CN110152020A - 一种免疫特异质肝损伤的评价模型及其应用 - Google Patents
一种免疫特异质肝损伤的评价模型及其应用 Download PDFInfo
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Abstract
本申请公开一种免疫特异质肝损伤的评价模型,所述评价模型包括NLRP3炎症小体以及促进NLRP3炎症小体活化的成分,其中,促进NLRP3炎症小体活化的成分为淫羊藿次苷II。本申请还提供所述评价模型的构建方法,所述构建方法包括:淫羊藿单体样品溶液的制备;LPS预处理小鼠原代骨髓巨噬细胞;将制备的样品溶液加入小鼠原代骨髓巨噬细胞中进行刺激;加入ATP进行刺激;进行Western blot检测、ELISA检测以及Caspase‑1活性测定,并进行统计学分析。在LPS和ATP的共同刺激作用下,NLRP3炎症小体处于活化状态,当给予致特异质肝毒性成分或药物时,促进了NLRP3炎症小体的进一步活化。
Description
技术领域
本申请涉及药物筛选领域,尤其但不限于涉及一种用于特异质肝损伤药物筛选评价模型及该模型在筛查淫羊藿中致特异质肝损伤成分的应用。
背景技术
近年来关于药物特异质肝损伤的报道大量增加,是导致药物撤市,增加黑框警示的主要原因。如果药物特异质肝损伤的问题解决不好,将严重影响相关产品的临床用药安全,并可能对产业健康发展带来巨大的不利影响。因此,亟待阐明致特异质肝损伤药物的相关科学机制,为临床合理用药、趋利避害提供科学依据。目前,特异质肝损伤药物的研究是非常困难的。一方面,鉴于特异质肝损伤的发生往往和剂量、用药时间无关,并且难以预测,实际上不可能在临床上进行前瞻性研究。此外,特异质肝损伤具有起病延迟,再次激发迅速发作的特点,是典型的免疫介导反应,组织学检查也强烈提示免疫介导的反应。目前研究发现NLRP3炎症小体在多种免疫相关疾病中发挥重要作用,特别是最近研究发现NLRP3炎症小体可能广泛参与了阿莫地喹(Amodiaquine)和奈韦拉平(nevirapine)等药物诱导的特异质肝损伤。寡聚化结构域样受体家族热蛋白结构域3(NOD-like receptor protein 3,NLRP3)炎症小体是由细胞内固有免疫受体蛋白NLRP3及下游的接头蛋白凋亡相关斑点样蛋白(Apoptosis associated speck like protein,ASC)和半胱氨酸天冬氨酸特异蛋白酶1(Cysteinyl aspartate specific proteinase,Caspase-1)作为核心组成的多蛋白复合物,其活化需要两条信号通路协同完成,首先TLRs受体(Toll-like receptor)与LPS等配体结合,诱导NF-κB信号通路活化,介导NLRP3、pro-IL-1β等NLRP3炎症小体组成蛋白表达,同时在ATP、mtDNA和Nigericin(尼日尼亚菌素)等损伤相关分子模式(damage associatedmolecular patterns,DAMPs)和病原相关分子模式(pathogen-associated molecularpatterns,PAMPs)作用下,NLRP3蛋白与ASC形成多聚化复合体,从而诱导pro-Caspase-1自我剪切活化,活化的Caspase-1一方面可以诱导细胞焦亡,另一方面则可诱导pro-IL-1β和pro-IL-18剪切活化形成IL-1β和IL-18,招募中性粒细胞等免疫细胞,从而参与人类众多疾病的发生发展。阿莫地喹(Amodiaquine)和奈韦拉平(nevirapine)等药物可以在THP-1细胞中直接激活NLRP3炎症小体诱导特异质肝损伤。然而,有关致特异质肝损伤的药物是否可以利用NLRP3炎症小体活化模型(即在LPS和NLRP3炎症小体刺激因子ATP刺激下,药物对NLRP3炎症小体的影响)来进行特异质肝损伤评价尚未见报道。
中药特异质肝损伤是指由中药本身及/或其代谢产物等所导致的肝脏损伤,是中药临床应用中常见的严重不良反应之一。尽管中药特异质肝损伤的发生率通常较低,但严重者可致急性肝衰竭甚至死亡,受到医药界、制药业、管理部门及公众的高度重视。然而,由于公众对中药存在“天然安全、无毒副作用”的认识误区,研发者和制药企业对中药不良反应亦存在重视不足的现象,在我国由于特异质肝损伤导致中药新药研发失败或上市后警示、撤市的实例亦不鲜见。中药因其自身成分复杂、研究基础薄弱、联合用药较普遍等因素,其肝损伤往往较为隐匿,导致中药特异质肝损伤评价比化药更加困难。淫羊藿作为传统无毒补益类中药,始载于《神农本草经》,列为中品,具有补肾阳、强筋骨、祛风湿的功效。然而,近年来淫羊藿及相关制剂致肝损伤现象屡见报道,其中壮骨关节丸、仙灵骨葆胶囊已被国家不良反应中心多次通报。此外更有临床罕见单味淫羊藿引起肝损害的报道。目前已有多个课题组开展了淫羊藿及其相关制剂肝毒性研究,其中一项研究发现单独灌胃小鼠3天淫羊藿即出现呕吐、纳差、活动减少,给药15天后可见肝脏脂肪变性。贾晓斌教授实验室利用斑马鱼模型研究发现淫羊藿总黄酮高剂量组有一定的毒性。王停教授则利用基原-用药部位-工艺-剂量-疗程结合数学方法对淫羊藿肝毒性进行研究,结果发现朝鲜淫羊藿及巫山淫羊藿存在潜在肝毒性,并与提取工艺、给药剂量和用药疗程存在密切关系。如何客观认识淫羊藿致肝损伤问题是当前中药安全性研究的难点。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
基于特异质肝损伤免疫介导反应的特点以及NLRP3炎症小体在特异质肝损伤中发挥的重要作用,我们建立一种基于NLRP3炎症小体激活模型的特异质肝损伤药物评价方法,以此开发出更好的预测特异质肝损伤药物的筛查试验,并将此方法用于淫羊藿中致特异质肝损伤成分的筛查。
具体地,本申请提供一种免疫特异质肝损伤的评价模型,所述评价模型包括NLRP3炎症小体以及促进NLRP3炎症小体活化的成分,其中,促进NLRP3炎症小体活化的成分为淫羊藿次苷II。
本申请还提供所述评价模型的构建方法,所述构建方法包括:
S100.细胞水平评价;
S101.淫羊藿单体样品溶液的制备;
S102.制备小鼠原代骨髓巨噬细胞,并加入LPS进行刺激;
S103.将步骤S101中制备的溶液加入小鼠原代骨髓巨噬细胞进行刺激;
S104.加入ATP进行刺激;
S105.Western blot检测NLRP3炎症小体相关蛋白的表达,ELISA检测细胞上清中IL-1β、TNF-α的含量,以及Caspase-1活性测定,并进行统计学分析。
在本申请中,所述淫羊藿可以选自朝霍定A、朝霍定A1、朝霍定B、朝霍定C、淫羊藿苷、淫羊藿素、淫羊藿次苷II和脱水淫羊藿黄素中的任一种或更多种。
在本申请中,步骤S100还可以包括:
筛选所述淫羊藿中促进NLRP3炎症小体活化的成分,并以1μM-5μM的浓度,进一步验证该成分对NLRP3炎症小体的活化作用。
在本申请中,在步骤S101中,取所述淫羊藿单体,用二甲基亚砜溶解成10mM,于-20℃储存,备用,临用前用Opti-MEM稀释至10μM。
在本申请中,所述构建方法还可以包括:
采用与步骤S100相同的操作步骤,用卡马西平对所述评价模型进行验证。
在本申请中,可以将卡马西平配制成10-40μM的浓度。
在本申请中,卡马西平可以促进NLRP3炎症小体活化。
在本申请中,所述构建方法可以包括:
S100.细胞水平评价;
S101.淫羊藿样品溶液的制备;
S102.制备小鼠原代骨髓巨噬细胞,并加入LPS进行刺激;
S103.将步骤S101中制备的溶液加入小鼠原代骨髓巨噬细胞进行刺激;
S104.加入ATP进行刺激;
S105.Western blot检测NLRP3炎症小体相关蛋白的表达,ELISA检测细胞上清中IL-1β、TNF-α的含量,以及Caspase-1活性测定,并进行统计学分析;
S200.动物水平评价;
S201.小鼠尾静脉注射LPS;
S202.腹腔注射促进NLRP3炎症小体活化的成分的溶液;
S203.测定血清中ALT、ASTI、L-1β、TNF-α的含量。
在本申请中,促进NLRP3炎症小体活化成分的小鼠给药浓度可以为25-100mg/kg。
本申请还提供一种免疫特异质肝损伤药物或成分的筛选方法,所述筛选方法包括使用所述评价模型进行筛选促进NLRP3炎症小体活化的成分。
本申请还提供NLRP3炎症小体用于特异质肝损伤中的用途。
本申请基于NLRP3炎症小体活化模型,提出一种免疫特异质肝损伤药物筛选评价模型及构建方法;基于此方法,筛查出淫羊藿致特异质肝毒性的化学成分,阐明淫羊藿特异质肝毒性的科学机制,为临床合理用药、趋利避害提供科学依据。
克服缺陷:1.以往关于药物特异质肝毒性的研究均是基于传统毒理学评价模式,而忽略机体免疫因素的影响。本方法在前期研究基础上确证了淫羊藿特异质肝毒性的免疫属性,并结合NLRP3炎症小体在多种肝脏疾病中发挥重要作用的报道,提出了基于NLRP3炎症小体激活的致特异质肝损伤药物筛选评价模型及方法;
2.目前基于正常动物对淫羊藿肝毒性的评价结果存在较大差异,而本方法联合NLRP3炎症小体激活模型及LPS诱导的特异质肝损伤模型,从细胞水平及动物水平两方面,评价淫羊藿特异质肝毒性;
NLRP3炎症小体能够被多种内源性或外源性因素激活,活化Caspase-1,引起IL-1β、IL-18等促炎因子的分泌,是机体免疫应答的重要组成部分,但是其过度激活将导致许多疾病的发生发展。目前研究发现NLRP3炎症小体在多种免疫相关疾病中发挥重要作用,特别是最近研究发现NLRP3炎症小体可能广泛参与了阿莫地喹(Amodiaquine)和奈韦拉平(nevirapine)等药物诱导的特异质肝损伤。因此,本方法基于NLRP3炎症小体活化模型,在细胞水平上初步筛查出具有促进NLRP3炎症小体激活的肝毒性成分或药物。进一步地,在动物水平上联合LPS诱导的特异质肝损伤评价模型验证筛查结果的准确性,进而准确评价药物特异质肝损伤。
一、细胞水平
1.具有疑似药物性肝损伤的药物或成分对NLRP3炎症小体激活的影响;在LPS及ATP共同刺激作用下,NLRP3炎症小体激活,在此基础上给予疑似免疫特异质肝损伤的目标成分或药物,研究目标成分或药物对NLRP3炎症小体的激活的影响,筛选出具有过度激活NLRP3炎症小体的药物或成分,进行下一步研究。
2.从1中筛选出的具有显著增强NLRP3炎症小体活性的单体成分或药物在不同浓度梯度下研究其对NLRP3炎症小体刺激因子ATP的响应情况;初步确定导致肝毒性的目标成分或药物。
二、动物水平
将初步筛查出的肝毒性目标成分或药物在LPS诱导的特异质肝损伤模型上进行验证,对炎症因子IL-1β、TNF-α及肝损伤相关指标ALT、AST进行检测,验证结果的准确性和客观性。
本申请的优点在于,在该模型方法中,LPS和ATP的共同刺激作用下,NLRP3炎症小体处于激活状态,当给予致特异质肝毒性目标成分时,NLRP3炎症小体进一步激活,促炎因子IL-1β大量增加,从而介导更为严重的免疫炎症反应,加重肝损伤。
该模型方法在之前并未用于药物免疫特异质肝损伤的预测及筛选评价。该方法可以用于一系列致免疫特异质肝损伤药物的预测及筛选评价。
本申请联合NLRP3炎症小体激活模型及LPS诱导的特异质肝损伤模型,用于免疫特异质肝损伤药物的筛选及评价,并在该方法的指导下,筛查出了淫羊藿中具有免疫特异质肝损伤的化学成分淫羊藿次苷II。
本申请提供的基于NLRP3炎症小体激活模型及LPS诱导的特异质肝损伤模型可以快速评价药物的免疫特异质肝毒性,为药物肝毒性评价模型、科学机制的研究以及临床应用提供参考。
本申请的其它特征和优点将在随后的说明书中阐述,并且,部分地从说明书中变得显而易见,或者通过实施本申请而了解。本申请的目的和其他优点可通过在说明书、权利要求书以及附图中所特别指出的结构来实现和获得。
附图说明
附图用来提供对本申请技术方案的进一步理解,并且构成说明书的一部分,与本申请的实施例一起用于解释本申请的技术方案,并不构成对本申请技术方案的限制。
图1:小鼠原代骨髓巨噬细胞中,脂多糖预处理后将培养基换成无血清opti-MEM配制而成的卡马西平(10,20,40μM),最后补加三磷酸腺苷(5mM)后收取细胞上清及细胞裂解液进行相关指标检测。细胞上清检测IL-1βp17和caspase-1 p20的蛋白表达,commassie为上样控制。细胞裂解液检测pro-IL-1 β、caspase-1 p45、NLRP3、ASC的蛋白表达,GAPDH为上样控制。柱状图分别为细胞上清中半胱天冬酶1活性、白细胞介素1β和肿瘤坏死因子α的含量。
图2:小鼠原代骨髓巨噬细胞中,脂多糖预处理细胞后将培养基换成无血清opti-MEM配制而成的淫羊藿各单体成分(10μM),最后补加三磷酸腺苷(5mM)后收取细胞上清及裂解液进行相关指标检测。柱状图分别为细胞上清中半胱天冬酶1活性、白细胞介素1β和肿瘤坏死因子α的含量。
图3:脂多糖预处理小鼠原代骨髓巨噬细胞,然后将培养基换成无血清opti-MEM配制而成的淫羊藿次苷II,最后补加三磷酸腺苷(5mM)后收取细胞上清及裂解液进行相关指标检测。上清检测IL-1βp17和caspase-1 p20的蛋白表达,commassie为上样控制。细胞裂解液检测pro-IL-1β、caspase-1 p45、NLRP3、ASC的蛋白表达,GAPDH为上样控制。
图4:小鼠经脂多糖(2mg/kg)尾静脉注射后,给予不同浓度的淫羊藿次苷II(25、50、100mg/kg)处理后测血清中谷丙转氨酶、谷草转氨酶、白细胞介素1β、肿瘤坏死因子α的含量。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚明白,下文中将结合附图对本申请的实施例进行详细说明。需要说明的是,在不冲突的情况下,本申请中的实施例及实施例中的特征可以相互任意组合。
样品:
卡马西平购自TargetMol公司,制成40mmol/L的储存浓度,溶于DMSO,储存于-20℃,备用。中药淫羊藿中八个单体成分(朝霍定A、朝霍定A1、朝霍定B、朝霍定C、淫羊藿苷、淫羊藿素、淫羊藿次苷II、脱水淫羊藿黄素)购自TargetMol公司,制成10mmol/L的储存浓度,溶于DMSO,储存于-20℃,备用,最终给药浓度:10μmol/L,由opti-Mem培养基稀释所得。
动物实验所需淫羊藿次苷II:购自成都普非德生物技术有限公司,临用前用含10%吐温80的PBS缓冲液溶解,分别配成25、50、100mg/kg。
动物实验所需LPS:购自美国Sigma Aldrich公司,临用前用PBS缓冲液配置成2mg/kg。
试剂、材料:
DMEM培养基、胎牛血清(美国HyClone公司);Opti-MEM无血清培养基(美国Gibco公司);胰蛋白酶、双抗(青霉素-链霉素溶液)(迈晨科技北京有限公司);三磷酸腺苷(ATP)、尼日利亚菌素(Nigericin)、脂多糖(LPS)、二甲基亚砜(DMSO)(美国Sigma Aldrich公司);NLRP3、ASC、Caspase-1、pro-Caspase-1、pro-IL-1β、GAPDH抗体(美国Cell SignalingTechnology公司);Caspase-1活性测定试剂盒(美国Promega公司);TNF-α、IL-1βELISA检测试剂盒(北京达科为生物技术有限公司);5×SDS蛋白电泳上样缓冲液、PM2510三色预染蛋白Marker(北京鼎国昌胜生物技术有限公司);电泳液、转印液、10×TBST缓冲液、考马斯亮蓝(北京博迈德基因技术有限公司);甲醇、乙酸(北京化工厂);RIPA裂解液(上海碧云天生物技术有限公司);A、B显影液、PVDF膜(美国Millipore公司)。
仪器:
二氧化碳培养箱(美国Thermo Scientific公司);酶标仪(美国Promega公司);低温高速离心机(美国Sigma公司);电泳槽、转印槽、凝胶电泳分析系统(美国Bio-Rad公司);细胞培养板振荡仪(德国IKA公司)。
基于NLRP3炎症小体活化模型,提出一种特异质肝损伤药物筛选评价模型及方法,即在NLRP3炎症小体激活的情况下,目标成分或药物进一步促进炎症小体的激活,导致炎症因子大量释放,介导更严重的免疫炎症反应,加重肝脏损伤。该模型和方法可以用于预测和评价药物免疫特异质肝毒性。
卡马西平具有特异质肝损伤,首先我们在该模型上评价卡马西平的致特异质肝损伤作用,以此说明模型的可靠性。然后再基于该模型和方法,筛查淫羊藿致免疫特异质肝损伤主要化学成分。
样品制备实施例:
1.卡马西平在免疫特异质肝损伤评价模型中激活NLRP3炎症小体情况
a.卡马西平的制备:卡马西平制成40mmol/L的储存浓度,溶于DMSO,储存于-20℃,备用。
b.小鼠原代骨髓巨噬细胞(BMDMs)的制备:取10周龄C57BL/6雌性小鼠,分离肱骨和股骨,用DMEM培养基(含10%胎牛血清、5%青霉素-链霉素溶液)反复抽取骨髓细胞并按1∶4000加入小鼠集落刺激因子(MCSF),分化5天后,消化细胞并按9×105/ml种板(24孔板,0.5ml/well),过夜贴壁后进行下一步实验。
c.LPS刺激:采取换液的方式,以50ng/ml的浓度(0.4ml/well)加入到BMDMs并刺激4h。
d.给药:将a中制备得到的卡马西平用Opi-Mem培养基稀释成10,20,40μM,吸走LPS,以换液的方式加入到BMDMs(0.4ml/well),刺激1h。
e.NLRP3刺激因子刺激:采取补液的方式,加入终浓度为5mM ATP(0.02ml/well)刺激1h。
f.收样:细胞收样,直接加入1×RIPA Loading裂解(120μL/孔),迅速晃匀,使Loading覆盖所有细胞,5mins后再如此,反复3次,将Loading吸入EP管中。细胞上清样品处理:室温4000rpm离心5mins,收上清,加入1/4体积三氯乙酸(TCA),4℃静置1h以上后,4℃,12000rpm离心15mins;弃上清,加入冰丙酮(500μL/孔)上下颠倒混匀,4℃,12000rmp离心15mins,弃上清,95℃金属浴挥干丙酮,待丙酮挥发后加1×RIPA Loading 35μL振荡混匀,水浴煮沸,冷却后作为上清样品。
g.Western blot:检测上清中IL-1βp17、caspase-1 p20蛋白表达水平,检测细胞裂解液中NLRP3、ASC、pro-Caspase-1,pro-IL-1β蛋白水平。细胞上清蛋白样品采用12%浓度的SDS-聚丙烯酰胺凝胶电泳,细胞裂解液样品采用10%浓度的SDS-聚丙烯酰胺凝胶电泳,接通电源,设定恒定电压80V,45min后电压改为135V,溴酚蓝到达分离胶底部时,结束电泳,安装转印装置。细胞上清结束电泳后,沿maker 60kD位置裁胶,60kD以上采用考马斯亮蓝染色30min,脱色液(水∶甲醇∶乙酸=5∶4∶1),至背景蓝色脱透明;转移槽的阴极铺4层滤纸,将60kD以下剩余凝胶平铺于滤纸上,并在胶上铺PVDF膜,将5层滤纸铺在膜的上方。接通电源,设定恒定电流,450mA,3h。转膜后,5%脱脂奶室温封闭1h,不同一抗(1∶1000)溶液4℃孵育过夜,PVDF膜经1%TBS-T溶液间隔5mins洗膜3次,再与相应标记的二抗(1∶5000)溶液进行反应1h,弃二抗,用1%TBS-T溶液间隔5mins洗膜3次,将显影液A和B两种试剂等体积混合后,立即加到膜上,暗室采用X光胶片压片曝光方式显影。
h.ELISA:测定细胞上清液中IL-1β、TNF-α的含量,操作步骤按说明书进行。在450nm波长读取OD值,根据标准曲线计算样品浓度。
i.Caspase-1活性测定:按照Caspase-1活性测定试剂盒说明书进行操作。先室温平衡Caspase-1缓冲液、Z-WEHD荧光素底物及MG-132抑制剂,然后在10mL Z-WEHD-氨基荧光素底物中加入60μL MG-132(120μM)抑制剂充分涡旋至彻底混合,再加入Caspase-1缓冲液,充分涡旋形成终浓度为60μM的荧光检测混合物,待用。96孔板中每孔加入50μL的细胞上清样本,再每孔加入30μL配制待用的荧光检测混合物,细胞培养板振荡器振荡1min,室温避光平衡使样品与荧光底物充分结合,1h后经酶标仪检测分析Caspase-1荧光值。
j.统计学方法:数据采用GraphPad Prism 6.0(GraphPad,San Diego,CA)统计软件进行处理,组间差异分析选用One-way ANOVA方法进行。P<0.05被认为存在统计学差异。
2.淫羊藿中各单体成分(主要包括朝霍定A、朝霍定A1、朝霍定B、朝霍定C、淫羊藿苷、淫羊藿素、淫羊藿次苷II、脱水淫羊藿黄素)在免疫特异质肝损伤评价模型中激活NLRP3炎症小体情况
a.淫羊藿中各单体成分制备:取朝霍定A、朝霍定A1、朝霍定B、朝霍定C、淫羊藿苷、淫羊藿素、淫羊藿次苷II、脱水淫羊藿黄素,用DMSO溶解成10mM。-20℃储存,备用。临用前将各单体成分用opti-MEM稀释至10μM,实验步骤同1项下b-j。
3.筛选出的单体成分的进一步验证
将2a中筛选出的具有促进NLRP3炎症小体激活的单体成分拉浓度梯度(1,2.5,5μM),进一步验证其对NLRP3炎症小体的激活作用。实验步骤同1项下b-j。
4.LPS特异质肝损伤模型评价筛选出的致特异质肝损伤成分的肝损伤作用
a.6-8周C57BL/6雌性小鼠尾静脉注射LPS(2mg/kg)
b.2h后腹腔注射筛选出的致特异质肝损伤成分(25、50、100mg/kg)
c.6h后,小鼠眼眶取血,3500rpm/min离心,测血清中IL-1β、TNF-α、ALT、AST的含量。
5.基于NLRP3炎症小体活化模型及LPS诱导的特异质肝损伤模型,确证淫羊藿致特异质肝损伤主要化学成分
结果:
1.致特异质肝损伤药物卡马西平对ATP刺激的NLRP3炎症小体活化的影响
在BMDMs细胞中,先用LPS(50ng/ml)处理细胞4h,然后将培养基换成无血清opti-MEM配制而成的卡马西平(10,20,40μM),处理1h,最后补加ATP(5mM),1h后收取细胞上清及裂解液进行相关指标检测。从图1中可以看出,LPS刺激下,细胞上清中caspase-1、IL-1β蛋白未表达,表明NLRP3炎症小体未激活;在LPS及ATP刺激下,细胞上清中caspase-l、IL-1β蛋白表达,caspase-l活性测定结果与单加LPS相比,显著增强(P<0.001),表明在LPS及ATP刺激下NLRP3炎症小体激活;在LPS、卡马西平及ATP共同刺激下,与LPS加ATP组相比,细胞上清caspase-1、IL-1β蛋白表达,caspase-1测活及ELISA试剂盒IL-1β的含量测定结果显示卡马西平呈剂量依赖性促进NLRP3炎症小体活性。说明该模型能评价特异质肝损伤药物或成分,并可用于致特异质肝损伤相关药物或成分的筛选及评价。
2.淫羊藿各单体成分对ATP刺激的NLRP3炎症小体活化的影响
在BMDMs细胞中,先用LPS(50-ng/ml)处理细胞4h,然后将培养基换成无血清opti-MEM配制而成的淫羊藿各单体成分(10μM),处理1h,最后补加ATP(5mM),1h后收取细胞上清及裂解液进行相关指标检测。从下图2可以看出,在LPS刺激下,细胞上清中caspase-1、IL-1β蛋白未表达,表明NLRP3炎症小体未激活;在LPS及ATP刺激下,细胞上清中caspase-1、IL-1β蛋白表达,caspase-1活性测定结果与单加LPS相比,显著增强(P<0.001),ELISA测定结果显示上清中IL-1β的含量与单加LPS相比,显著增加(P<0.001),表明在LPS及ATP刺激下NLRP3炎症小体激活;在LPS及ATP刺激的基础上,再给予淫羊藿中各单体成分,综合caspase-1活性及ELISA试剂盒IL-1β的含量,结果显示淫羊藿各单体成分中,淫羊藿次苷II具有显著促进NLRP3炎症小体活性的作用(P<0.001),而以上各单体成分对非炎症小体依赖的TNF-α的含量无影响。以上实验结果说明淫羊藿次苷II或为淫羊藿致特异质肝损伤的主要化学成分。
3.淫羊藿次苷II对ATP刺激的NLRP3炎症小体活化的梯度效应研究
选择淫羊藿次苷II以下浓度研究其对NLRP3炎症小体激活情况:1、2.5、5μ mol/L。先用LPS(50ng/ml)处理细胞4h,然后将培养基换成无血清opti-MEM配制而成的不同浓度的淫羊藿次苷II,处理1h,最后补加ATP(5mM),1h后收取细胞上清及裂解液进行相关指标检测。从图3中可以看出在LPS及ATP共同刺激作用下,western blot结果显示BMDMs细胞上清中,caspase-1 p20及IL-1βp17的蛋白表达量随着淫羊藿次苷II浓度的增加而增加;caspase-1活性测定结果表明BMDMs细胞上清中caspase-1的量随着淫羊藿次苷II浓度增加呈剂量依赖性增加;ELISA结果表明细胞上清中IL-1β的含量随着淫羊藿次苷II浓度增加呈剂量依赖性增加,而对非NLRP3炎症小体依赖的TNF-α的含量无影响。以上研究结果表明,淫羊藿次苷II在LPS和ATP刺激下,caspase-1进一步剪切活化,进而促进NLRP3炎症小体激活,导致炎症因子IL-1β大量增加,从而介导更为严重的免疫炎症反应,加重淫羊藿特异质肝损伤。
4.LPS诱导的特异质肝损伤模型进一步确证淫羊藿中致特异质肝损伤标志物--淫羊藿次苷II的肝损伤作用
6-8周龄雌性C57BL/6小鼠经LPS(2mg/kg)尾静脉注射2h后,给予不同浓度的淫羊藿次苷II(25、50、100mg/kg)处理6h,眼眶取血,3500rpm离心,测血清中IL-1β、TNF-α、ALT、AST的含量。如图4所示,与对照组相比,LPS组血清ALT、AST、IL-1β及TNF-α显著升高(P<0.05)。而单独给药组(25、50mg/kg)与对照组相比无显著性差异。单独给药组(100mg/kg)与对照组相比,血清ALT、AST显著升高(P<0.05),IL-1β及TNF-α无变化,提示该浓度下淫羊藿次苷II产生了肝脏直接毒性。而与LPS组相比,LPS+给药组(50、100mg/kg)血清ALT、AST、IL-1β及TNF-α显著升高(P<0.01),以此说明在LPS诱导的特异质肝损伤模型中,淫羊藿次苷II会加重肝脏损伤,且这种损伤伴随着炎症因子IL-1β及TNF-α的升高,表明淫羊藿次苷II确实是导致淫羊藿免疫特异质肝损伤的主要化学成分。
虽然本申请所揭露的实施方式如上,但所述的内容仅为便于理解本申请而采用的实施方式,并非用以限定本申请。任何本申请所属领域内的技术人员,在不脱离本申请所揭露的精神和范围的前提下,可以在实施的形式及细节上进行任何的修改与变化,但本申请的专利保护范围,仍须以所附的权利要求书所界定的范围为准。
Claims (12)
1.一种免疫特异质肝损伤的评价模型,所述评价模型包括NLRP3炎症小体以及促进NLRP3炎症小体活化的成分,其中,促进NLRP3炎症小体活化的成分为淫羊藿次苷II。
2.权利要求1所述的评价模型的构建方法,所述构建方法包括:
S100.细胞水平评价;
S101.淫羊藿单体样品溶液的制备;
S102.制备小鼠原代骨髓巨噬细胞,并加入LPS进行刺激;
S103.将步骤S101中制备的溶液加入小鼠原代骨髓巨噬细胞进行刺激;
S104.加入ATP进行刺激;
S105.Western blot检测NLRP3炎症小体相关蛋白的表达,ELISA检测细胞上清中IL-1β、TNF-α的含量,以及Caspase-1活性测定,并进行统计学分析。
3.根据权利要求2所述的构建方法,其中,所述淫羊藿单体选自朝霍定A、朝霍定A1、朝霍定B、朝霍定C、淫羊藿苷、淫羊藿素、淫羊藿次苷II和脱水淫羊藿黄素中的任一种或更多种。
4.根据权利要求2或3所述的构建方法,其中,在步骤S101中,取所述淫羊藿各单体,用二甲基亚砜溶解成10mM,于-20℃储存,备用,临用前用Opti-MEM稀释至10μM。
5.根据权利要求2或3所述的构建方法,其中,步骤S100还包括:
筛选出所述淫羊藿单体中促进NLRP3炎症小体活化的成分后,以1μM-5μM的浓度,进一步验证所述成分对NLRP3炎症小体的活化作用。
6.根据权利要求2所述的构建方法,其中,所述构建方法还包括:
采用与步骤S100相同的操作步骤,用卡马西平对所述评价模型进行验证。
7.根据权利要求6所述的构建方法,其中,将卡马西平配制成10-40μM的浓度。
8.根据权利要求6所述的构建方法,其中,卡马西平促进NLRP3炎症小体活化。
9.根据权利要求2所述的构建方法,其中,所述构建方法包括:
S100.细胞水平评价;
S101.淫羊藿样品溶液的制备;
S102.制备小鼠原代骨髓巨噬细胞,并加入LPS进行刺激;
S103.将步骤S101中制备的溶液加入小鼠原代骨髓巨噬细胞进行刺激;
S104.加入ATP进行刺激;
S105.Western blot检测NLRP3炎症小体相关蛋白的表达,ELISA检测细胞上清中IL-1β、TNF-α的含量,以及Caspase-1活性测定,并进行统计学分析;
S200.动物水平评价;
S201.小鼠尾静脉注射LPS;
S202.腹腔注射促进NLRP3炎症小体活化的成分的溶液;
S203.测定血清中ALT、ASTI、L-1β、TNF-α的含量。
10.根据权利要求9所述的构建方法,其中,促进NLRP3炎症小体活化成分的小鼠给药浓度为25-100mg/kg。
11.一种免疫特异质肝损伤药物或成分的筛选方法,所述筛选方法包括使用权利要求1至10中任一项所述的评价模型进行筛选促进NLRP3炎症小体活化的药物或成分。
12.NLRP3炎症小体活化模型用于评价免疫特异质肝损伤中的用途。
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