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。     

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.     

转化生长因子β1和Smad4在胃癌中的表达

目的胃癌预后极差,是最为常见的肿瘤致死病因.转化生长因子信号转导通路具有一系列重要的细胞调节功能,包括细胞的生长、分化、粘附、转移、细胞外基质的形成和免疫功能.探讨转化生长因子-β1(TGF-β1)和Smad4与胃癌发生发展的关系.方法采用免疫组化和分子原位杂交的方法,对49例胃癌、20例萎缩性胃炎和17例正常胃黏膜的TGF-β1蛋白和Smad4 mRNA表达进行检测.结果胃癌组织中TGF-β1蛋白表达明显增强,其阳性率(79.6%)明显高于正常胃黏膜组(35.3%)及萎缩性胃炎组(40.0%),Smad4 mRNA表达则明显降低,其阳性率(57.1%)明显低于正常胃黏膜组(94.1%)及萎缩性胃炎组(85.0%),差异均有显著性(P＜0.01).胃癌组织的分化程度越低,TGF-β1蛋白的阳性率越高,Smad4 mRNA阳性率越低.结论TGF-β1蛋白的高表达和Smad4 mRNA的低表达,对细胞的恶性转化及增殖可能有一定的生物学意义.     

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.

     

Smad7的表达对心肌梗死后大鼠心肌纤维化的影响

目的研究心肌梗死（MI）后大鼠心肌Smad7的表达及其与心肌纤维化的关系。方法结扎冠状动脉建立大鼠MI模型,并另设假手术组为对照组。8周后,测定心室的质量/体质量（HW/BW）。以多普勒超声评价心脏功能。以Mallory’s Trichrome染色法检测非梗死区胶原的含量。分别以RT-PCR和Western blot法检测非梗死区左室心肌转化生长因子（TGF）-β1、Smad7 mRNA及蛋白表达的水平。结果与假手术组比较,MI组的HW/BW（P<0.01）、左心室舒张末期内径（LVEDD,P<0.01）、E峰/A峰比值（E/A,P<0.01）、非梗死区胶原的含量（P<0.01）、非梗死区左室TGF-β1mRNA及蛋白的表达（P<0.01）均显著增加;Smad7 mRNA及蛋白的表达（P<0.01）、射血分数（EF,P<0.01）及短轴缩短率（FS,P<0.01）均显著降低。结论MI后大鼠心肌Smad7的表达降低,与MI后的心肌纤维化密切相关。     

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。