自噬和PI3K-Akt-mTOR信号转导通路在缺氧预处理对高糖心肌细胞缺氧/复氧损伤保护中的作用

目的 探讨自噬和磷脂酰肌醇3激酶（phosphatidylinositol 3 hydroxy kinase, PI3K）-蛋白激酶B（protein kinase B, Akt）-雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）信号转导通路在缺氧预处理（hypoxia preconditioning, HPC）对高糖心肌细胞缺氧/复氧（anoxia/reoxygenation, AR）损伤中的作用及机制。 方法 将采用高糖培养基培养72 h的心肌细胞用随机数字表法分为5组：空白对照组（S组）、AR损伤对照组（AR组）、HPC组、渥曼青霉素＋HPC组（Wo＋HPC组）、雷帕霉素＋HPC组（Ra＋HPC组）。采用乳酸脱氢酶（lactate dehydrogenase, LDH）检测试剂盒检测心肌细胞LDH漏出率,Annexin V/PI双染流式细胞术检测心肌细胞凋亡情况,蛋白印迹法检测心肌细胞微管相关蛋白1轻链3（microtubulesas sociated protein light, LC3）-Ⅱ、Beclin-1、mTOR、PI3K表达和磷酸化（phospho, p）蛋白激酶B/蛋白激酶B（p-Akt/Akt）比值。 结果 与AR组比较,Wo＋HPC组LDH漏出率、早期和晚期凋亡率降低（P〈0.05）,LC3-Ⅱ和Beclin-1表达降低（P〈0.05）,mTOR、PI3K表达和p-Akt/Akt比值升高（P〈0.05）。HPC组各指标与AR组比较,差异均无统计学意义（P〉0.05）。与HPC组比较,Wo＋HPC组LDH漏出率、早期和晚期凋亡率降低（P〈0.05）,Beclin-1表达降低（P〈0.05）,mTOR、PI3K表达和p-Akt/Akt比值升高（P〈0.05）。 结论 激活PI3K-Akt-mTOR信号转导通路抑制自噬可明显改善HPC对高糖心肌细胞AR损伤的保护作用。     

过氧化物酶体增殖物激活受体α激动剂非诺贝特抑制高糖条件下的肾脏系膜细胞增殖

目的探讨非诺贝特（Fenofibrate）对高糖条件下大鼠肾脏系膜细胞（mesangial cells,MCs）HBZY-1增殖的影响及其作用机制。方法将对数生长期大鼠肾脏系膜细胞分成正常糖组（NG组）、高糖组（HG组）和非诺贝特组（FN组）。NG组细胞体系常规培养,HG组细胞体系加入40mmol／L葡萄糖,FN组细胞培养体系中加入40mmol／L葡萄糖和非诺贝特100μmol／L。各组细胞培养48h后,利用MTT方法检测细胞增殖;realtime-PCR（实时定量PCR）检测各组细胞PPAR-α、JAK2和STAT3基因表达;Westernblotting检测细胞中过氧化物酶体增殖物激活受体Q激动剂（peroxisome proliferators activedreceptor-α,PPAR-α）、p-PPAR-a、JAK2、STAT3、p-JAK2和p-STAT3蛋白的变化。结果与NG组比较,HG组系膜细胞增殖率明显增高（P〈0．05）,JAK2与STAT3mRNA表达水平无统计学差异（P〉0．05）,PPAR-α、JAK2和STAT3蛋白无明显变化（P〉0．05）,但p-PPAR-α、p-JAK2、p-STAT3蛋白明显增加（P〈0．05）;与HG组比较,FN组系膜细胞增值率和JAK2、p-sTAT3蛋白表达均明显下降（P〈0．05）,PPAR-α、JAK2与STAT3mRNA和JAK2与STAT3mRNA表达均无统计学差异,但p-PPAR-α蛋白表达进一步增加（P〈0．05）。结论PPAR-α激动剂非诺贝特能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制JAK2／STAT3信号激活。     

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。     

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。     

大黄素对胆管癌QBC939细胞生长及磷酸肌醇3激酶/蛋白激酶B/雷帕霉素靶蛋白信号通路表达的影响

目的 观察大黄素在体外对胆管癌QBC939细胞生长的抑制作用及其对磷酸肌醇3激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)信号转导通路的影响.方法 用大黄素作为干预因素,时间效应组以含大黄素的培养液分别培养QBC939细胞不同时间,剂量效应组分别用不同浓度大黄素的培养液与QBC939细胞共培养;检测细胞增殖,逆转录-聚合酶链反应(RT-PCR)检测细胞中B淋巴细胞/白血病-2 (bcl-2) mRNA表达,Western blot检测细胞中bcl-2、Akt、磷酸化Akt (p-Akt)、核因子(NF)-κB、磷酸化NF-κB(p-NF-κB)、mTOR、磷酸化mTOR(p-mTOR)蛋白质的表达.结果 10、20、40、80 μmol/L大黄素对QBC939细胞增殖抑制率分别为17.3％、28.6％、46.5％和66.4％ (P ＜0.05),bcl-2、p-Akt、NF-κB、p-NF-κB、mTOR、p-mTOR表达明显下降,Akt表达无变化.结论 大黄素抑制QBC939细胞增殖,可能通过抑制PI3K/Akt/mTOR信号转导途径.     

miR-155靶向调节PIK3R1对类风湿关节炎大鼠模型PI3K/Akt/mTOR信号通路的影响

目的 探讨miR-155靶向调节PIK3R1对类风湿关节炎（rheumatoid arthritis,RA）大鼠PI3K/Akt/mTOR信号通路的影响。方法 SD大鼠随机分为对照组、模型组、miR-155 agomir组、miR-155 antagomir组、miR-155阴性对照组,诱导RA模型,分组处理后,观察关节症状,检测关节炎指数及后足趾容积;HE染色观察大鼠关节组织病理形态;使用试剂盒检测大鼠关节组织炎性因子IL-6、IL-17及IL-18水平;免疫印迹检测大鼠关节组织PI3K/Akt/mTOR信号通路蛋白表达;qRT-PCR实验检测大鼠关节组织miR-155及PIK3R1 mRNA水平;双荧光素酶报告基因实验检测miR-155对PIK3R1的靶向调控作用。结果 与对照组比较,模型组大鼠关节炎症状明显,关节炎指数、后足趾容积、关节组织炎性因子IL-6、IL-17及IL-18水平、关节组织p-PI3K/PI3K、p-Akt/Akt及p-mTOR/mTOR水平、关节组织miR-155水平明显升高（P＜0.05）,PIK3R1 mRNA水平明显降低（P＜0.05）。与模型组、miR-155阴性对照组比较,miR-155 antagomir组大鼠上述指标得到明显改善（P＜0.05）;miR-155 agomir组与miR-155 antagomir组趋势相反（P＜0.05）。结论 miR-155可靶向下调PIK3R1的表达,激活PI3K/Akt/mTOR信号,加重RA大鼠关节炎症损伤,下调miR-155表达,可抑制PI3K/Akt/mTOR信号激活及炎症反应发生发展,改善关节炎症状。     

高糖通过PI3K-AKT-mTOR信号通路增加足细胞自噬

目的 研究高糖引起足细胞自噬变化及其相关的信号机制.方法 培养的足细胞被分为6组,正常浓度葡萄糖(NG)组、高浓度葡萄糖(HG)组、NG+雷帕霉素(Rap)组、HG+Rap组、NG+LY294002组和HG+LY294002组.观察自噬增强剂Rap和PI3K抑制剂LY294002对高糖条件下培养的足细胞自噬和凋亡的影响.电镜和吖啶橙染色观察细胞内自噬体的形成;Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)和自噬血管基因Beclin-1的表达;通过阻断自噬的信号通路观察磷脂酰肌醇3激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白(PI3K-AKT-mTOR)相关蛋白AKT和mTOR的磷酸化水平的改变.结果 高糖可导致足细胞凋亡增加,促进足细胞内自噬体和自噬相关蛋白表达增加(均P＜ 0.05).与高糖组相比,HG+ Rap组LC3-Ⅱ和Beclin-1的表达增加(均P＜0.05);LY294002部分抑制高糖导致的LC3-Ⅱ和Beclin-1表达增加(均P＜0.05).与高糖组相比,HG+ LY294002组足细胞内AKT磷酸化的水平增加(P＜0.05),mTOR的磷酸化水平降低(P＜0.01);HG+ LY294002组足细胞的AKT和mTOR磷酸化水平较高糖组均降低(均P＜0.05).结论 高糖可促进足细胞的自噬和凋亡,推测高糖诱导的足细胞自噬作用部分通过PI3K-AKT-mTOR信号通路调节实现的.     

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。     

葡萄糖及吡格列酮对大鼠骨髓间充质干细胞脂肪分化的影响

目的观察不同糖浓度下吡格列酮对大鼠骨髓间充质干细胞（bone mesenchymal stem cells,BMSCs）向脂肪细胞分化的影响,探讨葡萄糖和吡格列酮对骨代谢的影响。方法采用体外细胞培养技术自大鼠股骨和胫骨中分离BMSCs进行纯化、培养,在诱导成脂培养基（地塞米松、3-异丁基-1-甲基黄嘌呤（IBMX）、胰岛素）中诱导BMSCs分化为脂肪细胞,实验分为高糖组（葡萄糖浓度为50mmol.L-1）及正糖组（葡萄糖浓度为25mmol.L-1）,两组分别用不同浓度的吡格列酮（0、0.1、1μg.mL-1）干预分化过程各21d,油红O（Oil Red O）染色鉴定分化后的脂肪细胞,光镜下观察橙红色脂滴沉着的细胞比例。实时荧光定量PCR测定脂肪细胞特异性标志LPL、PPARγmRNA的表达。结果诱导分化培养21d后,油红O染色结果显示随糖浓度增加脂肪细胞数量增多,PCR结果显示HG＋NC组比NG＋NC组LPL、PPARγmRNA表达分别增加1.40倍（P〈0.05）和1.63倍（P〈0.05）,在两种糖浓度下,分别加入吡格列酮后脂肪细胞分化均显著增加,数量明显增多,体积明显增大。与NG＋NC组相比,NG＋LP组LPL和PPARγmRNA表达分别增加1.43倍（P〈0.05）和1.50倍（P〈0.05）,NG＋GP组mRNA水平增加更明显,HG＋LP组和HG＋HP与HG＋NC组相比,LPL和PPARγmRNA表达增加更明显。结论高糖会促进BMSCs向脂肪细胞分化,可能为糖尿病性骨质疏松形成的重要机制。吡格列酮有显著增加BMSCs向脂肪细胞方向分化的作用,且随药物剂量的增加,其诱导成脂分化的效应越明显。吡格列酮可能通过诱导BMSCs向脂肪细胞分化增多而向成骨细胞分化减少从而导致成骨作用减弱,这可能是吡格列酮致骨质疏松的重要机制。     

P38 MAPK抑制剂在高糖诱导系膜细胞CTGF表达和基质蛋白分泌中的作用

目的:探讨P38 MAPK抑制剂SB203580在高糖诱导大鼠肾小球系膜细胞对丝裂原活化蛋白激酶磷酸酶-1(mitogen-activated protein kinase phosphatase-1,MKP-1)与结缔组织生长因子(connective tissue growth factor,CTGF)表达和细胞外基质蛋白分泌的作用。方法:体外培养大鼠肾小球系膜细胞分为4组:正常对照组(NG组,5. 5 mmol/L葡萄糖);渗透压对照组(NG+M组,5. 5 mmol/L葡萄糖+24. 5 mmol/L甘露醇);高糖组(HG组,30 mmol/L葡萄糖);高糖+SB203580组(HG+SB203580组,30 mmol/L葡萄糖+10μmol/L SB203580),分别给予不同刺激。48 h收集细胞,分别提取系膜细胞蛋白、RNA及细胞上清液。采用Western blot检测MKP-1、p38 MAPK及磷酸化p38MaPK的表达; RT-PCR检测p38 MAPK、MKP-1、CTGF和FN mRNA的表达; ELISA法测定细胞上清CTGF和纤维黏连蛋白(fibronectin,FN)含量,放免法测定细胞上清液Ⅳ型胶原的含量。结果:与NG组相比,HG组系膜细胞MKP-1表达下调,p38 MAPK蛋白表达无明显差别,但磷酸化的p38 MAPK表达明显升高,MKP-1 mRNA表达下降,p38 MAPK、CTGF和FN mRNA的表达增加,细胞上清液中CTGF、FN和Ⅳ型胶原含量增加;与HG组相比,HG+SB203580组MKP-1表达升高,p38 MAPK蛋白表达无明显差别,但磷酸化的p38MAPK表达明显下降,p38 MAPK、CTGF和FN mRNA的表达下降,系膜细胞上清液中CTGF、FN和Ⅳ型胶原含量下降。结论:p38 MAPK抑制剂SB203580通过增强MKP-1的表达,增强p38 MAPK去磷酸化,使p38 MAPK活性下降,从而阻断CTGF的合成和细胞外基质蛋白表达,在糖尿病肾病细胞外基质重构中发挥保护作用。     

Abated microRNA-21 attenuates high glucose-induced autophagy inhibition in rat mesangial cells by PTEN/Akt/mTOR pathway

Objective To investigate the effects of abated microRNA-21 (miRNA-21) on phosphatase and tensin homologue on chromosome ten protein (PTEN) and PI3K/Akt/mTOR pathway, as well as their further influence on the autophagy in high glucose (HG, 25.0 mmol/L) induced rat glomerular mesangial cells. Methods MiRNA-21 inhibitor and negative control were transfected by liposome 2000 into rat glomerular mesangial cells (HBZY-1). The cells were divided into normal glucose (5.5 mmol/L) group, normal glucose+negative control group, normal glucose+miRNA-21 inhibitor group, HG group, HG+negative control group and HG+miRNA-21 inhibitor group. Cell proliferation and hypertrophy were assayed by MTT and the ratio of total protein to cell number respectively. The miRNA-21 expression was detected using real time PCR. The expressions of PTEN/Akt/mTOR signaling signatures, autophagy-associated protein (p62 and LC3 Ⅱ) and collagen Ⅰ was detected by Western blotting and real time PCR. Autophagosomes were observed using electron microscopy. Results Compared with those in normal glucose group, in HG group cells had hypertrophy and proliferation, up-regulated miRNA-21 expression, and down-regulated PTEN protein and mRNA expressions (all P＜0.01). Also there were and up-regulated p-Akt, p-mTOR, p62 and collagen Ⅰ expression, and lower LC3 Ⅱ expression and autophagosomes (all P＜0.01). Further, compared with those in HG group, cells hypertrophy and proliferation in HG+miRNA-21 inhibitor group were reduced, expressions of p-Akt, p-mTOR, p62 and collagen Ⅰ were down-regulated, while expressions of PTEN and LC3 Ⅱ and autophagosomes were up-regulated (all P＜0.01). Conclusions MiRNA-21 inhibitor up-regulates PTEN expression, which inhibits the activation of Akt/mTOR signaling pathway, ameliorates cell hypertrophy, proliferation and enhances autophagy to reduce extracellular matrix accumulation.     

缬沙坦对高糖上调系膜细胞细胞外调节蛋白激酶表达的影响

目的观察在高糖刺激下，系膜细胞细胞外调节蛋白激酶（ERKI／2）的活性变化以及缬沙坦对其影响，探讨缬沙坦保护肾脏作用的可能机制。方法原代培养大鼠肾脏系膜细胞，随机分为4组：低糖组（NG，d-葡萄糖5．5mmol／L）、高糖组（HG，d-葡萄糖30mmol／L）、甘露醇组（MG，d-葡萄糖5．5mmol／L＋甘露醇24．5mmol／L）和缬沙坦组（HG＋Val，d-葡萄糖30mmol／L＋缬沙坦10μmol／L）。用免疫细胞化学法及Western印迹法对系膜细胞中磷酸化ERK1／2（p-ERK1／2）的表达进行定位及半定量分析；RT—PCR法检测细胞中TGF-β1 mRNA的表达；放射免疫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P-ERK1／2蛋白的表达较低糖组明显增高，并由胞质向胞核内转移，呈时间依赖方式（P〈0．01）；TGF-β1 mRNA及细胞上清液中Ⅳ型胶原水平均高于低糖组（P〈0．01）。而缬沙坦组上述指标均较同时相点高糖组显著降低，差异有统计学意义（P〈0．01）。甘露醇组与低糖组各指标间差异均无统计学意义。结论高糖可显著激活系膜细胞ERK信号通路，缬沙坦可抑制高糖的激活作用。     

乌索酸改善高糖诱导系膜细胞损伤的机制

目的 探讨乌索酸能否通过抑制高糖状态下系膜细胞内miRNA-21的过表达,上调其靶基因PTEN的表达,抑制磷脂酰肌醇-3-激酶(PI3K)-Akt-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路的激活,诱导自噬,减少细胞外基质堆积,发挥其肾脏保护作用.方法 高糖培养大鼠肾小球系膜细胞,以PI3K抑制剂LY294002以及不同剂量乌索酸进行干预,应用甲基噻唑基四唑(MTT)法观察细胞增殖能力,总蛋白/总细胞数测定细胞肥大,Western印迹和实时定量PCR检测PTEN-PI3K-Akt-mTOR信号通路活性、Ⅰ型胶原及自噬标志物.透射电镜观察自噬体的形成.结果 与正常对照组相比,高糖培养的系膜细胞出现显著的肥大、增殖,细胞内miRNA-21表达明显上调,PTEN蛋白及mRNA的表达明显下调,p85PI3K、磷酸化(p)-Akt、p-mTOR、Ⅰ型胶原、p62/SQSTMI表达明显增加,LC3 II表达明显降低,差异均有统计学意义(均P＜ 0.01).与高糖组相比,乌索酸干预组及LY294002组细胞肥大、增殖程度均明显降低,p85PI3K、p-Akt、p-mTOR、Ⅰ型胶原、p62/SQSTMI的表达均明显降低,LC3 II表达明显升高,差异均有统计学意义(均P＜ 0.01);但LY294002组细胞内miRNA-21和PTEN的表达与高糖组差异无统计学意义,而乌索酸干预组细胞内miRNA-21的表达明显下调,PTEN的表达明显上调,差异均有统计学意义(均P＜ 0.01).结论 乌索酸可能通过抑制高糖培养系膜细胞内miRNA-21的过表达,上调PTEN表达,抑制PI3K-Akt-mTOR信号通路异常活化,增强自噬从而减少细胞外基质堆积,减轻细胞的肥大、增殖.     

二甲双胍调控PI3K/Akt/mTOR通路减轻糖皮质激素诱导的成骨细胞凋亡

目的探讨二甲双胍(Met)调控磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对糖皮质激素地塞米松(Dex)诱导的成骨细胞凋亡的影响。方法将体外培养的小鼠胚胎成骨细胞前体细胞MC3T3-E1分为对照组(正常培养)、Dex组(以Dex处理)、Met组(以Dex和Met共同处理)、Met+IGF-1组(以Dex、Met和PI3K/Akt/m TOR通路活化剂IGF-1共同处理)、Met+NVP-BEZ235组(以Dex、Met和PI3K/Akt/m TOR通路抑制剂NVP-BEZ235共同处理),采用免疫印迹法(WB)检测MC3T3-E1细胞中PI3K、Akt、磷酸化(p)-Akt、m TOR和p-mTOR蛋白表达水平,通过噻唑蓝(MTT)法检测MC3T3-E1细胞存活率、流式细胞术检测MC3T3-E1细胞凋亡率、实时荧光定量PCR检测MC3T3-E1细胞中Bcl-2和Bax mRNA表达水平、Caspase-3活性测定试剂盒检测MC3T3-E1细胞Caspase-3活性、JC-1探针检测MC3T3-E1细胞线粒体膜电位变化。结果与对照组比较,Dex组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显降低,而细胞凋亡率、Bax mRNA表达水平和Caspase-3活性均明显升高(P0.05);与Dex组比较,Met组细胞中PI3K、p-Akt、p-mTOR蛋白表达水平和细胞存活率、Bcl-2 mRNA表达水平以及线粒体膜电位均明显升高,而细胞凋亡率、Bax mRNA表达水平、Caspase-3活性明显降低(P0.05);给予IGF-1作用后Met对MC3T3-E1细胞的作用效果明显增强,而给予NVP-BEZ235作用后Met对MC3T3-E1细胞的作用效果明显减弱(P0.05)。结论 Met可通过激活PI3K/Akt/m TOR通路抑制线粒体凋亡途径,减轻糖皮质激素Dex诱导的成骨细胞凋亡。     

Global adiponectin suppress the high expression of monocyte chemotactic protein-1 induced by high glucose in NRK52E cells

Objective To investigate the effect of globular adiponectin on the high expression of monocyte chemotactic protein-1 (MCP-1) induced by high glucose in rat renal tubular epithelial cells(NRK52E), and its relationship with adiponectin receptors and p38MAPK. Methods NRK52E cells were cultured in vitro and divided into six groups: normal glucose group (NG, 5.6 mmol/L glucose), high glucose group(HG, 25 mmol/L glucose), gAd group1 (HG+gAd 2 mg/L), gAd group2 (HG+gAd 5 mg/L), gAd group3 (HG+gAd 10 mg/L), p38MAPK antagonist group：(SB, HG+SB203580 10 μmol/L). The protein expression of phosphorylated p38MAPK (p-p38MAPK), total p38MAPK (t-p38MAPK), MCP-1 and AdipoR1/AdipoR2 were examined by western blotting. The mRNA expression of MCP-1 and AdipoR1/AdipoR2 were detected by RT-PCR and real-time PCR respectively. Results Compared with NG group, the mRNA and protein expression of MCP-1 increased significantly in HG group (all P＜0.05). The phosphorylation of p38MAPK increased (P＜0.05) with no change in t-p38MAPK protein. The addition of gAd or SB203580 inhibited the unregulation of MCP-1 and p-p38MAPK induced by HG. Two kinds of adipoR,adipoR1 and adipoR2,were all detectable in NG group, and mRNA and protein expression of adipoR1 was higher than that of adipoR2 (P＜0.01). Compared with NG group, the expression of adipoR decreased in HG group, but the difference had no statistical significance(P＞0.05). Compared to HG group, the mRNA and protein expression of adipoR1 increased in gAd groups (all P＜0.01). Conclusion The gAd can dose-dependently attenuate the overexpression of MCP-1 induced by high glucose, and this protective effect may be mediated by adipoR1 and p38MAPK.     

高糖环境下舒洛地特对大鼠近端肾小管上皮细胞增殖及细胞间黏附分子1表达的影响

目的探讨舒洛地特对高糖培养的大鼠近端肾小管上皮细胞（NRK52E）增殖和细胞间黏附分子1（ICAM-1）表达的影响。方法将NRK52E细胞用5．6mmol／L葡萄糖（NG组）、25mmol／L葡萄糖（HG组）、25mmol／L葡萄糖联合不同浓度舒洛地特（终浓度分别为0．5LRU／ml、1．0LRU／ml、2LRU／ml）于96孔板中分别培养24h、48h、72h后,运用MTT法测定细胞增殖变化。24h后,运用RT-PCR和Western blotting方法检测ICAM-1mRNA和蛋白质的表达。结果HG组细胞增殖及ICAM-1的表达均增强,舒洛地特组能抑制这种趋势并且呈剂量依赖性。结论舒洛地特能通过减少ICAM-1的表达而起到保护肾脏的作用。     

Effects of metformin on expression of AMP - activated protein kinase in rat glomerular mesangial cells

ObjectiveTo observe the effects of metformin on expression of Adenosine 5’- monophosphate (AMP)-activated protein kinase (AMPK), nuclear factor-κB (NF-κB) and transforming growth factor β1 (TGF - β1) in cultured rat glomerular mesangial cells (MCs), and explore its reno - protective mechanisms. Methods MCs were cultured in the medium with normal glucose (group NG, 5.6 mmol/L), high glucose (group HG, 25mmol/L) and different concentrations of metformin (group M1, M2, M3). After 48 h exposure, the supernatants and MCs were collected. The expression of NF-κB and TGF-β1 mRNA was analyzed by real time-PCR. Total-AMPK, phospho-Thr-172 AMPK (p-AMPK), NF -κB p65 and TGF-β1 were visualized by Western blot. ResultsThe real time-PCR and Western blot result showed MCs could express AMPK, NF-κB and TGF-β1 mRNA and protein. After stimulated by HG, the levels of intracellular NF - κB and TGF - β1 expressions were significantly increased compared with group NG (P＜0.05); The levels of NF-κB and TGF-β1 were significantly decreased in group M1, M2 and group M3 compared with group HG in a dose-dependent manner. After stimulated by HG, the level of intracellular p-AMPK were down-regulated compared with group NG(all P＜0.05); The expression of p-AMPK increased with the rising of metformin concentration, presenting the opposite trend (P＜0.05), while the level of total-AMPK protein was unchanged with exposure to HG or different concentrations of metformin(P＞0.05). ConclusionMetformin can suppress the expression of NF- κB and TGF-β1 of glomerular MCs induced by HG via AMPK activation, which may partly contribute to its reno-protection.     

霉酚酸对高糖环境下肾小球系膜细胞外基质的影响

目的：通过检测霉酚酸酯（MMF）的代谢产物霉酚酸（mycophenolic acid,MPA）对高糖环境下大鼠肾小球系膜细胞（mesangial cells,MCs）增殖、转化生长因子-β1（transformation growth factor-β1,TGF-β1）和细胞外基质的主要成分：纤维连接蛋白（fibrin,FN）、层黏连蛋白（laminin,LN）和胶原Ⅳ（typeⅣ collagen,ColⅣ）分泌的影响,探讨MPA对糖尿病肾病（diabetic nephropathy,DN）的保护机制。方法：四甲基偶氮唑盐微量酶反应比色法测定高糖及高糖加入不同浓度MPA（1～10μmol/L）后对大鼠MCs增殖的影响,ELASE的方法测定各组24h、48h、72hFN、LN、ColⅣ的表达,荧光定量多聚酶链反应的方法检测各组标本中TGF-β1 mRNA的表达,并进行统计学分析。结果：高糖可以诱导MCs增殖及TGF-β1、FN、LN、ColⅣ的表达,MPA抑制高糖环境下MCs增殖和FN、LN和ColⅣ分泌并呈剂量时间依赖性,MPA可以呈剂量依赖性抑制高糖环境下MCs分泌TGF-β1,各组之间有统计学差异（P〈0.05）。结论：MPA可以通过抑制高糖环境下MCs的增殖和TGF-β1的分泌,从而抑制系膜外基质增多、系膜区扩张,有效阻止细胞外基质积聚,从而防止肾小球硬化,延缓DN的发展。     

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.