罗格列酮抑制糖基化终产物诱导心肌成纤维细胞增殖和结缔组织生长因子及Smad的表达

目的 探讨糖基化终产物(AGE)对培养乳鼠心肌成纤维细胞增殖、结缔组织生长因子(CTGF)和Smad2、Smad4蛋白表达的影响及罗格列酮的干预作用.方法 采用胰酶消化法和差速贴壁分离法获取心肌成纤维细胞,应用MTT法、流式细胞仪法分别观察不同浓度AGE及罗格列酮对心肌成纤维细胞的细胞增殖、细胞周期的影响,ELISA方法 检测细胞培养上清中TGF-β1水平,Westem印迹技术检测CTGF及Smad2、Smad4蛋白质表达.结果 在一定浓度范围内,AGE干预心肌成纤维细胞,随浓度的增加,细胞增殖更加显著,TGF-β1分泌增加,CTGF蛋白表达增加.罗格列酮(0.1、1、10μmoVL)干预后,随浓度的增加,抑制心肌成纤维细胞增殖(分别为0.823±0.072、0.785±0.060、0.601±0.081对0.981±0.049,P<0.05)、抑制心肌成纤维细胞分泌TGF-β1(分别为257.77±9.09、230.29±6.56、200.84±10.26对300,68±8.56,P<0.01)、抑制CTGF蛋白表达的作用都更加显著(分别为0.769±O.108、0.590±0.095、0.534±0.115对1.021±0.113,P<0.01).罗格列酮(1和10μmol/L)干预后可显著减少AGE诱导的Smad2的蛋白表达(分别为0.424±0.059对0.572±0.073,P<0.05;0.396±0.080对0.572±0.073,P<0.01),同时可显著减少AGE诱导的Smad4的蛋白表达(分别为0.580±0.063对0.672±0.059,P<0.05;0.556±0.051对0.672±0.059,P<0.01).结论 AGE刺激心肌成纤维细胞增殖及分泌TGF-β1,同时诱导CTGF、Smad2及Smad4蛋白表达,罗格列酮在一定程度上抑制AGE上述作用,表明罗格列酮抑制心肌成纤维细胞增殖的效应与CTGF/Smad通路密切相关.     

罗格列酮抑制糖基化终产物诱导心肌成纤维细胞增殖和结缔组织生长因子及Smad的表达

Objective To investigate the effects of rosiglitazone on the proliferation,connective tissue growth factor and Smad expression in cultured cardiac fibroblasts induced by advanced glycosylation end-products (AGEs).Methods After being treated with various amounts of rosiglitazone,the cultured neonatal rat cardiac fibroblasts were incubated with AGEs.The status of cardiac fibroblasts proliferation and cell cycle were detected by 3-(4,5-dimethyhhiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTI) assay and flow cytometry.Furthermore,ELISA technique was applied to identify the level of TGF-β1.The protein expressions of CTGF and Smad in cardiac fibroblasts of neonatal SD rats were detected with Western blotting.Results The exposure of cardiac fibroblasts to AGEs at doses of 0-200 mg/L induced a dose-dependent increase in cell proliferation.At the concentration of rosiglitazooe (0.1,1,and 10 μmol/L),the cell proliferation was reduced compared with 200 mg/L AGEs group by O.823±0.072,0.785±0.060,0.601±0.081 vs 0.981±0.049,respectively (P < 0.05).The increased levels of TGF-β1 in supematants of cultured cardiac fibroblasts stimulated by AGEs were inhibited by rosiglitazone at the concentrations of 0.1,1,10μmol/L by 257.77±9.09,230.29±6.56,200.84±10.26 vs 300.68±8.56,respectively (vs 200 mg/L AGEs,P<0.01).Western blot indicated that pretreatment with rosiglitazone (0.1,1,and 10 μmol/L) inhibited CTGF protein production in a dose-dependent by 0.769±0.108,0.590±0.095,0.534±0.115 vs 1.021±0.113,respectively (vs 200 mg/L AGEs,P<0.01).It was also demonstrated that pretreatment with rosiglitazone (1 and 10 μmol/L) inhibited Smad2 protein production by 0.424±0.059,0.396±O.080 vs 0.572±0.073,respectively (vs 200 mg/L AGEs,P < 0.05 or P < 0.01).Meanwhile pretreatment with rosiglitazone (1 and 10 μmol/L) inhibited Smad4 protein production by 0.580±0.063,0.556±0.051 vs 0.672±0.059,respectively (vs 200 mg/L AGEs,P < 0.05 or P < 0.01).Conclusions The findings suggest that AGEs promote the proliferation of cardiac fibroblasts and stimulate the protein production of Smad and CTGF of cardiac fibroblasts.Rosiglitazone inhibits the above reaction.These results indicate that CTGF/Smad pathway may play an important role in the protective effect of rosiglitazone on myocardial fibrosis.     

罗格列酮对糖基化终产物干预的人肾系膜细胞fractalkine表达的影响

观察罗格列酮和糖基化终产物对人肾系膜细胞fractalkine(FKN)表达的影响.结果 显示糖基化终产物能上调人肾系膜细胞的FKN表达,而罗格列酮能抑制糖基化终产物引起的FKN表达增加.     

马来酸罗格列酮抑制糖基化终产物诱导的人肾系膜细胞RANTES高表达

目的探讨马来酸罗格列酮对糖基化终产物（AGEs）诱导的人肾系膜细胞（HRMC）趋化因子RANTES高表达的影响,及其对糖尿病大鼠血清糖基化终产物-肽（AGE-P）及肾脏RANTES表达的影响。方法运用糖基化修饰的牛血清白蛋白（AGE-BSA）与马来酸罗格列酮干预体外培养的HRMC,EusA法检测细胞培养上清中RANTEs蛋白水平,实时定量RT-PCR检测细胞中RANTES mRNA水平,Western blot检测RANTES蛋白表达。制备2型糖尿病大鼠模型,马来酸罗格列酮治疗10周。流动注射分析法测定血清AGEP,免疫组织化学检测肾皮质RANTES表达。结果马来酸罗格列酮干预组HRMC RANTES的mRNA和蛋白表达以及蛋白分泌明显低于AGE-BSA组;马来酸罗格列酮治疗组糖尿病大鼠血清AGE-P明显下降,肾皮质RANTES表达明显低于糖尿病非治疗组。结论马来酸罗格列酮可以抑制AGE-BSA诱导的HRMC RANTES的表达和分泌,还可降低糖尿病大鼠血清AGE-P及肾脏RANTES表达。     

糖基化终产物对大鼠主动脉平滑肌细胞结缔组织生长因子表达的影响

目的 研究糖基化终产物对结缔组织生长因子(CTGF mRNA)及蛋白质表达的影响,探讨糖基化终产物致动脉粥样硬化机制. 方法 采用组织块贴壁法培养大鼠主动脉平滑肌细胞.反转录聚合酶链反应、蛋白免疫印迹法检测血管平滑肌细胞结缔组织生长因子mRNA及蛋白质的表达. 结果 随着糖基化修饰的牛血清白蛋白(AGE-BSA)的干预浓度(100、200、400 mg/L)升高,CTGF mRNA的表达水平呈上升趋势,分别为(0.78±0.03)、(1.15±0.03)、(1.40±0.04)mg/L,与未干预(0.40±0.02)mg/L比较,差异有统计学意义(P<0.05),不同浓度间差异亦有统计学意义(P<0.05).以200 mg/L的AGE-BSA对平滑肌细胞分别干预0、4、8、16、24、48、72 h,发现4 h时CTGF mRNA表达即已升高(0.93±0.04)mg/L,8 h时最高(1.29±0.04)mg/L,以后虽略有下降,但仍维持较高水平. 结论 糖基化终产物促进血管平滑肌细胞表达结缔组织生长因子,可能是其致动脉粥样硬化机制中的一个重要方面.     

RNA干扰选择性下调心肌成纤维细胞结缔组织生长因子

目的 研究RNA干扰技术能否选择性下调乳鼠心肌成纤维细胞结缔组织生长因子(CTGF)及其诱导的下游纤维化成分的表达.方法 利用差速贴壁法分离培养乳鼠心肌成纤维细胞.用携带U6启动子和CTGF特异性短发夹RNA(shRNA)编码序列的质粒载体CTGF-shRNA,及含非特异性shRNA编码序列的对照质粒CTGF-con以lipofectamine TM2000为载体转染原代培养的乳鼠心肌成纤维细胞.加入含G418的DMEM培养液培养一月筛选后获得稳定转染的细胞.通过半定量RT-PCR检测细胞内转化生长因子(TGF-β1),CTGF及其下游纤维化成分纤维连接蛋白(FN)的表达,Western-blot法检测细胞内CTGF及其下游纤维化成分纤维连接蛋白(FN)的表达.结果 CTGF在心肌成纤维细胞中有基础表达,并在转化生长因子β1(TGF-β1)刺激下其含量增加,CTGF-shRNA使心肌成纤维细胞CTGF的mRNA和蛋白质的表达分别降低65%和78%,FN的mRNA和蛋白质的表达分别降低81%和63%,而TGF-β1mRNA的表达上调32%.转染对照质粒组较空白对照组与脂质体组CTGF,FN表达量差异无统计学意义.结论 RNA干扰技术能选择性下调乳鼠心肌成纤维细胞CTGF及下游纤维化成分FN的表达,进一步为拮抗心肌纤维化的基因治疗提供了依据.     

RNA干扰选择性下调心肌成纤维细胞结缔组织生长因子

目的研究RNA干扰技术能否选择性下调乳鼠心肌成纤维细胞结缔组织生长因子（CTGF）及其诱导的下游纤维化成分的表达。方法利用差速贴壁法分离培养乳鼠心肌成纤维细胞。用携带U6启动子和CTGF特异性短发夹RNA（shRNA）编码序列的质粒载体CTGF-shRNA，及含非特异性shRNA编码序列的对照质粒CTGF-con以lipofectamine ^TM2000为载体转染原代培养的乳鼠心肌成纤维细胞。加入含G418的DMEM培养液培养一月筛选后获得稳定转染的细胞。通过半定量RT-PCR检测细胞内转化生长因子（TGF-β1），CTGF及其下游纤维化成分纤维连接蛋白（FN）的表达，Western-blot法检测细胞内CTGF及其下游纤维化成分纤维连接蛋白（FN）的表达。结果CTGF在心肌成纤维细胞中有基础表达，并在转化生长因子β1（TGF-β1）刺激下其含量增加，CTGF-shRNA使心肌成纤维细胞CTGF的mRNA和蛋白质的表达分别降低65％和78％，FN的mRNA和蛋白质的表达分别降低81％和63％，而TGF-β1mRNA的表达上调32％。转染对照质粒组较空白对照组与脂质体组CTGF，FN表达量差异无统计学意义。结论RNA干扰技术能选择性下调乳鼠心肌成纤维细胞CTGF及下游纤维化成分FN的表达，进一步为拮抗心肌纤维化的基因治疗提供了依据。     

糖尿病大鼠阴茎组织糖基化终产物含量的变化和糖基化终产物受体的表达情况

目的研究糖尿病阴茎组织糖基化终产物(AGEs)含量的变化和糖基化终产物受体(RAGE)的表达情况。方法用葡萄糖和血红蛋白(Hb)在体外孵育形成Hb-AGEs,免疫新西兰兔,制备AGEs抗血清,建立竞争性ELISA法,测定大鼠阴茎组织AGEs含量。应用RT-PCR研究RAGEmRNA表达。结果各组糖尿病大鼠未经治疗时阴茎组织AGEs水平均较对照(NC)组明显升高(P<0.01),8周时糖尿病用胰岛素治疗(DMI)组及糖尿病用胰岛素和二氨基酚嗪(DAP)治疗(DMID)组的AGEs水平较糖尿病未治疗(DM)组降低(P<0.05～0.01),至16周DMID组上述指标较DMI组明显降低(P<0.01)。RAGEmRNA在DM组大鼠阴茎组织的表达明显高于NC组(P<0.01)。结论糖尿病大鼠阴茎组织AGEs水平明显升高,RAGEmRNA高表达,胰岛素和DAP治疗可降低AGEs水平。     

心肌成纤维细胞分泌生长因子在心肌重塑中的作用

本文论述心肌成纤维细胞在一些理化因素刺激下，多种生长因子分泌的变化及生长因子网络失衡在心肌重塑中的作用。     

罗格列酮对糖尿病大鼠胸主动脉钙化的影响

目的探讨罗格列酮对糖尿病大鼠胸主动脉钙化的影响。方法利用链脲佐菌素联合高能量饲料和维生素D3并尼古丁制备糖尿病血管钙化大鼠模型(DM+VDN组),并随机分为模型组、罗格列酮(RSG)治疗组和格列本脲(GLB)治疗组。检测大鼠一般代谢指标、血清糖基化终末产物(AGE)、丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性,胸主动脉钙含量及糖基化终末产物受体(RAGE)蛋白表达。结果 RSG治疗组和GLB治疗组血清AGE、MDA及SOD水平低于模型组,且RSG效果优于GLB(P0.05); RSG治疗组胸主动脉RAGE表达水平以及钙含量均低于模型组和GLB治疗组(P0.05)。结论罗格列酮可能通过降低AGE水平,下调RAGE表达,抑制氧化应激反应,延缓血管钙化进展。     

吡哆胺对动脉粥样硬化兔晚期糖基化终末产物及其受体的影响

目的观察吡哆胺对动脉粥样硬化兔晚期糖基化终末产物(AGEs)及其受体的影响。方法利用高脂饮食法诱导建立兔动脉粥样硬化模型。将新西兰白兔按随机数字表法随机分为正常对照组、动脉硬化组、吡哆胺小剂量组和吡哆胺大剂量组。正常对照组给予普通食物;动脉硬化组给予高脂饮食;吡哆胺小剂量组给予高脂饮食+吡哆胺100 mg/(kg·d);吡哆胺大剂量组给予高脂饮食+吡哆胺150 mg/(kg·d),共观察12周。实验12周末监测所有兔血糖、三酰甘油、总胆固醇、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C);应用酶联免疫吸附试验法检测血清AGEs水平;观察主动脉病理形态学改变;免疫组化方法检测主动脉AGEs受体(RAGE)含量,并进行各组间的比较。结果实验12周末,(1)与对照组比较,动脉硬化组兔三酰甘油、总胆固醇、LDL-C、HDL-C均明显增高(均P0.01),血糖无明显变化(P0.05);动脉硬化组的血清AGEs水平[(419±29)mg/L)]、动脉RAGE吸光度(0.88±0.14)及斑块面积[(67.4±5.8)%]均较对照组[分别为(141±11)mg/L、(0.19±0.04)及0%]明显增高(均P0.01);(2)与动脉硬化组相比,吡哆胺小剂量组和吡哆胺大剂量组兔三酰甘油、总胆固醇、LDL-C、HDL-C、血糖均无明显变化(均P0.05);吡哆胺小剂量组的血清AGEs水平[(278±22)mg/L]、动脉RAGE吸光度(0.43±0.06)及斑块面积[(43.2±2.4)%]和吡哆胺大剂量组的血清AGEs水平[(211±18)mg/L]、动脉RAGE吸光度(0.27±0.05)及斑块面积[(27.4±2.8)%]均较动脉硬化组明显减少(均P0.01);(3)吡哆胺大剂量组的血清AGEs水平、动脉RAGE吸光度及斑块面积较吡哆胺小剂量组低(均P0.01)。(4)主动脉病理形态学:显微镜下见动脉硬化组动脉内膜增厚,形成明显的粥样斑块;有大量的泡沫细胞聚集及较多的炎性细胞。吡哆胺小剂量组和吡哆胺大剂量组较动脉硬化组均有不同程度的减轻。结论吡哆胺能够抑制、减缓实验性高脂饮食兔的动脉粥样硬化形成,可抑制血清AGES的生成及动脉RAGE的表达。     

Connective tissue growth factor/IGF-binding protein-related protein-2 is a mediator in the induction of fibronectin by advanced glycosylation end-products in human dermal fibroblasts

Expansion of extracellular matrix with fibrosis occurs in many tissues, including skin, as part of the end-organ complications in diabetes. Advanced glycosylation end-products (AGEs) have been implicated as a pathogenic factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as IGF-binding protein-related protein-2, induces extracellular matrix. We have recently shown that CTGF mRNA and protein are up-regulated by AGE treatment of cultured human dermal fibroblasts. The aim of this study was to determine whether CTGF is an autocrine mediator in the induction of fibronectin (FN) by AGE. Primary cultures of nonfetal human dermal fibroblasts in confluent monolayer were treated with synthesized soluble AGE BSA, 0-200 microg/ml. Analysis of mRNA, by quantitative real-time RT-PCR and conditioned media from treated cultures, showed that FN mRNA was increased by approximately 4-fold at 48 h, and FN protein levels by Western immunoblot and FN ELISA were doubled, compared with control. In the same system, added recombinant human CTGF (0-500 ng/ml) induced FN mRNA and protein levels dose dependently and in a rapid time course. To test whether AGE BSA acts through cell-derived CTGF to induce FN, a CTGF neutralizing antibody was shown to significantly attenuate, but not fully inhibit, the AGE induction of FN mRNA. A pan-specific PKC inhibitor, GF109203X, at 0.2 microM, inhibited the induction of FN mRNA by AGE BSA. Although the same inhibitor did not significantly affect the induction of CTGF mRNA by AGE, it blocked the induction of FN mRNA by recombinant human CTGF. In summary, the induction of FN by AGE is partly mediated by the AGE-induced up-regulation of cell-derived CTGF and is dependent on PKC activity. These results have potential implications for the expansion of extracellular matrix in diabetes mellitus by advanced glycosylation end products.     

糖基化终产物对人血管内皮细胞基质金属蛋白酶-2表达和活性的影响

目的探讨糖基化终产物(AGEs)对人血管内皮细胞ECV304基质金属蛋白酶-2(MMP-2)表达和活性的影响。方法应用AGEs作用ECV304细胞后,采用RT-PCR法及明胶酶图分析方法分析MMP-2表达和活性与AGEs浓度和时间的关系。结果 RT-PCR结果显示,AGEs能够诱导ECV304细胞表达MMP-2,其诱导表达具有浓度和时间依赖性(P<0.01)。明胶酶图分析显示,MMP-2降解明胶的活性与AGEs具有浓度依赖性。结论 AGEs能够促进ECV304细胞MMP-2的表达和活性,推测AGEs调控MMP-2表达和活性可能在血管壁内皮细胞影响动脉粥样硬化的进展中发挥一定的作用。     

糖化终末产物对INS-1细胞基质金属蛋白酶2表达的影响

目的 探讨糖化终末产物(AGEs)对大鼠胰岛细胞株INS-1基质金属蛋白酶2(MMP-2)表达量的影响.方法 培养INS-1细胞,检测MMP-2表达;制备糖化胎牛血清,通过流式细胞仪检测细胞内活性氧簇(ROS)水平,通过逆转录PCR、实时定量PCR和Western印迹方法观察细胞中MMP-2表达量的变化.结果 INS-1细胞中存在MMP-2的表达;AGEs刺激后ROS明显升高(P<0.05),刺激组细胞内MMP-2mRNA表达量和蛋白表达水平均显著高于对照组(P<0.05).结论 提示胰岛β细胞内存在MMP-2表达,AGEs可诱导β细胞MMP-2表达量增高,MMP-2在介导胰岛氧化应激损伤中可能起着重要作用.     

Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis

Objective

Connective tissue growth factor (CTGF) is a cysteine‐rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor β (TGFβ) signaling or through distinct signal transduction pathways.

Methods

We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast‐specific collagen α2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis.

Results

Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK‐1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild‐type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFβ.

Conclusion

These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.