CN117180431A - Trpa1离子通道作为药物靶点在酰胺类除草剂中毒中的应用 - Google Patents
Trpa1离子通道作为药物靶点在酰胺类除草剂中毒中的应用 Download PDFInfo
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- CN117180431A CN117180431A CN202311090225.6A CN202311090225A CN117180431A CN 117180431 A CN117180431 A CN 117180431A CN 202311090225 A CN202311090225 A CN 202311090225A CN 117180431 A CN117180431 A CN 117180431A
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Abstract
本发明属于生物医药领域,涉及一种TRPA1离子通道作为药物靶点在酰胺类除草剂中毒中的应用。本发明的研究结果显示,TRPA1与酰胺类除草剂中毒均密切相关,抑制或敲除TRPA1靶点均能够通过降低酰胺类除草剂引起的TRPA1表达增加及钙离子内流,最终降低酰胺类除草剂引起的肺泡上皮细胞毒性。因此,本发明为酰胺类除草剂中毒诊断和基于TRPA1靶点的药物研发提供了广谱性治疗手段的新思路。
Description
本分案申请是2022年06月02日递交的发明名称为“TRPA1离子通道作为药物靶点在酰胺类除草剂中毒中的应用”的中国专利申请202210625128.1的分案申请。
技术领域
本发明属于生物医药领域,涉及一种TRPA1离子通道作为药物靶点在酰胺类除草剂中毒中的应用。
背景技术
酰胺类除草剂是一类含有典型苯环结构的除草农药,主要包括敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺和丁草胺等亲电有机小分子化合物,在我国年产量高达十万吨以上,具有高效、高选择性的除草效果得到广泛应用。酰胺类除草剂易在环境中发生迁移和富集,环境残留量高,水解半衰期长,可以通过食物链传递进入人体,干扰内分泌功能甚至导致肺肝等癌变,威胁人类健康和生态安全,但目前尚无应对酰胺类除草剂中毒的特效解毒药物。因而探究酰胺类除草剂的中毒机制,研发酰胺类除草剂靶点治疗药物对酰胺类除草剂中毒救治具有重要意义。
发明内容
一些实施方案中,本发明提供了一种以TRPA1作为药物作用靶点的药物在制备预防或治疗酰胺类化合物中毒引起的病症的药物中的应用。
一些实施方案中,所述病症包括肺损伤、肝损伤、甲状腺损伤、免疫毒性、或胚胎发育毒性等症状,但又不限于此。
一些实施方案中,所述的肺损伤包括肺泡上皮细胞损伤。
一些实施方案中,所述肺泡上皮细胞损伤包括细胞活力降低、细胞毒性增加、细胞钙内流增加。
一些实施方案中,所述的酰胺类化合物为酰胺类除草剂。
一些实施方案中,所述的酰胺类化合物包括敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺、丁草胺、扑草胺、乙氧苯草胺、双苯酰草胺、萘草胺、吡氟酰草胺、二甲噻草胺、萘氧丙草胺、敌草胺、稗草胺、萘丙酰草胺、克草胺、苯噻草胺、吡草胺、溴丁酰草胺、氟噻草胺、烯草胺、唑草胺、甲氧噻草胺、氟丁酰草胺、二甲噻草胺、高效二甲噻草胺、氟吡草胺、异噁草胺、高效异丙甲草胺、高效麦草伏甲酯或高效麦草伏丙酯,但又不限于此。
一些实施方案中,所述以TRPA1作为药物作用靶点的药物包括抑制TRPA1基因、TRPA1mRNA或TRPA1蛋白质的抑制剂。
一些实施方案中,所述抑制剂包括抑制TRPA1离子通道功能的抑制剂。
瞬时受体电位离子通道(transient receptor potential ion channels,TRPs)是近年来发现存在于细胞膜或胞内细胞器膜上的非选择性阳离子通道,对Ca2+等阳离子具有高通透性,与化学物质所诱导的痛觉或瘙痒有关。其中瞬时受体电位阳离子通道亚家族A成员1(transient receptor potential cation channel subfamily A member 1,TRPA1)广泛分布于肺、脑等器官细胞膜或细胞器膜上,被认为是“化学开关”,可以被肉桂醛、大蒜素、甲醛、丙烯醛、异硫氰酸烯丙酯等多种现有化合物激活,引起钙离子内流,产生炎症或疼痛反应。本发明提供TRPA1作为酰胺类除草剂中毒诊断和防治新靶点的用途。本发明的研究结果显示,TRPA1与酰胺类除草剂中毒均密切相关,抑制或敲除TRPA1靶点均能够通过降低酰胺类除草剂引起的TRPA1表达增加及钙离子内流,最终降低酰胺类除草剂引起的肺泡上皮细胞毒性。因此,本发明为酰胺类除草剂中毒诊断和基于TRPA1靶点的药物研发提供了广谱性治疗手段的新思路。
一些实施方案中,所述的抑制剂选自抑制TRPA1活性的物质、降解TRPA1的物质、降低TRPA1水平的基因工具组成的群组中的至少一种。
一些实施方案中,所述的抑制剂包括化合物、肽类拮抗剂、特异性针对TRPA1的第二种抗体、siRNA或特异性针对TRPA1的反义分子。
一些实施方案中,所述抑制或降解TRPA1的物质包括蛋白或化合物。
一些实施方案中,所述的降低TRPA1水平的基因工具包括RNA干扰、microRNA、基因编辑或基因敲除材料。
一些实施方案中,所述基因编辑或基因敲除使用的是crispr-cas9基因敲除技术。
一些实施方案中,所述的抑制剂包括TRPA1离子通道阻断剂或豆蔻明。
一些实施方案中,所述的TRPA1离子通道阻断剂包括HC-030031、A-967079、TCS5861528、或Chembridge-5861528中的至少一种。
一些实施方案中,所述的药物还包括药学上可接受的辅料和/或载体。
一些实施方案中,所述药物的剂型包括干粉针、注射剂、片剂、胶囊。
一些实施方案中,本发明提供了一种组合物在制备预防或治疗酰胺类化合物中毒引起的病症的药物中的用途,其特征在于,所述组合物包含有效量的药物,所述药物为以TRPA1作为药物作用靶点的药物。
一些实施方案中,所述病症包括肺损伤、肝损伤、甲状腺损伤、免疫毒性、或胚胎发育毒性等症状,但又不限于此。
一些实施方案中,所述的肺损伤包括肺泡上皮细胞损伤。
一些实施方案中,所述肺泡上皮细胞损伤包括细胞活力降低、细胞毒性增加、细胞钙内流增加。
一些实施方案中,所述的酰胺类化合物为酰胺类除草剂。
一些实施方案中,所述的酰胺类化合物包括敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺、丁草胺、扑草胺、乙氧苯草胺、双苯酰草胺、萘草胺、吡氟酰草胺、二甲噻草胺、萘氧丙草胺、敌草胺、稗草胺、萘丙酰草胺、克草胺、苯噻草胺、吡草胺、溴丁酰草胺、氟噻草胺、烯草胺、唑草胺、甲氧噻草胺、氟丁酰草胺、二甲噻草胺、高效二甲噻草胺、氟吡草胺、异噁草胺、高效异丙甲草胺、高效麦草伏甲酯或高效麦草伏丙酯中的至少一种。
一些实施方案中,所述以TRPA1作为药物作用靶点的药物包括抑制TRPA1基因、TRPA1mRNA或TRPA1蛋白质的抑制剂。
一些实施方案中,所述抑制剂包括抑制TRPA1离子通道功能的抑制剂。
一些实施方案中,所述的抑制剂选自抑制TRPA1活性的物质、降解TRPA1的物质、降低TRPA1水平的基因工具组成的群组中的至少一种。
一些实施方案中,所述化合物、肽类拮抗剂、特异性针对TRPA1的第二种抗体、siRNA或特异性针对TRPA1的反义分子。
一些实施方案中,所述抑制或降解TRPA1的物质包括蛋白或化合物。
一些实施方案中,所述的降低TRPA1水平的基因工具包括RNA干扰、microRNA、基因编辑或基因敲除材料。
一些实施方案中,所述基因编辑或基因敲除使用的是crispr-cas9基因敲除技术。
一些实施方案中,所述的抑制剂包括TRPA1离子通道阻断剂或豆蔻明。
一些实施方案中,所述的TRPA1离子通道阻断剂包括HC-030031、A-967079、TCS5861528、或Chembridge-5861528中的至少一种。
一些实施方案中,所述的药物还包括药学上可接受的辅料和/或载体。
一些实施方案中,所述药物的剂型包括干粉针、注射剂、片剂、胶囊。
一些实施方案中,本发明提供了一种TRPA1的检测试剂在制备检测酰胺类化合物中毒的诊断试剂中的应用。
一些实施方案中,所述检测试剂为检测TRPA1的基因表达量。
一些实施方案中,所述检测试剂为检测TRPA1的mRNA表达量。
一些实施方案中,所述检测试剂为检测TRPA1蛋白的表达量。
一些实施方案中,所述检测试剂包括荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针、抗体、抗体功能性片段和偶联抗体中的一种或多种。
在一些实施方案中,当怀疑患者农药或除草剂中毒、尤其是酰胺类除草剂中毒时,可以采用TRPA1检测试剂进行检测,如检测到TRPA1提高的表达量,则提示上述除草剂中毒的可能性大。在一些实施方案中,当怀疑或确认患者为酰胺类除草剂中毒时,可以施用TRPA1抑制剂进行治疗。在一些实施方案中,当怀疑患者除草剂中毒、尤其是酰胺类除草剂中毒时,可以先进行TRPA1检测,当结果显示出提高的TRPA1表达量时,施用TRPA1抑制剂。
附图说明
图1A-1G为本发明实验中7种酰胺类除草剂对肺上皮A549细胞活力的影响以及抑制或敲除TRPA1对酰胺类除草剂诱发细胞毒性的影响。图1A:敌稗;图1B:异丙草胺;图1C:异丙甲草胺;图1D:甲草胺;图1E:乙草胺;图1F:丙草胺;图1G:丁草胺。
图2A-2G为本发明实验中酰胺类除草剂诱发A549细胞损伤。图2A:敌稗;图2B:异丙草胺;图2C:异丙甲草胺;图2D:甲草胺;图2E:乙草胺;图2F:丙草胺;图2G:丁草胺。
图3A-3G为本发明实验中酰胺类除草剂对TRPA1 mRNA表达的影响以及抑制或敲除TRPA1对酰胺类除草剂引发TRPA1 mRNA表达的影响。图3A:敌稗;图3B:异丙草胺;图3C:异丙甲草胺;图3D:甲草胺;图3E:乙草胺;图3F:丙草胺;图3G:丁草胺。
图4A-4G为本发明实验中酰胺类除草剂对A549细胞钙离子内流的影响以及抑制或敲除TRPA1对酰胺类除草剂诱发细胞钙离子内流的影响。图4A:敌稗;图4B:异丙草胺;图4C:异丙甲草胺;图4D:甲草胺;图4E:乙草胺;图4F:丙草胺;图4G:丁草胺。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案,具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
除非另有定义,本文中所使用的所有技术与科学术语的定义与本领域技术人员所熟悉的定义相同。此外,任何与所记载内容相似或均等的方法和材料皆可应用于本发明方法中,具体实施方式中描述了优选的方法和材料。
文中所用的“一”和“一种”指语法上不定冠词的释义,表示“一个”、“一种”或“多个”、“多种"(即“至少一个”、“至少一种”)。例如“一要素”指一种或种要素。
如本文所使用的,除非另有说明,术语“预防”是指在症状发作前使用具有本文所提供的抑制剂治疗或将其给予至尤其是具有中毒患病风险和/或其他本文所述病症的患者。术语“预防”包括特定疾病的症状的抑制或减轻。此外,具有症状复发史的患者也是预防的潜在候选人。在这方面,术语“预防”可与术语“预防性治疗”互换使用。
如本文所使用的,除非另有说明,术语“治疗”是指疾病或病症,或与疾病或病症相关的一种或多种症状的消除或减轻。在某些实施方式中,该术语是指通过给予患有疾病或病症的对象一种或多种预防或治疗药物而使得该疾病或病症的蔓延或恶化最小化。
在本发明中,“组合物”中的组分可以是混合的形式存在,也可以被分开包装。分开的包装的组分也可以含有其各自的佐剂。所述的佐剂是指在药学中,可辅助药物疗效的手段。对于组合物中的组分在分开包装的情况下,各个分开包装的组分可以是同时施用或者是以任意的前后顺序施用,其中患者先用一种药物治疗,然后再给以另一种药物。所述的患者是指哺乳动物受治疗者,尤其是人类。
一些实施方案中,本发明组合物的组分可以单独提供,或者以单位剂型混合在一起,例如以干的形式提供在密闭容器如安瓿或香囊中冷冻干燥的粉末或无水浓缩物指示活性剂的量。当该组合物通过输液给药时,它可以与含有无菌药物级水或盐水的输液瓶一起使用。当该组合物通过注射给药时,可以提供注射用无菌水或盐水的安瓿,以便在给药之前混合这些成分。
“有效量”是足以实现有利或所需结果的量,例如增强的免疫应答,医学状况(疾病、感染等)的治疗、预防或改善。有效量可以在一次或多次施用、应用或剂量中施用。合适的剂量将依赖于体重、年龄、健康、待治疗的疾病或状况和施用途径而变。
在该公开内容中,“包含”、“包括”、“含有”、“具有”可指“囊括”、“涵盖”等,“基本由....组成”、“基本组成为”等具有专利法中赋予它们的含义,该术语为开放式的,允许多于所引用的事项的存在,只要所引用的事项的基本或新颖特征不因多于所引用的事项的存在而改变,但排除现有技术的实施方案。
药学上可接受的载体包括盐水、含水缓冲溶液、溶剂和/或分散介质。这种载体的使用是本领域公知的。优选地,该溶液在制造和储存的条件下是稳定的,并且通过例如对羟基苯甲酸酯类、氯丁醇、苯酚、抗坏血酸、硫柳汞等的使用,抵制细菌和真菌的微生物的污染作用而被保存。
实施例1
1.实验材料
1.1实验细胞:人Ⅱ型肺泡上皮细胞A549细胞,购自中国医学科学院基础医学研究所细胞资源中心;TRPA1 crispr-cas9基因敲除A549细胞(TRPA1-KO A549细胞),购自金斯瑞生物科技股份有限公司。
1.2McCoy’s 5A培养基、胎牛血清、0.25%Trypsin-EDTA,均购买自Gibco公司;酰胺类除草剂(敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺和丁草胺),均购自上海阿拉丁生化科技有限公司;TRPA1通道阻断剂HC-030031,购自Selleck Chemicals公司;CCK-8细胞活力检测试剂盒和LDH细胞损伤检测试剂盒,均购自Dojindo公司;反转录试剂盒和荧光实时定量RT-PCR反应试剂盒,均购自TaKaRa公司;Reagent和Fluo-4DirectTMCalcium试剂盒,均购自ThermoFisher Scientific公司。
2.实验方法
2.1细胞培养和处理
野生型(WT)的A549细胞和TRPA1基因敲除(TRPA1-KO)的A549细胞分别培养于含有10%FBS的McCoy’s 5A完全培养基中。分别接种5×104cells/mL WT A549细胞和TRPA1-KOA549细胞悬液,置于37℃,5%CO2,湿润培养箱中培养。
正常对照组(WTA549 cell组):完全培养基进行细胞换液,培养24h;
染毒组处理(TRPA1-KO A549 cell组):当WT/TRPA1-KO A549细胞处于对数生长期时,吸弃原培养基(含有10%FBS的McCoy’s 5A完全培养基),分别加入含有浓度为0、150、300、600、1200μM的酰胺类除草剂(敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺和丁草胺)的完全培养基,培养24h;
添加抑制剂组处理(WTA549 cell+HC-03003150μM组):当WTA549细胞处于对数生长期时,吸弃原培养基(含有10%FBS的McCoy’s 5A完全培养基),加入含有50μM HC-030031的完全培养基于37℃,5%CO2,培养箱中预培养0.5h;后再分别加入酰胺类除草剂,使得HC-030031终浓度为50mM,酰胺类除草剂浓度分别为0、150、300、600、1200μM,置于37℃,5%CO2,培养箱中培养24h。
2.2细胞活力测定
采用CCK-8试剂盒进行细胞活力的测定。取出96孔板,细胞吸弃上清液,将CCK-8试剂与完全培养基1:9体积比混合,100μL/孔添加。将96孔板放在37℃,5%CO2,培养箱中培养4h。用酶标仪测定在450nm处的吸光度,计算细胞活力百分比。
2.3细胞损伤测定
采用LDH试剂盒继续细胞损伤的测定。取出96孔板,在高对照孔中加入10μL/孔Lysis Buffer后,将96孔板放在37℃,5%CO2,湿润培养箱中培养30min。在每个孔中加入100μL/孔Working Solution后,常温、避光培养30min。在每孔中加入50μL/孔StopSolution后,立即用酶标仪测定在490nm处的吸光度,计算细胞损伤率。
2.4实时荧光定量RT-PCR
取出6孔板,吸弃培养基,加入1mL/样本TRIzol试剂。按照TRIzol说明书提取WT/TRPA1-KOA549细胞中的总RNA,用于接下来的反转录实验或-80℃冻存。cDNA逆转录参照反转录试剂盒说明书进行。反应条件分别为42℃,2min→4℃,∞(基因组DNA的除去反应)和37℃,15min→85℃,5s→4℃,∞(反转录反应)。实时荧光定量PCR反应参照TaKaRa公司实时荧光定量PCR试剂盒说明书进行。引物序列及产物长度大小分别为:hTRPA1(107bp)Forwardprimer 5’AGTATATTTGGGTATTGCAAAGAAGC 3’(SEQ ID NO:1),Reverse primer 5’ATGCCCGTCGTGTAGATAATCC 3’(SEQ ID NO:2);hβ-actin(184bp)Forward primer 5’AGAGCTACGAGCTGCCTGAC 3’(SEQ ID NO:3),Reverse primer 5’AGCACTGTGTTGGCGTACAG 3’(SEQ ID NO:4)。反应条件为95℃for 30s→(95℃for 5s→60℃for 30s→72℃for 30s+Plate Read)×40more times→Melt Curve65℃to 95℃,increment 0.5℃,for 5s+PlateRead→END.所有操作均在冰上进行。
2.5细胞钙成像
采用Fluo-4 DirectTMCalcium试剂盒进行细胞内钙离子含量测定。取出96孔板,将一瓶组分A与组分C混匀,加入200μL组分B工作液(77mg组分B完全溶于1mL组分C),涡旋混匀后得到Fluo-4 DirectTM钙试剂工作液。直接将工作液添加到细胞中,吹打3~4下。将96孔板放在37℃,5%CO2,湿润培养箱中孵育30min,取出后避光。通过共聚焦高内涵成像系统,单通道FITC荧光测定和数据化处理。
3.实验结果
3.1A549细胞活力结果
A549细胞活力结果如图1A-1G所示,分别与0μM酰胺类除草剂相比,敌稗(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、异丙草胺(150μM,P<0.01;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、异丙甲草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、甲草胺(150μM,P<0.001;300μM,P<0.001;600μM,P<0.0001;1200μM,P<0.0001)、乙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、丙草胺(150μM,P<0.001;300μM,P<0.001;600μM,P<0.001;1200μM,P<0.0001)和丁草胺(150μM,P<0.01;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)暴露后的A549细胞活力显著降低。
分别与相应组的150、300、600μM酰胺类除草剂相比,HC-030031(50μM)干预后,可显著提高敌稗(150μM,P<0.05;300μM,P<0.05;600μM,P<0.01)、异丙甲草胺(150μM,P<0.01;600μM,P<0.01)、甲草胺(300μM,P<0.0001;600μM,P<0.0001)、乙草胺(150μM,P<0.001;300μM,P<0.01;600μM,P<0.001)、丙草胺(600μM,P<0.05)和丁草胺(300μM,P<0.01)引起的A549细胞活力降低,也可以提高异丙草胺引起的A549细胞活力降低。
分别与相应组的150、300、600μM酰胺类除草剂相比,TRPA1基因敲除后,均可以可显著提高敌稗(300μM,P<0.05;600μM,P<0.01)、异丙草胺(150μM,P<0.05;600μM,P<0.01)、异丙甲草胺(150μM,P<0.001;600μM,P<0.01)、甲草胺(150μM,P<0.01;300μM,P<0.01;600μM,P<0.05)、乙草胺(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.05)、丙草胺(150μM,P<0.05)和丁草胺(150μM,P<0.01;300μM,P<0.001;600μM,P<0.01)引起的A549细胞活力降低。
3.2A549细胞损伤结果
A549细胞损伤结果如图2A-2G所示,分别与0μM酰胺类除草剂相比,敌稗(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、异丙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、异丙甲草胺(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、甲草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、乙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、丙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)和丁草胺(150μM,P<0.05;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)暴露后的A549细胞毒性显著升高。
结合图1和图2结果,发现7种AHs(酰胺类除草剂)均分别导致A549细胞活力显著降低,均存在显著的细胞毒性作用。
3.3TRPA1 mRNA表达的结果
TRPA1 mRNA表达的结果如图3A-3G所示。分别与0μM酰胺类除草剂相比,敌稗(300μM,P<0.05;600μM,P<0.01)、异丙草胺(300μM,P<0.01;600μM,P<0.01)、异丙甲草胺(300μM,P<0.01;600μM,P<0.0001)、甲草胺(300μM,P<0.01;600μM,P<0.01)、乙草胺(600μM,P<0.01)、丙草胺(600μM,P<0.05)和丁草胺(150μM,P<0.05;300μM,P<0.05;600μM,P<0.001)诱导的A549细胞TRPA1mRNA表达显著增加。
分别与相应组的150、300、600μM酰胺类除草剂相比,TRPA1特异性抑制剂HC-030031(50μM)干预后,可显著降低敌稗(150μM,P<0.01)、异丙甲草胺(150μM,P<0.05)、甲草胺(600μM,P<0.05)、乙草胺(150μM,P<0.01;600μM,P<0.05)和丁草胺(600μM,P<0.01)诱导的A549细胞TRPA1 mRNA表达增加,也可能降低异丙草胺、丙草胺诱导的TRPA1mRNA表达增加。而TRPA1-KO A549细胞暴露于各浓度的七类酰胺类除草剂后几乎不表达TRPA1 mRNA。
3.4A549细胞钙离子内流结果
A549细胞钙离子内流结果如图4A-4G所示。分别与0μM酰胺类除草剂相比,敌稗(150μM,P<0.01;300μM,P<0.001;600μM,P<0.001;1200μM,P<0.0001)、异丙草胺(150μM,P<0.01;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、异丙甲草胺(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、甲草胺(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、乙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、丙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)和丁草胺(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)导致的A549细胞钙离子内流显著增加。
分别与相应组的150、300、600、1200μM酰胺类除草剂相比,HC-030031(50μM)干预后,可显著降低敌稗(300μM,P<0.05;600μM,P<0.01)、异丙草胺(150μM,P<0.01;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.01)、异丙甲草胺(150μM,P<0.01;300μM,P<0.05;600μM,P<0.001;1200μM,P<0.0001)、甲草胺(150μM,P<0.01;300μM,P<0.0001;600μM,P<0.0001)、乙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、丙草胺(150μM,P<0.05;300μM,P<0.001;600μM,P<0.0001)和丁草胺(300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.05)导致的A549细胞钙离子内流增加。
分别与相应组的150、300、600、1200μM酰胺类除草剂相比,TRPA1基因敲除后,可显著降低敌稗(150μM,P<0.001;600μM,P<0.05;1200μM,P<0.0001)、异丙草胺(150μM,P<0.0001;300μM,P<0.001;600μM,P<0.0001;1200μM,P<0.001)、异丙甲草胺(150μM,P<0.0001;300μM,P<0.001;600μM,P<0.0001;1200μM,P<0.01)、甲草胺(150μM,P<0.001;300μM,P<0.0001;600μM,P<0.01;1200μM,P<0.0001)、乙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.0001)、丙草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.001;1200μM,P<0.0001)和丁草胺(150μM,P<0.0001;300μM,P<0.0001;600μM,P<0.0001;1200μM,P<0.01)导致的A549细胞钙离子内流增加。
本发明发现7种酰胺类除草剂均分别导致A549细胞活力显著降低和细胞损伤显著升高,而且TRPA1的mRNA表达显著增加;而TRPA1抑制剂HC-030031(50μM)干预或TRPA1crispr-cas9基因敲除可以提高酰胺类除草剂染毒的A549细胞活力和降低酰胺类除草剂诱发的TRPA1 mRNA表达增加;说明TRPA1与酰胺类除草剂引起的A549细胞毒性密切相关。
同时发现7种酰胺类除草剂均会引起A549细胞钙内流显著增加,而给与TRPA1抑制剂HC-030031(50μM)干预或TRPA1基因敲除可以显著抑制TRPA1介导的细胞钙内流增加,再次确证TRPA1为酰胺类除草剂诱发A549细胞毒性的重要靶点。
综上所述,本实验证明了TRPA1靶点与酰胺类除草剂中毒所致肺上皮细胞毒性密切相关,抑制或基因敲除TRPA1能够通过降低TRPA1表达增加及其介导的钙离子内流,从而降低酰胺类除草剂的细胞毒性。因此,本发明为酰胺类除草剂中毒诊断提供了新靶点,亦为基于TRPA1靶点的酰胺类除草剂中毒药物研发提供了新思路。
Claims (10)
1.以TRPA1作为药物作用靶点的药物在制备预防或治疗酰胺类化合物中毒引起的病症的药物中的应用。
2.如权利要求1所述的应用,其特征在于,所述病症包括肺损伤、肝损伤、甲状腺损伤、免疫毒性、或胚胎发育毒性。
3.如权利要求1所述的应用,其特征在于,所述的肺损伤包括肺泡上皮细胞损伤;
优选地,所述肺泡上皮细胞损伤包括细胞活力降低、细胞毒性增加、细胞钙内流增加;
优选地,所述肺泡上皮细胞选自人Ⅱ型肺泡上皮细胞;
优选地,所述人Ⅱ型肺泡上皮细胞选自A549细胞。
4.如权利要求1所述的应用,其特征在于,所述的酰胺类化合物为酰胺类除草剂。
5.如权利要求1所述的应用,其特征在于,所述的酰胺类化合物包括敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺、丁草胺、扑草胺、乙氧苯草胺、双苯酰草胺、萘草胺、吡氟酰草胺、二甲噻草胺、萘氧丙草胺、敌草胺、稗草胺、萘丙酰草胺、克草胺、苯噻草胺、吡草胺、溴丁酰草胺、氟噻草胺、烯草胺、唑草胺、甲氧噻草胺、氟丁酰草胺、二甲噻草胺、高效二甲噻草胺、氟吡草胺、异噁草胺、高效异丙甲草胺、高效麦草伏甲酯或高效麦草伏丙酯;
优选地,所述以TRPA1作为药物作用靶点的药物包括抑制TRPA1基因、TRPA1mRNA或TRPA1蛋白质的抑制剂;
优选地,所述抑制剂包括抑制TRPA1离子通道功能的抑制剂。
6.如权利要求1-6任一所述的应用,其特征在于,所述的抑制剂选自抑制TRPA1活性的物质、降解TRPA1的物质、降低TRPA1水平的基因工具组成的群组中的至少一种;
优选地,所述的抑制剂包括化合物、肽类拮抗剂、特异性针对TRPA1的第二种抗体、siRNA或特异性针对TRPA1的反义分子;
优选地,所述抑制或降解TRPA1的物质包括蛋白或化合物;
优选地,所述的降低TRPA1水平的基因工具包括RNA干扰、microRNA、基因编辑或基因敲除材料;
优选地,所述基因编辑或基因敲除使用的是crispr-cas9基因敲除技术;
优选地,所述的抑制剂包括TRPA1离子通道阻断剂;
优选地,所述的药物还包括药学上可接受的辅料和/或载体;
优选地,所述药物的剂型包括干粉针、注射剂、片剂、胶囊。
7.一种组合物在制备预防或治疗酰胺类化合物中毒引起的病症的药物中的用途,其特征在于,所述组合物包含有效量的药物,所述药物为以TRPA1作为药物作用靶点的药物;
优选地,所述病症包括肺损伤、肝损伤、甲状腺损伤、免疫毒性、或胚胎发育毒性;
优选地,所述的肺损伤包括肺泡上皮细胞损伤;
优选地,所述肺泡上皮细胞损伤包括细胞活力降低、细胞毒性增加、细胞钙内流增加;
优选地,所述的酰胺类化合物为酰胺类除草剂;
优选地,所述肺泡上皮细胞选自人Ⅱ型肺泡上皮细胞;
优选地,所述人Ⅱ型肺泡上皮细胞选自A549细胞;
优选地,所述的酰胺类化合物包括敌稗、异丙草胺、异丙甲草胺、甲草胺、乙草胺、丙草胺、丁草胺、扑草胺、乙氧苯草胺、双苯酰草胺、萘草胺、吡氟酰草胺、二甲噻草胺、萘氧丙草胺、敌草胺、稗草胺、萘丙酰草胺、克草胺、苯噻草胺、吡草胺、溴丁酰草胺、氟噻草胺、烯草胺、唑草胺、甲氧噻草胺、氟丁酰草胺、二甲噻草胺、高效二甲噻草胺、氟吡草胺、异噁草胺、高效异丙甲草胺、高效麦草伏甲酯或高效麦草伏丙酯中;
优选地,所述以TRPA1作为药物作用靶点的药物包括抑制TRPA1基因、TRPA1mRNA或TRPA1蛋白质的抑制剂;
优选地,所述抑制剂包括抑制TRPA1离子通道功能的抑制剂。
8.如权利要求7所述的用途,其特征在于,所述的抑制剂选自抑制TRPA1活性的物质、降解TRPA1的物质、降低TRPA1水平的基因工具组成的群组中的至少一种;
优选地,所述化合物、肽类拮抗剂、特异性针对TRPA1的第二种抗体、siRNA或特异性针对TRPA1的反义分子;
优选地,所述抑制或降解TRPA1的物质包括蛋白或化合物;
优选地,所述的降低TRPA1水平的基因工具包括RNA干扰、microRNA、基因编辑或基因敲除材料;
优选地,所述基因编辑或基因敲除使用的是crispr-cas9基因敲除技术;
优选地,所述的抑制剂包括TRPA1离子通道阻断剂;
优选地,所述的药物还包括药学上可接受的辅料和/或载体;
优选地,所述药物的剂型包括干粉针、注射剂、片剂、胶囊。
9.TRPA1的检测试剂在制备检测酰胺类化合物中毒的诊断试剂中的应用。
10.如权利要求9所述的应用,其特征在于,所述检测试剂为检测TRPA1的基因表达量的试剂;
优选地,所述检测试剂为检测TRPA1的mRNA表达量的试剂;
优选地,所述检测试剂为检测TRPA1蛋白的表达量的试剂;
优选地,所述检测试剂包括荧光定量PCR染料、荧光定量PCR引物、荧光定量PCR探针、抗体、抗体功能性片段和偶联抗体中的一种或多种。
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