表没食子儿茶素没食子酸酯对糖尿病大鼠肾脏细胞外调节蛋白激酶活性的影响

目的 观察表没食子儿茶素没食子酸酯（EGCG）对糖尿病大鼠肾脏细胞外调节蛋白激酶（ERK）活性的影响。方法 采用链脲佐菌素腹腔注射建立糖尿病大鼠模型，实验分3组：正常对照组、糖尿病模型组和EGCG干预组。EGCG干预组于模型成功后1周给予EGCG5mg·kg^-1·d^-1腹腔注射。以免疫组织化学方法检测肾组织磷酸化ERK（p-ERK）的表达。大鼠肾系膜细胞株分组：正常对照组（NG，葡萄糖5mmol／L），高糖组（HG，葡萄糖30mmol／L），HG＋EGCG1组（100μg／L），HG＋EGCG2组（200μg／L），HG＋EGCG4组（400μg／L），甘露醇组（5mmol／L葡萄糖＋25mmol／L甘露醇）。用四甲基偶氮唑蓝比色（MTT）法检测细胞增殖活性；常规生化分析系膜细胞氧化应激状态；Western印迹检测ERK、p-ERK和p27蛋白表达水平。结果 EGCG呈剂量和时间依赖方式抑制高糖时系膜细胞的增殖活性及抗氧化作用。EGCG干预后糖尿病大鼠肾脏p-ERK蛋白免疫组化染色明显减弱。高糖时系膜细胞p-ERK及p27蛋白表达上调，EGCG呈剂量和时间依赖方式下调高糖时p-ERK蛋白的表达；呈剂量依赖方式下调p27蛋白的表达。结论 EGCG能有效改善糖尿病肾损伤程度，其作用机制可能是通过调节ERK活性，抑制p27蛋白表达而减少糖尿病肾病损害。     

高糖对肾小球系膜细胞间通讯功能的影响

目的研究高糖对肾小球系膜细胞间隙连接蛋白（connexin 43）表达和细胞间通讯功能的影响。方法分离培养大鼠肾小球系膜细胞，调整培养液葡萄糖浓度为以下3组：正常葡萄糖组（5．5mmol／L葡萄糖）、高糖组（30mmol／L葡萄糖）和渗透压对照组（5．5mmol／L葡萄糖加24．5mmol／L甘露醇），于37℃5％CO2条件下培养24、48h后．利用激光共聚焦显微镜和荧光漂白恢复（FRAP）技术检测细胞间通讯功能，并应用Northern印迹和细胞免疫化学、Western印迹方法检测connexin 43 mRNA和蛋白质表达，比较3组之间的差异。结果正常葡萄糖浓度培养下系膜细胞表达丰富的connexin 43，细胞间通讯功能良好。高糖培养的系膜细胞细胞间通讯功能下降，荧光淬灭后的恢复比例和速度显著低于正常糖组（P〈0．05）。同时高糖环境下培养的系膜细胞connexin 43 mRNA和蛋白质表达均较正常糖组显著下降（P〈0．05）。渗透压对照组与正常糖组之间差异无统计学意义（P〉0．05）。结论高糖可抑制connexin 43的基因和蛋白质表达及细胞间通讯功能，可能是糖尿病肾病系膜细胞表型和功能异常的重要原因之一。     

megsin基因转染对高糖环境中系膜细胞胞外调节蛋白激酶表达的影响

目的 观察megsin基因转染对高糖环境中小鼠肾小球系膜细胞血小板源性生长因子BB（PDGF-BB）、磷酸化胞外调节蛋白激酶1/2（pERK1/2）、转化生长因子β1（TGF-β1）的表达及Ⅳ型胶原水平的影响;探讨megsin基因对细胞外信号调节激酶（ERK）通路的作用。 方法 将小鼠肾小球系膜细胞分为7组：低糖组（A组,5.5 mmol/L）、高糖组（B组,30 mmol/L）、高糖+空质粒组（C组）、高糖+megsin质粒组（D组）、高糖+megsin质粒+ERK通路抑制剂组（E组）、高糖+ megsin siRNA组（F组）和低糖+甘露醇组（24.5 mmol/L甘露醇,G组）。分别在培养12、24、48 h后,采用Western印迹法测定各组系膜细胞megsin、PDGF-BB、pERK1/2、TGF-β1的表达,放射免疫测定法（RIA）测定各组细胞上清液中Ⅳ型胶原浓度。 结果 与A组相比,高糖刺激可上调系膜细胞megsin、PDGF-BB、pERK1/2、TGF-β1的表达及上清液Ⅳ型胶原的水平（均P ＜ 0.05）,且有一定的时间依赖性。转染megsin基因可进一步上调高糖环境下系膜细胞PDGF-BB、pERK1/2、TGF-β1、Ⅳ型胶原的表达（均P ＜ 0.05）。应用ERK通路抑制剂后,与D组相比系膜细胞megsin 和PDGF-BB的表达无明显变化（均P ＞ 0.05）,而pERK1/2、TGF-β1及Ⅳ型胶原的表达则明显降低（均P ＜ 0.05）。转染megsin siRNA对系膜细胞megsin基因进行干扰后,系膜细胞PDGF-BB、pERK1/2、TGF-β1的表达及Ⅳ型胶原的含量较B组下调（均P ＜ 0.05）。 结论 高糖环境下,转染megsin基因可使小鼠系膜细胞megsin、PDGF-BB表达增高,其可能部分通过ERK通路使TGF-β1表达上调及Ⅳ型胶原合成与分泌增加。megsin siRNA干扰可使上述指标下调,megsin基因可能在糖尿病肾病发病中起重要作用。     

缬沙坦对高糖环境下系膜细胞GLUT1表达的影响

目的：探讨葡萄糖转运蛋白1（GLUT1）在糖尿病肾病发生、发展中的作用以及缬沙坦的肾脏保护机制。方法：体外培养大鼠肾小球系膜细胞,设正常对照组、甘露醇组、高糖非处理组及高糖＋缬沙坦组,Westernblot法检测GLUT1蛋白水平,RT-PCR法检测转化生长因子-β1（TGF-β1）mRNA水平,放免法测定细胞上清中Ⅳ型胶原含量。结果：高糖非处理组GLUT1蛋白、TGF-β1mRNA及细胞上清液Ⅳ型胶原含量均高于正常对照组（P〈0.01,P〈0.05）,缬沙坦干预后上述各指标明显降低（P〈0.01）。结论：高糖刺激使肾小球系膜细胞GLUT1蛋白表达增高;高糖状态下,缬沙坦具有不依赖于其调节肾脏血流动力学改变的肾脏保护作用,该作用可能与其抑制系膜细胞GLUT1过表达,减少TGF-β1与Ⅳ型胶原合成有关。     

高糖培养下系膜细胞JAK2／STAT3信号转导通路活性变化及与活性氧簇作用的研究

目的：通过观察高糖培养条件下大鼠肾小球系膜细胞P-STAT3的表达变化,探讨糖尿病状态下JAK2／STAT3途径活性的变化及该通路与活性氧簇（ROS之间的相互作用。方法：用传代培养的大鼠肾小球系膜细胞同步化后分组：（1）正常糖浓度组（含5．5mmol／L）,高糖浓度组（25mmol／L）,甘露醇组（5．5mmot／L糖＋19．5mmol甘露醇）,正常糖＋AG-490（浓度10／μmol／L）组,高糖＋AG-490（浓度10μmol／L）组。继续观察培养后用Westem Blot及细胞免疫化学方法检测系膜细胞STAT3、P-STAT3表达的变化。（2）正常糖浓度组（N）,高糖浓度组（H）,甘露醇组（M）,正常糖＋Apocynin组（N＋A,Apocynin浓度为100μmol／L）,高糖＋Apocynin组（H＋A,Atx）cynin浓度为100μmol／L）,收集上清液,用比色法检测系膜细胞ROS水平。（3）NADPH氧化酶抑制剂Apocynin预处理,分组同（2）,Apocynin提前1h加入,与正常糖或高糖同时培养后,用Westem Blot方法检测系膜细胞P-STAT3表达。结果：（1）高糖培养大鼠肾小球系膜细胞P-STAT3的表达较正常糖浓度组明显升高,甘露醇组与正常糖浓度组相比差异无统计学意义;各组之间STAT3表达差异无统计学意义。（2）高糖条件下,ROS产生明显升高,NADPH氧化酶抑制剂Apocynin可明显降低ROS的产生。（3）高糖条件下,Apocynin经预处理,在正常糖浓度和高糖浓度同时培养48h后,正常糖浓度组和正常糖＋Apocynin组对比P-NTAT3的表达差异无明显区别;高糖十Apocynin组较正常糖浓度组有明显区别,但与高糖组相比明显降低。结论：高糖通过磷酸化方式激活大鼠肾小球系膜细胞JAK2／STAT3信号转导通路;高糖作用下,肾小球系膜细胞ROS产生增加,并具有时间依赖性;高糖状态下ROS可激活肾小球系膜细胞的JAK2／STAL信号传导通路,证明ROS可能参与糖尿病肾病的发生和发展过程。     

Cbl相关蛋白在高糖刺激下肾小球系膜细胞中的作用

目的：探讨高糖对肾小球系膜细胞（GMC）Cbl相关蛋白（CAP）mRNA表达的影响,以及糖尿病肾病发生发展中葡萄糖转运蛋白4（GLUT4）的下游信号分子CAP的重要作用。方法：系膜细胞株分为8组,正常对照组,生理浓度胰岛素组（10^-9mol／L）,低浓度胰岛素组（10^-9mol／L）,高浓度胰岛素组（10^-6mol／L）,高糖组（30mmol／L）,甘露醇组,高糖加高浓度胰岛素组,高糖加生理浓度胰岛素组。采用RT—PCR法,观察高糖刺激与不同浓度胰岛素作用下,系膜细胞中CAPmRNA的表达及其变化。结果：正常对照组系膜细胞中（CAPmRNA有一定表达。高糖组CAPmRNA表达明显下降;低浓度胰岛素组和高浓度胰岛素组分别为对照组的1．91倍和2．15倍（P〈0．01）;高糖加入高浓度胰岛素组CAPmRNA表达为单纯高糖组的2．14倍（P〈0．01）。结论：（1）正常系膜细胞中CAPmRNA有一定表达;（2）高糖可抑制CAPmRNA表达;（3）胰岛素能部分拮抗高糖导致系膜细胞中CAPmRNA表达的下调作用;（4）CAP在糖尿病肾病发生发展过程中是其重要因子之一。     

蛋白激酶C在高糖诱导近端小管上皮细胞细胞外基质及转化生长因子β1高表达中的作用

目的：探讨高糖作用下近端肾小管上皮细胞蛋白激酶C（PKC）活性的变化,以及PKC 激活对近端肾小管上皮细胞外细胞基质（ECM）及转化生长因子β1(TGF-β1）表达的调控作用。方法：采用LLC－PK1细胞株,将细胞分为正常对照组NG(5.5mmol/L D－葡萄糖）、高糖组HG（25mmol/L D-葡萄糖）、高渗组HM（25mmol/L甘露醇）、NG＋PKC抑制剂（PKCI）组（10μmol/L chelerythrine chloride)、HG＋PKCI组、HM＋PKCI组。分别检测各组细胞PKC活性,并运用原位杂交和免疫细胞化学法检测各组Ⅳ胶原（Co1Ⅳ）、纤连蛋白（FN）及TGF-β1mRNA和蛋白表达。结果：HG组细胞胞膜PKC活性较NG组升高3．3倍。HG组细胞Co1Ⅳ、FN及TGF－β1mRNA和蛋白水平均较NG组显著升高,HM组无此变化。高糖导致的ECM和TGF－β1高表达可被PKC抑制剂chelerythrine chloride所阻断。结论：由高糖诱导的近端小管上皮细胞ECM和TGF－β1高表达是通过激活PKC通路所介导。     

高糖对系膜细胞胞外调节蛋白激酶表达及细胞外基质合成的影响

目的：探讨高糖环境下系膜细胞中胞外调节蛋白激酶（ERK）转导途径活性和转化生长因子-β1（TGF-β1）mRNA表达的变化。方法：分离培养大鼠肾脏系膜细胞,调整培养液浓度为以下3组,即正常糖组、高糖组、甘露醇组。分别予干预12h、24h、48h后用免疫细胞化学法和Western-blot法对系膜细胞中磷酸化ERK1/2（pERK1/2）的表达进行定位、定性及半定量分析,用RT-PCR法检测细胞中TGF-β1 mRNA的表达,放免法测定各组细胞上清中Ⅳ型胶原的含量。结果：高糖组系膜细胞中pERK1/2蛋白的表达较正常糖组明显增高并由胞浆向胞核内转移,随培养时间延长其表达呈上升趋势（P〈0.01）,高糖组TGF-β1 mRNA的表达及细胞上清液Ⅳ型胶原的含量均高于正常糖组（P〈0.01）,甘露醇组上述指标较正常糖组略有升高,但两组间无统计学意义（P〉0.05）。结论：高糖环境下系膜细胞中ERK信号转导通路被激活,TGF-β1 mRNA表达上调,可能是糖尿病肾病系膜细胞受损,肾小球硬化的机制之一。     

高糖对小鼠肾小球足细胞podocalyxin mRNA表达的影响

目的探讨高糖对小鼠肾小球足细胞podocalyxin mRNA表达的影响。方法以体外培养的小鼠足细胞为研究对象,将细胞分组,即正常对照组（NG组）：D-葡萄糖5．56mmol／L;高糖培养组（HG组）;D-葡萄糖25．0mmol／L。应用逆转录-PCR技术,检测高糖培养不同时间点对足细胞表达podocalyxin mRNA的影响;Western blot印迹分析检测细胞转录因子WT-1蛋白变化。结果①逆转录-PCR技术显示随着高糖培养时间增加,HG组肾小球足细胞与WG组比较、以及HG组（第6、12、24h）组间比较,podocalyxin mRNA表达均减少（P〈0．05）。②培养的足细胞表达基础水平的WT-1蛋白,但在经高糖培养2h后其表达即开始下降（P〈0．05）。结论高糖培养使小鼠肾小球足细胞podocalyxin mRNA和转录因子WT-1蛋白表达下降。     

高糖通过蛋白激酶C改变肾小球系膜细胞的间隙连接与细胞表型

目的观察高糖环境下，蛋白激酶C（PKC）活性的变化对肾小球系膜细胞间隙连接与细胞表型的影响。方法将体外培养的大鼠肾小球系膜细胞分为低糖组、高糖组、高糖＋PKC抑制剂十字孢碱（SP）组，测定细胞间隙连接蛋白-43（connexin 43）、α-平滑肌肌动蛋白（α—SMA）的表达。结果①与低糖组相比，高糖组细胞PKC活性、mSMA mRNA表达增高，connexin 43 mRNA表达下降，差异有统计学意义（P〈0．05）；②与高糖组相比，高糖＋SP组细胞PKC活性、α—SMA mRNA表达下降，connexin 43 mRNA表达增高，差异有统计学意义（P〈0．05）。结论高糖通过PKC改变肾小球系膜细胞的间隙连接与细胞表型。     

激活素A在高糖诱导的人肾小管上皮细胞中的表达变化

目的 通过体外高糖刺激的人近端肾小管上皮细胞株(HK-2),探讨激活素A(ACT A)的表达变化及卵泡抑素(FS)的干预作用.方法 HK-2细胞生长于DMEM培养基,待细胞生长至亚融合状态时,更换为无血清DMEM培养基使细胞生长同步化24h,然后将细胞分为以下5组:正常对照组(NG,5.5 mmol/L葡萄糖)、甘露醇对照组(MG,5.5 mmol/L葡萄糖+25 mmol/L甘露醇)、高糖组(HG,25 mmol/L葡萄糖)、HG+FS100组(25 mmol/L葡萄糖+100 μg/L FS)、HG+FS500组(25 mmol/L葡萄糖+500 μg/L FS).12、24、48 h后收集各组细胞及细胞上清液.Western印迹法检测各组细胞ACT A和p-Smad2/3的表达;ELISA检测各组细胞上清液转化生长因子β(TGF-β)和纤连蛋白(FN)的含量.结果 NG组细胞ACT A有微量表达,而HG组表达显著增加(P＜0.05),呈时间依赖性.与HG组相比,FS干预组ACT A表达显著减少(P＜0.05),呈剂量依赖性.与NG组相比,p-Smad2/3在HG组培养24 h后表达显著增加(P＜0.05);FS可抑制p-Smad2/3的高表达(P＜0.05).ELISA检测结果显示,与NG组相比,HG组培养12 h后TGF-β表达即显著增加(P＜0.05),呈时间依赖性,FS干预对高糖诱导的TGF-3高表达没有影响.与NG组相比,HG组培养24h后FN表达显著增高(P＜0.01),FS对此有明显抑制作用,呈剂量依赖性(P＜0.01).结论 高糖能刺激HK-2细胞ACT A表达增加,从而促进肾小管上皮细胞FN的合成;FS可通过阻断ACT A的活化,减轻肾小管上皮细胞FN的合成.     

Effects of benazepril on the expressions of intergrin-linked kinase and α-smooth muscle actin in glomerular mesangial cells exposed to high glucose

Objective To investigate the effect of benazepril on intergrin-linked kinase (ILK) and α-smooth muscle actin (α-SMA) expression in glomerular mesangial cells induced by high-glucose. Methods The mesangial cells from SD rat (HBZY-1) were cultured conventionally and randomly divided into four groups: normal glucose (D-glucose 5.5 mmol/L, group NG), mannitol-treated group (mannitol 20 mmol/L, group MG), high glucose (D-glucose 30 mmol/L, group HG), Benazepril-treated high glucose group (D-glucose 30 mmol/L＋Benazepril 10 μmol/L, group ACEI). Cells from NG, MG, HG, ACEI gronps were harvested after 3, 6, 12, 24, 48 and 72 hours of treatment respectively. The mRNA expressions of ILK and α-SMA were detected by RT-PCR. The protein levels of ILK and α-SMA were detected by Western blotting and immunofluorescence. Results The expressions of ILK mRNA and protein in HG group were significantly increased compared with those in NG group (all P＜0.05). The increased expressions of ILK and α-SMA in HG group were time-dependent and the expression reached the peak at 48 h (ILK, P＜0.05) or 72 h (α-SMA, P＜0.01). The expressions of ILK and α-SMA in ACEI group were lower than those in HG group (all P＜0.01), but failed to rescue to the same level as those in NG. There was no significant differences of ILK expressions between MG group and NG group at the same time point (P＞0.05). The expressions of α-SMA mRNA and protein in MG were higher than that in NG (P＜0.05), which suggest that high osmotic pressure could cause the increasing of α-SMA. Conclusions Benazepril can decrease the expressions of ILK and α-SMA to inhibit the process of fibrosis in DN and mediate the phenotypic transformation of glomerular mesangial cells. The phenotypic transformation of glomerular mesangial cells in glucose may also depend on high osmotic pressure in DN.     

虫草素通过下调TGF-β抑制高糖诱导的大鼠肾小管上皮细胞转分化

目的：探讨虫草素对高糖诱导的大鼠肾小管上皮细胞转分化的影响。方法：体外培养大鼠近端肾小管上皮细胞株（NRK52E细胞株）,分为正常对照组（葡萄糖5.5 mmol/L,NG组）、高糖组（葡萄糖30 mmol/L,HG组）、高糖＋虫草素组（葡萄糖30 mmol/L＋虫草素10μg/ml,HG＋C组）。分别于刺激12 h,24 h,48 h后收集细胞。应用定量RT-PCR测定NRK52E TGF-β,E-cadherin,α-SMA mRNA的表达;Western印迹方法检测TGF-β、E-cadherin、α-SMA蛋白的表达。结果：高糖刺激后NRK52E细胞的TGF-β和α-SMA mRNA及蛋白表达明显高于正常糖组（P〈0.01）,而虫草素组TGF-β和α-SMA mRNA及蛋白表达显著低于高糖组（P〈0.05）;高糖诱导的NRK52E细胞E-cadherin mRNA及蛋白水平明显降低（P〈0.01）;而虫草素组NRK52E细胞E-cadherin mRNA及蛋白水平显著高于高糖组（P〈0.05）。结论：虫草素可以明显抑制高糖诱导的大鼠肾小管上皮细胞转分化,其机制可能是通过下调TGF-β实现。     

MicroRNA-148b influences high glucose-induced endoplasmic reticulum stress in rat mesangial cell by targeting AMPKα1

Objective To observe the expression of microRNA-148b (miR-148b) induced by high glucose in rat mesangial cells, and to explore its effect on its target gene AMP-activated protein kinase α1 (AMPKα1) and extracellular matrix excretion. Methods Rat mesangial cells were divided into 3 groups: normal glucose (NG, 5.5 mmol/L glucose) group, hypertonic (MA, 5.5 mmol/L glucose+ 19.5 mmol/L mannitol) group and high-glucose (HG, 25.0 mmol/L glucose) group. MiR-148b expression was detected by real time PCR. Then miR-148b inhibitor was transfected to rat mesangial cells. Their protein expressions of AMPKα1, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), fibronectin (FN) and collagen Ⅳ were detected by Western blotting. The expression of AMPKα1 mRNA was detected by real time PCR. The expression of collagen Ⅳ was also detected by immunofluorescence. Results Compared with NG group, HG group showed up-regulated miR-148b expression, down-regulated AMPKα1 mRNA and protein expressions, and up-regulated CHOP, GRP78, collagen Ⅳ and FN expressions (all P＜0.05). HG-induced mesangial cells with miR-148b inhibitor had up-regulated AMPKα1 mRNA and protein expressions, and down-regulated CHOP, GRP78, collagen Ⅳ, FN expressions as compared with HG-induced cells without miR-148b inhibitor (all P＜0.05). Conclusions HG can up-regulate miR-148b expression and down-regulate AMPKα1 expression in rat mesangial cells, then activate endoplasmic reticulum stress to induce extracellular matrix excretion. MiR-148b inhibitor up-regulates AMPKα1 expression, inhibits endoplasmic reticulum stress and reduces extracellular matrix excretion.     

Angiotensin II activation of the JAK/STAT pathway in mesangial cells is altered by high glucose

BACKGROUND: Both high glucose (HG) and angiotensin II (Ang II) causes glomerular mesangial cell (GMC) growth and increased synthesis of matrix proteins like collagen IV contributing to diabetic nephropathy. We have recently found that exposure of vascular smooth muscle cells to HG augments the Ang II activation of the growth promoting JAK/STAT pathway. We hypothesized that Ang II activation of the JAK/STAT pathway is altered by HG in GMC, and that this pathway might be linked to the Ang II-induced growth and overproduction of collagen IV in GMC in HG conditions. METHODS: GMC were cultured under normal glucose (NG; 5.5 mmol/L) and HG (25 mmol/L) for 48 hours and stimulated with Ang II (0.1 micromol/L) for various times. GMC lysate was then immunoprecipitated and/or immunoblotted with SHP-1, SHP-2 and phosphospecific JAK2 and STAT antibodies. The HG and Ang II induced growth and collagen IV synthesis studies were performed in GMC transfected with JAK2 antisense or JAK2 sense. GMC growth was monitored via [3H]-thymidine incorporation, and collagen IV synthesis via ELISA. RESULTS: We found that Ang II-induced JAK2, STAT1, STAT3, STAT5A/B and SHP-2 phosphorylations were enhanced by HG, whereas that of SHP-1 was reduced. Ang II-induced growth and collagen IV synthesis also were increased under HG conditions. Transfection of GMC with JAK2 antisense oligonucleotides blocked the Ang II-induced growth and collagen IV synthesis in both NG and HG conditions. CONCLUSION: These results provide evidence that activation of the JAK/STAT pathway by HG or/and Ang II may be of importance in the increased GMC cell growth and collagen IV synthesis that is seen in diabetic nephropathy.     

黄连素通过PI3K/Akt/mTOR信号通路抑制高糖条件下的肾脏系膜细胞增殖

目的：探讨黄连素（berberine,BBR）对高糖条件下的大鼠肾脏系膜细胞 HBZY-1 cells （MCS）增殖的影响及其作用机制。方法将对数生长期细胞分成正常糖处理组（NG 组）,高糖组（HG组）和黄连素组（BBR组）。NG组细胞体系常规培养培养,HG 组细胞体系加入40 mmol/L 葡萄糖,BBR组细胞培养体系中加入40 mmol/L葡萄糖＋黄连素30μmol/L。各组细胞培养48 h 后,利用MTT检测各组HBZY-1细胞增殖;RT-PCR检测各组细胞p85,Akt和mTOR基因表达;West-ern blotting检测细胞中 p85、p-p85、Akt、p-Akt,mTOR 和 Collagen-Ⅳ蛋白的变化。结果 NG 组与HG组增值率分别为（25%±3%）和（75%±5%）,与 NG组增值率比较,HG组肾脏系膜细胞增殖率明显增高（P〈0.05）,p85与 Akt mRNA表达水平和蛋白水平均无明显变化（P＆gt;0.05）,但 p-p85、p-Akt,Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显增加（P〈0.05）;BBR 组增值率为（42%±5%）,与 HG 组比较,BBR 组肾脏系膜细胞增值率明显降低（P〈0.05）,但 p-p85、p-Akt、Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显下降（P〈0.05）,而 p85和Akt mRNA和蛋白表达水平均无统计学差异（P〈0.05）。结论黄连素能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制PI3K/Akt/mTOR信号通路激活。     

Effect of high glucose on the Notch signal pathway in mesangial cells and intervention of the cordyceps sinensis

Objective To investigate the expression of Notch signaling molecules, transforming growth factor-β (TGF-β) and fibronectin (FN) in mesangial cell induced by high glucose, and the underlying mechanism of cordyceps sinensis. Methods Rat glomerular mesangial cells were divided into following groups: normal control group (5.5 mmol/L glucose), hypertonic control group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), DAPT inhibitor group (25.0 mmol/L glucose+1 μmol/L DAPT), cordyceps sinensis intervention group (25.0 mmol/L glucose+10 mg/L cordyceps sinensis). Cell proliferation was detected by MTT. The protein and mRNA expression of Notch signaling molecules (Notch1, Jagged1 and Hes1), TGF-β and FN was detected by Western blotting and real time PCR. Results Compared with normal control group, high glucose induced mesangial cell proliferation, as well as the mRNA and protein expression of Notch1, Jagged1, Hes1, TGF-β1 and FN was up-regulated in high glucose group (all P＜0.05). Compared with that in high-glucose group, DAPT and cordyceps sinensis inhibited high glucose-induced mesangial cell proliferation and down-regulated the mRNA and protein expression of Notch pathway, TGF-β1 and FN (all P＜0.05). Conclusion By inhibiting the abnormal activation of Notch signaling pathway and TGF-β signaling pathway, cordyceps sinensis may alleviate high glucose-induced mesangial cell proliferation and reduce extracellular matrix accumulation, thus protecting kidney.     

高糖对大鼠肾成纤维细胞VEGF和PEDF表达的影响

目的探讨高糖对体外培养的大鼠肾成纤维细胞（NRK）血管内皮生长因子（vascular endothelial growth factor，VEGF）和色素上皮衍生因子（pigment epithelium derived factor，PEDF）表达的影响。方法采用RT-PCR分析方法，测定在不同浓度高糖、不同时间点NRK细胞VEGF与PEDF mRNA的表达。设5．6mmol／L葡萄糖组为正常对照组。结果与正常组相比，加入不同浓度高糖的各组NRK细胞VEGF mRNA的表达明显升高（P〈0．05），PEDF mRNA的表达明显下降（P〈0．05），呈剂量依赖性改变；随着各组作用时间的延长，VEGF mRNA的表达呈上升趋势（P〈0．05），PEDF mRNA的表达呈下降趋势（P〈0．05），呈时间依赖性改变；VEGF与PEDF之间呈明显负相关（r=-0．811，P〈0．05）。结论在高糖环境中，体外培养的NRK细胞VEGF和PEDF表达失衡，PEDF表达下降的同时VEGF表达增加，两者的表达失衡可能在糖尿病肾病（DN）的发生发展中起一定的作用。     

高糖对人肾小管上皮细胞和系膜细胞表达血清和糖皮质激素诱导蛋白激酶1的影响

目的研究高糖作用下人近端肾小管上皮细胞(HKC)和系膜细胞(HMC)中血清和糖皮质激素诱导蛋白激酶1(SGK1)的表达,并初步探讨SGK1在介导高糖致肾细胞(HKC和HMC)过度合成细胞外基质(ECM)中的作用。方法将HKC和HMC细胞分别分为正常对照组(NG组,5.5mmol/LD-葡萄糖)、高糖组(HG组,25mmol/LD-葡萄糖)和渗透浓度对照组(MG组,19.5mmol/L甘露醇和5.5mmol/LD-葡萄糖)。SGK1mRNA水平及蛋白水平的检测分别采用RT-PCR方法和Western印迹方法。培养液中纤连蛋白(FN)水平检测采用ELISA方法。结果HKC和HMC中均存在SGK1基因和蛋白的表达。HMC中SGK1的表达明显高于HKC(P<0.01)。高糖刺激8h后,两种细胞SGK1表达均明显升高(P<0.01);同时,甘露醇也上调HKC和HMCSGK1的表达(P<0.01),但其作用明显弱于高糖(P<0.05)。FN在高糖环境下表达上调,且高峰出现时间滞后于SGK1。结论高糖能促进近端肾小管上皮细胞和系膜细胞SGK1的表达,并可能通过SGK1介导的信号转导途径在糖尿病肾病ECM积聚中发挥重要作用。