CN109394748A - Tppu在治疗制备肥胖所致肾损伤药物中的应用 - Google Patents
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Abstract
本发明旨在提供化合物sEH抑制剂(TPPU)在制备治疗肥胖所致肾损伤药物中的作用,为肥胖导致的肾损伤提供新的候选化合物。实验结果表明,在肥胖所致肾损伤过程中Ampk和Pax2表达降低,而可溶性表氧水解酶(sEH)的表达和活性均升高。进而发现,TPPU显著缓解肥胖所致肾损伤,增加肾脏中环氧二十碳三烯酸(EETs)的浓度,并增高Ampk和Pax2在肾脏中的表达。进一步发现EETs可增加小鼠肾脏系膜细胞(mRMCs)Ampk和Pax2的表达,缓解脂肪酸对mRMCs造成的损伤。
Description
技术领域
本发明涉及TPPU在药物领域的应用,具体涉及其在制备治疗肥胖所致肾损伤药物中的应用。
背景技术
高脂饮食是现代西方饮食的重要组成部分,在我国也有越来越多的人偏好高脂饮食。高脂饮食易导致肥胖,脂肪肝,和肾损伤等疾病发生。而肾损伤进而可促进慢性肾脏病、糖尿病肾病以及其它疾病的发生与发展。因此探索肥胖所致肾损伤的防治方法具有很高的临床价值。
高脂饮食引起的肥胖所致肾损伤和巨自噬和伴侣介导的自噬(CMA)相关。5′-磷酸化腺苷酸活化蛋白激酶(AMPK)是巨自噬的重要介导因子,已有研究报道在高脂饮食引起的肥胖所致肾损伤过程中AMPK活性降低。成对盒基因2(PAX2)是CMA的重要调节因子,但其在肥胖所致肾损伤过程中的作用未见报道。
近来研究发现心脏组织和心肌细胞中的AMPK可被细胞色素P450 2J(CYP2J)以及其代谢产物环氧二十碳三烯酸(EETs)激活,并缓解心肌损伤。EETs不但是CYP2J的产物,还是可溶性表氧水解酶(sEH,其编码基因是EPHX2)的底物,sEH可催化EETs形成邻二羟基二十碳三烯酸(DHETs)。但是EETs和sEH对肾脏组织中的AMPK作用未见报导。EETs和sEH对PAX2的作用未见报导。另外,PAX2与AMPK间是否存在相关性也未见报道。
发明内容
本发明为治疗制备肥胖所致肾损伤的药物提供了新的化合物选择。
本发明提供了如下技术方案:
EETs在治疗制备肥胖所致肾损伤药物中的应用。
提高肾脏EETs浓度的药物可溶性环氧水解酶抑制剂在治疗制备肥胖所致肾损伤药物中的应用。
提高肾脏EETs浓度的药物CYP2C,2J激动剂在治疗制备肥胖所致肾损伤药物中的应用。
PAX2激动剂在治疗制备肥胖所致肾损伤药物中的应用。
采用动物和细胞模型,利用基于液相色谱联用的代谢组学技术、定量聚合酶链式反应(qPCR)、以及蛋白印迹技术,分析高脂饮食(HFD)或棕榈酸(PA)对小鼠肾脏和肾脏系膜细胞(mRMCs)的作用及其过程中Ampk、Pax2、和sEH、以及sEH调节的肾脏EETs和DHETs的变化。研究sEH抑制剂处理高脂饮食所致肥胖小鼠肾脏变化及其机制,并研究EETs对PA处理的mRMCs的作用与机制。并利用发卡RNA干扰技术和过表达构建质粒,研究Pax2和Ampk的可能关系。
如图1所示,HFD饮食饲喂的肥胖小鼠血浆肌酐显著升高(a),在mRNA水平上肾脏Mcp-1显著升高(b),标志肥胖导致小鼠肾损伤。同时肥胖小鼠肾脏EETs浓度显著降低(c),在蛋白水平上,肾脏sEH表达显著增高(d),肾脏Ampk(e)和Pax2(f)表达显著降低。当用sEH抑制剂TPPU同时饲喂肥胖小鼠时,能显著逆转由肥胖引起的肾损伤(a,b),以及肾脏EETs浓度(c),以及sEH(d)、Ampk(e)、和Pax2(f)表达的变化。说明增高sEH抑制剂TPPU能增高肾脏EETs浓度,重新激活Ampk和Pax2,防治肥胖所致肾损伤。
棕榈酸(PA)处理小鼠肾脏系膜细胞(mRMCs)是常用的高脂诱导肥胖所致肾损伤的体外模型。如图2所示,PA处理可引起mRMCs上Mcp-1表达显著增高(a),表明细胞受到损伤;同时显著降低Ampk(b)和Pax2(c)的表达。结果与小鼠实验结果一致。而利用14(15)EET处理PA处理的mRMCs,可以显著降低PA对mRMCs的损伤(a),并重新激活Ampk(b)和Pax2(c)。小鼠模型和细胞模型的实验均表明:增加EETs、激活Ampk和Pax2均可以防治肥胖所致肾脏损伤。
附图说明
图1 TPPU对高脂饮食导致的肥胖引起的肾损伤的防治作用。高脂饮食导致的肥胖引起的血肌酐(a)肾脏Mcp-1(b)显著升高,表明肾功能损伤;高脂饮食导致的肥胖引起肾脏EET显著降低(c),sEH表达显著升高(d),p-Ampkα(e),和Pax2(f)显著降低,而sEH抑制剂TPPU可以显著降低肥胖引起的肾损伤(a)和(b),显著降低肥胖引起的sEH升高(d),并显著逆转肥胖引起的EETs(c)、p-Ampkα(e)、和Pax2(f)降低。*0.01≤P<0.05,**0.001≤P<0.01,***0.0001≤P<0.001,****P<0.0001系经单因素方差检验并经Tukey’s或Games-Howell组间比较检验而得。
图2 14(15)-EET防治棕榈酸(PA)导致的小鼠肾脏系膜细胞(mRMCs)损伤。PA处理引起mRMCs Mcp-1显著升高(a),表明细胞功能损伤,同时导致Ampk(b)和Pax2(c)表达降低。而14(15)-EET能显著降低PA导致的Mcp-1升高,防治PA引起的细胞损伤(a);并同时显著增加Ampk(b)和Pax2(c)的表达。*0.01≤P<0.05,***0.0001≤P<0.001,****P<0.0001系经单因素方差检验并经Tukey’s或Games-Howell组间比较检验而得。
具体实施方式
所有动物实验均按照同济大学医学院上海第十人民医院动物使用与护理委员会批准的方案进行。雄性C57BL/6小鼠(7周龄)购自上海实验动物研究中心。实验小鼠置于在温度恒定的动物房内,房间设有12小时的亮暗周期,可以自由地获得食物和水。利用高脂饮食HFD(60kcal%脂肪,D12492J)饲喂小鼠构建肥胖模型,正常饮食(CTD,10kcal%脂肪,D12450J)作为对照。HFD和CTD均购自美国饮食研究公司(New Brunswick,NJ)。
小鼠被随机分为三组(N=10)。第1组小鼠喂食CTD作为正常对照,第2组小鼠喂食HFD构建肥胖模型,第3组小鼠喂食HFD并于饮用水中加入10mg/L TPPU研究药物疗效。经过8周高脂喂养后,处死小鼠分析样本。每只小鼠在处死前先经腹腔注射0.35毫升3.5%水合氯醛进行麻醉。小鼠的血液采集和血浆分离方法参照参考文献(Liu JY,et al.(2009)Pharmacokinetic optimization of four soluble epoxide hydrolase inhibitors foruse in a murine model of inflammation.Brit J Pharmacol 156(2):284-296.)。收集小鼠双肾,将左肾及1/2右肾置液氮中速冻,用于蛋白质免疫印迹和EETs浓度检测。在分析检测前,小鼠的肾脏和血浆存于-80摄氏度以下的深低温冰箱中保存备用。
用LC-MS/MS测量血浆肌酐水平,检测方法参照参考文献(Liu XY,Luo Y,Zhou CY,Peng A,&Liu JY(2017)A sensitive and accurate method to simultaneously measureuric acid and creatinine in human saliva by using LC-MS/MS.Bioanalysis 9(22):1751-1760.)。
肾脏EETs浓度的检测
根据Wang等人的报道(Wang W,et al.(2018)Lipidomic profiling revealssoluble epoxide hydrolase as a therapeutic target of obesity-induced colonicinflammation.Proc Natl Acad Sci U S A 115(20):5283-5288),EETs是从肾皮质中提取的。根据前面报道的分析方法(Luo Y,Wang L,Peng A,&lIU JY(2018)Metabolicprofiling of human plasma reveals the activation of 5-lipoxygenase in theacute attack of gouty arthritis.Rheumatology(Epub ahead of print,RHE-18-0568.R2).),使用安捷伦1260与AB Sciex QTrap 6500进行肾脏EETs的定量分析。
细胞培养实验
小鼠肾小球系膜细胞使用特殊细胞培养基培养(71.25%基础培养基DMEM+23.75%F-12培养基+5%胎牛血清+14mM HEPES+1%青霉素-链霉素),置于10cm2的组织培养瓶中,在5%CO2,37℃恒温恒湿的细胞培养箱中培养。细胞生长达到80%融合度后,用对照试剂,棕榈酸或14(15)-EET处理。棕榈酸和14(15)-EET的浓度以及细胞培养时间见图2。终浓度0.6%的二甲亚砜和终浓度0.32%的乙醇分别为对照和溶剂对照。收集细胞后用于定量聚合酶链式反应。所有实验均重复三次,见图2。
定量聚合酶链式反应
根据试剂说明书,对小鼠肾皮质组织和收获的小鼠肾小球系膜细胞进行定量聚合酶链式反应分析。组织和细胞样本制备方法参照参考文献(5)进行。目的基因引物序列如下:
Gapdh(上游引物:5′-AGGTCGGTGTGAACGGATTTG-3′;下游引物:5′-TGTAGACCATGTAGTTGAGGTCA-3′);
Mcp-1(上游引物:5′-CTTCTGGGCCTGCTGTTCA-3′;下游引物:5′-CCAGCCTACTCATTGGGATCA-3′)
蛋白质免疫印迹
根据试剂说明书,对小鼠肾皮质组织进行p-Ampkα,Pax2,sEH,和Gaphd进行蛋白质免疫印迹分析,。采用软件Image J(Java中的图像处理和分析)对蛋白质免疫印迹结果进行灰度测量。用Gapdh对蛋白条带光学灰度进行均一化处理。
实验结果显示,如图1所示,HFD饮食饲喂的肥胖小鼠血浆肌酐显著升高(a),在mRNA水平上肾脏Mcp-1显著升高(b),标志肥胖导致小鼠肾损伤。同时肥胖小鼠肾脏EETs浓度显著降低(c),在蛋白水平上,肾脏sEH表达显著增高(d),肾脏Ampk(e)和Pax2(f)表达显著降低。当用sEH抑制剂TPPU同时饲喂肥胖小鼠时,能显著逆转由肥胖引起的肾损伤(a,b),以及肾脏EETs浓度(c),以及sEH(d)、Ampk(e)、和Pax2(f)表达的变化。说明增高sEH抑制剂TPPU能增高肾脏EETs浓度,重新激活Ampk和Pax2,缓解肥胖所致肾损伤。
棕榈酸(PA)处理小鼠肾脏系膜细胞(mRMCs)是常用的高脂诱导肥胖所致肾损伤的体外模型。如图2所示,PA处理可引起mRMCs上Mcp-1表达显著增高(a),表明细胞受到损伤;同时显著降低Ampk(b)和Pax2(c)的表达。结果与小鼠实验结果一致。而利用14(15)EET处理PA处理的mRMCs,可以显著降低PA对mRMCs的损伤(a),并重新激活Ampk(b)和Pax2(c)。小鼠模型和细胞模型的实验均表明:增加EETs、激活Ampk和Pax2均可以防治肥胖所致肾脏损伤。
Claims (4)
1.EETs在治疗制备肥胖所致肾损伤药物中的应用。
2.提高肾脏EETs浓度的药物可溶性环氧水解酶抑制剂在制备治疗肥胖所致肾损伤药物中的应用。
3.提高肾脏EETs浓度的药物CYP2C,2J激动剂在制备治疗肥胖所致肾损伤药物中的应用。
4.PAX2激动剂在制备治疗肥胖所致肾损伤药物中的应用。
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Title |
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ANNIKA I. OSTERMANN, ET AL.: "Oral treatment of rodents with soluble epoxide hydrolase inhibitor 1-trifluoromethoxyphenyl -3 -(1-propionylpiperidin-4-yl) urea (TPPU): resulting drug levels and modulation of oxylipin pattern", 《PROSTAGLANDINS OTHER LIPID MEDIAT》 * |
BING-QING DENG, ET AL.: "Epoxide metabolites of arachidonate and docosahexaenoate function conversely in acute kidney injury involved in GSK3β signaling", 《PROC NATL ACAD SCI U S A》 * |
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