CN113384684A - Bmp2沉默改善肾草酸钙结石损伤的机制的应用 - Google Patents
Bmp2沉默改善肾草酸钙结石损伤的机制的应用 Download PDFInfo
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Abstract
本发明涉及生物医药领域，公开了BMP2沉默改善肾草酸钙结石损伤的机制的应用，所述机制如下：BMP2沉默通过抑制氧化应激反应来降低CaOx晶体沉积和肾细胞损伤。本发明证实BMP2沉默可减轻草酸钠诱导的细胞氧化应激和结晶，同时可抑制Smad1的磷酸化和NOX2和NOX4的表达，本发明证实BMP2沉默通过抑制氧化应激反应来降低CaOx晶体沉积和肾细胞损伤，表明BMP2沉默可改善肾小管上皮细胞氧化应激损伤，为草酸钙结石的发病机制研究指明了新的方向。
Description
技术领域
本发明涉及生物医药领域，具体涉及BMP2沉默改善肾草酸钙结石损伤的机制的应用。
背景技术
肾结石是泌尿外科常见病之一，严重威胁人类健康，肾结石是由肾盏和肾盂的矿物质沉积导致的。高尿酸血症是临床常见的代谢异常病，是草酸钙结石的主要危险因素，也是大多数结石的主要成分，其中高草酸尿是肾结石的重要诱因。报告显示，成人特发性尿钙增多症(idiopathichypercalciuria，IH)的发病率为5％～8％，IH患者泌尿系结石的发生率是正常人的5倍。虽然IH是肾结石的高危因素，但目前IH在肾结石形成中的作用和机制尚不清楚。肾上皮细胞损伤在肾结石的发生发展中起着决定性的作用，肾上皮细胞暴露于草酸和/或草酸钙(CaOx)晶体下会导致活性氧(ROS)产生，ROS可对核苷酸、脂类、蛋白质和碳水化合物产生损伤，并诱导氧化应激(OS)的产生，进而引起损伤和炎症。据报道，SD大鼠尿石症常出现OS升高，因此，防止ROS的产生和积累可为肾脏损伤/疾病的潜在治疗提供新的策略。
骨形态发生蛋白(bonemorphogeneticproteins，BMPs)与成骨分化和骨形成有关，骨形态发生蛋白2(BMP2)信号通路调控的异位钙化是多种结石的主要诱发因素，BMP2、runt相关转录因子2(RUNX2)、肌段同源框2(MSX2)和锌指结构转录因子(os-terix，Osx)是BMP2信号通路中的重要转录因子，在钙化血管平滑肌细胞(VSMCs)中高表达。因此，本发明采用乙二醇治疗的高钙尿症模型大鼠和草酸钠诱导的NRK-52E细胞模型，探讨BMP2信号通路在肾结石和OS形成中的调控机制。
发明内容
基于以上问题，本发明提供BMP2沉默改善肾草酸钙结石损伤的机制的应用，本发明为草酸钙结石的发病机制研究指明了新的方向。
为解决以上技术问题，本发明提供了BMP2沉默改善肾草酸钙结石损伤的机制的应用，所述机制如下：BMP2沉默通过抑制氧化应激反应来降低CaOx晶体沉积和肾细胞损伤。
进一步的，所述机制可应用于制备用于降低CaOx晶体沉积和肾细胞损伤的BMP2抑制剂中。
与现有技术相比，本发明的有益效果是：本发明证实BMP2沉默可减轻草酸钠诱导的细胞氧化应激和结晶，同时可抑制Smad1的磷酸化和NOX2和NOX4的表达，本发明证实BMP2沉默通过抑制氧化应激反应来降低CaOx晶体沉积和肾细胞损伤，表明BMP2沉默可改善肾小管上皮细胞氧化应激损伤，为草酸钙结石的发病机制研究指明了新的方向。
附图说明
图1为本发明的实施例测定的不同浓度草酸钠对CaOx晶体沉积和细胞氧化应激的影响结果图；
图2为本发明的实施例的草酸钠对NRK-52E细胞损伤及BMP2信号转导的影响结果图；
图3为本发明的实施例的BMP2沉默减轻草酸钠诱导的NRK-52E细胞损伤结果图；
图4为本发明的实施例的BMP2沉默抑制草酸钠诱导的NRK-52E细胞Smad1信号通路的激活和NOX2/NOX4的表达结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例和附图，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例：
本实施例准备了如下材料：SD雄性大鼠30只(SPF级，6周)，购自四川成都大硕动物实验有限公司，在四川大学华西动物实验中心饲养；饲养环境为25±1℃，相对湿度50％～60％，光/暗循环12h，SD大鼠可以自由进食和饮水，所有实验均经四川大学华西医院伦理委员会批准。
建立高草酸尿大鼠模型：将SD大鼠随机分为5组(n＝6)，即对照组，每只大鼠每天自由饮水70ml；0.1％乙二醇组；0.5％乙二醇组；1％乙二醇组；1.5％乙二醇组，乙二醇诱导组大鼠每天自由饮水1％(v/v)乙二醇溶液70ml，浓度分别为0.1％、0.5％、1％、1.5％；正常喂养4周，最后，用1％戊巴比妥钠(50mg/kg)麻醉大鼠并实施安乐死，移除肾组织并将其保存在-80℃中以供后续分析。
组织学和免疫组化染色：大鼠肾组织用4％多聚甲醛固定24h，石蜡包埋，用苏木精-伊红(H&E)染色对左肾进行组织病理学观察；为了评估晶体沉积，根据说明书在4μm肾脏上测量Von Kossa(中国北京Solarbio)染色，光镜下观察组织损伤和晶体沉积。
尿液化学检验：为了收集尿液，在研究结束时，大鼠被安置在代谢笼中进行尿液收集，用离子色谱法通过Thermo ScientificTM DionexTM ICS-6000HPIC TM系统(Dionex公司，加利福尼亚州桑尼维尔)检测草酸盐。
大鼠肾组织ROS活性的免疫荧光研究：采用二氢乙啶(DHE)染色法检测肾组织中ROS的含量，在荧光显微镜BX53(日本东京奥林巴斯)下观察细胞的红色发射图像，并用绿光激发拍摄，ROS阳性细胞呈红色。
免疫组化染色：4μm石蜡切片，IHC染色检测草酸钠肾结石模型大鼠肾组织蛋白表达，根据IHC方案的说明评估BMP2蛋白表达水平。
细胞培养：肾小管上皮细胞系(NRK-52E)购自上海细胞库(中国上海)，在37℃、5％CO2的加湿培养箱中，将细胞维持在添加10％胎牛血清(FBS；Gibco)的DMEM培养基中；根据制造商的说明，使用Lipofectamine 3000转染试剂将NC siRNA或BMP2 siRNA瞬时转染至NRK-52E细胞(1.0×105细胞/孔)，然后，将细胞与700μM草酸钠孵育24小时后用于BMP2表达分析或其他实验。
CCK-8测定：根据制造商的说明，使用细胞计数试剂盒8(CCK-8，Thermo FisherScientific)测量NRK-52E细胞的细胞活力，在450nm处记录吸光度。
流式细胞计数法：根据制造商的方案，使用膜联蛋白V-分析NRK-52E细胞的凋亡，简单地说，用PBS(Invitrogen，Carlsbad，CA，USA)洗涤细胞，并将细胞浓度调整为1.0×106cells/ml；随后将细胞悬浮在150μl缓冲溶液中。随后，用10μg/mlAnnexinV-FITC和5μlPI在4℃黑暗中染色20分钟。用BD-facselesta分析凋亡细胞TM流式细胞仪(Becton，Dickinson and Company)；同时，用草酸钠(700μM)处理NRK-52E细胞24h，转染或不转染NC-siRNA或BMP2-siRNA，流式细胞仪测定ROS含量。
蛋白质RNA水平的测定：使用
试剂(美国马萨诸塞州赛默飞世尔)分离总RNA，用逆转录试剂盒(Invitrogen，Carlsbad，CA，US)获得cDNA，采用SYBR-Pemix-Ex-Taq试剂盒(中国大连宝生物工程有限公司)进行qRT-PCR，获得目的基因RNA转录组的相对水平；逆转录反应条件如下：95℃30s，40个周期95℃5s，60℃30s；在加利福尼亚州福斯特市ABI软件上采用
法测定相对基因表达水平。
蛋白质印迹分析：使用RIPA缓冲液(信号技术公司)制备整个NRK-52E细胞裂解液，蛋白浓度由BCA试剂盒(Sigma-Aldrich；Merck-KGaA)测定，总蛋白(30μg/样品)通过10％SDS-PAGE分离，然后分离的蛋白质转移到硝化纤维素膜上，在4℃下用5％脱脂奶粉隔夜封闭膜，并用以下相应的蛋白抗体孵育：BMP2(Abcam，ab214821；1/1000)、磷(p)-Smad1(Abcam，ab226821；1/1000)、Smad1(Abcam，ab126761；1/1000)、NOX2(Abcam，ab129068；1/5000)、NOX4(Abcam，AB13303；1/1000)和β-肌动蛋白(Boster，BM0627；1/1000)；然后，用Tris缓冲盐水/0.1％吐温(TBST)清洗膜，并用HRP山羊抗兔IgG(Abcam，ab6721)孵育1.5小时；使用ECL系统(Affinity Biosciences，Cincinnati，Ohio，USA)显示条带，并使用β-肌动蛋白作为内部对照，使用Quantity One软件(Bio-Rad)测量净光密度。
酶联免疫吸附试验(ELISA)：用草酸钠对NRK52E细胞进行ELISA检测，按照制造商的说明，用ELISA试剂盒(日本Takara)测定NRK-52E细胞培养上清中MDA的含量，在450nm波长处测量吸光度，并使用酶联免疫监测仪(美国赛默飞世尔科学公司)进行估计，根据标准曲线计算样品中丙二醛的浓度。
见附图1，为测定的不同浓度草酸钠对CaOx晶体沉积和细胞氧化应激的影响结果图，其中草酸钠诱导肾内氧化钙晶体沉积(A)，给予乙二醇(0.1％、0.5％、1％和1.5％)后的H&E染色肾切片，200×放大倍率(B和C)，代表性Von Kossa染色显示CaOx晶体沉积并定量，200×放大倍数(D)，离子色谱法测定尿中草酸含量(E)，用DHE染色法测定ROS含量，并进行定量分析(F)，IHC染色检测肾组织BMP2的表达*与对照组相比，P<0.05和**P<0.01。图1A所示，肾组织中小管直径明显扩张，并观察到肾小管和肾小球损伤，同时高浓度乙二醇引起的肾脏损害最为明显。图1B所示，在乙二醇组的尿液中观察到草酸的浓度依赖性显著释放。见图1C和1D，Von Kossa染色显示，与生理盐水处理组相比，乙二醇浓度依赖性地诱导晶体形成，可见于皮质区和髓质区。见图1E，经乙二醇处理后，肾组织中的ROS含量呈剂量依赖性显著增加。发明人试图研究BMP2在肾组织中的表达水平，见图1F，IHC数据显示，在乙二醇诱导的肾组织中，BMP2表达显著上调。
发明人用不同浓度的草酸钠处理NRK-52E细胞24小时，然后通过CCK-8测定和流式细胞术分析进行评估，见附图2，为本实施例的草酸钠对NRK-52E细胞损伤及BMP2信号转导的影响结果图，其中草酸钠刺激的NRK-52E细胞损伤和BMP2表达(A)，CCK-8法检测细胞活力(B和C)，采用流式细胞术分析并量化ROS含量(D)，用光学显微镜观察了草酸晶体的形成(E)，通过RT-qPCR测定BMP2、MSX2、RUNX2和OSX的水平(F)，westernblot检测BMP2的表达*与对照组相比，P<0.05和**P<0.01。见图2A、2B和2C，与对照组相比，草酸钠处理的细胞活力以剂量依赖的方式逐渐降低，而草酸钠诱导的NRK-52E细胞中的ROS水平显著升高。同时，图2D表明，在草酸钠处理的细胞中，草酸晶体产量以浓度依赖的方式增加。此外，使用RT-qPCR和Westernblot分析，发明人量化了与BMP2信号通路(BMP2、Msx2、Runx2和Osx)相关的基因表达，发明人发现在草酸钠诱导的NRK-52E细胞中BMP2、Msx2、Runx2和Osx的mRNA水平显著增加，具体见图2E；见图2F，BMP2蛋白表达水平也相应升高。
见附图3，为BMP2沉默减轻草酸钠诱导的NRK-52E细胞损伤结果图，其中BMP2沉默减轻了草酸钠诱导的NRK-52E细胞损伤(A)，RT-qPCR检测BMP2的RNA水平(B)，CCK-8法检测细胞活力(C)，用光学显微镜观察了草酸晶体的形成(D和E)，采用流式细胞术分析并量化ROS含量(F)，酶联免疫吸附法测定丙二醛含量(流式细胞仪检测细胞凋亡**与对照组比较P<0.01##P<0.01比700μM草酸钠+NC-siRNA组。发明人还设计了一种siRNA来抑制草酸钠诱导的NRK-52E细胞中BMP2的表达的实验，用BMP2 siRNA转染NRK-52E细胞24小时，并用700μM草酸钠处理24小时，具体见附图3A。见图3B，CCK-8分析表明，通过BMP2 siRNA转染，草酸钠诱导的细胞活力抑制显著逆转，图3C表明BMP2沉默阻断了草酸钠诱导的NRK-52E细胞中草酸晶体生成的增加。采用流式细胞术分析BMP2的疗效是否与OS有关，图3D和3E表明，草酸钠处理后活性氧含量增加，经BMP2 siRNA处理后，ROS含量显著抑制。此外，发明人还测定了草酸钠诱导的NRK-52E细胞上清液中MDA的释放，如图3F所示，草酸钠诱导组与对照组相比MDA水平显著升高，经BMP2 siRNA处理后MDA水平显著降低。见图3G和H，进一步的流式细胞术分析显示，草酸钠处理促进了NRK-52E细胞的凋亡，而BMP2 siRNA则逆转了这种作用。
见附图4，为BMP2沉默抑制草酸钠诱导的NRK-52E细胞Smad1信号通路的激活和NOX2/NOX4的表达结果图，其中BMP2沉默抑制草酸钠诱导的NRK-52E细胞中Smad1信号通路的激活和NOX2/NOX4的表达(A)，RT-qPCR检测MSX2、RUNX2和OSX的表达水平(B)，westernblot法检测Smad1、p-Smad1、NOX2和NOX4蛋白水平**与对照组比较P<0.01##P<0.01比700μM草酸钠+NC-siRNA组(C)，研究表明BMP2沉默在草酸钠诱导的NRK-52E细胞晶体粘附改变中起重要作用。然而，Smad1的激活仍然不清楚，它是BMP2的下游调节器，介导RUNX2的表达。因此，发明人还检测了MSX2、RUNX2和OSX的表达水平，并发现草酸钠促进NRK-52E细胞中MSX2、RUNX2、OSX和p-Smad1的表达可以通过BMP2沉默来阻止。由于NADPH氧化酶(NOX)是包括肾结石在内的肾脏疾病中OS的主要来源，我们通过westernblot分析检测BMP2沉默是否调节NOX2-NOX4的表达。结果表明，草酸钠处理显著增加了NRK-52E细胞中NOX2和NOX4的RNA和蛋白质表达，而BMP2沉默显著抑制了这些表达。
近年来的研究表明，结石形成的机制和因素异位钙化，与骨矿化过程有着相似的调控过程。结果表明，乙二醇诱导的大鼠尿石症肾组织中BMP2的表达水平显著升高，本实施例的结果提示乙二醇诱导的肾结石可能是由BMP2升高引起的，所以发明人在本实施例中通过在草酸钠诱导的NRK-52E细胞中敲除BMP2来验证这一假设，结果表明BMP2敲除降低了草酸晶体的产生。
在BMP2信号通路中，BMP2触发Smad蛋白，然后直接或通过MSX2、RUNX2和OSX反式激活成骨基因。BMP2通过调控Smad1/5/8信号通路促进MSX2和RUNX2的表达，其中RUNX2是OSX的上游调节因子，参与骨细胞的形成和分化。利用siRNA技术沉默小鼠颅骨前成骨细胞(MC3T3-E1.4)OSX基因的表达，结果表明矿化过程被阻断。本实施例的研究表明，在草酸钠诱导的NRK-52E细胞中，BMP2、MSX2、RUNX2和OSX的表达水平呈浓度依赖性增加，这被BMP2siRNA转染阻断。本实施例还发现草酸钠显著促进p-Smad1、MSX2、RUNX2和OSX的表达，这些都被BMP2沉默所逆转。
目前的研究发现，CaOx肾晶体的形成往往会导致OS的发生和上皮细胞的损伤，这是结石形成的关键环节。草酸(Ox)和/或CaOx晶体激活NADPH氧化酶(NOX)，从而诱导肾上皮细胞产生大量ROS。在SD大鼠高草酸尿模型中，NOX亚基p47phox的表达显著增加，OS在肾脏的进展加重。临床研究还发现，在高草酸尿或肾结石患者中，血清中的氧化应激水平上调，表现为丙二醛水平升高。其他研究也表明NOX衍生的ROS在草酸肾病的发病机制中起关键作用。这些报道与我们的结果一致，即在高草酸尿大鼠的肾脏中观察到NOX衍生的ROS积聚，并伴随着草酸钠诱导的肾小管上皮细胞中NOX2和NOX4表达的增加。此外，BMP2沉默进一步阻断了NOX2和NOX4的表达和ROS的局灶性积累。另一方面，BMP2的过度表达抑制了地塞米松诱导的MC3T3-E1细胞ROS的产生和TNF-α、IL-6和M-CSF的分泌。与先前的研究相反，本实施例的结果表明，BMP2的敲除不仅抑制草酸钠诱导的草酸结晶，而且抑制草酸钠诱导的NRK-52E细胞中ROS的产生。
综上所述，本实施例表明，乙二醇剂量依赖性地增加了SD大鼠肾脏CaOx晶体沉积，激活了BMP2信号通路；此外，敲除BMP2可通过抑制OS在体内外显著降低CaOx晶体沉积和肾细胞损伤；本实施例的研究成果为草酸钙结石的发病机制研究指明了新的方向，本实施例的研究成果可应用于制备用于降低CaOx晶体沉积和肾细胞损伤的BMP2抑制剂中。
如上即为本发明的实施例。上述实施例以及实施例中的具体参数仅是为了清楚表述发明验证过程，并非用以限制本发明的专利保护范围，本发明的专利保护范围仍然以其权利要求书为准，凡是运用本发明的说明书及附图内容所作的等同结构变化，同理均应包含在本发明的保护范围内。
Claims (2)
1.BMP2沉默改善肾草酸钙结石损伤的机制的应用，其特征在于，所述机制如下：BMP2沉默通过抑制氧化应激反应来降低CaOx晶体沉积和肾细胞损伤。
2.根据权利要求1所述的应用，其特征在于，所述机制可应用于制备用于降低CaOx晶体沉积和肾细胞损伤的BMP2抑制剂中。
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