人皮肤成纤维细胞在紫外线B照射后的TGF—β和HSP70表达

目的和方法：采用人体皮肤成纤维细胞,观察了紫外线照射后细胞TGF－β表达与HSP70表达水平的相关性。结果：①经不同剂量的紫外线B照射后,TGF－βmRNA表达与HSP70表达水平呈正相关（r=0.906);②应用抗TGF－βⅡ型受体抗体后,在紫外线B照射后细胞培养上清液中的TGF－β含量与细胞HSP70表达水平呈负相关（r=-0.995)。结论：在紫外线B照射诱导人皮肤成纤维细胞表达HSP70的反应过程另TGF－β参与其信号转导。     

Role of Smad2/3 and p38 MAP kinase in TGF-β1-induced epithelial-mesenchymal transition of pulmonary epithelial cells

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-β1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-β1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.     

糖尿病大鼠血糖控制前后肾小管上皮细胞VEGF、TGF-β1及Smad蛋白表达的动态研究

目的动态观察链脲佐菌素（STZ）诱导的糖尿病大鼠血糖控制前后肾小管上皮细胞（TEC）中血管内皮生长因子（VEGF）、转化生长因子β1（TGF-β1）、Smad2/3、Smad4的表达情况,探讨四者在糖尿病大鼠TEC表型转变和肾间质纤维化中可能发挥的作用及相互关系。方法实验动物随机分为5组,依病程长短分为①A组（2周组）,②B组（4周组）,③C组（8周组）,④D组（16周组）,⑤E组（24周组）,每组分别设有正常对照组（N组）和糖尿病组（a组）;另外,16周、24周两组加设胰岛素治疗组（b组）。采用尾静脉注射STZ法复制糖尿病大鼠模型;免疫组织化学方法检测肾小管VEGF、TGF-β1、Smad2/3、Smad4及α-平滑肌肌动蛋白（-αSMA）和纤连蛋白（FN）的表达;Western blot检测肾皮质VEGF和TGF-β1蛋白;PAS染色光镜观察肾小管基底膜变化及细胞外基质沉积情况等形态学改变;生化方法测定血糖、血肌酐及24小时尿蛋白量。结果正常对照组VEGF、TGF-β1及Smad2/3、Smad4在肾小管均有少量表达,-αSMA在肾小管无表达;糖尿病组肾小管前述四者的表达均显著高于正常对照组,且从16周开始肾小管上皮细胞可见α-SMA蛋白阳性表达;糖尿病16周时肾小管VEGF、TGF-β1、Smad2/3、Smad4两两之间呈正相关;随糖尿病进展,α-SMA及FN在肾小管表达增多,24h尿蛋白增多,肾脏肥大指数增大,而VEGF、TGF-β1二者都分别和-αSMA、FN、24h尿蛋白及肾脏肥大指数呈正相关性;胰岛素治疗后,VEGF、TGF-β1、Smad2/3、Smad4及FN的表达都比糖尿病组明显下降,且各指标之间的正相关性依然存在,-αSMA蛋白则呈阴性表达。结论糖尿病肾病大鼠肾小管上皮细胞表达的VEGF、TGF-β1及Smad2/3、Smad4参与了TEC表型转变和肾间质纤维化的发生,并且VEGF和TGF-β1相互作用,共同促进了肾脏损害。胰岛素对DN大鼠TEMT和肾间质纤维化的影响可能部分是通过间接阻断VEGF、TGF-β1和Smad2/3、Smad4在TEC中的合成来实现的。     

人皮肤成纤维细胞在紫外线B照射后的TGF-β和HSP70表达

目的和方法:采用人体皮肤成纤维细胞,观察了紫外线照射后细胞TGF-β表达与HSP70表达水平的相关性.结果:①经不同剂量的紫外线B照射后,GF-βmRNA表达与HSP70表达水平呈正相关(r=0.906);②应用抗TGF-βⅡ型受体抗体后,在紫外线B照射后细胞培养上清液中的TGF-β含量与细胞HSP70表达水平呈负相关(r=-0.995).结论:在紫外线B照射诱导人皮肤成纤维细胞表达HSP70的反应过程中TGF-β参与其信号转导.     

基于TGF-β1/Smad信号通路初步探讨虎杖醇提物对腺嘌呤诱导小鼠肾间质纤维化的改善作用

目的 研究虎杖醇提物(ethanolic extract of Polygonum cuspidatum,PCE)对小鼠肾间质纤维化的保护作用及其潜在分子机制。方法 将50只雄性C57BL/6N小鼠随机分为5组(n=10):正常对照组、模型组、虎杖醇提物低、中、高(75、150、300 mg/kg)剂量组。除正常对照组外,其他各组灌胃腺嘌呤构建肾间质纤维化模型,虎杖醇提物低、中、高剂量组同时灌胃给予不同剂量的虎杖醇提物混悬液。各组经灌胃给药42 d后用生化试剂盒测定血清肌酐(serum creatinine, Scr)和尿素氮(blood urea nitrogen, BUN)水平,通过苏木精伊红染色和Masson三色染色分别观察小鼠肾组织病理学变化及胶原沉积情况,采用Western Blot法检测小鼠肾组织中转化生长因子-β1(transforming growth factor-β1,TGF-β1)、Smad6、α平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、I型胶原蛋白(Collagen I)的表达情况。结果 与正常对照组比较,模型组小鼠BUN、Sc...     

有氧运动和白藜芦醇对2型糖尿病大鼠肾脏JAK2及TGF-β1的影响

目的: 研究有氧运动和白藜芦醇对2型糖尿病大鼠肾脏Janus激酶2(JAK2)及转化生长因子-β1(TGF-β1)表达的影响,探讨运动与白藜芦醇改善糖尿病肾损伤的可能作用机制。方法: SD大鼠经5周高糖高脂饲料喂养加腹腔注射链脲佐菌素(STZ)建立糖尿病模型后,将糖尿病大鼠随机分为糖尿病安静组(DC组),糖尿病运动组(DE组),糖尿病药物组(DR组)和糖尿病运动药物组(DER组),每组各12只,另设正常对照组(NC组)。运动组大鼠进行8周的有氧运动(跑速为20 m/min),每天运动60 min,每周运动6 d;药物组大鼠进行8周的白藜芦醇灌胃(每天45 mg/kg,7天/周)。8周末,检测血糖、24 h尿白蛋白(24 h UA)、血肌酐(Scr)、血尿素氮(BUN)的变化;采用荧光定量PCR检测肾脏JAK2 mRNA的表达,免疫组织化学法和Western blot法检测肾脏JAK2和TGF-β1的表达。结果: 8周干预后,与NC组相比, DC组血糖浓度、24 h UA、Scr、BUN均显著上升(P＜0.05),肾组织病理损伤加重,肾组织TGF-β1、JAK2和JAK2 mRNA的表达均明显增加(P＜0.05)。与DC组相比,DE、DR和DER组血糖浓度、24 h UA、Scr、BUN均显著下降(P＜0.05),肾组织病理损伤减轻,肾组织TGF-β1、JAK2和JAK2 mRNA的表达均明显减少(P＜0.05),且DER组的降低更显著,与DE、DR组相比差异有显著性(P＜0.05)。结论: 有氧运动、白藜芦醇及联合干预可能通过下调肾脏JAK2 mRNA表达,抑制JAK2蛋白的合成,使TGF-β1表达减少,从而改善糖尿病大鼠肾脏损伤的病理性变化。有氧运动联合白藜芦醇干预减轻肾脏病理损伤的效果优于单一的有氧运动或白藜芦醇干预。     

支架蛋白GIT2通过与Smad3相互作用调节其转录活性

G蛋白偶联受体激酶相互作用蛋白2(G protein-coupled receptor kinase interacting proteins 2,GIT2)是一种信号支架蛋白,可募集多种信号通路的关键分子,参与肌动蛋白细胞骨架组装、整合素介导的细胞粘附、G蛋白偶联受体的内化及胞内信号传递等生物学过程. 采用酵母双杂交实验证明,TGF-β1信号通路的转录因子Smad3是GIT2的相互作用蛋白质,内、外源免疫共沉淀实验均证实,GIT2与Smad3存在蛋白质相互作用. 报告基因实验及免疫印迹结果表明,GIT2增加Smad3的转录活性并增强TGF-β1诱导的Smad3的磷酸化.研究还发现,Git2-/-小鼠骨髓间充质干细胞(MSC)的Smad3磷酸化受到抑制,其骨形成相关靶基因的表达水平也低于Git2+/+小鼠. 本研究表明,GIT2通过与Smad3的相互作用调节其转录活性并活化TGF-β1信号通路,可能参与调节骨髓间充质干细胞的分化.     

Catalpol inhibits TGF-β1-induced epithelial-mesenchymal transition in human non–small-cell lung cancer cells through the inactivation of Smad2/3 and NF-κB signaling pathways

Catalpol, one of the main active ingredients isolated from Rehmannia glutinosa, was reported to possess anticancer activity. However, the role of catalpol in transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human non–small-cell lung cancer (NSCLC) cells has not been elucidated. The objective of this study was to investigate the effect of catalpol on EMT in human NSCLC cells. Our results showed that catalpol significantly inhibited the TGF-β1-induced cell migration and invasion of A549 cells, as well as repressed matrix metalloproteinase (MMP)2 and MMP9 expression induced by TGF-β1 in A549 cells. In addition, catalpol markedly repressed the EMT process in A549 cells in response to TGF-β1. Furthermore, catalpol prevented the activation of Smad2/3 and nuclear factor κB (NF-κB) signaling pathways induced by TGF-β1 in A549 cells. In conclusion, these findings indicated that catalpol inhibits TGF-β1-induced EMT in human NSCLC cells through the inactivation of Smad2/3 and NF-κB signaling pathways. Thus, catalpol may be a promising agent for the treatment of NSCLC.     

Epigallocatechin-3-gallate prevents TGF-β1-induced epithelial-mesenchymal transition and fibrotic changes of renal cells via GSK-3β/β-catenin/Snail1 and Nrf2 pathways

Several lines of evidence have demonstrated anti-fibrotic property of epigallocatechin-3-gallate (EGCG) in many tissues/organs but with unclear mechanisms. This study thus aimed to define cellular mechanisms underlying such protective effect of EGCG. HK-2 renal cells were treated with 5 ng/ml TGF-β1 for 24 h with/without pretreatment by 5 μM EGCG for 1 h. The cells were then evaluated by morphological examination, immunofluorescence study, semi-quantitative RT-PCR, Western blotting, and atomic force microscopy (AFM). The results showed that TGF-β1-treated cells underwent epithelial mesenchymal transition (EMT) as evidenced by morphological change into fibroblast-like and increases in spindle index, mesenchymal markers (Snail1 and vimentin), extracellular matrix (fibronectin), cell stiffness (by AFM measurement) and actin stress fibers, whereas the epithelial markers (E-cadherin and ZO-1) were decreased. All of these features were abolished by EGCG pretreatment. Functional studies revealed that the anti-fibrotic property of EGCG was, at least in part, due to de-activation/stabilization of GSK-3β/β-catenin/Snail1 (EMT-triggering) signaling pathway that was activated by TGF-β1 as shown by maintaining phosphorylated GSK-3β, β-catenin and Snail1 to their basal levels. Additionally, Nrf2 knockdown by small interfering RNA could abolish the EGCG effect on β-catenin expression. These data indicate that EGCG attenuates TGF-β1-induced EMT in renal tubular cells through GSK-3β/β-catenin/Snail1 and Nrf2 pathways.     

红腹锦鸡肾的组织结构及EGFR、TGF-β、AQP-2在肾脏中的表达

为了解红腹锦鸡肾脏的结构特征,观察表皮生长因子受体(EGFR)、转化生长因子-β(TGF-β)、水通道蛋白-2(AQP-2)在肾脏中的表达情况,应用组织学方法及免疫组织化学方法,观察了5只红腹锦鸡肾脏的组织结构,检测了EGFR、TGF-β(TGF-β1,TGF-β2,TGF-β3)、AQP-2在肾中的表达。结果表明:红腹锦鸡肾脏主要由许多肾单位、集合管和少量结缔组织组成。肾单位是肾脏功能结构的基本单位,它由一个肾小体和一个与其连接的上皮性肾小管构成。血管球由一团迂回盘曲的毛细血管丛构成,结构简单;肾小囊足细胞的突起与毛细血管内皮细胞及基膜紧密相贴,三者构成肾小体的滤过屏障;近端小管由单层立方上皮组成,上皮细胞游离面有许多微绒毛,细胞界限不明显,侧面有许多指状突起彼此交错,质膜内褶较少;远端小管上皮细胞基底面有丰富的质膜内褶;集合管上皮细胞有明细胞和暗细胞两种类型;近端小管上皮细胞呈EGFR、TGF-β免疫反应阳性,集合管上皮细胞呈AQP-2免疫反应阳性。EGFR、TGF-β、AQP-2可能发挥着不同的功能,对肾单位的构筑、肾小管和集合管结构的稳定以及肾脏水的平衡等可能有重要的调节作用。     

青蒿琥酯基于TGF-β/Smad通路减轻2型糖尿病大鼠脾脏纤维化的机制研究CSCD

探讨青蒿琥酯(artesunate,ART)对2型糖尿病(type 2 diabetes mellitus,T2DM)大鼠脾脏纤维化的防治作用,并明确其相关分子机制,为T2DM脾脏损伤防治提供实验依据。50只SD实验大鼠分为正常组和模型组,模型组高糖高脂喂养8周后,注射链脲佐菌素构建T2DM模型,再随机分为糖尿病组、二甲双胍治疗组、青蒿琥酯治疗组、联合治疗组,连续用药干预4周后取大鼠脾脏,称脾脏重量;HE、Masson染色观察脾脏病理变化;免疫组织化学法和Western blot检测脾脏组织中转化生长因子β1(transforming growth factor-β,TGF-β1)、重组人蛋白2(smad family member 2,Smad2)、重组人蛋白7(smad family member 7,Smad7)、Ⅰ型胶原(CollagenⅠ)表达;qRT-PCR检测脾脏组织中TGF-β1、Smad2、Smad7、CollagenⅠ的mRNA表达量。与正常组比较,糖尿病组大鼠脾脏指数下降,脾脏细胞排列紊乱,大量纤维化增生。与糖尿病组比较,各用药干预组的脾脏损伤和纤维化减轻,脾脏组织中TGF-β1、Smad2、CollagenⅠ蛋白和mRNA含量显著降低、Smad7明显上升。以上结果说明ART能够减轻T2DM大鼠脾脏损伤,抑制脾脏纤维化增生,其机制可能与抑制TGF-β/Smad信号通路有关。     

Pathobiological mechanisms of peritoneal adhesions: The mesenchymal transition of rat peritoneal mesothelial cells induced by TGF-β1 and IL-6 requires activation of Erk1/2 and Smad2 linker region phosphorylation

Peritoneal adhesions, primarily caused by surgical procedures, are the leading cause of pelvic pain, bowel obstruction, and infertility. TGF-β1 and IL-6 have been found to be elevated in the peritoneal fluid of patients during/after abdominal surgery. However, it remains to be determined whether these cytokines interact and facilitate adhesion formation by promoting mesothelial to mesenchymal transition (MMT). In the present study, isolated rat peritoneal mesothelial cells were treated with TGF-β1 and/or IL-6 which elicited MMT as determined by morphologic and biochemical techniques. During this transition, cellular morphology changed from that of cobblestone polygonal cells to elongated/spindle-shaped fibroblast-like cells. There was decreased expression of genes characteristic of mesothelial cells, such as E-cadherin, and increased expression of genes characteristic of the myofibroblast phenotype, including α-smooth muscle actin and the EDA form of fibronectin, both of which appear to mediate the transfer of force to the extracellular matrix. Partial characterization of relevant signaling pathways identified Erk1/2 activation, which was enhanced by combined TGF-β1/IL-6 administration, as a crucial necessary factor in the transition. Erk1/2 activation as well as the phosphorylation of the linker region of Smad2 and MMT could be blocked by the MEK inhibitor, U0126, suggesting that such activation may be a potential pharmaceutical target to prevent MMT. In addition, the phenotypic transition could be prevented by hydrocortisone.     

MicroRNA-486-5p suppresses TGF-β<Subscript>2</Subscript>-induced proliferation,invasion and epithelial–mesenchymal transition of lens epithelial cells by targeting Smad2

The pathological development of lens epithelial cells (LECs) leads to posterior capsular opacification (PCO). This study was undertaken to investigate the effects of microRNA-486-5p (miR-486-5p) on TGF-β₂-induced proliferation, invasion and epithelial-mesenchymal transition (EMT) in the lens epithelial cell line SRA01/04, and to explore the underlying molecular mechanisms. The expression of miR-486-5p in TGF-β₂-induced SRA01/04 cells was down-regulated, and the expression of Smad2, p-Smad2 and p-Smad3 was up-regulated. A dual-luciferase reporter assay revealed that miR-486-5p directly targets the 3′-UTR of Smad2. MiR-486-5p mimic transfection markedly down-regulated the expression levels of Smad2, thus inhibiting the expression of p-Smad2 and p-Smad3. MiR-486-5p overexpression in SRA01/04 cells markedly suppressed TGF-β₂-induced proliferation and invasion, inhibited protein expression of CDK2 and CDK4, down-regulated fibronectin, α-SMA and vimentin and up-regulated E-cadherin; these effects were partly reversed by Smad2 overexpression. In short, these data show that miR-486-5p overexpression can inhibit TGF-β₂-induced proliferation, invasion and EMT in SRA01/04 cells by repressing Smad2/Smad3 signalling, implying that miR-486-5p may be an effective target to interfere in the progression of PCO.