贝前列素钠对高糖损伤大鼠系膜细胞的保护作用及其机制研究
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摘要
[背景]
糖尿病肾病(diabetic nephropathy, DN)是糖尿病(diabetes mellitus, DM)特有的微血管并发症之一,其特征性的病理改变包括系膜区细胞外基质(extracellular matrix, ECM)堆积、肾小球硬化及小管间质纤维化,最终导致进行性尿蛋白/白蛋白排泄的增加和肾功能的下降。DN的发病机制复杂,目前认为高血糖、肾血流动力学的异常、晚期糖基化终末产物(advanced glycation end products, AGEs)的增多、多元醇及蛋白激酶C (protein kinase C, PKC)途径的激活、氧化应激的产生等多种因素共同参与了DN的发生与发展。如何对糖尿病肾病进行有效地防治一直是临床关注的热点与难点。
肾小球系膜细胞(mesangial cell, MC)是肾小球最重要的固有细胞之一,对维持肾小球毛细血管床的完整性与系膜基质代谢平衡起到了重要的作用。在DN的发病中,各种病理因素直接作用于MC,引起MC肥大与过度增殖,系膜区胶原纤维(collagen)、纤维连接蛋白(fibronectin, FN)等ECM成分异常的聚集沉积,构成了DN肾小球硬化重要的病理基础。持续的细胞外高糖,以及高糖介导的各种病理因素还可以通过肾小球内皮细胞、足细胞、炎症细胞与MC相互作用,产生细胞因子与炎症介质,通过“自分泌”、“旁分泌”等方式影响MC,介导并促进了肾小球纤维化向肾小球硬化的进展。
在DN肾小球硬化的病理过程中,转化生长因子β1(transforming growth factor β1, TGFβ1)是重要的肾脏致纤维化的标志之一。MC能够分泌TGFβ1,并具有高亲和力的TGFβ1受体。因而TGFβ1能够通过“自分泌”、“旁分泌”的方式发挥其促进肾脏纤维化的作用。细胞外高糖以及高糖介导的各种病理因素均可作为促纤维化因素作用于MC促进TGFβ1的合成与分泌,激活TGFβ1下游Smads信号转导分子家族,促进ECM的产生并抑制其降解。
除了TGFβ1/Smads介导的DN肾纤维化经典途径外,丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)通路可以独立的或与TGFβ1/Smads通路相互作用,参与DN的发病。MAPK通路主要包括细胞外信号调控的蛋白激酶(Extracellular signal-regulated kinase mediates, ERK)、c-Jun N端激酶(c-jun amino-terminal kinase, JNK)/应激激活蛋白激酶(stress-activated protein kinase, SAPK)、p38MAPK等途径,是细胞将信号从细胞外传递到细胞内的主要通路。糖尿病高糖状态下,多元醇途径的激活、蛋白质非酶糖化,PKC途径的激活以及氧化应激水平的增加均可导致MAPK的激活,磷酸化下游转录因子,调节基因的表达,参与DN的发生发展。
糖尿病肾病的病理状态下,在MC过度增殖、系膜区ECM异常增多的同时,还伴有多种细胞因子和炎症介质分泌的增加,导致局部炎症反应的激活,加速了肾小球硬化的过程和肾病的进展。糖尿病本身是一个慢性炎症的过程并由此引起巨噬细胞在多个组织中聚集。这种巨噬细胞的聚集与糖尿病疾病状态、炎症的激活及DN肾损伤的进展关系密切。大量研究表明,单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)在糖尿病组织巨噬细胞浸润及炎症中起到了关键作用。
MCP-1是一种能被多种细胞表达的,对单核细胞、T淋巴细胞等具有趋化作用的分泌型蛋白。MCP-1与其受体结合后,诱导下游黏附分子的表达,使单核细胞／巨噬细胞等向病变部位聚集而发挥其病理作用。高糖培养系膜细胞能够通过PKC与氧化应激的激活促进MCP-1的表达。阻断MCP-1与其受体结合能减少糖尿病小鼠肾组织巨噬细胞的浸润及TGFβ1、Ⅳ胶原的表达,有效地阻止DN肾小球硬化的进展。
前列环素(prostacyclin, PGI2)是前列腺素(prostaglandin, PG)家族中的重要成员,主要由血管内皮细胞产生,具有强大的舒张血管平滑肌、扩张血管及抗血小板凝聚的作用。体内的细胞膜磷脂被磷脂酶A2水解为花生四烯酸,后者进一步在环氧合酶与前列环素合成酶的作用下逐步合成PGI2。PGI2合成后通过与两类受体结合发挥其生物学作用：1)与细胞膜上G蛋白偶联的膜受体IP结合,主要激活下游的环磷酸腺苷(cAMP)／蛋白激酶A (protein kinase A, PKA)途径发挥舒张血管、抗血小板凝聚及抗纤维化等作用；2)与细胞核过氧化物酶体增殖激活受体β/δ (peroxisome proliferator-activated receptor β/δ, PPARβ/δ)结合,通过PPAR特异的反应元件PPRE (PPAR responsive element)调控靶基因的转录。PGI2的性质十分不稳定,37℃时半衰期仅4min,很快转化为稳定的无活性的代谢产物6-酮-前列腺素。目前主要通过与PGI2有相似药理作用的人工合成的各种PGI2类似物来研究PGI2的作用机制。
贝前列素钠(beraprost sodium, BPS)将PGI2结构中的外烯醇醚结构替换为环戊烷苯并呋喃酮结构,体内半衰期延长至60min,口服途径给药仍保留了与PGI2相似的药理作用。体外研究表明,BPS通过增加系膜细胞内cAMP水平抑制机械迁张拉力对MAPK的激活从而抑制FN的表达,减轻肾小球内高压对MC的损伤作用；还能够抑制高糖诱导的MC的增殖、Ⅳ胶原的合成并提高MC的抗氧化能力。动物实验结果显示,BPS喂养OLETF大鼠后,其肾小球体积及硬化指数明显低于对照组,肾小球内AGEs的形成及巨噬细胞浸润明显减少,同时伴有尿蛋白排泄率及血清尿素氮的降低。在STZ诱导的DM大鼠中,BPS通过调节入球小动脉内皮细胞一氧化氮合酶活性抑制了DM状态下入球小动脉的扩张,减轻了肾小球的高滤过,同时还抑制了巨噬细胞的浸润。早期的临床研究发现,BPS能够抑制糖尿病患者血清血栓调节蛋白水平,通过减轻AngⅡ对出球小动脉的收缩作用、缓解肾小球的高滤过状态,降低糖尿病患者尿白蛋白的排泄。这些结果提示BPS具有一定的肾脏保护作用。因而,BPS作为一种可以口服的PGI2类似物,在糖尿病肾病的防治中具有广泛地应用前景,进一步研究BPS肾脏保护的作用机制,具有十分重要的临床意义。
因此在本研究中,我们探讨了PGI2类似物贝前列素钠(BPS)对高糖环境下大鼠系膜细胞株HBZY-1细胞ECM合成与降解的作用,对趋化因子MCP-1表达的影响,以及对细胞内信号通路的影响,以阐明贝前列素钠对肾脏保护作用的可能机制,为贝前列素钠用于糖尿病肾病的临床防治提供部分理论依据。
第一章贝前列素钠对高糖环境下大鼠系膜细胞ECM合成的影响
[目的]
1.观察不同浓度BPS对高糖环境下大鼠细胞系膜细胞株HBZY-1增殖活性的影响。
2.观察不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1、FN及MMP-2蛋白质表达的影响。
3.观察不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1、FN及MMP-2mRNA水平表达的影响。
4.探讨p38MAPK、ERK信号通路是否参与了BPS对高糖环境下HBZY-1细胞ECM合成的调节。
5.探讨BPS对高糖环境下HBZY-1细胞TGFβ1/Smad3信号通路的影响。
[方法]
1.细胞培养：采用含10%胎牛血清的DMEM培养基,于37℃、5%CO2培养箱内传代培养大鼠肾小球系膜细胞株HBZY-1。
2.细胞处理分组：正常葡萄糖浓度组(NG组,葡萄糖浓度5.6mmol/L),高葡萄糖浓度组(HG组,葡萄糖浓度30mmol/L), HG联合不同浓度的BPS组：BPS药物处理浓度分别为0.1umol/L(uM),0.5uM,1uM,2uM,5uM,10uM。
3.不同处理组(NG组,HG组,HG分别联合不同浓度的BPS组)体外培养HBZY-1细胞,于24h,48h用MTT法测定细胞增殖活性。
4.不同处理组体外培养HBZY-1细胞,于24h,48h收集细胞培养上清液,用Elisa法测定细胞培养上清液中TGFβ1、FN及MMP-2蛋白质的表达量。
5.不同处理组体外培养HBZY-1细胞,于24h收集细胞,用实时荧光定量(real-time) PCR法测定细胞TGFβ1、FN及MMP-2mRNA的相对表达量(2-△△Ct)。
6.应用免疫印迹法分别检测Omin,15min,30min,60min,90min不同时间点HG培养条件下HBZY-1细胞p38MAPK、ERK磷酸化蛋白的表达。
7.分别选取HG诱导HBZY-1细胞p38MAPK、ERK磷酸化蛋白表达最显著的时间点,应用免疫印迹法分别检测该时间点HG联合不同浓度BPS培养HBZY-1细胞p38MAPK、ERK磷酸化蛋白的表达。
8.应用免疫印迹法分别检测Omin,15min,30min,60min,90min不同时间点HG培养条件下HBZY-1细胞smad3磷酸化蛋白的表达。
9.选取HG诱导HBZY-1细胞smad3磷酸化蛋白表达最显著的时间点,应用免疫印迹法检测该时间点HG联合不同浓度BPS培养HBZY-1细胞smad3磷酸化蛋白的表达。
10.统计学处理：实验数据采用SPSS13.0软件进行统计学分析,各组计量资料数据采用均数±标准差(x±s)表示。采用析因方差分析方法进行主效应和交互效应的分析；多组均数比较采用单向方差分析(One-way ANOVA),多重比较采用LSD法(Least-significant difference test);方差不齐时,用Welch法校正,多重比较采用Dunnett's T3法；两独立样本比较采用t检验。P≤0.05被认为差异具有统计学意义。
[结果]
1.不同浓度BPS对高糖环境下HBZY-1细胞增殖活性的影响：
不同处理组分别培养24h,48h后各组细胞O.D.值均有显著差异(24h: F=5.977, P<0.001;48h:F=25.585, P<0.001)。与NG组相比,HG组培养24h及48h后HBZY-1细胞O.D.值均显著增加(P均<0.001)。HG联合不同浓度BPS培养细胞24h及48h,与HG组相比,除HG+0.1uMBPS组外,其余5个BPS浓度处理组O.D.值均显著低于HG组(24h:P=0.044;48h:P<0.001)。
根据本组实验的结果,选取0.5、1、2、5uM等4个浓度作为后续实验中BPS的处理浓度。
2.不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞TGFβ1蛋白表达量差异有统计学意义(24h:F=26.441, P<0.001;48h:F=14.939, P<0.001)。与NG组相比,HG组分别培养24h及48h后]HBZY-1细胞TGFβ1蛋白表达量均显著增加(P<0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞TGFβ1蛋白表达量均显著降低(P=0.001)；HG联合不同浓度BPS培养细胞48h后,与HG组相比,各浓度BPS组细胞TGFβ1蛋白表达量呈浓度依赖性下降(P=0.011)。
3.不同浓度BPS对高糖环境下HBZY-1细胞FN蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞FN蛋白表达量差异有统计学意义(24h:F=5.445, P=0.008;48h:F=10.736, P<0.001)。与NG组相比,HG组分别培养24h及48h后HBZY-1细胞FN蛋白表达量均显著增加(P=0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+2uMBPS组及HG+5uMBPS组细胞FN蛋白表达量均显著降低(P值分别为0.018,0.007)；HG联合不同浓度BPS培养细胞48h后,与HG组相比,HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞FN蛋白表达量均显著降低(P=0.005)。
4.不同浓度BPS对高糖环境下HBZY-1细胞MMP-2蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞MMP-2蛋白表达量差异有统计学意义(24h:F=7.232, P=0.002;48h:F=4.610, P=0.014)。与NG组相比,HG组分别培养HBZY-1细胞24h及48h后,细胞MMP-2蛋白表达量均显著下降(P=0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+2uMBPS组及HG+5uMBPS组细胞MMP-2蛋白表达量显著增加(P值分别为0.017,0.001)；HG联合不同浓度BPS培养细胞48h后,与HG组相比, HG+2uMBPS组及HG+5uMBPS组细胞MMP-2蛋白表达量显著增加(P值分别为0.019,0.007)。
5.不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1mRNA表达的影响：
与NG组相比,HG组细胞TGFβ1mRNA相对表达量明显增加(2.45±0.22vs.1.00±0.00,HG组均数的95%置信区间为[1.92,2.99])。与HG组相比,HG+0.5uMBPS组、HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞TGFβ1mRNA表达水平逐渐下降,差异具有统计学意义(P=0.008)。
6.不同浓度BPS对高糖环境下HBZY-1细胞FN mRNA表达的影响：
与NG组相比,HG组细胞FN mRNA表达相对量明显增加(1.93±0.23vs.1.00±0.00,HG组均数的95%置信区间为[1.36,2.51])。与HG组相比,HG+2uMBPS组及HG+5uMBPS组细胞FN mRNA相对表达量显著下降,差异具有统计学意义(P值分别为0.006,<0.001)。
7.不同浓度BPS对高糖环境下HBZY-1细胞MMP-2mRNA表达的影响：
与NG组相比,HG组细胞MMP-2mRNA表达相对量显著下降(0.528±0.132vs.1.000±0.000, HG组均数的95%置信区间为[0.199,0.857])。与HG组相比,HG联合不同浓度BPS组细胞MMP-2mRNA相对表达量有上升趋势。但统计学分析提示,HG组与HG联合不同BPS浓度组,各组细胞MMP-2mRNA相对表达量差异无统计学意义(F=0.906,P=0.497),可能与样本量偏小,或检测MMP-2mRNA时间点的选择有关。
8.不同时间点高糖诱导HBZY-1细胞p38MAPK磷酸化蛋白的表达：
HG Omin组细胞有少量磷酸化p38MAPK蛋白表达,HG刺激15min后细胞p38MAPK磷酸化蛋白表达最明显,刺激30min后p38MAPK磷酸化蛋白的表达开始减弱,刺激90min磷酸化p38MAPK蛋白信号仍未消失。
9.不同浓度BPS对高糖环境下HBZY-1细胞p38MAPK蛋白磷酸化的影响：
不同处理组刺激细胞15min后,HG组HBZY-1细胞p38MAPK磷酸化蛋白表达与NG组相比明显增多(P<0.001)；与HG组比较,HG联合不同浓度的BPS培养细胞,随着BPS药物浓度的增加,细胞p38MAPK磷酸化蛋白表达逐渐减少(P=0.006)。
10.不同时间点高糖诱导HBZY-1细胞ERK磷酸化蛋白的表达：
HG Omin细胞有少量磷酸化ERK蛋白表达,HG刺激15min后细胞ERK磷酸化蛋白表达明显增多,刺激30min后ERK磷酸化蛋白的表达开始减弱,刺激至90min磷酸化p38MAPK蛋白条带仍未消失。
11.不同浓度BPS对高糖环境中HBZY-1细胞ERK蛋白磷酸化的影响：
HG刺激15min后,HG组HBZY-1细胞ERK磷酸化蛋白表达比NG组明显增多(P<0.001)；与HG组比较,HG联合不同浓度的BPS培养细胞,随着BPS药物浓度的增加,细胞ERK磷酸化蛋白表达水平逐渐减少(P<0.001)。
12.不同时间点高糖诱导HBZY-1细胞Smad3蛋白磷酸化的表达：
HG Omin及HG15min组未能检测到细胞Smad3磷酸化蛋白的表达；HG30min组Smad3磷酸化蛋白开始表达,HG刺激60min时Smad3磷酸化蛋白表达最明显,刺激90min时明显减弱。
13.不同浓度BPS对高糖环境中HBZY-1细胞Smad3蛋白磷酸化的影响
HG刺激60min后,HG组HBZY-1细胞Smad3磷酸化蛋白与NG组比较表达增多(P=0.007)；与HG组相比,HG+2uMBPS组与HG+5uMBPS组细胞Smad3磷酸化蛋白表达水平明显下降(P值分别为0.012,0.004)。
[结论]
1.BPS能够抑制高糖环境下HBZY-1细胞的增殖；
2.BPS能够抑制高糖环境下HBZY-1细胞TGFβ1、FN蛋白质的合成及mRNA的表达,并增加了HBZY-1细胞MMP-2蛋白质的合成；
3.BPS能够抑制高糖环境下HBZY-1细胞p38MAPK、ERK磷酸化蛋白表达与Smad3蛋白的磷酸化。BPS可能通过不同信号转导通路间的相互作用,对高糖环境下HBZY-1细胞ECM合成与代谢过程进行保护性的调控。
第二章贝前列素钠对高糖环境下大鼠系膜细胞MCP-1表达的影响
[目的]
1.观察不同浓度BPS对高糖环境下HBZY-1细胞MCP-1蛋白质表达的影响。
2.观察不同浓度BPS对高糖环境下HBZY-1细胞MCP-1mRNA表达的影响。
3.探讨NFκB信号通路是否参与了BPS对高糖环境下HBZY-1细胞MCP-1表达的调控。
[方法]
1.细胞培养同第一章
2.细胞处理分组：NG组,HG组,HG联合不同浓度BPS (0.5uM,1uM,2uM与5uM)组,增加HG+PDTC (1uM)组。
3.不同处理组体外培养HBZY-1细胞,于24h,48h收集细胞培养上清液,用Elisa法测定细胞培养上清液中MCP-1蛋白质的表达量。
4.不同处理组体外培养HBZY-1细胞,于24h收集细胞,用实时荧光定量(real-time) PCR法测定细胞MCP-1mRNA的相对表达量(2-△△Ct)。
5.用免疫印迹法分别检测Omin,15min,30min,60min,90min不同时间点HG培养条件下HBZY-1细胞NFκB p65亚基核转位的情况。
6.选取HG诱导HBZY-1细胞NFκB p65亚基核转位最显著的时间点,应用免疫印迹法检测改时间点HG联合不同浓度BPS培养HBZY-1细胞NFκBp65亚基核转位的变化情况。
7.用细胞免疫荧光染色法观察不同处理组NFκB p65亚基核转位的变化。
8.统计学处理：同第一章。
[结果]
1.不同浓度BPS对高糖环境下HBZY-1细胞MCP-1蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞MCP-1蛋白表达量差异有统计学意义(24h:F=6.973, P=0.001;48h:F=10.436, P<0.001)。与NG组相比,HG组分别培养24h及48h后HBZY-1细胞MCP-1蛋白表达量均显著增加(P<0.001)。与HG组比较,HG+PDTC组分别培养细胞24h、48h后,细胞MCP-1蛋白表达量均显著下降(P<0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞MCP-1蛋白表达量显著降低(P=0.006)；HG联合不同浓度BPS培养细胞48h后,与HG组相比,HG+luMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞MCP-1蛋白表达量显著降低(P=0.003)。
2.不同浓度BPS对高糖环境下HBZY-1细胞MCP-1mRNA表达的影响：
与NG组相比,HG组细胞MCP-1mRNA表达相对量明显增加(4.62±0.12vs1.00±0.00,HG组均数的95%置信区间为[4.32,4.93])。与HG组相比,HG+PDTC组细胞MCP-1mRNA相对表达量显著下降(P<0.001)；HG联合不同浓度BPS组细胞MCP-1mRNA相对表达量较HG组均显著下降(P=0.011)。
3.不同时间点高糖诱导的HBZY-1细胞NFκB p65核转位的变化
HG Omin组细胞核内检测到少量NFκB p65蛋白的表达,HG刺激5min后即可检测到核内NFκB p65蛋白表达的增加；随着刺激时间的延长,核内NFκBp65蛋白的表达量逐渐增加,于刺激90min时最为明显,刺激120min时开始减弱。
胞浆内NFκB p65蛋白在HG0min表达最明显,HG刺激5min后胞浆内NFκB p65蛋白表达量开始减少；随着刺激时间的延长,胞浆内NFκB p65蛋白的表达逐渐减少,至90min时表达量最少。
4.免疫印迹法检测不同浓度BPS对高糖诱导的HBZY-1细胞NFκB p65核转位的影响
HG刺激90min后,HG组HBZY-1细胞核内NFκB p65蛋白表达与NG组比较明显增多。HG联合不同浓度的BPS培养细胞,与HG组相比,HG+luMBPS组、HG+2uMBPS组与HG+5uMBPS组胞核NFκB p65蛋白表达逐渐减少(P=0.001)。
HG刺激90min后,HG组HBZY-1细胞胞浆NFκB p65蛋白表达较NG组减少。HG联合不同浓度的BPS培养细胞,与HG组相比,HG+2uMBPS组与HG+5uMBPS组胞浆NFκB p65蛋白表达逐渐增多(P分别为0.049,0.034)。
5.细胞免疫荧光法观察BPS对高糖诱导的HBZY-1细胞NFκB p65核转位的影响
各不同处理组培养细胞90min后,NG组NFκB p65红色荧光均显示于胞浆中,胞核中未见NFκB p65红色荧光。HG组NFκB p65红色荧光集中于胞核中,胞浆中仅有少量NFκB p65红色荧光。用PDTC预先处理细胞1h抑制NFκB的活化,HG+PDTC组细胞NFκB p65红色荧光在胞核中的表达明显减少。与HG组比较,HG+2uMBPS组NFκB p65红色荧光在核内显示明显减少,在胞浆内显示明显增多,与HG+PDTC组结果相似。
[结论]
1.BPS能够抑制高糖环境下HBZY-1细胞MCP-1蛋白质的合成及mRNA的表达；
2.BPS能够抑制高糖环境下HBZY-1细胞NFκB p65亚基的核转位,从而抑制了高糖诱导的NFκB的激活,可能参与了BPS对高糖环境下HBZY-1细胞MCP-1表达的调控。
第三章PPARβ/δ在贝前列素钠减轻高糖对系膜细胞损伤中的作用
[目的]
1.观察不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ蛋白表达的影响。
2.观察不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ mRNA表达的影响。
3.观察选择性PPARβ/δ受体拮抗剂GSK0660对高糖环境下HBZY-1细胞TGFβ1、FN与MMP-2合成的影响。
4.观察GSK0660对高糖环境下HBZY-1细胞MAPK信号通路的影响。
5.观察GSK0660对高糖环境下HBZY-1细胞MCP-1表达的影响。
6.观察GSK0660对高糖环境下HBZY-1细胞NFκB-DNA结合活性的影响。
[方法]
1.细胞培养同第一、二章。
2.细胞处理分组：
NG组,HG组,HG+2uMBPS组,HG+GSK0660(5uM)组, HG+2uMBPS+GSK0660(5uM)组,HG+PDTC (1uM)组。
3.不同处理组体外培养HBZY-1细胞,于24h收集细胞,用免疫印迹法检测细胞PPARβ/δ蛋白质的表达量。
4.不同处理组体外培养HBZY-1细胞,于12h收集细胞,用实时荧光定量(real-time) PCR法测定细胞PPARβ/δ mRNA的相对表达量(2-△△Ct)。
5.正式刺激前,提前用GSK0660预处理细胞1h拮抗PPARβ/δ受体的作用,然后按上述处理分组继续培养细胞24h。
6.收集细胞培养上清液用Elisa法测TGFβ1、FN及MMP-2的蛋白质表达。
7. HBZY-1细胞p38MAPK、Erk1/2磷酸化蛋白表达测定同第一章。
8.收集细胞培养上清液用Elisa法测MCP-1的蛋白质表达量。
9.应用EMSA法检测NFκB p65-DNA结合活性变化。
10.统计学处理：同第一、二章。
[结果]
1.不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ蛋白表达的影响
NG组HBZY-1细胞核内存在PPARβ/δ蛋白的表达。与NG组相比,HG培养24h后胞核PPARβ/δ蛋白表达量减少(P=0.028)。HG联合不同浓度BPS培养细胞24h后,各组胞核PPARβ/δ蛋白表达量均较HG组增加(P=0.009)。在不同浓度BPS组中,HG+2uMBPS组PPARβ/δ/Lamin B1蛋白表达相对量升高最明显(P<0.001vs. HG+0.5uMBPS); HG+5uMBPS组PPARβ/δ/Lamin B1蛋白表达相对量较HG+2uMBPS组下降(P=0.017vs. HG+2uMBPS)。
2.不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ mRNA表达的影响：
与NG组相比,HG组细胞PPARβ/δ mRNA表达相对量明显下降(0.69±0.02vs.1.00±0.00,HG组均数的95%置信区间为[0.64,0.73])。与HG组相比,HG联合不同浓度BPS组细胞PPARβ/δ mRNA相对表达量均显著上升(P<0.001)。在不同浓度BPS组中,HG+2uMBPS组PPARβ/δ mRNA表达相对量升高最明显(P<0.001vs. HG+0.5uMBPS); HG+5uMBPS组PPARβ/δ mRNA表达相对量较HG+2uMBPS组下降(P<0.001vs. HG+2uMBPS)。
3.GSK0660对高糖环境下HBZY-1细胞TGFβ1合成的影响：
不同处理组培养细胞24h后,各组细胞TGFβ1蛋白表达量有显著差异(F=16.429,P<0.001)。与NG组相比,HG培养24h后细胞TGFβ1蛋白表达量显著增加(P<0.001)。与HG组比较,HG+2uMBPS组培养24h后细胞TGFβ1蛋白表达量显著下降(P=0.001)。HG+2uMBPS+GSK0660组细胞TGFβ1蛋白表达量与HG+2uMBPS组比较显著上升(P=0.049)；而HG+GSK0660组与HG组比较,两组细胞TGFβ1蛋白表达量差异无统计学意义(P=0.621)。
4.GSK0660对高糖环境下HBZY-1细胞FN合成的影响：
不同处理组培养细胞24h后,各组细胞FN蛋白表达量有显著差异(F=26.493,P<0.001)。与NG组相比,HG培养24h后细胞FN蛋白表达量显著增加(P<0.001)；与HG组比较,HG+2uMBPS组培养24h后细胞FN蛋白表达量显著下降(P=0.001)。HG+2uMBPS+GSK0660组细胞FN蛋白表达量与HG+2uMBPS组相比显著上升(P=0.047)；而HG+GSK0660组与HG组比较,两组FN蛋白表达量变化不明显,差异无统计学意义(P=0.592)。
5.GSK0660对高糖环境下HBZY-1细胞MMP-2合成的影响：
不同处理组培养细胞24h后,各组细胞MMP-2蛋白表达量有显著差异(F=10.269,P=0.001)。与NG组相比,HG组细胞MMP-2蛋白表达量显著下降(P<0.001)；与HG组比较,HG+2uMBPS组细胞MMP-2蛋白表达量显著上升,(P=0.017)。HG+2uMBPS+GSK0660组细胞MMP-2蛋白表达量与HG+2uMBPS组比较变化不明显,差异无统计学意义(P=0.956); HG+GSK0660组与HG组比较,两组细胞MMP-2蛋白表达量变化不明显,差异无统计学意义(P=0.958)。
6.GSK0660对HG高糖环境下HBZY-1细胞p38MAPK信号通路的影响：
HG刺激15min后,与NG组比较,细胞内p38磷酸化蛋白表达显著增加(P<0.001)。HG+2uMBPS组与HG组比较,细胞内磷酸化p38蛋白表达减少(P<0.001)。与HG+2uMBPS组比较,HG+2uMBPS+GSK0660组细胞内磷酸化p38蛋白表达明显增加(P<0.001)。HG+GSK0660组与HG组比较,细胞磷酸化p38蛋白表达变化不明显(P=0.061)。
7.GSK0660对HG高糖环境下HBZY-1细胞ERK信号通路的影响：
HG刺激15min后细胞内ERK磷酸化蛋白表达较NG组显著增加(P<0.001)。与HG组比较,HG+2uMBPS组细胞内ERK磷酸化蛋白表达明显下降(P<0.001)。HG+2uMBPS+GSK0660组细胞内ERK磷酸化蛋白表达较HG+2uMBPS组有所增加(P<0.001)。HG+GSK0660组与HG组比较,细胞内磷酸化ERK蛋白表达水平无明显变化(P=0.093)。
8.GSK0660对高糖环境下HBZY-1细胞MCP-1合成的影响：
不同处理组培养细胞24h后,各组细胞MCP-1蛋白表达量有显著差异(F=11.402,P=0.001)。与NG组相比,HG组细胞MCP-1蛋白表达量显著增加(P<0.001)。与HG组比较,HG+2uMBPS组细胞MCP-1蛋白表达量显著下降(P=0.004)。HG+2uMBPS+GSK0660组MCP-1蛋白表达量与HG+2uMBPS组相比显著上升(P=0.042)；而HG+GSK0660组与HG组比较,两组MCP-1蛋白表达量变化不明显,差异无统计学意义(P=0.847)。
9.GSK0660对高糖环境下HBZY-1细胞NFκB p65-DNA结合活性的影响：
NG组未见NFκB-DNA的结合条带,HG组可见明显的NFκB-DNA结合条带,说明HG导致NFκB的激活,并且促进了NFκB与DNA的结合。用PDTC预处理细胞后,HG+PDTC组NFκB-DNA结合条带比HG组明显减弱,HG+GSK0660组NFκB-DNA结合条带与HG组比较变化不明显,提示GSK0660拮抗RRARβ/δ受体的作用后对HG导致的NFκB的激活没有影响。
HG+2uMBPS组的NFκB-DNA结合条带比HG组明显减弱,与HG+PDTC组的结果相似,说明2uM BPS能够抑制HG诱导的NFκB与DNA的结合。HG+2uMBPS+GSK0660组的NFκB-DNA结合条带比HG+2uMBPS组有所增强,提示BPS能够通过与RRARβ/δ受体作用,抑制HG诱导的NFκB与DNA的结合。
[结论]
1.BPS能够上调高糖环境下HBZY-1细胞PPARβ/δ蛋白与1mRNA的表达；
2.GSK0660能够逆转BPS对高糖环境下HBZY-1细胞TGFβ1、FN合成的抑制作用；而对BPS增加细胞MMP-2的合成没有影响,提示BPS能够通过与PPARβ/δ受体结合,调节高糖环境下HBZY-1细胞TGFβ1、FN的合成；而BPS对细胞MMP-2的合成调节作用可能不通过PPARβ/δ受体途径；
3.GSK0660能够拮抗BPS对高糖环境下HBZY-1细胞内p38MAPK、ERK蛋白磷酸化的抑制作用；提示BPS可以通过与PPARβ/δ受体结合,抑制HG诱导的p38MAPK与ERK的激活；
4.GSK0660能够逆转BPS对高糖环境下HBZY-1细胞MCP-1合成的抑制作用,提示BPS可以通过与PPARβ/δ受体结合,调节高糖环境下HBZY-1细胞的MCP-1的表达；
5.GSK0660能够拮抗BPS对高糖环境下HBZY-1细胞NFκB-DNA结合活性的抑制作用,提示BPS通过与PPARβ/δ受体结合,抑制了HG诱导的NFκB与DNA的结合。以上结果说明,BPS可以通过与细胞核受体PPARβ/δ的结合,对高糖环境下的大鼠系膜细胞HBZY-1起到了的一定保护作用。
Background
Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus (DM). The characteristic pathological changes of DN include mesangial extracellular matrix (ECM) accumulation, glomerulosclerosis and tubule interstitial fibrosis, which eventually lead to the progressive increase of urinary protein/albumin excretion and decline of renal function. The pathogenesis of DN is complicated. Multiple mechanisms contribute to the development and outcomes of diabetic nephropathy, such as the interactions among hyperglycemia induced metabolic and hemodynamic changes, increased production of advanced glycation end products (AGEs), enhanced polyol pathway, activation of protein kinase C (PKC), and oxidative stress. How to prevent and treat DN effectively has been concerned considerably by clinicians.
Mesangial cells (MCs) is one of the most important intrinsic glomerular cells, maintaining structural integrity of the glomerular microvascular bed and mesangial matrix homeostasis. In response to metabolic or hemodynamic injury induced by hyperglycemia, MCs undergo hypertrophy, proliferation with excessive production of ECM, such as collagen and fibronectin (FN). MCs may also respond to injury that primarily involves podocytes and endothelial cells or to abnormalities of the glomerular basement membrane, to produce growth factors, chemokines and cytokines. These factors exert autocrine and paracrine effects on MCs or on other glomerular cells, facilitating the progress of glomerulosclerosis.
Transforming growth factor-β1(TGFβ1) is one of the crucial mediators of renal fibrosis. MCs are able to secrete TGFβ1, and express the receptor of TGFβ1. Thus, TGFβ1exerts direct effects on MCs through autocrine and paracrine way, promoting the glomerulosclerosis and the renal fibrosis. In the diabetic state, a variety of profibrosis factors can induce the TGFβ1synthesis and activation by MCs, in turn leading Smads protein phosphorylation, to promote ECM generation and inhibit ECM degradation.
Mitogen activated protein kinase (MAPK) pathway is another important signaling pathway playing roles in pathogenesis of DN. MAPK pathway mainly includes extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38, transducting extracellular signals into cells. Hyperglycemia, AGEs and the activation of PKC and oxidative stress may induce MAPKs activation. Once be activated, MAPKs phosphorylate their downstream transcription factors, regulating expression of the target genes. MAPK pathway may have direct effects, or interact with TGFβ1/Smads pathway involving in the pathogenesis of DN.
The development of diabetes involves metabolic, endocrine, and hemodynamic abnormalities which can promote a state of chronic inflammation. In the kidney, this can lead to the accumulation of kidney macrophages, promoting the development of renal injury, kidney fibrosis and the renal function decline. Recent evidence has highlighted the production of monocyte chemoattractant protein-1(MCP-1) by diabetic kidneys as a major factor influencing macrophage accumulation in this disease. High levels of glucose have been shown to stimulate MCP-1production by mesangial cells through ways of the activation of PKC, increased levels of oxidative stress, and the activation of the transcription factor nuclear factor-icB (NFκB). Selective inhibition of MCP-1has been proven to be an effective reduction of kidney macrophages infiltration and TGFβ1expression, preventing the progress of glomerulosclerosis in animal models.
Prostacyclin (PGI2), belonging to the prostaglandin (PG) family, is an end product derived from the sequential metabolism of arachidonic acid via cyclooxygenase-2(COX-2) and prostacyclin synthase (PGIS). It has been well known that PGI2has the ability to inhibit platelet aggregation and has powerful vasodilatory effects via relaxation of smooth muscle since first reported in1976. There are two recognized receptors that mediate the effects of PGI2:1) binding to the cell surface G protein-coupled IP receptor and activate cAMP/PKA second messenger system,2) binding to the nuclear peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and alter target gene transcription. The character of PGI2is labile, and its half life is only4min at37degrees C. Therefore, many synthetic stable PGI2analogues are very useful to help us to study the biological effects of PGI2in vivo and in vitro.
Beraprost sodium (BPS) is the only one stable PGI2analogue taken as a tablet. In vitro studies, BPS, which inhibited the stretch-induced FN overexpression through the inhibition of the stretch-induced activation of MAPKs in cAMP/PKA dependent manner, may work protectively against the injury from glomerular hypertension in MCs. It also has been shown that BPS inhibited high glucose-induced cellular proliferation and the generation of ROS, and improved the antioxidant capacities of MCs. In animal models, BPS attenuated glomerular hyperfiltration by modulating vasomotion of glomerular afferent arterioles, inhibited macrophages infiltration and AGEs formation in diabetic glomeruli, and ameliorated renal function by reduing creatinine clearance and urinary excretion of albumin. In the type2diabetic patients with incipient nephropathy, urinary albumin excretion was significantly decreased after18months of BPS administration, and the level of creatinine clearance was significantly reduced in BPS group after24months. Therefore, BPS may have promising prospects of its applications in the prevention of diabetic nephropathy. To study the potential renoprotective mechamism of BPS may have great clinical significance.
In this study, we explored the effects of beraprost sodium on ECM metabolism and MCP-1expression in a rat mesangial cell line under high glucose conditions, and its potential mechanisms, serving as rationale for beraprost sodium in the prevention of diabetic nephropathy.
Chapter One Effects of Beraprost Sodium on ECM synthesis induced by high glucose in rat mesangial cells
[Objectives]
1. To investigate into the effect of Beraprost Sodium (BPS) on proliferative activity of HBZY-1cells induced by high glucose (HG).
2. To investigate into the effects of BPS on TGFβ1, FN and MMP-2protein expressions induced by HG in HBZY-1cells.
3. To investigate into effects of BPS on mRNA expressions of TGFβ1, FN and MMP-2induced by HG in HBZY-1cells.
4. To explore whether BPS can regulate p38and ERK MAPK signalling pathway in HBZY-1cells cultured under high glucose conditions.
5. To explore whether BPS can regulate TGFβ1/Smad3signalling pathway in HBZY-1cells cultured under high glucose conditions.
[Method]
1. Cell culture:HBZY-1cells were cultured at37℃,5%C02with DMEM containing10%FCS, penicillin(100units/mL), and streptomycin(100xg/mL).
2. HBZY-1cells were grouped according to different intervention: Normal glucose (NG) group, the glucose concentration was5.5mmol/L (mM), High glucose (HG) group, the glucose concentration was30mM, HG combined with different concentrations of BPS groups, the BPS concentration were0.1umol/L (uM),0.5uM,1uM,2uM,5uM, and10uM, respectively.
3. MTT assay:to investigate into the proliferative activity of HBZY-1cells
4. Elisa assaay:to investigate into protein expressions of TGFβ1, FN and MMP-2in the cell culture supernatants.
5. Relative mRNA expressions of TGFβ1, FN and MMP-2were detected by real-time PCR.
6. Expressions of p38and ERK MAPK phosphorylated protein were analysed by Western blots.
7. Expression of Smad3phosphorylated protein were analysed by Western blots.
8. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3. P≤0.05was considered as significant.
[Results]
1. Proliferative activity of HBZY-1cells by MTT assay:compared with NG group, OD values of HBZY-1cells were significantly increased in HG group (P<0.001) after24h and48h culture, respectively. Compared with HG group, except HG+O.luMBPS group, OD values of HBZY-1cells in HG+0.5uMBPS, HG+luMBPS, HG+2uMBPS, HG+5uMBPS and HG+lOuMBPS group were significantly decrease after24h (P=0.044) and48h (P<0.001) culture, respectively.
According to the results of this experiment, we selected the concentration of0.5,1.2,5uM as BPS concentration processing in the follow-up experiments.
2. Protein expression of TGFβ1:compared with NG group, TGFβ1protein expression of HBZY-1cells was significantly increased in HG group (P<0.001) after24h and48h culture, respectively. After24h culture, TGFβ1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.001). After48h culture, TGFβ1protein expressions in HG+0.5uMBPS, HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.011).
3. Protein expression of FN:compared with NG group, FN protein expression of HBZY-1cells were significantly increased in HG group (P=0.001) after24h and48h culture, respectively. After24h culture, FN protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly decreased compared with HG group (P=0.018,0.007). After48h culture, FN protein expressions in HG+1uMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.005).
4. Protein expression of MMP-2:compared with NG group, MMP-2protein expression of HBZY-1cells were significantly decreased in HG group (P=0.001) after24h and48h culture, respectively. After24h culture, MMP-2protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly increased compared with HG group (P=0.017,0.001). After48h culture, MMP-2protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly increased compared with HG group (P=0.019,0.007).
5. Relative mRNA expression of TGFβ1:After24h cultures, compared with NG group, the relative mRNA expression of TGFβ1in HG group was significantly increased (2.45±0.22vs.1.00±0.00,95%CI for Mean [1.92,2.99]). Compared with HG group, the relative mRNA expressions of TGFβ1in HG+0.5uMBPS, HG+1uMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased (P=0.008).
6. Relative mRNA expression of FN:After24h cultures, compared with NG group, the relative mRNA expression of FN in HG group was significantly increased (1.93±0.23vs.1.00±0.00,95%CI for Mean [1.36,2.51]). Compared with HG group, the relative mRNA expressions of FN in HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.006,<0.001).
7. Relative mRNA expression of MMP-2:After24h cultures, compared with NG group, the relative mRNA expression of MMP-2in HG group was significantly decreased (0.528±0.132vs.1.000±0.000,95%CI for Mean [0.199,0.857]). Compared with HG group, there were no difference among the HG group and HG combined with different concentration BPS groups (F=0.906, P=0.497).
8. p38MAPK protein phosphorylation:after15min stimulation, expression of phosphorylated p38MAPK protein in HG group was significantly higher compared with NG group (P<0.001). Compared with HG group, expressions of phosphorylated p38MAPK protein in all BPS groups were significantly decreased (P=0.006).
9. ERK protein phosphorylation:after15min stimulation, expression of phosphorylated ERK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expressions of phosphorylated ERK protein in all BPS groups were significantly decreased (P<0.001).
10. Smad3protein phosphorylation:after60min stimulation, expression of phosphorylated Smad3protein in HG group was significantly higher compared with NG group (P=0.007). Compared with HG group, expressions of phosphorylated Smad3protein in HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.012,0.004).
[Conclusions]
1. BPS inhibited cell proliferation induced by high glucose in HBZY-1cells.
2. BPS inhibited TGFβ1and FN protein and mRNA expressions induced by high glucose in HBZY-1cells, and incresed MMP-2protein synthesis in HBZY-1cells cultured under high glucose conditions.
3. BPS inhibited p38and ERK MAPK protein phosphorylation, and Smad3protein phosphorylation, which indicated that BPS could regulate ECM synthesis and metabolic processes in HBZY-1cells cultured under high glucose conditions through different signal pathway interaction.
Chapter Two Effects of Beraprost Sodium on MCP-1expression induced by high glucose in rat mesangial cells
[Objectives]
1. To investigate into the effects of BPS on MCP-1protein expression induced by HG in HBZY-1cells.
2. To investigate into the effects of BPS on MCP-1mRNA expression induced by HG in HBZY-1cells.
3. To explore whether BPS can regulate NFκB signalling pathway in HBZY-1cells cultured under high glucose conditions.
[Method]
1. HBZY-1cells were grouped according to different intervention:
NG group, HG group, HG combined with different concentrations of BPS groups, the BPS concentration were0.5uM,1uM,2uM, and5uM, respectively, and HG combined with PDTC (1uM) group.
2. Elisa assaay:to investigate into MCP-1protein expression in cell culture supernatants.
3. Relative mRNA expression of MCP-1was detected by real-time PCR.
4. Nuclear translocation of NFκB p65protein was analysed by Western blots and immunofluoscence technique.
5. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3. P=≤0.05was considered as significant.
[Results]
1. Protein expression of MCP-1:compared with NG group, MCP-1protein expression of HBZY-1cells were significantly increased in HG group (P<0.001) after24h and48h culture, respectively. Compared with HG group, MCP-1protein expression in HG+PDTC group was significantly decreased (P<0.001) after24h and48h culture, respectively. After24h culture, MCP-1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.006). After48h culture, MCP-1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.003).
2. Relative mRNA expression of MCP-1:After24h cultures, compared with NG group, the relative mRNA expressions of MCP-1in HG group were significantly increased (4.62±0.12vs.1.00±0.00,95%CI for Mean [4.32,4.93]). Compared with HG group, the relative mRNA expression of MCP-1in HG+PDTC group was significantly decreased (P<0.001), and the relative mRNA expressions of MCP-1in all BPS groups were significantly decreased (P=0.011).
3. Nuclear translocation of NFκB p65:after90min stimulation, compared with NG group, NFκB p65protein expression in nucleus in HG group was was significantly increased (P<0.001). Compared with HG group, NFκB p65protein expressions in nucleus in HG+luMBPS, HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.001). In the mean time, NFκB p65protein expression in cytoplasm in HG group was significantly decreased after90min stimulation compared with NG group (P=0.001). NFκB p65protein expressions in cytoplasm in HG+2uMBPS and HG+5uMBPS group were significantly increased (P=0.049,0.034) compared with HG group.
4. Immunofluoscence images of NFκB p65nuclear translocation:in NG group, the red fluorescence of NFκB p65was concentrated in the cytoplasm of HBZY-1cells, while the red fluorescence of NFκB p65was concentrated in the nucleus of HBZY-1cells in HG group. The images of red fluorescence of NFκB p65were similar between HG+2uMBPS group and HG+PDTC group. Compared with HG group, the red fluorescence of NFκB p65was reduced in the nucleus in HG+2uMBPS and HG+PDTC groups, which was mainly displayed in the cytoplasm of HBZY-1cells.
[Conclusion]
1. BPS inhibited MCP-1protein and mRNA expressions induced by high glucose in HBZY-1cells.
2. BPS could inhibit NFκB p65subunit nuclear translocation in HBZY-1cells induced by high glucose, thereby inhibiting the activation of NFκB induced by high glucose, which was the potential mechmism of BPS in the regulation of MCP-1synthesis in HBZY-1cells culture in high glucose environment.
Chapter Three
Effects of PPARβ/δ on Beraprost Sodium pretection rat mesangial cells in high glucose enviroment
[Objectives]
1. To investigate into the effect of BPS on PPARβ/δ protein expression in HBZY-1cells cultured in HG environment.
2. To investigate into the effect of BPS on PPARβ/δ mRNA expression in HBZY-1 cells cultured in HG environment.
3. To explore whether PPARβ/δ was involved in BPS inhibition of TGFβ1, FN and MMP-2expressions induced by HG in HBZY-1cells.
4. To explore whether PPARβ/δ was involved in BPS inhibition of p38and ERK MAPK activation induced by HG in HBZY-1cells.
5. To explore whether PPARβ/δ was involved in BPS inhibition of MCP-1expression induced by HG in HBZY-1cells.
6. To explore whether PPARβ/δ was involved in BPS inhibition of NFκB-DNA binding activity induced by HG in HBZY-1cells.
[Method]
1. HBZY-1cells were grouped according to different intervention: Normal glucose (NG) group, High glucose (HG) group, HG+2uMBPS groups, HG+2uMBPS+GSK0660(5uM) groups, and HG+GSK0660group.
2. Protein expression of PPARβ/δ was detected by Western blots.
3. The relative mRNA expression of PPARβ/δ was detected by real-time PCR.
4. Elisa assaay:to investigate into the protein expressions of TGFβ1, FN, MMP-2and MCP-1in the cell culture supernatants.
5. Expressions of p38and ERK MAPK phosphorylated protein were analysed by Western blots.
6. EMSA:to detect the NFκB-DNA binding activtity.
7. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3. P≤0.05was considered as significant.
[Results]
1. Protein expression of PPARβ/δ:after24h culture, compared with NG group, PPARβ/δ protein expression of HBZY-1cells in HG group was significantly decreased (P=0.028). Compared with HG group, PPARβ/δ protein expression in all BPS groups were significantly increased (P=0.009). PPARβ/δ protein expression in HG+2uMBPS group was the highest among the BPS groups (P<0.001vs. HG+0.5uMBPS), and PPARβ/δ protein expression in HG+5uMBPS group was lower than that in HG+2uMBPS group (P=0.017vs. HG+2uMBPS).
2. Relative mRNA expression of PPARβ/δ:After12h cultures, compared with NG group, the relative mRNA expressions of PPARβ/δ in HG group were significantly decreased (0.69±0.02vs.1.00±0.00,95%CI for Mean [0.64,0.73]). Compared with HG group, the relative mRNA expressions of PPARβ/δ in all BPS groups were significantly increased (P<0.001). The relative mRNA expression of PPARβ/δ in HG+2uMBPS group was the highest among the BPS groups (P<0.001vs. HG+0.5uMBPS), and the relative mRNA expression of PPARβ/δ in HG+5uMBPS group was lower than that in HG+2uMBPS group (P<0.001vs. HG+2uMBPS).
3. Protein expression of TGFβ1:after24h culture, TGFβ1protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. TGFβ1protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.001). TGFβ1protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.049). There was no difference between HG+GSK0660group and HG group (P=0.621).
4. Protein expression of FN:after24h culture, FN protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. FN protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.001). FN protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.047). There was no difference between HG+GSK0660group and HG group (P=0.592).
5. Protein expression of MMP-2:after24h culture, MMP-2protein expression of HBZY-1cells in HG group was significantly decreased (P<0.001) compared with NG group. MMP-2protein expression in HG+2uMBPS group was significantly increased compared with HG group (P=0.017). There was no difference between HG+2uMBPS+GSK0660group and HG+2uMBPS group (P=0.956), and no difference between HG+GSK0660group and HG group (P=0.958).
6. p38MAPK protein phosphorylation:after15min stimulation, expression of phosphorylated p38MAPK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expression of phosphorylated p38MAPK protein in HG+2uMBPS group was significantly decreased (P<0.001). Compared with HG+2uMBPS group, expression of phosphorylated p38MAPK protein in HG+2uMBPS+GSK0660group was significantly increased (P<0.001). There was no difference between HG+GSK0660group and HG group (P=0.061).
7. ERK protein phosphorylation:after15min stimulation, expression of phosphorylated ERK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expression of phosphorylated ERK protein in HG+2uMBPS group was significantly decreased (P<0.001). Compared with HG+2uMBPS group, expression of phosphorylated ERK in HG+2uMBPS+GSK0660group was significantly increased (P<0.001). There was no difference between HG+GSK0660group and HG group (P=0.093).
8. Protein expression of MCP-1:after24h culture, MCP-1protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. MCP-1protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.004). MCP-1protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.042). There was no difference between HG+GSK0660group and HG group (P=0.847).
9. NFκB-DNA binding activity:There was no NFκB-DNA binding band detected in NG group. Strong band was detected in HG group, which indicated that HG could induce NFκB bind to DNA by NFκB activation. The grey value of NFκB-DNA binding band was reduced in HG+PDTC group compared with HG group, because PDTC could inhibit the activation of NFκB. The grey value of NFkB-DNA binding band in HG+2uMBPS group was also decreased compared with HG group, which indicated that2uMBPS could reduce the NFκB-DNA binding activity. In HG+2uMBPS+GSK0660group, the grey value of NFκB-DNA binding band was increased compared with HG+2uMBPS group.
[Conclusion]
1. BPS could increase PPARβ/δ protein and mRNA expression in HBZY-1cells cultured under high glucose conditions.
2. GSK0660reversed the inhibiton of BPS on TGFβ1and FN protein expression in HBZY-1cells induced by high glucose, but no changes in the MMP-2protein expression, suggesting that BPS could regluate TGFβ1and FN expression through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
3. GSK0660reversed the inhibiton of BPS on p38and ERK MAPK activation induced by high glucose in HBZY-1cells, suggesting that BPS could regluate p38and ERK MAPK pathway through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
4. GSK0660reversed the inhibiton of BPS on MCP-1protien expression in HBZY-1cells induced by high glucose, suggesting that BPS could regluate MCP-1expression through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
5. BPS could inhibit the NFκB-DNA binding activity induced by high glucose in HBZY-1cells, while GSK0660could reverse this effect. It was indicated that BPS could regluate NFκB-DNA binding activity through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
糖尿病肾病(diabetic nephropathy, DN)是糖尿病(diabetes mellitus, DM)特有的微血管并发症之一,其特征性的病理改变包括系膜区细胞外基质(extracellular matrix, ECM)堆积、肾小球硬化及小管间质纤维化,最终导致进行性尿蛋白/白蛋白排泄的增加和肾功能的下降。DN的发病机制复杂,目前认为高血糖、肾血流动力学的异常、晚期糖基化终末产物(advanced glycation end products, AGEs)的增多、多元醇及蛋白激酶C (protein kinase C, PKC)途径的激活、氧化应激的产生等多种因素共同参与了DN的发生与发展。如何对糖尿病肾病进行有效地防治一直是临床关注的热点与难点。
肾小球系膜细胞(mesangial cell, MC)是肾小球最重要的固有细胞之一,对维持肾小球毛细血管床的完整性与系膜基质代谢平衡起到了重要的作用。在DN的发病中,各种病理因素直接作用于MC,引起MC肥大与过度增殖,系膜区胶原纤维(collagen)、纤维连接蛋白(fibronectin, FN)等ECM成分异常的聚集沉积,构成了DN肾小球硬化重要的病理基础。持续的细胞外高糖,以及高糖介导的各种病理因素还可以通过肾小球内皮细胞、足细胞、炎症细胞与MC相互作用,产生细胞因子与炎症介质,通过“自分泌”、“旁分泌”等方式影响MC,介导并促进了肾小球纤维化向肾小球硬化的进展。
在DN肾小球硬化的病理过程中,转化生长因子β1(transforming growth factor β1, TGFβ1)是重要的肾脏致纤维化的标志之一。MC能够分泌TGFβ1,并具有高亲和力的TGFβ1受体。因而TGFβ1能够通过“自分泌”、“旁分泌”的方式发挥其促进肾脏纤维化的作用。细胞外高糖以及高糖介导的各种病理因素均可作为促纤维化因素作用于MC促进TGFβ1的合成与分泌,激活TGFβ1下游Smads信号转导分子家族,促进ECM的产生并抑制其降解。
除了TGFβ1/Smads介导的DN肾纤维化经典途径外,丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)通路可以独立的或与TGFβ1/Smads通路相互作用,参与DN的发病。MAPK通路主要包括细胞外信号调控的蛋白激酶(Extracellular signal-regulated kinase mediates, ERK)、c-Jun N端激酶(c-jun amino-terminal kinase, JNK)/应激激活蛋白激酶(stress-activated protein kinase, SAPK)、p38MAPK等途径,是细胞将信号从细胞外传递到细胞内的主要通路。糖尿病高糖状态下,多元醇途径的激活、蛋白质非酶糖化,PKC途径的激活以及氧化应激水平的增加均可导致MAPK的激活,磷酸化下游转录因子,调节基因的表达,参与DN的发生发展。
糖尿病肾病的病理状态下,在MC过度增殖、系膜区ECM异常增多的同时,还伴有多种细胞因子和炎症介质分泌的增加,导致局部炎症反应的激活,加速了肾小球硬化的过程和肾病的进展。糖尿病本身是一个慢性炎症的过程并由此引起巨噬细胞在多个组织中聚集。这种巨噬细胞的聚集与糖尿病疾病状态、炎症的激活及DN肾损伤的进展关系密切。大量研究表明,单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)在糖尿病组织巨噬细胞浸润及炎症中起到了关键作用。
MCP-1是一种能被多种细胞表达的,对单核细胞、T淋巴细胞等具有趋化作用的分泌型蛋白。MCP-1与其受体结合后,诱导下游黏附分子的表达,使单核细胞／巨噬细胞等向病变部位聚集而发挥其病理作用。高糖培养系膜细胞能够通过PKC与氧化应激的激活促进MCP-1的表达。阻断MCP-1与其受体结合能减少糖尿病小鼠肾组织巨噬细胞的浸润及TGFβ1、Ⅳ胶原的表达,有效地阻止DN肾小球硬化的进展。
前列环素(prostacyclin, PGI2)是前列腺素(prostaglandin, PG)家族中的重要成员,主要由血管内皮细胞产生,具有强大的舒张血管平滑肌、扩张血管及抗血小板凝聚的作用。体内的细胞膜磷脂被磷脂酶A2水解为花生四烯酸,后者进一步在环氧合酶与前列环素合成酶的作用下逐步合成PGI2。PGI2合成后通过与两类受体结合发挥其生物学作用：1)与细胞膜上G蛋白偶联的膜受体IP结合,主要激活下游的环磷酸腺苷(cAMP)／蛋白激酶A (protein kinase A, PKA)途径发挥舒张血管、抗血小板凝聚及抗纤维化等作用；2)与细胞核过氧化物酶体增殖激活受体β/δ (peroxisome proliferator-activated receptor β/δ, PPARβ/δ)结合,通过PPAR特异的反应元件PPRE (PPAR responsive element)调控靶基因的转录。PGI2的性质十分不稳定,37℃时半衰期仅4min,很快转化为稳定的无活性的代谢产物6-酮-前列腺素。目前主要通过与PGI2有相似药理作用的人工合成的各种PGI2类似物来研究PGI2的作用机制。
贝前列素钠(beraprost sodium, BPS)将PGI2结构中的外烯醇醚结构替换为环戊烷苯并呋喃酮结构,体内半衰期延长至60min,口服途径给药仍保留了与PGI2相似的药理作用。体外研究表明,BPS通过增加系膜细胞内cAMP水平抑制机械迁张拉力对MAPK的激活从而抑制FN的表达,减轻肾小球内高压对MC的损伤作用；还能够抑制高糖诱导的MC的增殖、Ⅳ胶原的合成并提高MC的抗氧化能力。动物实验结果显示,BPS喂养OLETF大鼠后,其肾小球体积及硬化指数明显低于对照组,肾小球内AGEs的形成及巨噬细胞浸润明显减少,同时伴有尿蛋白排泄率及血清尿素氮的降低。在STZ诱导的DM大鼠中,BPS通过调节入球小动脉内皮细胞一氧化氮合酶活性抑制了DM状态下入球小动脉的扩张,减轻了肾小球的高滤过,同时还抑制了巨噬细胞的浸润。早期的临床研究发现,BPS能够抑制糖尿病患者血清血栓调节蛋白水平,通过减轻AngⅡ对出球小动脉的收缩作用、缓解肾小球的高滤过状态,降低糖尿病患者尿白蛋白的排泄。这些结果提示BPS具有一定的肾脏保护作用。因而,BPS作为一种可以口服的PGI2类似物,在糖尿病肾病的防治中具有广泛地应用前景,进一步研究BPS肾脏保护的作用机制,具有十分重要的临床意义。
因此在本研究中,我们探讨了PGI2类似物贝前列素钠(BPS)对高糖环境下大鼠系膜细胞株HBZY-1细胞ECM合成与降解的作用,对趋化因子MCP-1表达的影响,以及对细胞内信号通路的影响,以阐明贝前列素钠对肾脏保护作用的可能机制,为贝前列素钠用于糖尿病肾病的临床防治提供部分理论依据。
第一章贝前列素钠对高糖环境下大鼠系膜细胞ECM合成的影响
[目的]
1.观察不同浓度BPS对高糖环境下大鼠细胞系膜细胞株HBZY-1增殖活性的影响。
2.观察不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1、FN及MMP-2蛋白质表达的影响。
3.观察不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1、FN及MMP-2mRNA水平表达的影响。
4.探讨p38MAPK、ERK信号通路是否参与了BPS对高糖环境下HBZY-1细胞ECM合成的调节。
5.探讨BPS对高糖环境下HBZY-1细胞TGFβ1/Smad3信号通路的影响。
[方法]
1.细胞培养：采用含10%胎牛血清的DMEM培养基,于37℃、5%CO2培养箱内传代培养大鼠肾小球系膜细胞株HBZY-1。
2.细胞处理分组：正常葡萄糖浓度组(NG组,葡萄糖浓度5.6mmol/L),高葡萄糖浓度组(HG组,葡萄糖浓度30mmol/L), HG联合不同浓度的BPS组：BPS药物处理浓度分别为0.1umol/L(uM),0.5uM,1uM,2uM,5uM,10uM。
3.不同处理组(NG组,HG组,HG分别联合不同浓度的BPS组)体外培养HBZY-1细胞,于24h,48h用MTT法测定细胞增殖活性。
4.不同处理组体外培养HBZY-1细胞,于24h,48h收集细胞培养上清液,用Elisa法测定细胞培养上清液中TGFβ1、FN及MMP-2蛋白质的表达量。
5.不同处理组体外培养HBZY-1细胞,于24h收集细胞,用实时荧光定量(real-time) PCR法测定细胞TGFβ1、FN及MMP-2mRNA的相对表达量(2-△△Ct)。
6.应用免疫印迹法分别检测Omin,15min,30min,60min,90min不同时间点HG培养条件下HBZY-1细胞p38MAPK、ERK磷酸化蛋白的表达。
7.分别选取HG诱导HBZY-1细胞p38MAPK、ERK磷酸化蛋白表达最显著的时间点,应用免疫印迹法分别检测该时间点HG联合不同浓度BPS培养HBZY-1细胞p38MAPK、ERK磷酸化蛋白的表达。
8.应用免疫印迹法分别检测Omin,15min,30min,60min,90min不同时间点HG培养条件下HBZY-1细胞smad3磷酸化蛋白的表达。
9.选取HG诱导HBZY-1细胞smad3磷酸化蛋白表达最显著的时间点,应用免疫印迹法检测该时间点HG联合不同浓度BPS培养HBZY-1细胞smad3磷酸化蛋白的表达。
10.统计学处理：实验数据采用SPSS13.0软件进行统计学分析,各组计量资料数据采用均数±标准差(x±s)表示。采用析因方差分析方法进行主效应和交互效应的分析；多组均数比较采用单向方差分析(One-way ANOVA),多重比较采用LSD法(Least-significant difference test);方差不齐时,用Welch法校正,多重比较采用Dunnett's T3法；两独立样本比较采用t检验。P≤0.05被认为差异具有统计学意义。
[结果]
1.不同浓度BPS对高糖环境下HBZY-1细胞增殖活性的影响：
不同处理组分别培养24h,48h后各组细胞O.D.值均有显著差异(24h: F=5.977, P<0.001;48h:F=25.585, P<0.001)。与NG组相比,HG组培养24h及48h后HBZY-1细胞O.D.值均显著增加(P均<0.001)。HG联合不同浓度BPS培养细胞24h及48h,与HG组相比,除HG+0.1uMBPS组外,其余5个BPS浓度处理组O.D.值均显著低于HG组(24h:P=0.044;48h:P<0.001)。
根据本组实验的结果,选取0.5、1、2、5uM等4个浓度作为后续实验中BPS的处理浓度。
2.不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞TGFβ1蛋白表达量差异有统计学意义(24h:F=26.441, P<0.001;48h:F=14.939, P<0.001)。与NG组相比,HG组分别培养24h及48h后]HBZY-1细胞TGFβ1蛋白表达量均显著增加(P<0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞TGFβ1蛋白表达量均显著降低(P=0.001)；HG联合不同浓度BPS培养细胞48h后,与HG组相比,各浓度BPS组细胞TGFβ1蛋白表达量呈浓度依赖性下降(P=0.011)。
3.不同浓度BPS对高糖环境下HBZY-1细胞FN蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞FN蛋白表达量差异有统计学意义(24h:F=5.445, P=0.008;48h:F=10.736, P<0.001)。与NG组相比,HG组分别培养24h及48h后HBZY-1细胞FN蛋白表达量均显著增加(P=0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+2uMBPS组及HG+5uMBPS组细胞FN蛋白表达量均显著降低(P值分别为0.018,0.007)；HG联合不同浓度BPS培养细胞48h后,与HG组相比,HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞FN蛋白表达量均显著降低(P=0.005)。
4.不同浓度BPS对高糖环境下HBZY-1细胞MMP-2蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞MMP-2蛋白表达量差异有统计学意义(24h:F=7.232, P=0.002;48h:F=4.610, P=0.014)。与NG组相比,HG组分别培养HBZY-1细胞24h及48h后,细胞MMP-2蛋白表达量均显著下降(P=0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+2uMBPS组及HG+5uMBPS组细胞MMP-2蛋白表达量显著增加(P值分别为0.017,0.001)；HG联合不同浓度BPS培养细胞48h后,与HG组相比, HG+2uMBPS组及HG+5uMBPS组细胞MMP-2蛋白表达量显著增加(P值分别为0.019,0.007)。
5.不同浓度BPS对高糖环境下HBZY-1细胞TGFβ1mRNA表达的影响：
与NG组相比,HG组细胞TGFβ1mRNA相对表达量明显增加(2.45±0.22vs.1.00±0.00,HG组均数的95%置信区间为[1.92,2.99])。与HG组相比,HG+0.5uMBPS组、HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞TGFβ1mRNA表达水平逐渐下降,差异具有统计学意义(P=0.008)。
6.不同浓度BPS对高糖环境下HBZY-1细胞FN mRNA表达的影响：
与NG组相比,HG组细胞FN mRNA表达相对量明显增加(1.93±0.23vs.1.00±0.00,HG组均数的95%置信区间为[1.36,2.51])。与HG组相比,HG+2uMBPS组及HG+5uMBPS组细胞FN mRNA相对表达量显著下降,差异具有统计学意义(P值分别为0.006,<0.001)。
7.不同浓度BPS对高糖环境下HBZY-1细胞MMP-2mRNA表达的影响：
与NG组相比,HG组细胞MMP-2mRNA表达相对量显著下降(0.528±0.132vs.1.000±0.000, HG组均数的95%置信区间为[0.199,0.857])。与HG组相比,HG联合不同浓度BPS组细胞MMP-2mRNA相对表达量有上升趋势。但统计学分析提示,HG组与HG联合不同BPS浓度组,各组细胞MMP-2mRNA相对表达量差异无统计学意义(F=0.906,P=0.497),可能与样本量偏小,或检测MMP-2mRNA时间点的选择有关。
8.不同时间点高糖诱导HBZY-1细胞p38MAPK磷酸化蛋白的表达：
HG Omin组细胞有少量磷酸化p38MAPK蛋白表达,HG刺激15min后细胞p38MAPK磷酸化蛋白表达最明显,刺激30min后p38MAPK磷酸化蛋白的表达开始减弱,刺激90min磷酸化p38MAPK蛋白信号仍未消失。
9.不同浓度BPS对高糖环境下HBZY-1细胞p38MAPK蛋白磷酸化的影响：
不同处理组刺激细胞15min后,HG组HBZY-1细胞p38MAPK磷酸化蛋白表达与NG组相比明显增多(P<0.001)；与HG组比较,HG联合不同浓度的BPS培养细胞,随着BPS药物浓度的增加,细胞p38MAPK磷酸化蛋白表达逐渐减少(P=0.006)。
10.不同时间点高糖诱导HBZY-1细胞ERK磷酸化蛋白的表达：
HG Omin细胞有少量磷酸化ERK蛋白表达,HG刺激15min后细胞ERK磷酸化蛋白表达明显增多,刺激30min后ERK磷酸化蛋白的表达开始减弱,刺激至90min磷酸化p38MAPK蛋白条带仍未消失。
11.不同浓度BPS对高糖环境中HBZY-1细胞ERK蛋白磷酸化的影响：
HG刺激15min后,HG组HBZY-1细胞ERK磷酸化蛋白表达比NG组明显增多(P<0.001)；与HG组比较,HG联合不同浓度的BPS培养细胞,随着BPS药物浓度的增加,细胞ERK磷酸化蛋白表达水平逐渐减少(P<0.001)。
12.不同时间点高糖诱导HBZY-1细胞Smad3蛋白磷酸化的表达：
HG Omin及HG15min组未能检测到细胞Smad3磷酸化蛋白的表达；HG30min组Smad3磷酸化蛋白开始表达,HG刺激60min时Smad3磷酸化蛋白表达最明显,刺激90min时明显减弱。
13.不同浓度BPS对高糖环境中HBZY-1细胞Smad3蛋白磷酸化的影响
HG刺激60min后,HG组HBZY-1细胞Smad3磷酸化蛋白与NG组比较表达增多(P=0.007)；与HG组相比,HG+2uMBPS组与HG+5uMBPS组细胞Smad3磷酸化蛋白表达水平明显下降(P值分别为0.012,0.004)。
[结论]
1.BPS能够抑制高糖环境下HBZY-1细胞的增殖；
2.BPS能够抑制高糖环境下HBZY-1细胞TGFβ1、FN蛋白质的合成及mRNA的表达,并增加了HBZY-1细胞MMP-2蛋白质的合成；
3.BPS能够抑制高糖环境下HBZY-1细胞p38MAPK、ERK磷酸化蛋白表达与Smad3蛋白的磷酸化。BPS可能通过不同信号转导通路间的相互作用,对高糖环境下HBZY-1细胞ECM合成与代谢过程进行保护性的调控。
第二章贝前列素钠对高糖环境下大鼠系膜细胞MCP-1表达的影响
[目的]
1.观察不同浓度BPS对高糖环境下HBZY-1细胞MCP-1蛋白质表达的影响。
2.观察不同浓度BPS对高糖环境下HBZY-1细胞MCP-1mRNA表达的影响。
3.探讨NFκB信号通路是否参与了BPS对高糖环境下HBZY-1细胞MCP-1表达的调控。
[方法]
1.细胞培养同第一章
2.细胞处理分组：NG组,HG组,HG联合不同浓度BPS (0.5uM,1uM,2uM与5uM)组,增加HG+PDTC (1uM)组。
3.不同处理组体外培养HBZY-1细胞,于24h,48h收集细胞培养上清液,用Elisa法测定细胞培养上清液中MCP-1蛋白质的表达量。
4.不同处理组体外培养HBZY-1细胞,于24h收集细胞,用实时荧光定量(real-time) PCR法测定细胞MCP-1mRNA的相对表达量(2-△△Ct)。
5.用免疫印迹法分别检测Omin,15min,30min,60min,90min不同时间点HG培养条件下HBZY-1细胞NFκB p65亚基核转位的情况。
6.选取HG诱导HBZY-1细胞NFκB p65亚基核转位最显著的时间点,应用免疫印迹法检测改时间点HG联合不同浓度BPS培养HBZY-1细胞NFκBp65亚基核转位的变化情况。
7.用细胞免疫荧光染色法观察不同处理组NFκB p65亚基核转位的变化。
8.统计学处理：同第一章。
[结果]
1.不同浓度BPS对高糖环境下HBZY-1细胞MCP-1蛋白表达的影响：
不同处理组分别培养细胞24h,48h后各组细胞MCP-1蛋白表达量差异有统计学意义(24h:F=6.973, P=0.001;48h:F=10.436, P<0.001)。与NG组相比,HG组分别培养24h及48h后HBZY-1细胞MCP-1蛋白表达量均显著增加(P<0.001)。与HG组比较,HG+PDTC组分别培养细胞24h、48h后,细胞MCP-1蛋白表达量均显著下降(P<0.001)。HG联合不同浓度BPS培养细胞24h后,与HG组相比,HG+1uMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞MCP-1蛋白表达量显著降低(P=0.006)；HG联合不同浓度BPS培养细胞48h后,与HG组相比,HG+luMBPS组、HG+2uMBPS组及HG+5uMBPS组细胞MCP-1蛋白表达量显著降低(P=0.003)。
2.不同浓度BPS对高糖环境下HBZY-1细胞MCP-1mRNA表达的影响：
与NG组相比,HG组细胞MCP-1mRNA表达相对量明显增加(4.62±0.12vs1.00±0.00,HG组均数的95%置信区间为[4.32,4.93])。与HG组相比,HG+PDTC组细胞MCP-1mRNA相对表达量显著下降(P<0.001)；HG联合不同浓度BPS组细胞MCP-1mRNA相对表达量较HG组均显著下降(P=0.011)。
3.不同时间点高糖诱导的HBZY-1细胞NFκB p65核转位的变化
HG Omin组细胞核内检测到少量NFκB p65蛋白的表达,HG刺激5min后即可检测到核内NFκB p65蛋白表达的增加；随着刺激时间的延长,核内NFκBp65蛋白的表达量逐渐增加,于刺激90min时最为明显,刺激120min时开始减弱。
胞浆内NFκB p65蛋白在HG0min表达最明显,HG刺激5min后胞浆内NFκB p65蛋白表达量开始减少；随着刺激时间的延长,胞浆内NFκB p65蛋白的表达逐渐减少,至90min时表达量最少。
4.免疫印迹法检测不同浓度BPS对高糖诱导的HBZY-1细胞NFκB p65核转位的影响
HG刺激90min后,HG组HBZY-1细胞核内NFκB p65蛋白表达与NG组比较明显增多。HG联合不同浓度的BPS培养细胞,与HG组相比,HG+luMBPS组、HG+2uMBPS组与HG+5uMBPS组胞核NFκB p65蛋白表达逐渐减少(P=0.001)。
HG刺激90min后,HG组HBZY-1细胞胞浆NFκB p65蛋白表达较NG组减少。HG联合不同浓度的BPS培养细胞,与HG组相比,HG+2uMBPS组与HG+5uMBPS组胞浆NFκB p65蛋白表达逐渐增多(P分别为0.049,0.034)。
5.细胞免疫荧光法观察BPS对高糖诱导的HBZY-1细胞NFκB p65核转位的影响
各不同处理组培养细胞90min后,NG组NFκB p65红色荧光均显示于胞浆中,胞核中未见NFκB p65红色荧光。HG组NFκB p65红色荧光集中于胞核中,胞浆中仅有少量NFκB p65红色荧光。用PDTC预先处理细胞1h抑制NFκB的活化,HG+PDTC组细胞NFκB p65红色荧光在胞核中的表达明显减少。与HG组比较,HG+2uMBPS组NFκB p65红色荧光在核内显示明显减少,在胞浆内显示明显增多,与HG+PDTC组结果相似。
[结论]
1.BPS能够抑制高糖环境下HBZY-1细胞MCP-1蛋白质的合成及mRNA的表达；
2.BPS能够抑制高糖环境下HBZY-1细胞NFκB p65亚基的核转位,从而抑制了高糖诱导的NFκB的激活,可能参与了BPS对高糖环境下HBZY-1细胞MCP-1表达的调控。
第三章PPARβ/δ在贝前列素钠减轻高糖对系膜细胞损伤中的作用
[目的]
1.观察不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ蛋白表达的影响。
2.观察不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ mRNA表达的影响。
3.观察选择性PPARβ/δ受体拮抗剂GSK0660对高糖环境下HBZY-1细胞TGFβ1、FN与MMP-2合成的影响。
4.观察GSK0660对高糖环境下HBZY-1细胞MAPK信号通路的影响。
5.观察GSK0660对高糖环境下HBZY-1细胞MCP-1表达的影响。
6.观察GSK0660对高糖环境下HBZY-1细胞NFκB-DNA结合活性的影响。
[方法]
1.细胞培养同第一、二章。
2.细胞处理分组：
NG组,HG组,HG+2uMBPS组,HG+GSK0660(5uM)组, HG+2uMBPS+GSK0660(5uM)组,HG+PDTC (1uM)组。
3.不同处理组体外培养HBZY-1细胞,于24h收集细胞,用免疫印迹法检测细胞PPARβ/δ蛋白质的表达量。
4.不同处理组体外培养HBZY-1细胞,于12h收集细胞,用实时荧光定量(real-time) PCR法测定细胞PPARβ/δ mRNA的相对表达量(2-△△Ct)。
5.正式刺激前,提前用GSK0660预处理细胞1h拮抗PPARβ/δ受体的作用,然后按上述处理分组继续培养细胞24h。
6.收集细胞培养上清液用Elisa法测TGFβ1、FN及MMP-2的蛋白质表达。
7. HBZY-1细胞p38MAPK、Erk1/2磷酸化蛋白表达测定同第一章。
8.收集细胞培养上清液用Elisa法测MCP-1的蛋白质表达量。
9.应用EMSA法检测NFκB p65-DNA结合活性变化。
10.统计学处理：同第一、二章。
[结果]
1.不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ蛋白表达的影响
NG组HBZY-1细胞核内存在PPARβ/δ蛋白的表达。与NG组相比,HG培养24h后胞核PPARβ/δ蛋白表达量减少(P=0.028)。HG联合不同浓度BPS培养细胞24h后,各组胞核PPARβ/δ蛋白表达量均较HG组增加(P=0.009)。在不同浓度BPS组中,HG+2uMBPS组PPARβ/δ/Lamin B1蛋白表达相对量升高最明显(P<0.001vs. HG+0.5uMBPS); HG+5uMBPS组PPARβ/δ/Lamin B1蛋白表达相对量较HG+2uMBPS组下降(P=0.017vs. HG+2uMBPS)。
2.不同浓度BPS对高糖环境下HBZY-1细胞PPARβ/δ mRNA表达的影响：
与NG组相比,HG组细胞PPARβ/δ mRNA表达相对量明显下降(0.69±0.02vs.1.00±0.00,HG组均数的95%置信区间为[0.64,0.73])。与HG组相比,HG联合不同浓度BPS组细胞PPARβ/δ mRNA相对表达量均显著上升(P<0.001)。在不同浓度BPS组中,HG+2uMBPS组PPARβ/δ mRNA表达相对量升高最明显(P<0.001vs. HG+0.5uMBPS); HG+5uMBPS组PPARβ/δ mRNA表达相对量较HG+2uMBPS组下降(P<0.001vs. HG+2uMBPS)。
3.GSK0660对高糖环境下HBZY-1细胞TGFβ1合成的影响：
不同处理组培养细胞24h后,各组细胞TGFβ1蛋白表达量有显著差异(F=16.429,P<0.001)。与NG组相比,HG培养24h后细胞TGFβ1蛋白表达量显著增加(P<0.001)。与HG组比较,HG+2uMBPS组培养24h后细胞TGFβ1蛋白表达量显著下降(P=0.001)。HG+2uMBPS+GSK0660组细胞TGFβ1蛋白表达量与HG+2uMBPS组比较显著上升(P=0.049)；而HG+GSK0660组与HG组比较,两组细胞TGFβ1蛋白表达量差异无统计学意义(P=0.621)。
4.GSK0660对高糖环境下HBZY-1细胞FN合成的影响：
不同处理组培养细胞24h后,各组细胞FN蛋白表达量有显著差异(F=26.493,P<0.001)。与NG组相比,HG培养24h后细胞FN蛋白表达量显著增加(P<0.001)；与HG组比较,HG+2uMBPS组培养24h后细胞FN蛋白表达量显著下降(P=0.001)。HG+2uMBPS+GSK0660组细胞FN蛋白表达量与HG+2uMBPS组相比显著上升(P=0.047)；而HG+GSK0660组与HG组比较,两组FN蛋白表达量变化不明显,差异无统计学意义(P=0.592)。
5.GSK0660对高糖环境下HBZY-1细胞MMP-2合成的影响：
不同处理组培养细胞24h后,各组细胞MMP-2蛋白表达量有显著差异(F=10.269,P=0.001)。与NG组相比,HG组细胞MMP-2蛋白表达量显著下降(P<0.001)；与HG组比较,HG+2uMBPS组细胞MMP-2蛋白表达量显著上升,(P=0.017)。HG+2uMBPS+GSK0660组细胞MMP-2蛋白表达量与HG+2uMBPS组比较变化不明显,差异无统计学意义(P=0.956); HG+GSK0660组与HG组比较,两组细胞MMP-2蛋白表达量变化不明显,差异无统计学意义(P=0.958)。
6.GSK0660对HG高糖环境下HBZY-1细胞p38MAPK信号通路的影响：
HG刺激15min后,与NG组比较,细胞内p38磷酸化蛋白表达显著增加(P<0.001)。HG+2uMBPS组与HG组比较,细胞内磷酸化p38蛋白表达减少(P<0.001)。与HG+2uMBPS组比较,HG+2uMBPS+GSK0660组细胞内磷酸化p38蛋白表达明显增加(P<0.001)。HG+GSK0660组与HG组比较,细胞磷酸化p38蛋白表达变化不明显(P=0.061)。
7.GSK0660对HG高糖环境下HBZY-1细胞ERK信号通路的影响：
HG刺激15min后细胞内ERK磷酸化蛋白表达较NG组显著增加(P<0.001)。与HG组比较,HG+2uMBPS组细胞内ERK磷酸化蛋白表达明显下降(P<0.001)。HG+2uMBPS+GSK0660组细胞内ERK磷酸化蛋白表达较HG+2uMBPS组有所增加(P<0.001)。HG+GSK0660组与HG组比较,细胞内磷酸化ERK蛋白表达水平无明显变化(P=0.093)。
8.GSK0660对高糖环境下HBZY-1细胞MCP-1合成的影响：
不同处理组培养细胞24h后,各组细胞MCP-1蛋白表达量有显著差异(F=11.402,P=0.001)。与NG组相比,HG组细胞MCP-1蛋白表达量显著增加(P<0.001)。与HG组比较,HG+2uMBPS组细胞MCP-1蛋白表达量显著下降(P=0.004)。HG+2uMBPS+GSK0660组MCP-1蛋白表达量与HG+2uMBPS组相比显著上升(P=0.042)；而HG+GSK0660组与HG组比较,两组MCP-1蛋白表达量变化不明显,差异无统计学意义(P=0.847)。
9.GSK0660对高糖环境下HBZY-1细胞NFκB p65-DNA结合活性的影响：
NG组未见NFκB-DNA的结合条带,HG组可见明显的NFκB-DNA结合条带,说明HG导致NFκB的激活,并且促进了NFκB与DNA的结合。用PDTC预处理细胞后,HG+PDTC组NFκB-DNA结合条带比HG组明显减弱,HG+GSK0660组NFκB-DNA结合条带与HG组比较变化不明显,提示GSK0660拮抗RRARβ/δ受体的作用后对HG导致的NFκB的激活没有影响。
HG+2uMBPS组的NFκB-DNA结合条带比HG组明显减弱,与HG+PDTC组的结果相似,说明2uM BPS能够抑制HG诱导的NFκB与DNA的结合。HG+2uMBPS+GSK0660组的NFκB-DNA结合条带比HG+2uMBPS组有所增强,提示BPS能够通过与RRARβ/δ受体作用,抑制HG诱导的NFκB与DNA的结合。
[结论]
1.BPS能够上调高糖环境下HBZY-1细胞PPARβ/δ蛋白与1mRNA的表达；
2.GSK0660能够逆转BPS对高糖环境下HBZY-1细胞TGFβ1、FN合成的抑制作用；而对BPS增加细胞MMP-2的合成没有影响,提示BPS能够通过与PPARβ/δ受体结合,调节高糖环境下HBZY-1细胞TGFβ1、FN的合成；而BPS对细胞MMP-2的合成调节作用可能不通过PPARβ/δ受体途径；
3.GSK0660能够拮抗BPS对高糖环境下HBZY-1细胞内p38MAPK、ERK蛋白磷酸化的抑制作用；提示BPS可以通过与PPARβ/δ受体结合,抑制HG诱导的p38MAPK与ERK的激活；
4.GSK0660能够逆转BPS对高糖环境下HBZY-1细胞MCP-1合成的抑制作用,提示BPS可以通过与PPARβ/δ受体结合,调节高糖环境下HBZY-1细胞的MCP-1的表达；
5.GSK0660能够拮抗BPS对高糖环境下HBZY-1细胞NFκB-DNA结合活性的抑制作用,提示BPS通过与PPARβ/δ受体结合,抑制了HG诱导的NFκB与DNA的结合。以上结果说明,BPS可以通过与细胞核受体PPARβ/δ的结合,对高糖环境下的大鼠系膜细胞HBZY-1起到了的一定保护作用。
Background
Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus (DM). The characteristic pathological changes of DN include mesangial extracellular matrix (ECM) accumulation, glomerulosclerosis and tubule interstitial fibrosis, which eventually lead to the progressive increase of urinary protein/albumin excretion and decline of renal function. The pathogenesis of DN is complicated. Multiple mechanisms contribute to the development and outcomes of diabetic nephropathy, such as the interactions among hyperglycemia induced metabolic and hemodynamic changes, increased production of advanced glycation end products (AGEs), enhanced polyol pathway, activation of protein kinase C (PKC), and oxidative stress. How to prevent and treat DN effectively has been concerned considerably by clinicians.
Mesangial cells (MCs) is one of the most important intrinsic glomerular cells, maintaining structural integrity of the glomerular microvascular bed and mesangial matrix homeostasis. In response to metabolic or hemodynamic injury induced by hyperglycemia, MCs undergo hypertrophy, proliferation with excessive production of ECM, such as collagen and fibronectin (FN). MCs may also respond to injury that primarily involves podocytes and endothelial cells or to abnormalities of the glomerular basement membrane, to produce growth factors, chemokines and cytokines. These factors exert autocrine and paracrine effects on MCs or on other glomerular cells, facilitating the progress of glomerulosclerosis.
Transforming growth factor-β1(TGFβ1) is one of the crucial mediators of renal fibrosis. MCs are able to secrete TGFβ1, and express the receptor of TGFβ1. Thus, TGFβ1exerts direct effects on MCs through autocrine and paracrine way, promoting the glomerulosclerosis and the renal fibrosis. In the diabetic state, a variety of profibrosis factors can induce the TGFβ1synthesis and activation by MCs, in turn leading Smads protein phosphorylation, to promote ECM generation and inhibit ECM degradation.
Mitogen activated protein kinase (MAPK) pathway is another important signaling pathway playing roles in pathogenesis of DN. MAPK pathway mainly includes extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38, transducting extracellular signals into cells. Hyperglycemia, AGEs and the activation of PKC and oxidative stress may induce MAPKs activation. Once be activated, MAPKs phosphorylate their downstream transcription factors, regulating expression of the target genes. MAPK pathway may have direct effects, or interact with TGFβ1/Smads pathway involving in the pathogenesis of DN.
The development of diabetes involves metabolic, endocrine, and hemodynamic abnormalities which can promote a state of chronic inflammation. In the kidney, this can lead to the accumulation of kidney macrophages, promoting the development of renal injury, kidney fibrosis and the renal function decline. Recent evidence has highlighted the production of monocyte chemoattractant protein-1(MCP-1) by diabetic kidneys as a major factor influencing macrophage accumulation in this disease. High levels of glucose have been shown to stimulate MCP-1production by mesangial cells through ways of the activation of PKC, increased levels of oxidative stress, and the activation of the transcription factor nuclear factor-icB (NFκB). Selective inhibition of MCP-1has been proven to be an effective reduction of kidney macrophages infiltration and TGFβ1expression, preventing the progress of glomerulosclerosis in animal models.
Prostacyclin (PGI2), belonging to the prostaglandin (PG) family, is an end product derived from the sequential metabolism of arachidonic acid via cyclooxygenase-2(COX-2) and prostacyclin synthase (PGIS). It has been well known that PGI2has the ability to inhibit platelet aggregation and has powerful vasodilatory effects via relaxation of smooth muscle since first reported in1976. There are two recognized receptors that mediate the effects of PGI2:1) binding to the cell surface G protein-coupled IP receptor and activate cAMP/PKA second messenger system,2) binding to the nuclear peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and alter target gene transcription. The character of PGI2is labile, and its half life is only4min at37degrees C. Therefore, many synthetic stable PGI2analogues are very useful to help us to study the biological effects of PGI2in vivo and in vitro.
Beraprost sodium (BPS) is the only one stable PGI2analogue taken as a tablet. In vitro studies, BPS, which inhibited the stretch-induced FN overexpression through the inhibition of the stretch-induced activation of MAPKs in cAMP/PKA dependent manner, may work protectively against the injury from glomerular hypertension in MCs. It also has been shown that BPS inhibited high glucose-induced cellular proliferation and the generation of ROS, and improved the antioxidant capacities of MCs. In animal models, BPS attenuated glomerular hyperfiltration by modulating vasomotion of glomerular afferent arterioles, inhibited macrophages infiltration and AGEs formation in diabetic glomeruli, and ameliorated renal function by reduing creatinine clearance and urinary excretion of albumin. In the type2diabetic patients with incipient nephropathy, urinary albumin excretion was significantly decreased after18months of BPS administration, and the level of creatinine clearance was significantly reduced in BPS group after24months. Therefore, BPS may have promising prospects of its applications in the prevention of diabetic nephropathy. To study the potential renoprotective mechamism of BPS may have great clinical significance.
In this study, we explored the effects of beraprost sodium on ECM metabolism and MCP-1expression in a rat mesangial cell line under high glucose conditions, and its potential mechanisms, serving as rationale for beraprost sodium in the prevention of diabetic nephropathy.
Chapter One Effects of Beraprost Sodium on ECM synthesis induced by high glucose in rat mesangial cells
[Objectives]
1. To investigate into the effect of Beraprost Sodium (BPS) on proliferative activity of HBZY-1cells induced by high glucose (HG).
2. To investigate into the effects of BPS on TGFβ1, FN and MMP-2protein expressions induced by HG in HBZY-1cells.
3. To investigate into effects of BPS on mRNA expressions of TGFβ1, FN and MMP-2induced by HG in HBZY-1cells.
4. To explore whether BPS can regulate p38and ERK MAPK signalling pathway in HBZY-1cells cultured under high glucose conditions.
5. To explore whether BPS can regulate TGFβ1/Smad3signalling pathway in HBZY-1cells cultured under high glucose conditions.
[Method]
1. Cell culture:HBZY-1cells were cultured at37℃,5%C02with DMEM containing10%FCS, penicillin(100units/mL), and streptomycin(100xg/mL).
2. HBZY-1cells were grouped according to different intervention: Normal glucose (NG) group, the glucose concentration was5.5mmol/L (mM), High glucose (HG) group, the glucose concentration was30mM, HG combined with different concentrations of BPS groups, the BPS concentration were0.1umol/L (uM),0.5uM,1uM,2uM,5uM, and10uM, respectively.
3. MTT assay:to investigate into the proliferative activity of HBZY-1cells
4. Elisa assaay:to investigate into protein expressions of TGFβ1, FN and MMP-2in the cell culture supernatants.
5. Relative mRNA expressions of TGFβ1, FN and MMP-2were detected by real-time PCR.
6. Expressions of p38and ERK MAPK phosphorylated protein were analysed by Western blots.
7. Expression of Smad3phosphorylated protein were analysed by Western blots.
8. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3. P≤0.05was considered as significant.
[Results]
1. Proliferative activity of HBZY-1cells by MTT assay:compared with NG group, OD values of HBZY-1cells were significantly increased in HG group (P<0.001) after24h and48h culture, respectively. Compared with HG group, except HG+O.luMBPS group, OD values of HBZY-1cells in HG+0.5uMBPS, HG+luMBPS, HG+2uMBPS, HG+5uMBPS and HG+lOuMBPS group were significantly decrease after24h (P=0.044) and48h (P<0.001) culture, respectively.
According to the results of this experiment, we selected the concentration of0.5,1.2,5uM as BPS concentration processing in the follow-up experiments.
2. Protein expression of TGFβ1:compared with NG group, TGFβ1protein expression of HBZY-1cells was significantly increased in HG group (P<0.001) after24h and48h culture, respectively. After24h culture, TGFβ1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.001). After48h culture, TGFβ1protein expressions in HG+0.5uMBPS, HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.011).
3. Protein expression of FN:compared with NG group, FN protein expression of HBZY-1cells were significantly increased in HG group (P=0.001) after24h and48h culture, respectively. After24h culture, FN protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly decreased compared with HG group (P=0.018,0.007). After48h culture, FN protein expressions in HG+1uMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.005).
4. Protein expression of MMP-2:compared with NG group, MMP-2protein expression of HBZY-1cells were significantly decreased in HG group (P=0.001) after24h and48h culture, respectively. After24h culture, MMP-2protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly increased compared with HG group (P=0.017,0.001). After48h culture, MMP-2protein expressions in HG+2uMBPS and HG+5uMBPS group were significantly increased compared with HG group (P=0.019,0.007).
5. Relative mRNA expression of TGFβ1:After24h cultures, compared with NG group, the relative mRNA expression of TGFβ1in HG group was significantly increased (2.45±0.22vs.1.00±0.00,95%CI for Mean [1.92,2.99]). Compared with HG group, the relative mRNA expressions of TGFβ1in HG+0.5uMBPS, HG+1uMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased (P=0.008).
6. Relative mRNA expression of FN:After24h cultures, compared with NG group, the relative mRNA expression of FN in HG group was significantly increased (1.93±0.23vs.1.00±0.00,95%CI for Mean [1.36,2.51]). Compared with HG group, the relative mRNA expressions of FN in HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.006,<0.001).
7. Relative mRNA expression of MMP-2:After24h cultures, compared with NG group, the relative mRNA expression of MMP-2in HG group was significantly decreased (0.528±0.132vs.1.000±0.000,95%CI for Mean [0.199,0.857]). Compared with HG group, there were no difference among the HG group and HG combined with different concentration BPS groups (F=0.906, P=0.497).
8. p38MAPK protein phosphorylation:after15min stimulation, expression of phosphorylated p38MAPK protein in HG group was significantly higher compared with NG group (P<0.001). Compared with HG group, expressions of phosphorylated p38MAPK protein in all BPS groups were significantly decreased (P=0.006).
9. ERK protein phosphorylation:after15min stimulation, expression of phosphorylated ERK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expressions of phosphorylated ERK protein in all BPS groups were significantly decreased (P<0.001).
10. Smad3protein phosphorylation:after60min stimulation, expression of phosphorylated Smad3protein in HG group was significantly higher compared with NG group (P=0.007). Compared with HG group, expressions of phosphorylated Smad3protein in HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.012,0.004).
[Conclusions]
1. BPS inhibited cell proliferation induced by high glucose in HBZY-1cells.
2. BPS inhibited TGFβ1and FN protein and mRNA expressions induced by high glucose in HBZY-1cells, and incresed MMP-2protein synthesis in HBZY-1cells cultured under high glucose conditions.
3. BPS inhibited p38and ERK MAPK protein phosphorylation, and Smad3protein phosphorylation, which indicated that BPS could regulate ECM synthesis and metabolic processes in HBZY-1cells cultured under high glucose conditions through different signal pathway interaction.
Chapter Two Effects of Beraprost Sodium on MCP-1expression induced by high glucose in rat mesangial cells
[Objectives]
1. To investigate into the effects of BPS on MCP-1protein expression induced by HG in HBZY-1cells.
2. To investigate into the effects of BPS on MCP-1mRNA expression induced by HG in HBZY-1cells.
3. To explore whether BPS can regulate NFκB signalling pathway in HBZY-1cells cultured under high glucose conditions.
[Method]
1. HBZY-1cells were grouped according to different intervention:
NG group, HG group, HG combined with different concentrations of BPS groups, the BPS concentration were0.5uM,1uM,2uM, and5uM, respectively, and HG combined with PDTC (1uM) group.
2. Elisa assaay:to investigate into MCP-1protein expression in cell culture supernatants.
3. Relative mRNA expression of MCP-1was detected by real-time PCR.
4. Nuclear translocation of NFκB p65protein was analysed by Western blots and immunofluoscence technique.
5. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3. P=≤0.05was considered as significant.
[Results]
1. Protein expression of MCP-1:compared with NG group, MCP-1protein expression of HBZY-1cells were significantly increased in HG group (P<0.001) after24h and48h culture, respectively. Compared with HG group, MCP-1protein expression in HG+PDTC group was significantly decreased (P<0.001) after24h and48h culture, respectively. After24h culture, MCP-1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.006). After48h culture, MCP-1protein expressions in HG+luMBPS, HG+2uMBPS, and HG+5uMBPS group were significantly decreased compared with HG group (P=0.003).
2. Relative mRNA expression of MCP-1:After24h cultures, compared with NG group, the relative mRNA expressions of MCP-1in HG group were significantly increased (4.62±0.12vs.1.00±0.00,95%CI for Mean [4.32,4.93]). Compared with HG group, the relative mRNA expression of MCP-1in HG+PDTC group was significantly decreased (P<0.001), and the relative mRNA expressions of MCP-1in all BPS groups were significantly decreased (P=0.011).
3. Nuclear translocation of NFκB p65:after90min stimulation, compared with NG group, NFκB p65protein expression in nucleus in HG group was was significantly increased (P<0.001). Compared with HG group, NFκB p65protein expressions in nucleus in HG+luMBPS, HG+2uMBPS and HG+5uMBPS group were significantly decreased (P=0.001). In the mean time, NFκB p65protein expression in cytoplasm in HG group was significantly decreased after90min stimulation compared with NG group (P=0.001). NFκB p65protein expressions in cytoplasm in HG+2uMBPS and HG+5uMBPS group were significantly increased (P=0.049,0.034) compared with HG group.
4. Immunofluoscence images of NFκB p65nuclear translocation:in NG group, the red fluorescence of NFκB p65was concentrated in the cytoplasm of HBZY-1cells, while the red fluorescence of NFκB p65was concentrated in the nucleus of HBZY-1cells in HG group. The images of red fluorescence of NFκB p65were similar between HG+2uMBPS group and HG+PDTC group. Compared with HG group, the red fluorescence of NFκB p65was reduced in the nucleus in HG+2uMBPS and HG+PDTC groups, which was mainly displayed in the cytoplasm of HBZY-1cells.
[Conclusion]
1. BPS inhibited MCP-1protein and mRNA expressions induced by high glucose in HBZY-1cells.
2. BPS could inhibit NFκB p65subunit nuclear translocation in HBZY-1cells induced by high glucose, thereby inhibiting the activation of NFκB induced by high glucose, which was the potential mechmism of BPS in the regulation of MCP-1synthesis in HBZY-1cells culture in high glucose environment.
Chapter Three
Effects of PPARβ/δ on Beraprost Sodium pretection rat mesangial cells in high glucose enviroment
[Objectives]
1. To investigate into the effect of BPS on PPARβ/δ protein expression in HBZY-1cells cultured in HG environment.
2. To investigate into the effect of BPS on PPARβ/δ mRNA expression in HBZY-1 cells cultured in HG environment.
3. To explore whether PPARβ/δ was involved in BPS inhibition of TGFβ1, FN and MMP-2expressions induced by HG in HBZY-1cells.
4. To explore whether PPARβ/δ was involved in BPS inhibition of p38and ERK MAPK activation induced by HG in HBZY-1cells.
5. To explore whether PPARβ/δ was involved in BPS inhibition of MCP-1expression induced by HG in HBZY-1cells.
6. To explore whether PPARβ/δ was involved in BPS inhibition of NFκB-DNA binding activity induced by HG in HBZY-1cells.
[Method]
1. HBZY-1cells were grouped according to different intervention: Normal glucose (NG) group, High glucose (HG) group, HG+2uMBPS groups, HG+2uMBPS+GSK0660(5uM) groups, and HG+GSK0660group.
2. Protein expression of PPARβ/δ was detected by Western blots.
3. The relative mRNA expression of PPARβ/δ was detected by real-time PCR.
4. Elisa assaay:to investigate into the protein expressions of TGFβ1, FN, MMP-2and MCP-1in the cell culture supernatants.
5. Expressions of p38and ERK MAPK phosphorylated protein were analysed by Western blots.
6. EMSA:to detect the NFκB-DNA binding activtity.
7. Statistical analysis:Statistical analyses were carried out with the SPSS13.0, all data are present as means±standard deviation (x±s). Statistical significance of differences among groups was evaluated by one-way ANOVA. Multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed, and multiple comparisons was analyzed by Dunnett's T3. P≤0.05was considered as significant.
[Results]
1. Protein expression of PPARβ/δ:after24h culture, compared with NG group, PPARβ/δ protein expression of HBZY-1cells in HG group was significantly decreased (P=0.028). Compared with HG group, PPARβ/δ protein expression in all BPS groups were significantly increased (P=0.009). PPARβ/δ protein expression in HG+2uMBPS group was the highest among the BPS groups (P<0.001vs. HG+0.5uMBPS), and PPARβ/δ protein expression in HG+5uMBPS group was lower than that in HG+2uMBPS group (P=0.017vs. HG+2uMBPS).
2. Relative mRNA expression of PPARβ/δ:After12h cultures, compared with NG group, the relative mRNA expressions of PPARβ/δ in HG group were significantly decreased (0.69±0.02vs.1.00±0.00,95%CI for Mean [0.64,0.73]). Compared with HG group, the relative mRNA expressions of PPARβ/δ in all BPS groups were significantly increased (P<0.001). The relative mRNA expression of PPARβ/δ in HG+2uMBPS group was the highest among the BPS groups (P<0.001vs. HG+0.5uMBPS), and the relative mRNA expression of PPARβ/δ in HG+5uMBPS group was lower than that in HG+2uMBPS group (P<0.001vs. HG+2uMBPS).
3. Protein expression of TGFβ1:after24h culture, TGFβ1protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. TGFβ1protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.001). TGFβ1protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.049). There was no difference between HG+GSK0660group and HG group (P=0.621).
4. Protein expression of FN:after24h culture, FN protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. FN protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.001). FN protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.047). There was no difference between HG+GSK0660group and HG group (P=0.592).
5. Protein expression of MMP-2:after24h culture, MMP-2protein expression of HBZY-1cells in HG group was significantly decreased (P<0.001) compared with NG group. MMP-2protein expression in HG+2uMBPS group was significantly increased compared with HG group (P=0.017). There was no difference between HG+2uMBPS+GSK0660group and HG+2uMBPS group (P=0.956), and no difference between HG+GSK0660group and HG group (P=0.958).
6. p38MAPK protein phosphorylation:after15min stimulation, expression of phosphorylated p38MAPK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expression of phosphorylated p38MAPK protein in HG+2uMBPS group was significantly decreased (P<0.001). Compared with HG+2uMBPS group, expression of phosphorylated p38MAPK protein in HG+2uMBPS+GSK0660group was significantly increased (P<0.001). There was no difference between HG+GSK0660group and HG group (P=0.061).
7. ERK protein phosphorylation:after15min stimulation, expression of phosphorylated ERK protein in HG group was significantly increased compared with NG group (P<0.001). Compared with HG group, expression of phosphorylated ERK protein in HG+2uMBPS group was significantly decreased (P<0.001). Compared with HG+2uMBPS group, expression of phosphorylated ERK in HG+2uMBPS+GSK0660group was significantly increased (P<0.001). There was no difference between HG+GSK0660group and HG group (P=0.093).
8. Protein expression of MCP-1:after24h culture, MCP-1protein expression of HBZY-1cells in HG group was significantly increased (P<0.001) compared with NG group. MCP-1protein expression in HG+2uMBPS group was significantly decreased compared with HG group (P=0.004). MCP-1protein expression in HG+2uMBPS+GSK0660group was significantly increased compared with HG+2uMBPS group (P=0.042). There was no difference between HG+GSK0660group and HG group (P=0.847).
9. NFκB-DNA binding activity:There was no NFκB-DNA binding band detected in NG group. Strong band was detected in HG group, which indicated that HG could induce NFκB bind to DNA by NFκB activation. The grey value of NFκB-DNA binding band was reduced in HG+PDTC group compared with HG group, because PDTC could inhibit the activation of NFκB. The grey value of NFkB-DNA binding band in HG+2uMBPS group was also decreased compared with HG group, which indicated that2uMBPS could reduce the NFκB-DNA binding activity. In HG+2uMBPS+GSK0660group, the grey value of NFκB-DNA binding band was increased compared with HG+2uMBPS group.
[Conclusion]
1. BPS could increase PPARβ/δ protein and mRNA expression in HBZY-1cells cultured under high glucose conditions.
2. GSK0660reversed the inhibiton of BPS on TGFβ1and FN protein expression in HBZY-1cells induced by high glucose, but no changes in the MMP-2protein expression, suggesting that BPS could regluate TGFβ1and FN expression through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
3. GSK0660reversed the inhibiton of BPS on p38and ERK MAPK activation induced by high glucose in HBZY-1cells, suggesting that BPS could regluate p38and ERK MAPK pathway through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
4. GSK0660reversed the inhibiton of BPS on MCP-1protien expression in HBZY-1cells induced by high glucose, suggesting that BPS could regluate MCP-1expression through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
5. BPS could inhibit the NFκB-DNA binding activity induced by high glucose in HBZY-1cells, while GSK0660could reverse this effect. It was indicated that BPS could regluate NFκB-DNA binding activity through PPARβ/δ receptor in HBZY-1cells cultured under high glucose conditions.
引文
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[3]. Scindia, Y M, U S Deshmukh and H Bagavant, Mesangial pathology in glomerular disease:targets for therapeutic intervention. Adv Drug Deliv Rev, 2010.62(14):p.1337-1343.
[4]. Qian, Y, E Feldman and S Pennathur, et al., From fibrosis to sclerosis: mechanisms of glomerulosclerosis in diabetic nephropathy. Diabetes,2008. 57(6):p.1439-1445.
[5]. Derynck, R and Y E Zhang, Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature,2003.425(6958):p.577-584.
[6]. Lan, H Y, Diverse roles of TGF-beta/Smads in renal fibrosis and inflammation. Int J Biol Sci,2011.7(7):p.1056-1067.
[7]. Schnaper, H W, T Hayashida and S C Hubchak, et al., TGF-beta signal transduction and mesangial cell fibrogenesis. Am J Physiol Renal Physiol,2003, 284(2):p. F243-F252.
[8]. Heldin, C H, K Miyazono and D P Ten, TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature,1997.390(6659):p. 465-471.
[9]. Flanders, K C, Smad3 as a mediator of the fibrotic response. Int J Exp Pathol, 2004.85(2):p.47-64.
[10].Li, J H, X R Huang and H J Zhu, et al., Role of TGF-beta signaling in extracellular matrix production under high glucose conditions. Kidney Int, 2003.63(6):p.2010-2019.
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