DHE染色的代表性图像;D为DHE阳性细胞数量统计,n=4;E为免疫荧光检测RPE细胞中的ZO-1紧密连接;F为ZO-1阳性细胞数量统计,n=6。
图6为蓝光诱导RPE细胞中的Nrf2/HO-1通路活化。其中:A为Western blot检测RPE细胞中HO1、NQO1、NRF2和FPRL的蛋白水平;B为Western Blot的定量统计图,N=5;C为实时荧光定量PCR检测各组RPE细胞HO-1基因表达,N=7;D为免疫荧光分析各组RPE细胞中的Nrf2核易位;E为Nrf2核易位的定量统计,N=5;F为Western blot检测RPE细胞胞浆和胞核的Nrf2蛋白水平;G为F图Western Blot的统计图,N=5。
图7为LXA4促进蓝光诱导的RPE细胞中Nrf2/HO1的通路。其中:A为RPE细胞中Nrf2与Keap1结合的Co-IP分析;B为Nrf2与Keap1结合作用统计图,N=3;C为Western blot检测RPE细胞中HO-1和NQO1的Western blot的蛋白水平;D为RPE细胞中HO-1和NQO1蛋白水平的相应定量分析,N=3;E为免疫荧光染色分析各组RPE细胞中的NRF2核易位;F为RPE细胞中Nrf2核易位的定量分析,N=6;G为EMSA检测ARE结合活性;H为RPE细胞ARE结合活性进行相应的统计学分析,N=3。
图8为阻断NRF2-HO1通路逆转LXA4对蓝光诱导的RPE细胞氧化应激的保护作用;其中:A为Western blot检测RPE细胞质中Nrf2、NQO1和HO-1的蛋白水平;B为RPE细胞中Nrf2、NQO1和HO-1的定量分析,N=3;C为Western blot检测RPE细胞核中Nrf2蛋白水平;D为Nrf2的定量分析,N=3;E为为实时荧光定量PCR检测各组RPE细胞HO-1基因表达;F为DHE染色RPE细胞的代表性图像;G为各组RPE细胞中ROS阳性细胞比例的定量分析,N=6。
具体实施方式
下面结合实施例对本发明作进一步的描述,但本发明的实施方式不限于此。
如图1所示,本发明分别以蓝光诱导的光损伤动物模型和人源视网膜色素上皮细胞作为研究对象,从组织功能到细胞水平全方位进一步探究(1)LXA4对蓝光诱导的光损伤动物的治疗作用及(2)LXA4调控蓝光诱导的视网膜色素上皮细胞氧化损伤的分子机制。
实施例1
1、实验动物:本发明使用的实验动物为5周龄健康雄性Balb-c小鼠(SPF级)小鼠,饲养于无锡市人民医院实验动物中心。
2、光损伤动物模型的建立
健康SPF级5周雄性C57BL/6小鼠经过眼科裂隙灯显微镜和间接眼底镜检查,排除眼部疾病。在黑暗环境中适应 24小时后,非麻醉状态下暴露于蓝色发光二极管光照(波长:480nm 10000 lux)每天1小时,持续2周。)
3、动物模型的建立
(1)干预1:光损伤模型小鼠予LXA4 (0.1 mg/kg)尾静脉注射。
(2)干预2:光损伤模型小鼠予阿司匹林饲喂(10 mg·kg-1·d-1)。
(3)溶剂对照组:光损伤模型小鼠予无水酒精处理并充分挥发完后的饲料饲喂(10mg·kg-1·d-1)。
分组:空白对照组、光损伤模型组、干预组(干预1和干预2)和溶剂对照组。
采用免疫荧光染色检测视网膜氧化及抗氧化基因表达,OCT及眼底成像检测小鼠视网膜结构改变,HE染色比较各组视网膜厚度,亚甲蓝染色评价RPE层形变,脉络膜铺片免疫荧光染色判断RPE细胞形态。
如图2和图3所示,给与LXA4干预后,视网膜厚度较光照组显著增加,并部分恢复RPE层结构。HE染色显示LXA4干预组可改善蓝光照射引起的节细胞丢失和视网膜厚度变薄,逆转蓝光照射诱导的RPE层的排列紊乱。
实施例2
细胞模型:
(1)光照模型:ARPE19细胞采用A2E 25 nmol/L加蓝光(波长:480 nm,强度2000lux),光照时间分为1小时和15小时。
(2)干预:ARPE19细胞予LXA4(50 nmol/L、100 nmol/L、500nmol/L)共培养24h
分组:正常对照组、A2E组、A2E加光照组及A2E加光照加LXA4干预组。
培养人源的视网膜色素上皮细胞,LXA4预处理后,采用MTT、WST检测细胞活力,荧光免疫染色检测细胞ROS及紧密连接蛋白ZO-1表达,流式细胞术检测凋亡细胞比例。RT-PCR和Western Blot检测Nrf2及下游抗氧化因子mRNA及蛋白表达水平,免疫共沉淀检测Nrf2与Keap1蛋白结合。定量PCR、Western Blot和免疫共沉淀检测LXA4对Nrf2抗氧化通路的影响,并采用小干扰RNA技术考察Nrf2沉默对LXA4抗氧化效应的影响。
如图4和图5所示,光损伤可增加RPE细胞中ROS的产生,破坏RPE细胞形态并抑制细胞活力,而这种现象在LXA4组得到明显逆转,说明LXA4的存在能够改善光照诱导的视网膜结构破坏的情况,从而抑制RPE细胞损伤。
实施例3
揭示LXA4抑制RPE细胞光损伤的作用机制研究。
1)通过Western-blot、免疫荧光及定量PCR验证光损伤是否影响Nrf2核转位;Co-IP检测Nrf2和Keap1的结合率,Western-blot检测HO-1、NQO1的蛋白水平,EMSA检测ARE转录活性。揭示了LXA4促进RPE细胞中抗氧化通路的分子机制。
2)通过siRNA阻断Nrf2信号通路,检测Nrf2对LXA4作用的影响。
如图6和图7所示,在视网膜色素上皮细胞中,LXA4可进一步激活Nrf2,诱导Nrf2与Keap1分离,促进细胞的抗氧化反应,从而导致HO-1和NQO1的水平的升高。
如图8所示,在视网膜色素上皮细胞中,予siNrf2干预后RPE细胞浆中NQO1及HO-1的蛋白表达量以及HO-1mRNA的转录水平较LXA4干预组显著下降,ROS阳性细胞数较LXA4干预组显著上调,进一步证实予siNrf2干预后可显著抑制LXA4的抗氧化作用。
通过上述实施例可知,脂氧素A4通过激活Nrf2/ARE通路,促进Nrf2的核转位,增加下游抗氧化因子的表达,发挥抗氧化作用,从而减少RPE细胞损伤和凋亡。LXA4可改善光照或碘酸钠诱导的视网膜结构破坏,并抑制RPE细胞损伤从而干预AMD发生发展,具有潜在的临床应用前景。
本发明首次将内源性脂质介质引入年龄相关性黄斑变性的治疗领域,LXA4干预年龄相关性黄斑变性的研究,将为年龄相关性黄斑变性的诊断和治疗提供科学依据,为进一步开发脂氧素A4治疗年龄相关性黄斑变性提供新思路。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (8)
1.脂氧素A4在制备治疗黄斑变性的药物中的应用。
2.根据权利要求1所述的脂氧素A4在制备治疗黄斑变性的药物中的应用,其特征在于,所述黄斑变性为年龄相关性黄斑变性。
3.脂氧素A4在制备抑制RPE细胞损伤或凋亡的药物中的应用。
4.根据权利要求2所述的脂氧素A4在制备抑制RPE细胞损伤或凋亡的药物中的应用,其特征在于,所述RPE细胞损伤或凋亡由光照或碘酸钠诱导产生。
5.一种治疗年龄相关性黄斑变性的药物组合物,其特征在于,所述药物包括脂氧素A4,以及任选的药学可接受的载体和/或辅料。
6.根据权利要求5所述的一种治疗年龄相关性黄斑变性的药物组合物,其特征在于,药物组合物呈选自如下的剂型:溶液、悬液、乳剂、丸剂、片剂、胶嚢、粉末、控制释放或持续释放制剂。
7.一种抑制RPE细胞损伤或凋亡的药物组合物,其特征在于,所述药物包括脂氧素A4,以及任选的药学可接受的载体和/或辅料。
8.根据权利要求7所述的一种抑制RPE细胞损伤或凋亡的药物组合物,其特征在于,药物组合物呈选自如下的剂型:溶液、悬液、乳剂、丸剂、片剂、胶嚢、粉末、控制释放或持续释放制剂。
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