PPARγ配体对高糖诱导大鼠腹膜间皮细胞结缔组织生长因子和纤溶酶原激活抑制因子1表达的影响

目的 研究过氧化物酶体增殖物活化受体γ（PPARγ）天然配体15d-PGJ2及人工合成配体吡格列酮（pioglitazone）对高糖诱导大鼠腹膜间皮细胞（RPMC）表达结缔组织生长因子（CTGF）和纤溶酶原激活抑制因子1（PAI-1）的影响。 方法 胰蛋白酶消化法分离培养RPMC,经鉴定分组：（1）0.1%、1.5%、2.5%、4.25%葡萄糖作用24 h组;（2）2.5%葡萄糖作用0、6、12、24、36、48、72 h组 ;（3）0.1%、1.5%、2.5%、4.25%甘露醇作用24 h组;（4）15d-PGJ2（5、15 μmol/L）及吡格列酮（5、15 μmol/L）分别预孵育2 h,加2.5%葡萄糖再作用24 h。RT-PCR检测CTGF和PAI-1 mRNA表达;Western印迹检测PPARγ、CTGF及PAI-1 蛋白表达。 结果 正常RPMC有PPARγ表达。1.5%葡萄糖使RPMC的PPARγ蛋白表达减少（P < 0.05）,而4.25%葡萄糖作用最大（P < 0.01）;2.5%葡萄糖作用6 h,RPMC的PPARγ蛋白表达减少（P < 0.05）,而72 h达高峰（P < 0.01）。各种浓度的甘露醇作用24 h,RPMC的PPARγ蛋白表达均无明显变化（P > 0.05）。2.5%葡萄糖作用后RPMC的CTGF和PAI-1 mRNA和蛋白表达均显著增加（P < 0.01）。5 μmol/L的吡格列酮显著降低CTGF和PAI-1 mRNA和蛋白表达（均P < 0.05）,而15 μmol/L作用更强（P < 0.01）。5 μmol/L的15d-PGJ2显著降低RPMC的CTGF mRNA和蛋白表达以及PAI-1 mRNA的表达（均P < 0.05）,但不影响PAI-1蛋白表达（P > 0.05）,15 μmol/L的15d-PGJ2对CTGF和PAI-1 mRNA和蛋白表达均有抑制作用（P < 0.05或P < 0.01）。 结论 葡萄糖以时间和剂量依赖方式调节RPMC PPARγ的表达,其作用与高渗透浓度无关。PPARγ配体可显著抑制高糖诱导的CTGF和PAI-1 的表达,提示激活PPARγ可能成为防治腹膜透析相关腹膜纤维化的新途径之一。     

过氧化物酶增殖物激活受体γ减少血管紧张素Ⅱ诱导的系膜细胞外基质的积聚及其作用机制

目的 探讨过氧化物酶增殖物激活受体(PPAR)γ1对血管紧张素Ⅱ(AngⅡ)诱导的系膜细胞外基质积聚的抑制作用及其机制。方法 脂质体转染质粒pIRES2-EGFP-PPARγ1/WT(野生型)于系膜细胞，给予AngⅡ(10-7 mol/L)刺激48 h。采用RT-PCR检测TGF-β1、PAI-1、c-fos、c-jun mRNA表达水平。采用ELISA法检测细胞上清液中TGF-β1和FN浓度。Western印迹观察胞浆中I-κB、NF-κB及胞核中NF-κB、pERK蛋白表达水平。利用PPARγ与PPRE结合活性测定PPARγ1在系膜细胞中的活性。同时以PPARγ激动剂吡格列酮(6 μmol/L)、不具有目的基因PPARγ1的空白质粒pIRES2-EGFP和表达功能缺陷-突变型(DN)PPARγ1的质粒pIRES2-EGFP-mPPARγ1/DN作为对照。结果 PPARγ1过表达能显著性抑制AngⅡ诱导的系膜细胞TGF-β1、PAI-1的mRNA高表达(P < 0.05)，同时下调c-fos和c-jun mRNA高表达(P < 0.05)。转染PPARγ1/WT能显著降低AngⅡ 48 h刺激下细胞上清液中FN和TGF-β1浓度(P < 0.05)。在AngⅡ作用下系膜细胞AT1表达增加(P < 0.05)， PPARγ1可显著性减少AT1的高表达(P < 0.05)。AngⅡ诱导的系膜细胞pERK表达明显升高， PPARγ1可显著性减少pERK表达(P < 0.05)。转染PPARγ1/WT能上调AngⅡ刺激下系膜细胞胞浆中I-κB低表达，同时下调NF-κB由胞浆向细胞核的转移(P < 0.05)。转染PPARγ1/DN并无上述作用。吡咯列酮具有与PPARγ1相同的显著性效应。PPARγ1/WT转染组PPARγ1的活性明显高于其他组(P < 0.05)。结论 PPARγ1过表达能够抑制AngⅡ刺激下系膜细胞外基质的聚积，降低AT1受体蛋白表达，具有直接抗硬化的非代谢性效应，其机制可能是通过抑制ERK/AP-1及NF-κB等信号分子的传递。     

过氧化物酶体增殖物活化受体γ减少高糖诱导系膜细胞外基质的积聚及其机制

目的探讨过氧化物酶体增殖物活化受体(PPAR)γ对高糖诱导肾小球系膜细胞(GMC)外基质(ECM)积聚的抑制作用及其机制。方法以表达野生型小鼠PPARγ1的质粒pIRES2-EGFP-mPPARγ1/WT转染系膜细胞(WT),然后予30mmol/L的高糖(HG)刺激48h,以ELISA法检测细胞上清液中TGF-β1、纤连蛋白(FN)的浓度。GMC中c-fos、c-jun、葡萄糖转运蛋白1(GLUT-1)mRNA的表达检测采用半定量RT-PCR法,并以Western印迹检测胞浆中核因子抑制物(Ⅰ-κB)、核因子(NF-κB)及胞核中NF-κB、磷酸化细胞外调节激酶(p-ERK)的蛋白表达水平。同时以2μmol/L的PPARγ激活剂吡咯列酮(Pio)、转染表达功能缺陷的突变型(dominantnegative,DN)PPARγ1的质粒pIRES2-EGFP-mPPARγ1/DN(DN)或空白质粒pIRES2-EGFP作为对照。PPARγ的活性以其与PPARγ反应元件(PPRE)的结合能力表示。结果HG培养时系膜细胞的PPARγ活性较正常糖浓度(5mmol/L)培养时明显上升(P<0.01),HG WT组的PPARγ活性显著高于HG DN、HG pIRES2-EGFP和HG Pio组。与HG DN、HG pIRES2-EGFP相比,WT转染所致的PPARγ1高表达能显著抑制高糖诱导GMC的TGFβ-1、FN生成增多,减轻c-fos和c-jun的mRNA表达的上调,改善p-ERK蛋白水平的增加、胞浆Ⅰ-κB表达的降低以及NF-κB由胞浆向胞核转移的增加(P均<0.0     

吡格列酮对系膜细胞基质合成和降解的影响

目的观察吡格列酮对高糖状态下系膜细胞（MCs）增殖、细胞外基质合成和降解的影响。方法以大鼠MCs为研究对象,给予吡格列酮干预,并以苯那普利作为阳性对照。MTT法观察细胞增殖情况,ELISA法测定细胞上清液中转化生长因子-β1（TGF-β1）、基质金属蛋白酶9（MMP-9）、金属蛋白酶组织抑制剂-1（TIMP-1）、Ⅳ型胶原以及肝细胞生长因子（HGF）的含量,RT-PCR法检测细胞TGF-β1、MMP-9、TIMP-1及HGFmRNA的表达。结果①高糖可刺激MCs增殖,吡格列酮呈剂量和时间依赖性抑制该增殖作用。②高糖促进MCsⅣ型胶原分泌,吡格列酮则显著抑制Ⅳ型胶原分泌。③高糖状态下,MCsMMP-9分泌减少,TIMP-1分泌增加。吡格列酮则促进MMP-9分泌及其mRNA的表达,抑制TIMP-1分泌。④高糖可促进MCsTGF-β1分泌,抑制HGF分泌。吡格列酮则抑制TGF-β1分泌,促进HGF分泌及其mRNA的表达。结论吡格列酮可抑制高糖诱导的MCs增殖,促进MMP9分泌,抑制TIMP-1分泌,抑制TGF-β1分泌,促进HGF分泌,从而抑制胶原合成、促进其降解,发挥抗糖尿病肾小球硬化作用。     

血管紧张素Ⅱ诱导Toll样受体4在腹膜间皮细胞的表达及其对脂多糖诱导CD40表达的影响

目的 观察血管紧张素Ⅱ（AngⅡ）对原代培养大鼠腹膜间皮细胞（RPMC）Toll样受体4（TLR4）表达的影响及其在脂多糖（LPS）诱导的核转录因子κB （NF-κB）活化及CD40表达中的作用。 方法 分离及培养RPMC。用不同浓度AngⅡ（10－9、10－8、10－7、10－6 mol/L）刺激细胞及用10-7 mol/L AngⅡ刺激细胞不同时间（mRNA为1、2、4、8、12、24、48 h和蛋白为6、12、24、36、48 h），观察血管紧张素1型受体（AT1R）阻滞剂洛沙坦（10－5 mol/L）和血管紧张素2型受体（AT2R）阻滞剂PD123177（10-5 mol/L）对AngⅡ诱导TLR4表达的影响。将细胞随机分为下列4组：对照组、AngⅡ (10－7 mol/L)组、LPS(1 mg/L)组、AngⅡ (10－7 mol/L)+LPS(1 mg/L)组，观察AngⅡ对LPS诱导的NF-κB激活和CD40表达的影响。RT-PCR检测TLR4、CD40 mRNA表达；Western印迹检测TLR4、IκBα、磷酸化IκBα（p-IκBα）、NF-κB p65、磷酸化NF-κB（p-p65）蛋白表达；免疫荧光检测细胞NF-κB p65亚单位的表达及分布。 结果 (1)10－9、10－8、10－7、10－6 mol/L AngⅡ作用RPMC 12 h，TLR4 mRNA表达分别增加70.5%、89.5%、102.9%和121.9%；作用24 h TLR4蛋白表达分别增加12.1%、27.7%、51.2%和41.6%。AngⅡ（10－7 mol/L）作用RPMC不同时间，TLR4 mRNA表达高峰为8 h和12 h（P < 0.01），蛋白表达高峰为12 h和24 h（P < 0.01）。(2)洛沙坦阻断后，AngⅡ诱导的TLR4表达与未阻断组比较，下调33.5%（P < 0.05）。PD123177对AngⅡ诱导的TLR4表达无显著影响（P > 0.05）。(3) 与正常对照组比较，LPS作用60 min p-IκBα/IκBα、p-p65/p65表达分别上调362.6%（P < 0.01）和67.4%（P < 0.05）；作用4 h CD40 mRNA表达上调299.9%（P < 0.01）；与LPS组比较，AngⅡ预刺激24 h加LPS作用60 min，p-IκBα/IκBα、p-p65/p65表达分别上调49.1%（P < 0.01）和29.3%（P < 0.05）；作用4 h CD40 mRNA表达上调56.8%（P < 0.01）。(4)免疫荧光结果显示正常对照组与AngⅡ组细胞中，p65信号定位于细胞胞质；LPS作用60 min，p65信号从胞质进入胞核；AngⅡ+LPS组NF-κB p65胞核信号显著增强。 结论 AngⅡ呈浓度、时间依赖性诱导RPMC TLR4表达，并显著增强LPS诱导NF-κB激活及CD40的表达。提示腹膜组织局部产生的AngⅡ可能对LPS诱导的腹膜组织炎性反应具有放大作用。     

TLR4、NF-κB和AP-1在乳腺癌中的表达及其与临床病理特征的相关性研究

目的 探讨Toll 样受体4（TLR4）、核因子κB（NF-κB）和激活因子蛋白1（AP-1）在乳腺癌组织中的表达及其与临床病理参数的关系.方法 采用免疫组织化学方法 检测106 例乳腺癌组织中TLR4、NF-κB 和AP-1 的表达.分析三者与乳腺癌临床病理因素的关系,以及TLR4与NF-κB、AP-1 的相关性.结果 随着乳腺癌患者T 分期、N 分期、M 分期和临床分期的增加,乳腺癌组织TLR4、NF-κB 及AP-1 的表达均升高（P 均〈 0.05）.随着乳腺癌组织分化程度的降低,NF-κB 的表达升高（P 〈 0.05）.乳腺癌组织中TLR4 和AP-1 的表达呈正相关（P 〈 0.05）.结论 乳腺癌组织TLR4 高表达,激活AP-1 信号通路,促进癌细胞的增殖、浸润及转移,进而影响患者的预后.     

过氧化物酶体增殖激活物受体γ配体诱导肾癌细胞的凋亡

目的 观察过氧化物酶体增殖激活物受体γ(PPARγ)配体对肾癌细胞凋亡的影响.方法 通过ELISA方法 观察0～100 μmol/L浓度的吡格列酮和曲格列酮(TZDs)对肾癌细胞786-0、A498及正常肾细胞HK-2、HMCC凋亡的影响,Heochst 33342荧光染色、DNA片段化分析检测70.00 μmol/L吡格列酮或80.00 μmol/L曲格列酮处理后肾癌细胞的凋亡,Western blot观察TZDs处理后肾癌细胞凋亡调控蛋白bcl-2/bax的表达变化及Caspase-3活性的改变.结果 超过50.00 μmol/L的TZDs可诱导肾癌细胞出现凋亡,LC_(50)值分别为65.11 μmol/L(曲格列酮/786-O)、67.73 μmol/L(吡格列酮J/786.0)、63.91 μmol/L(曲格列酮/A498)和78.12 μmol/L(吡格列酮/A498),但对正常肾细胞没有影响.荧光染色显示80.00 μmol/L的吡格列酮及70.00 μmol/L的曲格列酮处理后肾癌细胞明显凋亡,伴随bcl-2蛋白的表达水平降低和bax蛋白的增加,Caspase-3的活性明显增强,在48 h可达到基础值的3.5-4.5倍.结论 激活PPARγ可以诱导肾癌细胞的凋亡,PPARγ有可能成为肾细胞癌新的治疗靶点.     

血管紧张素1-7对高糖诱导人肾小管上皮细胞转分化的影响及其机制

目的 研究血管紧张素1-7（Ang 1-7）对高糖诱导人肾小管上皮细胞（HK-2）转分化的影响及其可能机制。 方法 培养HK-2细胞分组如下：对照组（N组）、高糖组（H组）、高糖+Ang 1-7组（A组）、高糖+Ang 1-7+A779组（D组）、高糖+吡格列酮组（P组）。Western印迹检测各组HK-2细胞过氧化物酶体增殖物激活受体γ（PPAR-γ）及α平滑肌肌动蛋白（α-SMA）的蛋白表达;实时定量PCR检测HK-2细胞PPAR-γ及α-SMA的mRNA表达;免疫荧光检测α-SMA表达。 结果 Ang 1-7可上调高糖刺激下HK-2细胞PPAR-γ蛋白及mRNA表达（P < 0.05）;抑制高糖刺激的α-SMA蛋白及mRNA表达（P < 0.05）。这种作用与PPAR-γ激动剂吡格列酮类似。给予Mas受体抑制剂A779后,Ang 1-7的上述作用可被部分抑制。 结论 Ang 1-7在体外可通过上调PPAR-γ表达,从而部分抑制高糖诱导的α-SMA表达,实现其抑制转分化的作用,而这种作用部分通过Mas受体所介导。     

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。     

虫草菌液对高糖作用下大鼠腹膜间皮细胞转化生长因子β_1和纤维连接蛋白表达的影响

目的：研究虫草菌液（HS）对高糖作用下体外培养的大鼠腹膜间皮细胞（RPMCs）转化生长因子β1（TGF-β1）和纤维连接蛋白（FN）表达的影响。方法：将原代培养的第3代RPMCs随机分为6组：对照组、1.5%葡萄糖组、2.5%葡萄糖组、单纯虫草组（10mg/ml）、1.5%葡萄糖＋10mg/ml虫草组、2.5%葡萄糖＋10mg/ml虫草组。同步培养72h后,以RTPCR法检测各组细胞TGF-β1和FN mRNA表达情况;ELISA法检测细胞上清液中TGF-β1和FN的蛋白质水平。结果：高糖作用下RPMC的TGF-β1、FN mRNA和蛋白表达水平均明显高于对照组（P〈0.05）,且增高的程度与葡萄糖浓度呈依赖关系。与相应浓度的高糖组相比,经虫草菌液干预后RPMC的TGF-β1、FN mRNA和蛋白表达水平均显著降低（P〈0.05）。结论：虫草菌液可下调高糖作用下RPMCs的TGF-β1和FN的表达,具有抗腹膜纤维化的作用。     

CCN3 inhibits deposition of extracellular matrix of glomerular mesangial cells via promoting the expression of microRNA-29a

Objective To investigate the effects of nephroblastoma over-expressed protein (CCN3) on the formation of extracellular matrix (ECM) induced by transforming growth factor -β1 (TGF-β1) in human mesangial cells (HMCs) and its underlying signal transduction mechanism related with microRNA-29(miRNA-29). Methods HMCs were pretreated with different doses of exogenous CCN3 (5 μg/L, 50 μg/L and 500 μg/L) or transfected with pcDNA3.1(+)-CCN3 before exposed to TGF-β1(2 μg/L), to observe the expression of fibronectin (FN), type Ⅰ collagen (COLⅠ) and miRNA-29a, b and c. The mimics or inhibitor of the miRNA-29a were transfected into HMCs to analyze whether miRNA-29a affect CCN3. The expressions of FN mRNA, COLⅠmRNA and miRNA-29 family were detected by real time PCR. The protein expressions of FN and COLⅠ were detected by Western blotting and cell immunofluorescence. Results (1) Compared with the normal control group, the expressions of FN and COLⅠ were up-regulated in TGF-β1 group, while the expressions of miRNA-29a, b, c were down-regulated in TGF-β1 group (all P＜0.05). (2) Compared with the TGF-β1 group, the expressions of FN and COLⅠ were decreased when pretreated with the different doses of exogenous of CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P＜0.05). Meanwhile, the expression of miRNA-29a was significantly increased when pretreated with 50 μg/L and 500 μg/L CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P＜0.05); whereas miRNA-29b and c had no statistical difference (all P＞0.05). (3) Compared with TGF-β1+CCN3 group, the expressions of FN and COLⅠ were decreased in CCN3+TGF-β1+miRNA-29a mimics group (all P＜0.05), whereas the expressions of FN and COLⅠ in CCN3+TGF-β1+miRNA-29a inhibitors group were increased (all P＜0.05). Conclusions CCN3 reduces the TGF-β1-induced production of ECM by the up-regulation of miRNA-29a.     

Effect of MG132 on the expression of ERK1/2 and connective tissue growth factor in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of MG132 on the expression of extracellular regulated kinase 1/2 (ERK1/2) and connective tissue growth factor (CTGF) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose (1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 0, 12, 24, 48 hours,incubated with MG132 (0.5, 1, 2 μmol/L) for half an hour and then with high glucose (2.5%) for 24 hours. ERK1/2 protein was detected by Western blotting, and CTGF protein in supernatant was detected by ELISA. Results Compared with the control group, the expression of p-ERK1/2 was significantly increased in the groups stimulated by high glucose (P＜0.01), reached the peak at 24th hour (P＜0.01), and then the expression decreased at 48th hour, but still was higher than that in the normal control group (P＜0.01). CTGF protein expression of RPMCs induced by high glucose increased, in time- and dose-dependent manner (P＜0.05). MG132 could significantly decrease the expression of ERK1/2 and CTGF induced by high glucose (P＜0.05). Conclusions MG132 can decrease the expression of p-ERK1/2 and CTGF in RPMCs induced by high glucose. The ubiquitin proteasome pathway participates in the development of peritoneal fibrosis, and blocking the way may contribute to the prevention of peritoneal fibrosis.     

罗格列酮通过核因子κB抑制脂多糖诱导的大鼠腹膜间皮细胞CD40和胞间黏附分子1的表达

目的 探讨罗格列酮对脂多糖（LPS）诱导的体外培养大鼠腹膜间皮细胞CD40和胞间黏附分子1（ICAM-1）表达的影响以及调节机制。 方法 分离及培养大鼠原代腹膜间皮细胞。将细胞随机分为正常对照组、LPS（5 mg/L）组、BAY11-7085（NF-κB抑制剂）组（5 μmol/L预刺激3 h后加入LPS作用3 h）、不同浓度罗格列酮（过氧化物酶体增殖蛋白激活性受体γ配体）组（10、20 μmol/L分别预处理3 h再加入LPS 5 mg/L）、GW9662（过氧化物酶体增殖蛋白激活性受体γ拮抗剂）预处理组（预处理3 h后加入罗格列酮10 μmol/L,3 h后再加入LPS 5 mg/L）和溶媒对照组。加入LPS后 1 h收集细胞检测核因子κB（NF-κB） p65水平;3 h收集细胞分别检测CD40和ICAM-1基因表达;24 h收集细胞分别检测CD40和ICAM-1蛋白表达。RT-PCR法检测基因表达;Western印迹和免疫荧光方法检测蛋白表达及核因子磷酸化。 结果 （1）常规培养的腹膜间皮细胞表达基础量CD40和ICAM-1,LPS显著上调其表达（P < 0.05）;LPS作用1 h时腹膜间皮细胞磷酸化NF-κB p65活化水平显著增高,与对照组差异有统计学意义 (1.10±0.17比0.55±0.06,P < 0.05)。（2）NF-κB抑制剂BAY11-7085预处理后LPS诱导的磷酸化NF-κB p65水平、CD40 和ICAM-1表达显著低于LPS组（0.22±0.11比1.10±0.17,P < 0.01;0.34±0.02 比 0.50±0.06,P < 0.05;0.35±0.16 比0.74±0.03,P < 0.05）。（3）罗格列酮预处理后,LPS诱导的磷酸化NF-κB p65水平、CD40以及ICAM-1蛋白表达亦显著低于LPS组（0.77±0.08比0.90±0.10,P < 0.01;0.79±0.16 比0.99±0.06,P < 0.05;0.83±0.20比1.22±0.13,P < 0.05）。GW9662和罗格列酮联合预处理后,LPS诱导的磷酸化NF-κB p65水平与罗格列酮预处理组差异无统计学意义,但CD40和ICAM-1表达显著高于罗格列酮预处理组（0.95±0.19比0.79±0.16;1.04±0.24比0.83±0.20,均P < 0.05）。 结论 NF-κB信号通路参与调节LPS诱导的腹膜间皮细胞表达CD40和ICAM-1。罗格列酮通过 NF-κB途径下调CD40和ICAM-1表达,从而发挥抗炎作用。     

DDR2RNA干扰对CCl4诱导肝纤维化动物模型的实验研究

目的 探讨沉默大鼠盘状结构域受体2(discoidin domain receptor 2,DDR2)基因表达对CCl4诱导大鼠肝纤维化的影响及其机制.方法 SD大鼠随机分为正常组(8)、纤维化组(18)、阴性对照组(18)和治疗组(18),每组又据干预时间不同平均分为4周、6周两组.化学合成经胆固醇修饰的siRNA-DDR2尾静脉体内注射,干扰CCl4诱导大鼠纤维化模型DDR2基因表达.用荧光实时定量PCR及Western blot分别检测干扰DDR2基因后DDR2、MMP-2和Ⅰ型胶原纤维(COLⅠ)的表达;同时行肝脏组织学观察及肝功能检测.结果 经siRNA-DDR2干扰后,纤维化组大鼠DDR2、MMP-2和COL Ⅰ的mRNA水平(t4=6.78,t6=9.02;t4=4.71,t6=6.37;t4=8.81,t6=6.50,均P＜0.01)及蛋白表达水平(t=6.11,t=4.39,t=5.23,均P＜0.01,4周;t=7.82,t=4.80,t=7.64,均P＜0.01,6周)均显著降低;同时,肝脏的组织学病变及肝功能指标也显著改变.结论 尾静脉注射化学合成经胆固醇修饰的抗DDR2siRNA能显著降低DDR2基因表达,促进细胞外基质降解,具有潜在的抗肝纤维化作用.

Abstract：

Objective To explore the effects of silencing DDR2 expression by siRNA on CCl4-induced liver fibrosis and its mechanism in rats. Methods Liver fibrosis model was induced by intraperitoneal injection of CCl4 twice a week for 6 consecutive weeks. Some rats were administered siRNA targeting DDR2 (0. 3 mg/kg), saline or control siRNA every three days from the beginning of CCl4 injection via tail vein injection, while other rats were treated in the same pattern after 2-week CCl4 injection. Quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot were used to detect the mRNA and protein expressions of DDR2, MMP-2 and COL Ⅰ . Meanwhile, the pathological changes of liver tissues and the levels of liver function were also observed. Results QRT-PCR showed that the DDR2, MMP-2 and COL Ⅰ mRNA in the chemically synthetic cholesterol-modified siRNADDR2 group were significantly decreased as compared with those in the control group (P＜0.01) ,and the protein expressions of DDR2, MMP-2 and COL Ⅰ were also significantly decreased (P＜0. 01,4 wand 6w). In addition, in comparison with those in the control group, the pathological changes of liver tissues in the siRNA-DDR2 treated group were markedly attenuated, and the levels of ALT(1356.17 ±83.80 nkat/L vs 2532. 70±145.11 nkat/L,4w,1367. 60±321.76 nkat/L vs 2604.37±255.02 nkat/L,6w,P＜0. 01 ) and AST (2460. 80 ± 207. 58 nkat/L vs 3983. 70 ± 253. 08 nkat/L, 4w, P＜ 0. 01,2383.27±290.16 nkat/L vs 3227.70±353. 34 nkat/L,6w,P＜0. 05)were also significantly lowered,while the level of TBIL (7. 97 ± 1.60 μmol/L vs 3.80± 0.60 μmol/L, 4w, 10.40±1.61 μmol/L vs 6.10±0.79 μmol/L,6w,P＜0. 01)was markedly increased. Conclusion Systemic administration of cholesterol-modified siRNA targeting DDR2 could significantly suppress the expression of DDR2, decrease the contents of the extracellular matrix,and thus has a potential antifibrotic effect.     

Fluorofenidone reduces renal fibrosis in diabetic kidney disease mice

Objective To investigate the effects of fluorofenidone (AKF-PD) on diabetic kidney disease in db/db mice and its possible mechanisms. Methods (1) Fifty-six mice aged 8 weeks (half male and half female), including 42 db/db mice and 14 wild-type mice were studied. Forty-two db/db mice randomly were divided into model group (mock-treated diabetic db/db mice), AKF-PD (250 mg?kg-1?d-1) treatment group and losartan (20 mg?kg-1?d-1) treatment group. Wild-type mice and model mice were treated with vehicle (0.5% sodium carboxymethylcellulose), while the treatment groups received either AKF-PD or losartan. After 18 weeks, the blood glucose and urinary albumin were measured, the pathological changes of kidney were observed by PAS staining. The protein expressions of type Ⅳ collagen and fibronectin (FN) in kidney tissue were detected by immunohistochemistry. (2) Mouse glomerular mesangial cells (MES-13 cells) were divided into six groups: normal glucose group (5.5 mmol/L glucose), hypertonic group (5.5 mmol/L glucose+19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), AKF-PD group (25.0 mmol/L glucose+400 mg/L AKF-PD) and losartan group (25.0 mmol/L glucose+2 μmol/L losartan). After 72 h treatment, the expressions of type Ⅰ collagen, type Ⅳ collagen and transforming growth factor-β1 (TGF-β1) mRNA were detected by real-time PCR, and the content of TGF-β1 protein in the culture supernatant was detected by ELISA. Results (1) Compared with the wild type mice, model mice had increased weight, blood glucose and glomerulosclerosis index (all P＜0.01), accompanied with heavy albuminuria, glomerular hypertrophy, mesangial area expansion and deposition of collagen type Ⅳ and FN (all P＜0.01). Compared with model mice, in AKF-PD and losartan groups 24 h urinary albumin and glomerulosclerosis index decreased (all P＜0.01), glomerular hypertrophy and mesangial area expansion alleviated, and the protein expressions of collagen type Ⅳ and FN were inhibited (all P＜0.01). (2) Compared with the normal glucose group, the mRNA expressions of type Ⅰ collagen and type Ⅳ collagen increased in high glucose group, meanwhile the mRNA and protein expressions of TGF-β1 increased (all P＜0.01). In AKF-PD and losartan groups the expressions of type Ⅰ collagen, type Ⅳ collagen and TGF-β1 were inhibited as compared with high glucose group (all P＜0.05). Conclusion Fluorofenidone may play an anti-fibrotic effect in db/db mice by reducing the expression of TGF-β1 and inhibiting collagen synthesis in glomerular mesangial cells.     

吡咯烷二硫代氨基甲酸盐增强肿瘤坏死因子α诱导人胃癌细胞株SGC-7901凋亡作用的研究

目的研究核转录因子-κb（NF—κb）抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）对肿瘤坏死因子α（TNF—α）诱导的人胃癌细胞株SGC-7901生长抑制及凋亡的影响,并探讨其作用机制。方法应用噻唑蓝（MTr）法检测不同浓度的PDTC和TNF-α以及两者联合应用对SGC-7901细胞增殖的抑制率：采用Hoechst检测SGC-7901细胞凋亡情况：Westernblot检测SGC-7901细胞survivin和easpase-3蛋白的表达。结果PDTC在15、30、60和100μmol／L浓度时．对SGC-7901的细胞生长抑制率分别为（12．14±0．91）％、（20．00±1．11）％、（37．63±1．01）％和（41.46±1．07）％．均可抑制细胞增殖（P〈0．01）。TNF-α为25mg／L时,对SGC．7901细胞的生长抑制率为（2．38±0．67）％,与对照组（1．50±0．81）％相比,差异无统计学意义（F=28．28,P〉0．05）：而在50、100和150mg／L浓度时,对SGC-7901细胞的生长抑制率分别为（4．53±0．85）％、（4．43±0．70）％和（4．74±1．07）％,与对照组相比,差异有统计学意义（P〈0．05）。PDTC15μmol／L分别与25、50、100和150mg／L的TNF．仅联合应用时,对SGC-7901的细胞生长抑制率则分别为（18．94±1．10）％、（30．23±0．89）％、（41．55±0．94）％和（53．34±0．98）％,与单用TNF—α或单用15μmol／LPDTC比较,细胞生长抑制率增加（P〈0．01）。Hoechst检测结果显示,TNF-α100mg／L组、PDTC15μmol／L组及两者联合应用组细胞凋亡率均显著增加（P〈0．01）,且联合用药组细胞凋亡率增高最为显著（P〈0．01）。PDTC（15μmol／L）与TNF-α（100mg／L）联合用药与单用TNF—α（100mg／L）比较,细胞survivin蛋白表达明显降低（P〈0．01）,与单用PDTC（15μmol／L）比较,差异无统计学意义（P〉0．05）;但caspase-3蛋白的表达联合用药组较两者分别单用时显著增加（P〈0．01）。结论PDTC可增强TNF-α对人SGC-7901细胞的促凋亡作用,其机制可能与PDTC阻断TNF-α诱导的NF—κb活性、下调survivin表达并最终上调凋亡蛋白easpase-3的表达有关。     

Effects of silibinin on expression of integrin linked kinase,transforming growth factor β1 and α-smooth muscle actin in rat peritoneal mesothelial cells induced by high glucose

Objective To observe the effect of silibinin on the expression of integrin linked kinase (ILK), transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat peritoneal mesothelial cells (RPMCs) induced by high glucose. Methods RPMCs were isolated, cultured and passaged by trypsin, then identified. The second generation of cultured RPMCs were used in the experiment. RPMCs were divided into normal control group, high glucose(1.5%, 2.5%, 4.25%) for 24 hours, high glucose (2.5%) for 12, 24, 48, 72 hours, high glucose (2.5%) for 24 hours after silibinin (5, 10, 20 mg/L) preincubate for 2 hours. ILK and α-SMA mRNA were detected by real-time PCR. ILK protein was detected by Western blotting. TGF-β1 protein in supernatants was detected by ELISA. Results Compared with the control group, the expresssion of ILK, TGF-β1 and α-SAM was significantly increased in groups stimulated by high glucose (all P＜0.05). Silibinin could significantly decrease the expression of ILK, TGF-β1 and α-SMA induced by high glucose (all P＜0.05). Conclusions High glucose can up-regulate the expression of ILK, TGF-β1 and α-SMA. Silibinin can reverse these changes.     

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。