CN114796224A - 吡喹酮在抗肾纤维化疾病中的应用 - Google Patents
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Abstract
本发明公开了吡喹酮在抗肾纤维化疾病中的应用,涉及生物医学领域。本发明应用吡喹酮能够降低血吸虫感染引起的胶原沉积,逆转肾脏组织中高表达结缔组织生长因子(CTGF)和胶原分子Col1、Col3和Col4,从而改善肾纤维化的病变;本发明公开的吡喹酮具有良好的抗肾纤维化疾病的应用前景。
Description
技术领域
本发明涉及生物医学领域,特别是涉及吡喹酮在抗肾纤维化疾病中的应用。
背景技术
肾脏疾病影响着全球约10%的人口,是一个急需解决的全球健康问题。肾纤维化(renal fibrosis)是肾脏疾病最终导致终末肾功能衰竭的共同基础。导致肾纤维化发生的疾病有:糖尿病、高血压、系统性红斑狼疮、慢性肾小球肾炎、慢性肾盂肾炎、药物及感染性疾病等。肾脏由于受到创伤、感染、炎症、血循环障碍,以及免疫反应等多种致病因素刺激,持续存在的炎症环境以及组织的异常修复引起细胞外基质大量堆积,发生部位以肾小球和肾小管间质为主,最终进展为肾纤维化,若不及时进行干预可进展为肾衰竭。
血吸虫病是世界上仅次于疟疾的第二大寄生虫病,全球感染者约有2亿,严重危害全球人民的健康。血吸虫病造成的主要病理损伤是沉积在肝脏的虫卵形成的肉芽肿反应以及持续慢性感染引起的组织损伤修复过度,进而形成肝纤维化。此外,肾脏也是感染血吸虫后易受损伤的靶器官之一。1968年Andrade报道了曼氏血吸虫病患者的肾脏存在炎症;随后多篇研究报道血吸虫病患者中伴有血吸虫肾病的占比较高;我们前期研究中构建了日本血吸虫感染小鼠模型,观察血吸虫感染后肾脏损伤的发生进程以及纤维化相关指标的评价,明确血吸虫感染性肾损伤的发生和发展过程;首次证实血吸虫感染可引起小鼠肾脏纤维化的发生。
吡喹酮(Praziquantel,PZQ)作为一种副作用小,安全性高的抗血吸虫药物,已投入临床使用了40多年。然而,吡喹酮针对肾纤维化是否同样有逆转作用,尚未有相关评价数据。
发明内容
针对现有技术的不足,本发明的目的是提供吡喹酮在抗肾纤维化疾病中的应用。
为实现上述目的,本发明提供了如下方案:
第一方面,本发明保护吡喹酮在制备抗肾纤维疾病的相关药物中的应用。
作为本申请的优选技术方案,所述肾纤维化疾病由糖尿病、高血压、系统性红斑狼疮、慢性肾小球肾炎、慢性肾盂肾炎、药物及感染性疾病中的任一种引起。
作为本申请的优选技术方案,所述感染由血吸虫感染引起。
第二方面,吡喹酮与第二治疗药物联合在制备抗肾纤维化疾病的药物中的应用。
作为本申请的优选技术方案,所述吡喹酮与第二治疗药物采用同时给药、或不分次序先后单独给药。
作为本申请的优选技术方案,所述治疗药物为适于口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌肉内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
优选的,治疗药物为适于口服或肌肉内任意给药方式的制剂。
更优选的,所述肌肉内给药方式为肌肉注射。
第三方面,本发明还保护一种药物组合物,所述药物组合物包括吡喹酮,以及药学上可接受的载体或辅料。
作为本申请的优选技术方案,所述药物组合物的剂型包括滴剂、口服液、片剂、胶囊剂、颗粒剂、冲剂、膜剂、凝胶剂、散剂、乳剂、自乳化制剂、滴丸剂、栓剂、气雾剂、喷雾剂、粉雾剂、贴剂、贴膏剂、溶液剂、软膏剂或乳膏剂等等剂型。
有益效果
本发明中公开的吡喹酮,能够降低血吸虫感染引起的肾脏组织的胶原沉积。
本发明中公开的吡喹酮,能够逆转血吸虫病肾脏组织中结缔组织生长因子(CTGF)和胶原分子Col1、Col3和Col4的高表达。
本发明中公开的吡喹酮,能够降低肾纤维化程度,改善肾功能,可为改善血吸虫肾纤维化疾病及其他类型纤维化的治疗提供新的候选药物。
附图说明
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的前提下,还可以根据这些附图获得其他的附图。
图1.血吸虫病慢性期经吡喹酮抗纤维化治疗后小鼠肾脏外观和肾功能变化。(A)慢性期吡喹酮抗纤维化治疗后小鼠肾脏外观。(B)慢性期吡喹酮抗纤维化治疗后小鼠尿蛋白含量。(C)小鼠尿蛋白含量统计。(D)慢性期吡喹酮抗纤维化治疗后小鼠血清肌酐水平。(ig:口服灌胃,im:肌肉注射)(ns,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001。)
图2.血吸虫病慢性期经吡喹酮抗纤维化治疗后小鼠肾脏组织Masson染色。(A)慢性期吡喹酮抗纤维化治疗后小鼠肾脏Masson染色,100×。(B)定量分析Masson染色胶原纤维阳性区域面积。(ig:口服灌胃,im:肌肉注射)(ns,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001。)
图3.血吸虫病慢性期吡喹酮抗纤维化治疗后小鼠肾脏组织病理变化—慢性期吡喹酮抗纤维化治疗后小鼠肾脏HE染色,100×。(ig:口服灌胃,im:肌肉注射)(ns,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001。)
图4.血吸虫病慢性期经吡喹酮抗纤维化治疗后小鼠肾脏相关的纤维化指标。(A)慢性期吡喹酮抗纤维化治疗后qRT-PCR检测纤维化指标CTGF、Col1、Col3和Col4的基因表达水平。(B)慢性期吡喹酮抗纤维化治疗后CTGF、Col1和的GAPDH蛋白表达。(C)定量分析CTGF、Col1和GAPDH的表达情况。(ig:口服灌胃,im:肌肉注射)(ns,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001。)
图5.口服/肌肉注射方式对血吸虫感染造成的肾纤维化的治疗效果。感染者+吡喹酮治疗组与仅感染组纤维化指标的基因表达水平比值。(ig:口服灌胃,im:肌肉注射)(ns,P>0.05;*,P<0.05;**,P<0.01;***,P<0.001。)
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明构建了血吸虫感染模型,并于感染的慢性期(第10周)给予吡喹酮治疗4周,评价吡喹酮对血吸虫病肾纤维化干预的效果。另外,由于肝脏在生理上具有首过效应,吡喹酮传统的口服给药方式必然会受到限制从而影响其血药浓度的变化,所以综合考虑后采取肌肉注射和口服药物两种方式进行给药治疗,观察了肌注和口服两种给药方式对血吸虫感染引起的肾损伤的影响。本发明评价吡喹酮在血吸虫肾纤维化模型上的治疗效果,为改善其他类型纤维化的治疗提供新的候选药物。
实施例1吡喹酮混悬液的配制
①1%羧甲基纤维素溶液:分析天平精准秤取1g羧甲基纤维素粉末溶于100mlddH2O,用涡旋器充分震荡,置于4℃冰箱中的摇床上过夜,配制透明的1%羧甲基纤维素溶液,4℃冰箱摇床可保存一周。
②吡喹酮混悬液:分析天平精准秤取适量吡喹酮粉末,用1%羧甲基纤维素溶液充分混匀,配制成300mg/kg浓度的吡喹酮混悬液,4℃冰箱可保存一周。
实施例2吡喹酮抗日本血吸虫感染慢性期小鼠肾脏纤维化的治疗
感染日本血吸虫10周的小鼠(处于感染慢性期)及正常小鼠随机分组,每组6~8只。给予抗纤维化剂量的吡喹酮治疗,分为口服灌胃和肌肉注射两种给药方式。具体分组及处理方式如下:①正常对照组(Normal):正常小鼠;②正常口服吡喹酮组[Normal+PZQ(ig)]:同期正常小鼠以300mg/kg浓度的吡喹酮100μl口服灌胃,每12小时一次;③正常肌注吡喹酮组[Normal+PZQ(im)]:同期正常小鼠以20%浓度的吡喹酮以300mg/kg浓度300μl腿部肌肉注射,每12小时一次;④感染组(Infected):日本血吸虫感染小鼠;⑤口服吡喹酮治疗组[Infected+PZQ(ig)]:同期感染小鼠以300mg/kg浓度的吡喹酮100μl口服灌胃,每12小时一次;⑥肌注吡喹酮治疗组[Infected+PZQ(im)]:同期感染小鼠以20%浓度的吡喹酮以300mg/kg浓度300μl腿部肌肉注射,每12小时一次。分别在给药后的第1、14和28天处理小鼠。
实施例3吡喹酮抗纤维化治疗小鼠肾脏外观和肾功能检测
①尿蛋白含量半定量:收集小鼠晨尿,均匀滴在试纸条的检测区域,2秒后用纸巾吸去多余尿液,在60~120秒内与标准比色卡对比,完成比色拍照。
②血清肌酐水平检测:小鼠麻醉后,内眦静脉取血,分离血清,各样本血清肌酐水平用Olympus AU5400全自动生化分析仪检测。
本发明的应用结果显示:与感染组相比,给药组小鼠肾功能指标尿蛋白含量和血清肌酐水平在给药14天后降低,给药28天后基本恢复到正常组水平,治疗效果显著(图1)。
实施例4小鼠肾脏Masson染色
将小鼠肾脏组织置于5ml 10%的福尔马林溶液中固定,常规脱水石蜡包埋;切片厚约3μm,温水脱蜡固定于载破片上;切片入Bouin液媒染后天青石蓝染色,苏木素染色后乙醇分化;丽春红品红染色后磷钼酸溶液处理,苯胺蓝染色;无水乙醇脱水,二甲苯透明,中性树脂封片,静置干燥。显微镜观察拍照,用Image-Pro Plus 6.0软件分析阳性面积百分比。
采用了上述Masson染色检测各组小鼠肾脏组织中胶原纤维比例,与正常对照组相比,给药组中小鼠肾脏切片Masson染色的阳性区域面积即胶原纤维沉积比例较感染组减少,且治疗28天后胶原纤维沉积比例减少约70%(图2)。
实施例5小鼠肾脏HE染色
将肾脏组织置于5ml 10%的福尔马林溶液中固定,常规脱水石蜡包埋,切片为约3μm厚的薄片,温水脱蜡,用苏木素和伊红进行染色,随后无水乙醇脱水,二甲苯透明,中性树脂封片,静置干燥。在普通光学显微镜下观察拍照。
应用本发明中吡喹酮干预小鼠肾纤维化的肾脏切片的HE染色,结果提示:给药14天后,肾脏组织中浸润的炎性细胞减少,肾小球系膜细胞的增生情况也有明显改善,给药28天后肾脏组织中未见明显的细胞浸润(图3)。
实施例6肾脏纤维化相关指标检测
①纤维化相关基因mRNA检测
取适量肾脏组织加1ml TRIzol,按常规方法提取mRNA并逆转成cDNA;实时荧光定量PCR检测纤维化相关指标的mRNA水平,反应体系如下(总体积20μl):2×SYBR Green Mix10μl,cDNA模板8.8μl,上游引物0.6μl,下游引物0.6μl。在LightCycler96 PCR仪上进行PCR反应,程序如下:预变性95℃,900秒;二步法扩增(45个循环)95℃,10秒;60℃,30秒;熔解曲线分析:95℃,10秒;65℃,60秒;97℃,1秒。采用2-△△Ct法分析数据:计算F值:F=2-△△Ct。各个引物序列根据美国生物信息中心(NCBI)小鼠mRNA序列用Primer 5.0设计引物,具体如下:
Mus-GAPDH:
F5’-AGGTCGGTGTGAACGGATTTG-3’,
R5’-TGTAGACCATGTAGTTGAGGTCA-3’;
Mus-CTGF:
F5’-AACAGTGGAGATGCCAGGAG-3’,
R5’-TAATTTCCCTCCCC GGTTAC-3’;
Mus-Col1α1:
F5’-GTCGAGGGCCAAGACGAAG-3’,
R5’-CAGATCACGTCATCGCACAAC-3’;
Mus-Col3α1:
F5’-TGGTCCCCAAGGTG TCAAAG-3’,
R5’-GGGGGTCCTGGGTTACCATTA-3’;
Mus-Col4α1:
F5’-TTC AGA TTCCGCAGTGCCCTA-3’,
R5’-TTCTCATGCACACTTGGCAGC-3’。
本发明中检测小鼠肾脏纤维化相关指标发现:各时间点中,结缔组织生长因子(CTGF)和胶原分子Col1、Col3和Col4的基因水平在感染未治疗组中显著上升,证实血吸虫感染引起肾脏纤维化的发生;而经吡喹酮治疗后,肾脏纤维化相关指标的基因表达水平均降低,且在治疗28天后基本恢复至正常水平(图4A)。
②纤维化相关基因蛋白表达水平检测
剪取各组小鼠肾脏组织块少量,加400μl蛋白裂解液,按常规方法提取总蛋白,BCA法测蛋白浓度,行SDS-PAGE后转膜,脱脂乳封闭2小时。用1×的TBST将一抗稀释至适当浓度(常用稀释倍数为1:1000),4℃冰箱孵育过夜;TBST洗涤PVDF膜5遍;用1×的TBST将二抗稀释至适当浓度(常用稀释倍数为1:5000),置于摇床上,室温避光孵育1小时;弃去二抗,用TBST洗涤PVDF膜5遍。ECL发光液均匀滴加至PVDF膜表面,置于CD Touch凝胶电泳成像系统中曝片。
在前述基因水平上确认了吡喹酮治疗后逆转了纤维化相关指标发生显著改变后,本发明进一步检测了CTGF和Col1的蛋白表达情况,发现感染组中的蛋白表达均较正常组增加,且随感染时间延长表达也逐渐增多,证实血吸虫感染引起肾脏纤维化的发生。而在吡喹酮干预治疗后,CTGF和Col1的蛋白表达在给药组中显著降低(图4B)。以上结果表明在血吸虫感染模型上,吡喹酮能降低肾脏纤维化指标的表达,具有抗肾脏纤维化的作用。
实施例7口服/肌肉注射给药方式对血吸虫病肾纤维化的治疗效果
为了对比口服灌胃和肌肉注射两种方式的治疗效果,前述实施例2中分别使用口服吡喹酮治疗组和肌注吡喹酮干预纤维化的效果。
在本发明中发现肌肉注射的给药方式降低血吸虫感染小鼠纤维化指标的基因表达比口服更快(图5)。这表明肌肉注射吡喹酮的给药治疗发挥抗血吸虫感染性肾脏炎症和纤维化作用比口服发挥作用更快。
本发明证实吡喹酮具有抗肾纤维化作用,为抗肾纤维化治疗药物的研发提供了新的候选药物。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.吡喹酮在制备抗肾纤维化疾病的相关药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述肾纤维化疾病由糖尿病、高血压、系统性红斑狼疮、慢性肾小球肾炎、慢性肾盂肾炎、药物及感染性疾病中的任一种引起。
3.吡喹酮与第二治疗药物联合在制备抗肾纤维化疾病的药物中的应用。
4.根据权利要求3所述的应用,其特征在于,所述吡喹酮与第二治疗药物采用同时给药、或不分次序先后单独给药。
5.根据权利要求1-4任一所述的应用,其特征在于,所述治疗药物为适于口服、肠胃外、腹膜内、静脉内、动脉内、透皮、舌下、肌肉内、直肠、透颊、鼻内、吸入、阴道、眼内、局部、皮下、脂肪内、关节内、腹膜内或鞘内任意给药方式的制剂。
6.根据权利要求5所述的应用,其特征在于,所述治疗药物为适于口服或肌肉内任意给药方式的制剂。
7.一种药物组合物,所述药物组合物包括吡喹酮,以及药学上可接受的载体或辅料。
8.根据权利要求7所述的一种药物组合物,其特征在于,所述药物组合物的剂型包括滴剂、口服液、注射剂、片剂、胶囊剂、颗粒剂、冲剂、膜剂、凝胶剂、散剂、乳剂、自乳化制剂、滴丸剂、栓剂、气雾剂、喷雾剂、粉雾剂、贴剂、贴膏剂、溶液剂、软膏剂或乳膏剂。
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