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。     

糖肾平通过TGF-β1-Smad2/3-ILK信号通路干预高糖＋LPS诱导足细胞上皮间质转分化的分子机制研究

也页目的：探讨糖肾平对高糖环境下脂多糖（ lipopolysaccharide,LPS）刺激足细胞上皮间质转分化的影响,并探讨其作用机制。方法：以体外培养大鼠肾小球足细胞为研究对象,以高糖（25 mmol/L）、LPS（1μg/mL）刺激足细胞建立模型,分为正常组、高糖组、高糖＋LPS组、厄贝沙坦组、抑制剂组、糖肾平小、中、大剂量组。采用Western blotting及RT-PCR方法检测足细胞中转化生长因子-β1（ transforming growth factor-β1,TGF-β1）、Smad2/3、整合素连接激酶（ integrin-linked kinase, ILK）、CD2相关蛋白（CD2AP）、α-平滑肌肌动蛋白（α-Smooth muscle actin,α-SMA）的表达水平。结果：与正常组比较,高糖组和高糖＋LPS组足细胞TGF-β1、ILK、α-SMA蛋白及其mRNA表达明显增加（P〈0.01）,P-Smad2/3蛋白及Smad2/3 mRNA表达明显增加（P〈0.01）,CD2AP蛋白及其mRNA表达明显减少（P〈0.01）;与高糖＋LPS组比较,厄贝沙坦组足细胞P-Smad2/3、α-SMA蛋白表达明显减少（P〈0.01）,TGF-β1蛋白表达减少（P〈0.05）,TGF-β1,Smad2/3,α-SMAmRNA表达明显减少（P〈0.01）,ILK mRNA表达减少（P〈0.05）,CD2AP蛋白及其mRNA表达明显增加（P〈0.01）;糖肾平大、中、小各剂量组足细胞TGF-β1蛋白表达明显减少（P〈0.01）,糖肾平大剂量组足细胞TGF-β1 mRNA表达减少（P〈0.05）,小、中剂量组表达明显减少（P〈0.01）;糖肾平小、中、大各剂量组足细胞P-Smad2/3蛋白及Smad2/3mRNA表达均明显减少（P〈0.01）;糖肾平小、大剂量组足细胞ILK mRNA表达减少（P〈0.05）,中剂量组表达明显减少（P〈0.01）;糖肾平大剂量组足细胞α-SMA蛋白及其mRNA表达减少（P〈0.05）;糖肾平小、中、大各剂量组足细胞CD2AP蛋白及其mRNA表达均明显增加（P〈0.01）。结论：糖肾平能够降低足细胞TGF-β1、ILK蛋白及mRNA表达,降低P-Smad2/3蛋白及Smad2/3 mRNA表达,升高足细胞标志物CD2AP蛋白及mRNA表达,降低间充质细胞标志物α-SMA蛋白及mRNA表达,通过抑制TGF-β1-Smad2/3-ILK信号通路的激活减少足细胞转分化,保护足细胞,可能是其防治糖尿病肾病的作用机制之一。     

白细胞介素1β对人肾小球系膜细胞LOX-1和ABCA1表达的影响

目的 研究白细胞介素1β（IL-1β）对人肾小球系膜细胞株（HMCL）植物血凝素样氧化低密度脂蛋白受体1（LOX-1）以及腺苷三磷酸结合盒转运体A1（ABCA1）表达的影响，及其与细胞胆固醇稳态的关系。 方法 实时定量PCR和Western印迹法检测IL-1β对人肾小球系膜细胞LOX-1、ABCA1表达的影响。 结果 人肾小球系膜细胞表达LOX-1 mRNA和蛋白。IL-1β促进人肾小球系膜细胞LOX-1 mRNA和蛋白表达，5 μg/L IL-1β刺激细胞0~24 h，LOX-1 mRNA表达于6 h达高峰，为对照的6.87倍；LOX-1蛋白24 h达高峰，为对照的1.88倍。IL-1β降低脂质负荷的人肾小球系膜细胞 ABCA1 mRNA和蛋白表达。5 μg/L IL-1β刺激细胞0~48 h，48 h时ABCA1 mRNA和蛋白下降最明显，分别为对照的19.0％和50.62％。 结论 IL-1β促进人肾小球系膜细胞LOX-1表达，抑制ABCA1的表达，导致细胞内胆固醇的失衡，促使其变成泡沫细胞，可能加重肾小球硬化和肾病的进展。     

高糖诱导足细胞发生上皮-间叶细胞转分化

目的 通过观察高糖是否引起小鼠足细胞发生上皮-间叶细胞转分化现象,探讨糖尿病肾小球损伤的可能机制。 方法 以小鼠永生化足细胞株为研究对象,予不同浓度葡萄糖（12.5、25、50 mmol/L）处理该细胞36 h,并设低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理组为对照组。采用蛋白免疫印迹和间接免疫荧光染色方法检测α平滑肌肌动蛋白 （α-SMA）、迁连蛋白（FN）、CD2相关蛋白（CD2AP）和Wilms’肿瘤1基因（WT-1）蛋白的表达。 结果 低糖（5.6 mmol/L）和甘露醇（50 mmol/L）处理条件下小鼠足细胞表达WT-1、CD2AP,基本不表达α-SMA和FN;而予不同浓度葡萄糖处理36 h后,足细胞中α-SMA和FN表达水平呈剂量依赖性上调（P < 0.05）。间接免疫荧光染色结果也显示,与低糖组相比,高糖组中 α-SMA阳性的足细胞比例显著增加（P < 0.05）。同时,蛋白免疫印迹结果还表明高糖可呈剂量依赖性方式下调WT-1和CD2AP的表达（P < 0.05）。 结论 在高糖条件下,足细胞发生向间叶细胞表型的转分化可能是引起足细胞功能失调,进而引起糖尿病肾小球损伤发生的作用机制之一。     

高糖对大鼠肾系膜细胞泛素及泛素化蛋白表达的影响

目的 探讨高糖对大鼠肾系膜细胞泛素及泛素化蛋白表达的影响。 方法 将大鼠HBZY-1肾系膜细胞进行体外培养,高糖作为刺激因子,分别设正常对照组(5.6 mmol/L葡萄糖)、高糖组（10、20、30 mmol/L葡萄糖）、甘露醇组。刺激12、24、48 h后,分别用实时定量PCR法和Western印迹法检测各组系膜细胞泛素mRNA和泛素化蛋白的表达。 结果 泛素化蛋白多以大分子蛋白为主。与正常对照组比较,30 mmol/L葡萄糖刺激24 h和48 h后泛素化蛋白的表达分别增加36％和52％（均P ＜ 0.01）;20 mmol/L和30 mmol/L葡萄糖作用48 h泛素化蛋白的表达分别增加31％和52％（均P ＜ 0.01）;高糖作用24、48 h,泛素mRNA的表达显著增加（均P ＜ 0.05）。 结论 高糖呈时间浓度依赖性地促进大鼠肾系膜细胞泛素mRNA及泛素化蛋白的表达,增加泛素蛋白酶体途径活性。     

黏着斑激酶在转化生长因子β1诱导的人肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF）β1诱导的正常人近端肾小管上皮细胞（HK-2）转分化（EMT）过程中黏着斑激酶（FAK）的表达及下调FAK的表达后对TGF-β1诱导的HK-2细胞转分化进程的影响。 方法 应用TGF-β1（10 μg/L）刺激HK-2细胞,采用RT-PCR、Western印迹和免疫荧光方法分别检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）、FAK mRNA和蛋白的表达及磷酸化（p）-FAK（Tyr397）的蛋白表达。应用Lipofectmine2000将FAK siRNA转染HK-2细胞,采用Western印迹观察下调表达FAK对上述指标的影响。 结果 TGF-β1刺激后,HK-2细胞α-SMA蛋白和mRNA水平上调,E-cadherin蛋白和mRNA表达下调。FAK蛋白和mRNA随时间的延长表达逐渐增多,48 h达到高峰。p-FAK（Tyr397）蛋白表达趋势与FAK相同。脂质体转染siRNA后FAK的mRNA和蛋白分别下调了50%和41%,下调表达FAK后可以显著抑制TGF-β1诱导的HK-2细胞α-SMA蛋白的上调表达,逆转 E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导的HK-2细胞转分化进程FAK蛋白表达上调,敲低FAK蛋白表达后可以部分减轻EMT的程度,提示FAK在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中发挥一定的作用。     

Ox-LDL对血管内皮细胞ABCA1及炎症因子表达的研究

目的：研究在致动脉粥样硬化（AS）因子氧化低密度脂蛋白胆固醇（Ox-LDL）刺激下,人血管内皮细胞ECV304中ATP-结合盒转运子A1（ABCA1）对炎性细胞因子ICAM-1及IL-1β水平调节作用,阐明ABCA1基因在AS发生中的可能机制。方法：培养人血管内皮细胞株ECV304,加入Ox-LDL（30ng／ml）刺激3、6、12、24h,以荧光定量逆转录-聚合酶链式反应（RT—PCR）检测ABCA1、ICAM1 mRNA表达量,Western blot和酶联免疫吸附（ELISA）法检测ABCA1、ICAM1及IL-1β蛋白表达量;ABCA1的反义寡核苷酸（100nmol／L）转染人血管内皮细胞,按相同方法给予Ox-LDL刺激细胞,同样的方法测定上述指标的改变。阴性对照为未加Ox-LDL刺激并同时培养的细胞。结果：与阴性对照组比较,人血管内皮细胞株ECV304在给予Ox-LDL刺激不同时间后,AB—CA1、ICAM-1的mRNA和蛋白及IL-1β蛋白水平均增高;给予ABCA1反义寡核苷酸转染后各时间点ABCA1、ICAM-1 mRNA表达均降低,ABCA1、ICAM-1及IL-1β蛋白表达水平也降低。结论：人管内皮细胞在Ox-LDL作用下,其ABCA1可能通过增加IL-1β的表达,促进炎症细胞因子ICAM-1释放,在AS的早期发生中发挥作用。     

Par-3蛋白在转化生长因子?茁1介导的大鼠肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF)β1诱导的正常大鼠近端肾小管上皮细胞（NRK52E）转分化（EMT）过程中细胞极性蛋白Par-3的表达及上调Par-3蛋白表达对TGF-β1诱导NRK52E细胞转分化进程的影响。 方法 应用TGF-β1 (10 μg/L)刺激NRK52E细胞，采用RT-PCR、Western印迹和免疫荧光方法分别检测E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白（α-SMA）、Par-3 mRNA和蛋白的表达；应用Lipofectmine 2000将pKH3-HA-Par-3质粒瞬时转染NEK52E细胞，采用Western印迹观察上调表达Par-3对上述指标的影响。 结果 TGF-β1刺激后，NRK52E细胞α-SMA蛋白和mRNA水平上调，E-cadherin蛋白和mRNA的表达下调；Par-3蛋白表达呈时间依赖模式下调，72 h TGF-β1刺激组与对照组比较，差异有统计学意义（P < 0.05）。但Par-3 mRNA水平在各时间点差异均无统计学意义（P > 0.05）。脂质体转染外源性质粒pKH3-HA-Par-3上调表达Par-3可显著抑制TGF-β1诱导NRK52E细胞α-SMA蛋白的上调表达；逆转E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导NRK52E细胞转分化进程中细胞极性Par-3蛋白表达下调，基因转染上调表达Par-3可部分减轻EMT的程度，提示Par-3蛋白在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中可发挥保护性作用。     

RhoA-Rock信号通路在转化生长因子β1诱导大鼠腹膜间皮细胞转分化中的作用

目的　探讨RhoA-Rock信号通路在转化生长因子β1（TGF-β1）诱导大鼠腹膜间皮细胞（RPMC）转分化中的作用。 方法　体外培养SD大鼠原代腹膜间皮细胞,静止24 h后,采用随机数字表法随机分为以下4组：正常对照组、TGF-β1（10 μg/L）刺激组、TGF-β1（10 μg/L）＋Y-27632（Rock特异性抑制剂,10 μmol/L）组（Y-27632预处理2 h）、Y-27632（10 μmol/L）组。用TGF-β1（10 μg/L）刺激RPMC不同时间,观察α平滑肌肌动蛋白（α-SMA）,E钙黏素（E-cadherin）、Ⅰ型胶原（ColⅠ）的表达。RT-PCR法检测E-cadherin、α-SMA 和ColⅠmRNA表达。Western印迹法检测RhoA（包括总RhoA及活化的RhoA）、E-cadherin、α-SMA、ColⅠ和波形蛋白（vimentin）表达。活化的RhoA由膜蛋白提取试剂盒提取。 结果 （1）TGF-β1（10 μg/L）刺激RPMC能诱导RhoA活化,于10 min开始出现活性升高,为对照组的（2.57±0.52）倍（P < 0.05）;1 h达高峰,为对照组的（4.35±0.41）倍（P < 0.05）。（2）TGF-β1（10 μg/L）刺激RPMC能导致E-cadherin mRNA和蛋白表达下调,α-SMA、ColⅠmRNA和蛋白表达上调,呈时间依赖性。（3）Rock特异性抑制剂Y-27632能显著下调α-SMA、ColⅠmRNA的表达,较TGF-β1刺激组各降低了53.8%和55.7%（均P < 0.05）,并且能下调α-SMA、ColⅠ和vimentin蛋白的表达,较TGF-β1刺激组分别降低了42.6%、60.1%和58.1%（均P < 0.05）,但不能上调E-cadherin mRNA和蛋白的表达。 结论　TGF-β1可通过RhoA-Rock信号通路介导大鼠腹膜间皮细胞转分化,抑制该通路可作为防治腹膜纤维化的潜在靶点。     

高糖刺激下大鼠肾脏系膜细胞Smad7的表达及姜黄素对其表达的影响

目的：观察高糖刺激下大鼠肾脏系膜细胞（MC）smad7的表达及姜黄素对Smad7表达的影响。方法：MC分6组，对照组给予正常培养液，4组给予含30mmol／L葡萄糖的培养液，分别于6、12、24、48h后收集细胞，另1组给予含30mmol／L葡萄糖和33．33μmol／L姜黄素的培养液共孵育6h后收集细胞。分别用逆转录多聚酶链反应（RT—PCR）和Western杂交分析法检测各组系膜细胞Smad7 mRNA和蛋白的表达情况。结果：正常MC有丰富的Smad7 mRNA表达，高糖刺激后表达明显增加，于6h达到高峰，Smad7蛋白的变化趋势与mRNA相同。MC加姜黄素共孵育后，Smad7 mRNA表达增加，高糖刺激6h组加或不加姜黄素，其吸光度相对值分别为1．113±0．124、2．235±0．356，Smad7蛋白吸光度值分别为12556±1335、24336±1866。结论：高糖刺激下，系膜细胞Smad7mRNA和蛋白表达呈时间依赖性，姜黄素可诱导高糖刺激下的系膜细胞Smad7表达，从而抑制转化生长因子-β（transforming growth factor-β,TGF-β）目的基因的转录。     

Effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells

Objective To evaluate the effects of KIM-1 on high glucose induced the expression of MCP-1 and FN in rat tubular epithelial cells and to explore the possible mechanisms of KIM-1 involved in renal interstitial fibrosis of DN. Methods The rat renal tubular epithelial cells (NRK52E) were cultured in vitro and divided into five groups: Normal control group (D-glucose 5.6 mmol/L), Hypertonic group (D-glucose 5.6 mmol/L＋D-mannitol 24.4 mmol/L), High glucose group (D-glucose 30 mmol/L), Control siRNA group,KIM-1 siRNA group. ELISA assay was used to assess the levels of MCP-1 and FN in the cells supernatant; Western blotting was used to detect the protein expression of KIM-1; RT-PCR was used to detect mRNA expression of KIM-1, MCP-1 and FN. Results Compared with the control group, the protein and mRNA expression of KIM-1 in the high glucose group were increased at 12 h (P＜0.05), and reached the peak at 48 h (P＜0.05); the protein and mRNA expression of MCP-1 and FN in high glucose group were increased at 24 h significantly (P＜0.05), and peaked at 48 h (P＜0.05). Compared with the high glucose group, the protein and mRNA expressions of MCP-1 and FN in KIM-1 siRNA group were decreased (P＜0.05). Conclusions Down-regulating the expression of KIM-1 can significantly inhibit the expression of MCP-1 and FN, which suggests that KIM-1 may be involved in renal interstitial fibrosis of DN by regulating expression of MCP-1 and FN.     

TAK1信号通路在高糖诱导骨髓来源巨噬细胞激活中的作用

Objective To investigate the role of transforming growth factor-β activated kinase-1 (TAK1) signaling pathway in the activation of bone marrow derived macrophages (BMDM) induced by high glucose. Methods Purity of mouse BMDM was detected by flow cytometry. The mice macrophages cultured in vitro were stimulated by high glucose and treated with TAK1 specific inhibitor 5Z-7-oxozeaenol. Cells were divided into normal control group (RPMI 1640), osmolality control group (25 mmol/L mannitol), high glucose group (33 mmol/L D-glucose) and inhibitor group (33 mmol/L D-glucose+300 nmol/L 5Z-7-oxozeaenol). Immunocytochemistry and flow cytometry were used to detect macrophage subtype. The expression of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis Factor-α (TNF-α) mRNA were determined by real time PCR. Expressions of p-TAK1, TAK1 binding protein (TAB1), p-JNK, p-p38 MAPK and NF-κB p65 proteins were analyzed by Western blotting. Results The purity of BMDM was about 99.36%. Compared with normal control group, high glucose group had increased percentage of M1 macrophages, increased expression of MCP-1 and TNF-α mRNA (all P＜0.05). Moreover, p-TAK1, TAB1, p-JNK, p-p38 MAPK and NF-κB p65 proteins expression also increased significantly in high glucose group (all P＜0.05). After treatment with inhibitor 5Z-7-oxozeaenol, the effects induced by high glucose were inhibited (P＜0.05). Conclusions High glucose can induce M1 macrophage activation and expression of inflammatory cytokine of BMDM, which can be inhibited 5Z-7-oxozeaenol through inhibiting TAK1/MAPK and TAK1/NF-κB pathway.     

Insights into cyclosporine A-induced atherosclerotic risk in transplant recipients: macrophage scavenger receptor regulation

Clinical monitoring of organ-transplant recipients suggests that administration of cyclosporine (CsA) may increase the risk of atherosclerosis when compared with the general population. The purpose of this work is to demonstrate the utility of the in vitro Tamm-Horsfall protein (THP)-1 human monocyte cell culture model for determining drug-related atherosclerotic potential in macrophages. The effect of CsA on the mRNA expression of macrophage scavenger receptor genes including CD36, CD68, scavenger receptor (SR)-A, SR-BII, and lectin-like oxidized low-density lipoprotein receptor (LOX-1); the nuclear hormone receptors, including peroxisome-proliferator activated receptor (PPAR)gamma and liver-X-receptor (LXR)alpha; and the cholesterol efflux pump ABCA1 were investigated as markers of atherosclerotic progression. The THP-1 cells were cultured and differentiated into macrophages. The macrophages were then treated with CsA to assess gene expression. Time- (1, 2, 4, 8, and 24 hours) and dose- (concentrations [mg/L] corresponding to the trough [0.5], peak [1.25] and 4x peak [5]) dependency of CsA was assessed. The treated macrophage mRNA gene expression of CD36, CD68, and PPARgamma were up-regulated in the presence of CsA. Interestingly, SR-A, SR-BII, LOX-1, and LXRalpha expression appeared to be slightly down-regulated, and ABCA1 was relatively unchanged. Immunoblotting studies demonstrated that the protein expression of CD36 was unchanged or increased, PPARgamma was unchanged, and ABCA1 was unchanged or decreased at 4 and 8 hours. The results document CsA-induced mRNA and protein changes in receptors relevant to lipid-laden foam cell formation and demonstrate the utility of THP-1 macrophages for screening of atherosclerotic risk potential.     

The role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells

Objective To investigate the role and mechanism of macrophage activation induced by exosomes from high glucose-treated renal tubular epithelial cells. Methods (1) The supernatant of renal tubular epithelial cells which were cultured in normal glucose control group (5.5 mmol/L D-glucose) or high glucose group (30.0 mmol/L D-glucose) for 48 h were collected and ultracentrifuged to harvest exosomes. Exosomes were identified by transmission electron microscope and Western blotting. (2) Exosomes were labeled with the green lipophilic fluorescent dye PKH67 and cultured with THP-1 macrophage to investigate whether HK2-derived exosomes could be internalized by THP-1 macrophage. Observing the morphology microscopically and detecting the chemotaxis function of THP-1 macrophages in Transwell chamber after co-cultured with exosomes. The expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) in cells and supernatants were separately detected by quantitative Real-time PCR (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), and the expression of p-c-Jun NH2-terminal kinase (p-JNK), mitogen-activated protein kinase p-p38 (p-p38MAPK) and nuclear factor κB p65 (NF-κB p65) in THP-1 macrophages were detected by Western blot. Results (1) Vesicles that harvested by ultracentrifugation ranged in size from 30 nm to 100 nm and expressed exosomal marker CD63, TSG101 but absence of calnexin which is a marker of endoplasmic reticulum, suggesting that the exosomes were not contaminated with cells. (2) Results from laser scanning confocal microscope showed that each group of exosomes can be internalized by THP-1 macrophages. Compared with normal glucose exosomes group, high glucose exosomes had increased the expression of iNOS, TNF-α, IL-1β and MCP-1 in THP-1 macrophages (all P＜0.01), moreover, p-JNK, p-p38 MAPK and NF-κB p65 proteins level also increased significantly (all P＜0.01). Conclusions Exosomes from high glucose-treated HK2 cells can induce THP-1 macrophage activation and functional changes through MAPK/NF-κB pathway.     

罗格列酮对高糖环境中大鼠系膜细胞活性氧及单核细胞化学吸引蛋白质1的抑制作用

目的 观察罗格列酮对高糖培养基中大鼠系膜细胞活性氧（ROS）及单核细胞化学吸引蛋白质1（MCP-1）mRNA、蛋白表达的抑制作用,并探讨相关机制。 方法 将培养的大鼠系膜细胞分为以下6组：对照组（C组,普通MEM培养基,含5.6 mmol/L葡萄糖）、甘露醇组（M组,24.2 mmol/L甘露醇＋C组）、高糖组（H组,30 mmol/L高糖MEM培养基）、小剂量罗格列酮干预组（R1组,H组＋10 μmol/L罗格列酮）、大剂量罗格列酮干预组（R2组,H组＋20 μmol/L罗格列酮）、N-乙酰半胱氨酸（NAC）干预组（N组,H组＋5 mmol/L NAC）。采用激光共聚焦法检测ROS水平,分别采用RT-PCR和ELISA法检测MCP-1 mRNA及蛋白含量。 结果 C组和M组间ROS、MCP-1的表达差异无统计学意义,H组ROS水平较C组增高4.1倍（P < 0.01）,分别采用罗格列酮（20 μmol/L）和NAC干预后,ROS水平显著降低（P < 0.01）;罗格列酮（20 μmol/L）和NAC均可显著抑制高糖环境中MCP-1 mRNA的高表达（P < 0.01）。H组中MCP-1蛋白水平[（940.9±20.3） ng/L]显著高于C组[（403.0±8.1） ng/L]（P < 0.01）,而R2组和N组的MCP-1蛋白水平[（562.5±15.3） ng/L,（539.8±8.3） ng/L]显著低于H组（P < 0.01）。 结论 罗格列酮可通过减少ROS而降低高糖所诱导的MCP-1表达,而这可能是罗格列酮发挥直接肾脏保护作用的机制之一。     

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.     

色素上皮衍生因子对高糖诱导人肾小球系膜细胞p38丝裂原活化蛋白激酶-cAMP反应元件结合蛋白通路及纤连蛋白的影响

目的 探讨色素上皮衍生因子(PEDF)对高糖条件下人肾小球系膜细胞( HMC )p38丝裂原活化蛋白激酶(p38MAPK)、cAMP反应元件结合蛋白(CREB)及细胞外基质成分纤连蛋白(FN)的影响.方法 体外予正常糖(5.6 mmol/L)、甘露醇(24.4 mmol/L)、高糖(30 mmol/L)、高糖+PEDF(30 mmol/L葡萄糖+10 nmol/L、40 nmol/L、100 nmol/L PEDF)培养基培养HMC 24 h,观察p38MAPK、CREB活性(Western印迹)及FN mRNA( RT-PCR)和蛋白( ELISA)的变化.结果 与正常糖及甘露醇组相比,高糖组p-p38MAPK、p-CREB、FN的表达均显著升高(均P＜ 0.01);与高糖组相比,PEDF干预组上述指标的表达均显著下降(均P＜0.05).结论 PEDF可能通过p38MAPK-CREB通路抑制糖尿病肾病的纤维化.     

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。