CN116003290A - 一种3-苯基戊二酸衍生物lonp1激活剂及其在慢性肾脏病防治药物中的应用 - Google Patents
一种3-苯基戊二酸衍生物lonp1激活剂及其在慢性肾脏病防治药物中的应用 Download PDFInfo
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- CN116003290A CN116003290A CN202111613874.0A CN202111613874A CN116003290A CN 116003290 A CN116003290 A CN 116003290A CN 202111613874 A CN202111613874 A CN 202111613874A CN 116003290 A CN116003290 A CN 116003290A
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Abstract
本发明提供了一种3‑苯基戊二酸衍生物LonP1激活剂及其在慢性肾脏病防治药物中的应用。本发明发现一种结构新颖的3‑苯基戊二酸衍生物类化合物可以有效激活LonP1的蛋白酶活性,并改善慢性肾脏病模型小鼠的肾脏纤维化,逆转肾小管上皮细胞向肌成纤维细胞的转分化,抑制间质成纤维细胞活化与基质生成,减轻足细胞损伤,从而起到改善慢性肾脏病的作用;可用于制备各种原因引起的慢性肾病的防治药物。
Description
技术领域
本发明涉及医药领域,具体涉及一种3-苯基戊二酸衍生物小分子作为LonP1激活剂,及其在制备慢性肾脏病防治药物中的应用。
背景技术
全球改善肾脏病预后组织(Kidney Disease:Improving global outcomes,KDIGO)把慢性肾脏病(chronic kidney disease,CKD)为肾脏的结构或功能异常达到或超过3个月并影响健康。近年来,慢性肾脏病这一公共健康问题在世界范围内的发病率逐年增高,成为世界性的公共健康问题。而目前临床上仍然缺乏满意的CKD干预手段,相当一部分CKD患者最终进展为终末期肾脏病 (End-stage renal disease,ESRD),需要依赖肾脏替代治疗以维持生命,给家庭和社会带来沉重的负担,因此亟需寻找延缓和治疗CKD的有效手段。
从CKD发展到ESRD是进行性的病理生理过程,其病理生理机制主要包括肾小管间质纤维化和肾小球的损伤。肾小管间质纤维化与肾小球硬化相互交织、相互作用,共同导致ESRD的发生。在肾小管间质损伤中,肾小管上皮细胞向间充质细胞的转分化和肾间质成纤维细胞的活化与增殖,共同促进了细胞外基质在肾间质的过度积聚,在肾小管间质纤维化过程中发挥重要作用。与此同时,足细胞损伤在肾小球硬化过程中扮演重要的角色,足细胞损伤后后基底膜脱落,肾小球滤过屏障破坏,产生蛋白尿,进一步诱导肾脏多种细胞的损伤。
线粒体离子肽酶1(lon peptidase 1,LonP1)又称为Lon蛋白酶,是一种高度保守的线粒体蛋白酶。LonP1是由核DNA编码的,前体通过线粒体靶向序列导向转运至线粒体基质中,其主要功能是酶解线粒体内错误折叠的蛋白、激活功能蛋白前体、修复线粒体DNA 等以维持线粒体的稳态及功能。大量文献报道,肾小管上皮细胞向间充质细胞转分化及肾间质成纤维细胞活化均与线粒体功能密切相关。此外,研究还证明局灶节段性肾小球硬化(focal segmental glomerular sclerosis,FSGS)及微小病变肾病(minimal changedisease,MCD)慢性肾脏病患者肾小球中LonP1表达水平下调,且LonP1表达水平与尿蛋白水平呈负相关。
据此,对CKD患者使用LonP1小分子激活剂治疗可通过激活LonP1的酶活性,维持线粒体稳态,抑制肾小管上皮细胞转分化和肾间质成纤维细胞活化、减轻足细胞损伤,进而延缓CKD向ESRD的转变。因此,本发明提供的3-苯基戊二酸衍生物类LONP1激活剂对各种病因引起的慢性肾脏病患者具有重要的应用价值。目前尚未见LonP1激活剂的报道,更未见LonP1激活剂用于肾脏疾病防治药物的报道。
发明内容
本发明的目的在于克服现有技术的缺失,提供了一种3-苯基戊二酸衍生物LonP1激活剂,其特征在于可以剂量依赖性地激活LonP1的酶活性,并在较高剂量下提高细胞中LonP1 的表达水平。
本发明的另一目的是克服现有技术的缺失,提供所述3-苯基戊二酸衍生物LonP1激活剂在制备慢性肾脏病防治药物中的应用。我们的研究发现,优选的3-苯基戊二酸衍生物 84-B10改善单侧输尿管梗阻(unilateral ureteral occlusion,UUO)模型小鼠的肾脏纤维化,抑制组织生长因子1(TGF-β1)诱导的肾小管上皮细胞转分化和肾间质成纤维细胞活化,减轻炎症因子导致的足细胞损伤,从而提供了一类慢性肾脏病防治的新型药物。
为实现上述目的,本发明采用了如下技术方案:
一种3-苯基戊二酸衍生物类LonP1激活剂,其特征在于,所述3-苯基戊二酸衍生物类 LonP1激活剂为84-B10,用于激动LonP1的蛋白酶活性,并在较高浓度下上调细胞中的LonP1的表达水平,其中所述84-B10的化学名为:
5-[2-(4-methoxyphenoxy)-5-(trifluoromethyl)anilino]-5-oxo-3-phenylpentanoicacid,分子式为C25H22F3NO5,CAS号为698346-43-9,化学结构为:
一种3-苯基戊二酸衍生物类LonP1激活剂,其特征在于,所述3-苯基戊二酸衍生物类 LonP1激活剂包括MQ2(分子式为C19H18F3NO3)、MQ4(分子式为C17H17NO3)、MQ5(分子式为C19H18F3NO4)、MQ6(分子式为C18H19NO3)和MQ7(分子式为 C17H23NO3),均具有LonP1的蛋白酶活性的激动效应;
所述MQ2化合物结构式为:
所述MQ4化合物结构式为:
所述MQ5化合物结构式为:
所述MQ6化合物结构式为:
所述MQ7化合物结构式为:
一种3-苯基戊二酸衍生物类LonP1激活剂在慢性肾脏病防治药物中的应用,其特征在于,将所述3-苯基戊二酸衍生物类LonP1激活剂用于制备防治各种原因引起的慢性肾脏病药物。
所述3-苯基戊二酸衍生物类LonP1激活剂用于制备抑制肾小管上皮细胞转分化的药物。
所述3-苯基戊二酸衍生物类LonP1激活剂用于制备抑制肾间质成纤维细胞活化的药物。
所述3-苯基戊二酸衍生物类LonP1激活剂用于制备减轻足细胞损伤的药物。
本发明提供一种LonP1小分子激活剂,为3-苯基戊二酸衍生物类化合物,该类化合物均具有LonP1激动效应。
优选的,本发明选择上述3-苯基戊二酸衍生物类LonP1激活剂中的一种84-B10为代表,通过实验验证,84-B10可在20μM浓度下上调肾小管上皮细胞中LonP1的表达,在动物模型中减轻慢性肾脏病的肾脏病理损伤和纤维化,可用于制备慢性肾脏病防治药物。
具体的,所述3-苯基戊二酸衍生物类LonP1激活剂可用于制备抑制肾小管上皮细胞- 间充质转化的药物。
所述3-苯基戊二酸衍生物类LonP1激活剂84-B10可用于制备抑制肾间质成纤维细胞活化的药物。
所述3-苯基戊二酸衍生物类LonP1激活剂84-B10可用于制备减轻足细胞损伤的药物。
与现有技术相比,本发明的有益效果在于:
对于局灶节段性肾小球硬化肾病、微小病变肾病、IgA肾病等导致的慢性肾脏病患者而言,其肾脏LonP1的表达水平下调,且LonP1表达水平与尿蛋白水平呈负相关。本发明首次发现并证明3-苯基戊二酸衍生物类LonP1激活剂可有效激活线粒体LonP1活性,优选的该类激活剂中的84-B10可减轻肾间质纤维化模型小鼠的肾脏病理损伤及纤维化,抑制肾小管上皮细胞-间充质转分化、肾间质成纤维细胞的活化并减轻足细胞损伤,说明该类 3-苯基戊二酸衍生物类LonP1激活剂可以是一种潜在的慢性肾脏病防治药物。将所述3-苯基戊二酸衍生物类LonP1激活剂应用于药物中可以起到改善慢性肾脏病的作用,具有良好的开发应用前景。
附图说明
图1为显示3-苯基戊二酸衍生物类LonP1激活剂可激活LonP1的蛋白酶活性的示意图;
图2为显示优选的3-苯基戊二酸衍生物类LonP1激活剂84-B10在20μM浓度下可增加小鼠肾小管上皮细胞中LonP1的蛋白表达的示意图;
图3为显示治疗剂量的84-B10对假手术(Sham)及UUO小鼠无心、肝、肾毒副作用的示意图;
图4为显示在小鼠UUO模型中,马松染色表明低剂量(LD,0.5mg/kg/d)与高剂量(HD,7.5mg/kg/d)的84-B10治疗均可显著减轻肾脏病理的损伤程度,改善肾间质纤维化的示意图;
图5为显示在小鼠UUO模型中,低剂量(LD,0.5mg/kg/d)与高剂量(HD,7.5mg/kg/d)的84-B10治疗均可显著降低肾脏组织中纤维化指标的蛋白和mRNA水平的示意图;
图6为显示在体外培养小鼠肾小管上皮细胞(mPTC)中,84-B10剂量依赖性地降低了TGF-β1诱导的肾小管上皮细胞-间充质转分化指标的蛋白表达水平的示意图;
图7为显示在体外培养的大鼠成纤维细胞(NRK-49F)中,84-B10剂量依赖性地降低了TGF-β1诱导的肾间质成纤维细胞的活化与基质生成指标的蛋白表达水平的示意图;
图8为显示在体外培养的人足细胞(HPC)中,84-B10剂量依赖性地改善了细胞因子混合物诱导的足细胞损伤的示意图。
具体实施方式
下面将结合本发明的附图,对本发明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的一部分实施例,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做出创造性劳动前提下所获得的所有其他实施例,都属于本发明的保护范围。
本实施例中涉及的技术方案介绍如下:
(1)3-苯基戊二酸衍生物类LonP1激活剂对LonP1蛋白酶活性的作用检测
本实施例使用的重组人LonP1蛋白从大肠杆菌中表达并纯化,可激发荧光二肽底物 (Z-AA)2-Rh110购自Anaspec(货号AS-60320)。
检测方法为LonP1(800nM)与相应化合物(浓度均为1μM)或溶剂对照(Ctrl)在反应缓冲液(150mM NaCl,50mM Hepes pH 8.0,10mM MgCl2)中37℃预孵育30min,随后加入(Z-AA)2-Rh110(6μM)和ATP(2mM)启动酶解反应,反应体系于37℃静置1 h。(Z-AA)2-Rh110被LonP1酶解前无荧光,酶解后可释放出红色荧光的Rh110,其最大吸收峰/最大发射峰分别为497nm/527nm。
(2)3-苯基戊二酸衍生物类LonP1激活剂84-B10对LonP1蛋白酶表达的影响
小鼠肾小管上皮细胞(mPTC)培养于含10%FBS的DEME/F12培养基中,置于37℃、5%CO2的细胞培养箱内培育。当细胞生长密度达到90%时,使用0.25%的胰酶(含有0.02%的EDTA)消化,并传均匀铺于6孔板。待6孔板细胞密度60~70%时,给予84-B10(1, 5,10μM)或溶剂对照(0μM)处理24h。免疫印迹(Western blot)法检测LONP1的蛋白水平。
(3)小鼠饲养
本实施例使用的C57BL/6雄性小鼠(购买时7周龄,体重20-23 g)购自江苏集萃药康生物科技股份有限公司,饲养于南京医科大学实验动物中心SPF级屏障环境中,每笼5 只小鼠,进食标准鼠粮,自由饮水,恒温恒湿控制室,维持12:12h光暗节律,适应性饲养1周后进行实验。
(4)单侧输尿管梗阻性(unilateral ureteral obstruction,UUO)模型建立与药物干预 UUO模型是目前应用最为广泛的研究慢性肾脏病肾间质纤维化的实验动物模型。通过结扎单侧输尿管引起梗阻侧肾脏引流系统的阻塞,诱导慢性肾脏结构的损害,模拟临床上的肾间质损伤。将小鼠适应性饲养1周后随机分为Sham组,UUO组,低剂量治疗组 UUO+LD和高剂量治疗组UUO+HD,每组小鼠数量为8-10只。小鼠仰卧位胶布固定四肢于相对无菌操作台,吸入性异氟烷麻醉后,沿腹中线剪开约1 cm纵行切口,依次打开皮层、肌层,进入腹腔,钝性分离左侧输尿管,模型组(UUO组)和给药组(UUO+LD及UUO+HD 组)均结扎输尿管,假手术(Sham)组无需结扎输尿管,然后逐层关闭腹腔。
化合物84-B10,给药时采用的溶媒为10%DMSO/生理盐水。UUO+LD组小鼠84-B10的剂量为0.5 mg/kg/d,UUO+HD组小鼠84-B10的剂量为7.5 mg/kg/d,给药方式为腹腔注射。在UUO手术前1天及手术前2 h预给药2次,之后每天定时给药1次,Sham组和UUO 组注射给予同样体积的上述溶媒,小鼠于UUO造模1周后处死。
(5)小鼠肾小管上皮细胞mPTC与大鼠肾间质成纤维细胞NRK-49F细胞培养与给药
小鼠肾小管上皮细胞(mPTC)及大鼠成纤维细胞(NRK-49F)分别培养于含10%FBS的DEME/F12培养基和DEME培养液基中,置于37℃、5%CO2的细胞培养箱内培育。当细胞生长密度达到90%时,使用0.25%的胰酶(含有0.02%的EDTA)消化,并传均匀铺于6孔板。待6孔板细胞密度60~70%时,给予84-B10(2.5,5,10μM)预处理2 h后, mPTC给予的与相应浓度的84-B10继续处理24 h,NRK-49F给予的与相应浓度的84-B10继续处理24 h。
(6)人足细胞HPC培养与给药
HPC正常培养于含10%FBS及10%胰岛素铁硒传递蛋白(Insulin TransferrinSelenium, ITS)的RPMI-1640培养基中,置于33℃、5%CO2的细胞培养箱内培育。待细胞长至 90%密度后,使用0.25%的胰酶(含有0.02%的EDTA)消化,并传均匀铺于6孔板,置于37℃、5%CO2的细胞培养箱中分化7天后,给予84-B10(2.5,5μM)预处理2h,再予以细胞因子混合物(Cytokines)(IL-1β,TNF-α,IFN-α,IFN-γ,浓度均为10ng/mL) 与相应浓度的84-B10继续处理24h。
(7)免疫印迹(Westernblot)
蛋白裂解液(含蛋白酶抑制剂)提取细胞和组织蛋白,用BCA试剂盒测定蛋白浓度,将吸取的上清按比例加入5×上样缓冲液,混匀后100℃金属浴煮沸10min。采用雅酶PAGE凝胶快速制备试剂盒制备10%聚丙烯酰胺凝胶,每个样本上样量为30或50μg。恒压100V,电泳1.5h至溴酚蓝指示剂至分离胶底部,恒流300mA湿转1.5h至PVDF膜上。PVDF 膜于5%脱脂奶粉/TBST中室温封闭1h后孵育一抗(4℃摇床过夜),一抗LonP1 (ProteinTech,货号66043-1-Ig),alpha smooth muscle actin(α-SMA)(ProteinTech,货号14395-1-AP),vimentin(Abcam,货号ab92547),fibronectin1(FN1)(Abcam,货号ab2413),collagen III(Bioss,货号bs-0549R),podocin(Bioss,货号bs-6597R),WT-1 (Santa Cruz,货号sc-7385),GAPDH(ProteinTech,货号60004-1-Ig);再孵育二抗(室温摇床孵育1h),二抗购自碧云天生物技术有限公司,1:2000稀释于TBST中。孵育完成后,滴加ECL化学发光液并置于凝胶成像系统显影,采用ChemiDoc XRS+对显影所得条带进行灰度值定量。
(8)实时荧光定量PCR(Quantitative Real-time PCR,RT-PCR)
加入Trizol提取细胞和组织总RNA,用分光光度法测RNA浓度,取质量1μg的RNA 即刻逆转录成cDNA,反应体系为20μL,见表1。
表1:逆转录反应体系
逆转录条件:37℃15min,85℃5s,4℃保持。
采用罗氏SYBR green PCR方法,在罗氏LightCycler96实时荧光定量PCR仪中进行扩增,反应体系为10μL,见表2。
表2:Real-time PCR反应体系
PCR反应程序:预变性95℃10min,随后95℃15s和60℃1min循环40次。
(9)Masson染色
参照Masson三色染色液试剂盒(Servicebio公司)说明书操作,步骤如下:
石蜡切片脱蜡至水;切片浸入A液中浸泡过夜;于65℃烤箱中加热30min,同时将D液和F液放于65℃烤箱内预热;自来水洗1min;B液和C液等量混合,浸染切片1min,流水稍洗;1%盐酸酒精分化30s;流水稍洗,D液浸染8min;将多余水分沥干,E液浸染1min;不水洗,沥干多余E液,直接加入F液染色30s;1%冰醋酸分化3次,每次约 8s;无水乙醇、二甲苯脱水透明;中性树脂封片,风干过夜。
(10)统计分析:
使用均值±SEM表示数据。多组间比较用方差分析(AN0VA),两组间数据比较用T检验。p<0.05为具有统计学意义;相对于Sham组或Ctrl组,p<0.05标记为*;p<0.01标记为**;p<0.001标记为***;相对于UUO组或TGF-β1组或Cytokines组,p<0.05标记为 #;p<0.01标记为##;p<0.001标记为###。
下面结合具体实施例详细阐述本发明:
实施例1
3-苯基戊二酸衍生物类LonP1激活剂对LonP1的蛋白酶活性激动效应:
在无细胞酶解反应体系中,LonP1(800nM)与1μM浓度的84-B10及MQ2,MQ4-7 在反应缓冲液预孵育30min,随后加入(Z-AA)2-Rh110(6μM)和ATP(2mM)于37℃反应1h。(Z-AA)2-Rh110经LonP1酶解后可释放出具有红色荧光的Rh110,因此可以通过检测红色荧光(497nm/527nm)强度,来检测LonP1酶活性。
结果显示,3-苯基戊二酸衍生物84-B10及84-B10及MQ2,MQ4-7相较溶剂组(Ctrl)均可显著激活LONP1的蛋白酶活性(图1A-B),其中84-B10为优选的LonP1激活剂。
实施例2
优选的3-苯基戊二酸衍生物类LonP1激活剂84-B10对细胞LonP1表达的影响
在体外培养的小鼠肾小管上皮细胞(mPTC)中,使用5,10,20μM的84-B10处理细胞24h,Western Blot法检测LonP1蛋白酶的表达水平。
结果显示,5和10μM的84-B10对LonP1的表达水平没有影响,而20μM的84-B10 可明显上调LonP1的表达水平(图2)。说明84-B10不仅可以激动LonP1的蛋白酶活性,还可以在较高浓度下增加细胞内LonP1的蛋白表达水平。
实施例3
治疗剂量的84-B10对Sham及UUO小鼠无心、肝、肾毒副作用:
小鼠随机分为Sham组、UUO组、低剂量84-B10给药组(UUO+LD)及高剂量84-B10 给药组(UUO+HD组),其中UUO+LD组小鼠84-B10的给药剂量为0.5mg/kg/d,UUO+HD 组小鼠84-B10的给药剂量为7.5mg/kg/d,给药方式为腹腔注射。在UUO手术前1天及手术前2h预给药2次,构建UUO模型,之后每天定时给药1次,Sham组和UUO组注射给予同样体积的溶媒,小鼠于UUO造模1周后处死并收集各组别的小鼠全血样本于EDTA 抗凝管中,室温离心机4000rpm,15min,移取血清样本,测定肾功能标志物尿素氮(blood urea nitrogen,BUN)、血肌酐(serum creatinine,sCr),肝功能标志物谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST),和心功能标志物乳酸脱氢酶(lactatedehydrogenase,LDH)、肌酸激脢(creatine kinase-muscle/brain,CK-MB)水平。
实验结果如下:
Sham组、UUO组和低剂量84-B10给药组(UUO+LD)及高剂量84-B10给药组(UUO+HD组)小鼠的肾功能标志物BUN(图3A)和sCr(图3B)、肝功能标志物ALT (图3C)和AST(图3D)、心功能标志物LDH(图3E)和CK-MB(图3F)水平无明显差异,说明上述剂量对Sham及UUO小鼠的肾脏、肝脏和心脏均无毒副作用。
实施例4
治疗剂量的84-B10可改善UUO小鼠肾脏纤维化:
Sham组、UUO组、低剂量84-B10给药组(UUO+LD)及高剂量84-B10给药组(UUO+HD组)小鼠同实施例3造模与给药方法,末次给药结束后处死小鼠,取Sham组左侧肾组织,以及UUO组和给药组患侧肾组织,4%多聚甲醛1mL室温固定组织24h,石蜡包埋切片,进行Masson染色。
实验结果:
Masson染色后,肾脏组织胶原纤维呈蓝色,而肌纤维呈红色。光镜下观察,UUO小鼠与Sham组相比其肾小管间质出现明显的胶原沉积(蓝色),给药组(UUO+LD及 UUO+HD组)小鼠肾小管间质胶原沉积较UUO组明显减轻(图4A)。定量结果显示, 84-B10给药可剂量依赖性地减少肾脏组织纤维化面积,其差异均具有统计学意义(图4B)。
实施例5
治疗剂量的84-B10可显著降低UUO小鼠肾脏组织纤维化指标的蛋白与mRNA水平:
Sham组、UUO组、低剂量84-B10给药组(UUO+LD)及高剂量84-B10给药组(UUO+HD组)小鼠同实施例3造模与给药方法,末次给药结束后处死小鼠,取Sham组左侧肾组织,以及UUO组和给药组患侧肾组织,各组分别取适量肾组织,采用Western Blot及RT-PCR 法分别检测各组纤维化指标的蛋白(α-SMA、Vimentin、FN1、collagen I)及mRNA(α-SMA、Vimentin、FN1、collagen III)水平。
实验结果
UUO小鼠的肾组织中,α-SMA、Vimentin、FN1、collagen I的蛋白表达较Sham组均明显升高,其差异有统计学意义,表明纤维化程度较重,而低剂量与高剂量84-B10均可降低纤维化相关指标的高表达,除低UUO+LD组collagen I表达水平以外,其余蛋白灰度值差异均有统计学意义(图5A-B)。
同样地,与Sham组相比,UUO小鼠的肾组织中纤维化指标α-SMA、Vimentin、FN1、collagen III的mRNA表达明显升高,其差异有统计学意义,而低剂量与高剂量84-B10均可显著降低各纤维化指标的高表达,其差异有统计学意义(图5C-F)。
因此,本部分结果与实施例3、4共同说明了,治疗剂量的84-B10可减轻UUO导致的肾脏纤维化。
实施例6
84-B10剂量依赖性地抑制TGF-β1诱导的肾小管上皮细胞-间充质转分化:
为研究84-B10对肾小管上皮细胞转分化的影响,本实验采用TGF-β1在体外诱导小鼠肾小管上皮细胞(mPTC)转分化,模拟CKD过程中的肾小管上皮细胞的转分化。将mPTC 在无血清培养基中加入终浓度分别为2.5μM、5μM和10μM的84-B10预处理2h,然后加入终浓度为10ng/mL的TGF-β1,24h后收集细胞提取总蛋白,Western blot法检测肾小管上皮细胞转分化指标(α-SMA、vimentin、FN1、collagen III)的蛋白表达水平。
实验结果:
在TGF-β1诱导下,mPTC向间充质转分化,表现为α-SMA、vimentin、FN1、collagenIII的蛋白表达水平明显升高,而84-B10可剂量依赖性地改善转分化指标蛋白的高表达,其中10μM的84-B10处理组与TGF-β1组的4个指标蛋白表达差异均有统计学意义,表现出较好的抗肾小管上皮细胞转分化作用(图6A-E)。
实施例7
84-B10剂量依赖性地抑制TGF-β1诱导的肾间质成纤维细胞的活化与基质生成:
为研究84-B10对肾间质成纤维细胞活化与基质生成的影响,本实验采用TGF-β1在体外诱导大鼠肾间质成纤维细胞(NRK-49F),以模拟CKD过程中的肾间质成纤维细胞的活化。将NRK-49F在无血清培养基中加入终浓度分别为2.5μM、5μM和10μM的84-B10 预处理2h,然后加入终浓度为5ng/mL的TGF-β1,24h后收集细胞提取总蛋白,Western blot法检测肾间质成纤维细胞活化指标α-SMA和细胞外基质成份FN1的蛋白表达水平。
实验结果:
在TGF-β1诱导下,NRK-49F发生活化且基质生成增加,表现为α-SMA、FN1的蛋白表达水平明显升高,而84-B10可剂量依赖性地改善转分化指标蛋白的高表达,其中10μM 的84-B10处理组与TGF-β1组的2个指标蛋白表达差异均有统计学意义,表现出较好的抗肾间质成纤维细胞活化作用(图7A-C)。
实施例8
84-B10减轻细胞因子混合物导致的足细胞损伤
为研究84-B10对人足细胞(HPC)损伤的影响,本实验采用细胞因子混合物(IL-1β,TNF-α,IFN-α,IFN-γ)在体外作用于分化成熟的足细胞,以模拟CKD过程中复杂的炎症微环境对足细胞的损伤作用。将HPC在无血清培养基中加入终浓度分别为2.5μM和5μM 的84-B10预处理2h,然后加入细胞因子混合物(Cytokines)(IL-1β,TNF-α,IFN-α,IFN-γ,浓度均为10ng/mL),24h后收集细胞提取总蛋白,Western blot法检测足细胞特异性标志物Podocin和WT-1的蛋白表达水平。
实验结果:
在细胞因子混合物(Cytokines)作用下,足细胞受到严重损伤,表现为特异性标志物 Podocin、WT-1蛋白表达水平均显著下调。其中Podocin位于肾小球裂孔膜,对维持肾小球的滤过屏障十分重要。84-B10可显著地恢复Podocin、WT-1蛋白表达,改善足细胞结构损伤,表现出较好的足细胞保护作用(图8A-C)。
因此,实施例6、7、8共同说明了,治疗剂量的84-B10可改善慢性肾脏病病理环境下3种重要的参与细胞的表型:抑制TGF-β1诱导的肾小管上皮细胞-间充质转分化、抑制肾间质成纤维细胞的活化与基质生成、减轻足细胞损伤。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的仅为发明的优选例,并不用来限制本发明,在不脱离本发明新型精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
3.一种3-苯基戊二酸衍生物类LonP1激活剂在慢性肾脏病防治药物中的应用,其特征在于,将权利要求1或权利要求2中所述3-苯基戊二酸衍生物类LonP1激活剂用于制备防治各种原因引起的慢性肾脏病药物。
4.根据权利要求3所述的3-苯基戊二酸衍生物类LonP1激活剂在慢性肾脏病防治药物中的应用,其特征在于:所述3-苯基戊二酸衍生物类LonP1激活剂用于制备抑制肾小管上皮细胞转分化的药物。
5.根据权利要求3所述的3-苯基戊二酸衍生物类LonP1激活剂在慢性肾脏病防治药物中的应用,其特征在于:所述3-苯基戊二酸衍生物类LonP1激活剂用于制备抑制肾间质成纤维细胞活化的药物。
6.根据权利要求3所述的3-苯基戊二酸衍生物类LonP1激活剂在慢性肾脏病防治药物中的应用,其特征在于:所述3-苯基戊二酸衍生物类LonP1激活剂用于制备减轻足细胞损伤的药物。
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