参仁活血颗粒对四氯化碳诱导的大鼠肝纤维化TGF-β1/Smad信号通路的影响

目的观察参仁活血颗粒对四氯化碳诱导的大鼠肝纤维化TGF-β1/Smad信号通路的影响,探讨参仁活血颗粒治疗肝纤维化的作用机制。方法 50只雄性SD大鼠随机分为正常组、模型组和参仁活血颗粒低、中、高剂量组,正常组予以生理盐水1 mL/kg腹腔注射,模型组及参仁活血颗粒各组予以40%四氯化碳0.2 mL/100 g腹腔注射,2次/周,连续8周,于第4周起分别予以参仁活血颗粒,低剂量组每日1.575 g/(kg?bw)、中剂量组每日3.15 g/(kg?bw)、高剂量组每日6.3 g/(kg?bw)剂量灌胃,连续4周,于第8周牺牲大鼠。HE染色及Masson染色检测大鼠肝纤维化程度;免疫组化检测大鼠肝collagen I、collagen III、TGF-β1的表达;real-time PCR和Western blot检测大鼠肝TGF-β1、Smad3、Smad7的表达。结果模型组与正常组相比可见肝小叶结构破坏,纤维明显增多,形成大小不一的假小叶;而参仁活血颗粒各剂量组较模型组肝纤维化程度明显改善,以参仁活血颗粒高剂量组疗效最显著。②模型组大鼠肝collagen I、collagen III、TGF-β1、Smad 3表达较正常组明显升高(P0.05),Smad7表达明显降低(P0.05),而参仁活血颗粒各剂量组collagen I、collagen III、TGF-β1、Smad3表达较模型组降低(P0.05),Smad7表达较模型组升高,以参仁活血颗粒高剂量组疗效最好(P0.05)。结论参仁活血颗粒能显著改善大鼠肝纤维化,其机制可能与其能下调大鼠肝组织中TGF-β1、Smad3的表达、上调Smad7的表达,从而减少Collagen I、Collagen III的合成相关。     

藏红花酸对TGF-β1刺激的人肝星状细胞LX-2信号转导通路的影响

目的:研究藏红花酸(crocetin)对转化生长因子-β1(TGF-β1)刺激的人肝星状细胞LX-2信号转导通路的影响。方法:体外培养LX-2细胞,随机设立空白对照组,模型组,藏红花酸低剂量组,藏红花酸中剂量组,藏红花酸高剂量组,用含10%血清的1640培养液培养48 h,MTT法测各组细胞增殖,蛋白免疫印迹测定各组细胞α-SMA蛋白的表达,实时荧光定量PCR法测各组细胞Smad2、Smad3、Smad7mRNA的表达。结果:与对照组比较,TGF-β1刺激后人肝星状细胞LX-2增殖作用明显,且上调α-SMA蛋白、Smad2mRNA、Smad3mRNA表达,下调Smad7mRNA表达(P0.01)与模型组比较,藏红花酸组均能抑制LX-2细胞增殖,并呈剂量依赖性,高、中剂量组作用明显,能够明显下调α-SMA蛋白、Smad2mRNA、Smad3mRNA表达,上调Smad7mRNA表达,差异具有统计学意义(P0.05)。结论:藏红花酸对TGF-β1刺激的LX-2细胞中Smad7mRNA具有上调作用,对Smad2、Smad3mRNA具有下调作用,其抗肝纤维化作用可能与抑制TGF-β1/Smads信号转导通路有关。     

LncRNA Terc在心肌纤维化过程中的功能研究

摘要 目的：探讨lncRNA端粒酶RNA组分（Terc）在心肌纤维化（MF） 过程中的作用。方法：使用不同浓度的TGF-β1诱导心肌成纤维细胞（CFs）转分化，通过免疫荧光染色、western blot检测α-SMA、Vimentin、Collagen I、Collagen III蛋白的表达水平，qRT-PCR检测lncRNA Terc表达水平。过表达和敲减Terc后，通过western blot、CCK-8和流式细胞术观察模型细胞的胞外基质产生、细胞增殖、凋亡和Smads信号传导情况。皮下注射异丙肾上腺素（ISO）构建小鼠心肌纤维化模型，并使用Terc敲低慢病毒干预，其后多普勒超声仪检测小鼠的心脏射血分数（EF）和左室短轴缩短率（FS），称量小鼠心脏湿重，HE、Masson染色检测小鼠心脏的病理改变，IHC检测α-SMA、Vimentin蛋白的表达水平，qRT-PCR检测lncRNA Terc表达水平。结果：TGF-β1处理增加CFs的α-SMA、Vimentin、Collagen I、Collagen III的蛋白表达以及Terc水平；过表达Terc促进α-SMA、Vimentin、Collagen I、Collagen III的蛋白表达以及Smad2/3的磷酸化水平，同时还可促进CFs的增殖、抑制CFs的凋亡；敲减Terc则起相反的作用；动物模型中，ISO可抑制EF和FS，增加心脏湿重，加重心肌的病理损伤，而敲减Terc可有效缓解上述过程。结论：LncRNA Terc可通过促进Smads信号传导，加速心肌成纤维细胞转分化和心肌纤维化进展。     

miR-101a在乙型肝炎病毒相关性肝纤维化患者中的表达及对肝星状细胞的影响

摘要 目的：探究miR-101a在乙型肝炎病毒（HBV）相关性肝纤维化患者中的表达及对肝星状细胞（HSC）的影响。方法：根据肝纤维化程度将HBV相关性肝纤维化患者进行分组（S0组、S1组、S2组、S3组和S4组）,健康受试者作为健康对照组。通过RT-PCR检测肺组织中miR-101a的表达,并分析miR-101a与疾病严重程度的关系。使用重组人TGF-β1处理人肝星状细胞系LX-2,并对LX-2细胞转染阴性对照 miRNA模拟物（NC-mimic组）、miR-101a模拟物（miR-101a-mimic组）、阴性对照 miRNA抑制剂（NC-inhibitor组）或miR-101a抑制剂（miR-101a-inhibitor组）,未转染的细胞作为对照组,然后通过RT-PCR或蛋白质印迹检测激活HSC及ECM产生的关键基因（α-SMA、COL1A1、COL1A2和COL3A1）和蛋白（a-SMA、collagen I和collagen III）的表达水平。将SD大鼠随机分为4组：对照组、CCl4组、Ad-control组和Ad-miR-101a组,对大鼠腹腔注射CCl₄（1 mL/kg体重）诱导肝纤维化模型,每周3次,共4周。然后将5×10⁹感染单位的携带miR-101a的重组腺病毒（Ad-miR-101a）或对照腺病毒（Ad-control）经尾静脉注射到大鼠中。4周后,通过苏木精和伊红（H＆E）和Masson三色染色评估肝脏形态和纤维化,通过免疫组化染色评估肝脏α-SMA、E-cadherin、vimentin、Smad4或p-Smad2/3的表达。结果：与健康受试者相比,HBV相关肝纤维化患者肝组织中miR-101a的表达水平明显降低,并且miR-101a的表达水平随着患者的严重程度升高而降低（P<0.05）。与未处理的细胞相比,miR-101a在TGF-β1处理的LX-2细胞中以浓度和时间依赖性方式显著下降（P<0.05）。与未处理的细胞相比,5 ng/mL TGF-β1处理LX-2细胞中的α-SMA、COL1A1、COL1A2和COL3A1 mRNA表达水平及a-SMA、collagen I和collagen III 蛋白表达水平均显著升高（P<0.05）。与对照组相比,miR-101a-mimic组的α-SMA、COL1A1、COL1A2、COL3A1和TGF-β1 mRNA和a-SMA、collagen I、collagen III、TGF-β1、Smad3和p-Smad3蛋白表达均下调（P<0.05）。与对照组相比,Ad-miR-101a组大鼠肝组织中E-cadherin的表达上调,但α-SMA、vimentin、Smad4和p-Smad2/3的表达下调（P<0.05）;Ad-miR-101a组大鼠的肝组织形态基本恢复正常,肝组织纤维化程度低于CCl₄组。结论：miR-101a水平与乙型肝炎病毒相关性肝纤维化严重程度相关,上调miR-101a可能通过抑制HSC的活化及上皮间质转化发挥抗纤维化作用。     

表没食子儿茶素没食子酸酯对心力衰竭小鼠心肌损伤的影响

为了评估表没食子儿茶素没食子酸酯(EGCG)对心力衰竭小鼠心肌损伤的保护作用,本研究通过主动脉弓缩窄术建立了心力衰竭的昆明小鼠模型,然后应用EGCG (20 mg/kg)处理小鼠4周。采用苏木精和伊红染色评价心肌组织的病理改变。采用ELISA法检测血清脑钠肽(BNP)、N末端-脑钠肽前体(NT-proBNP)、白细胞介素-1β(IL-1β)、白细胞介素-6 (IL-6)、肿瘤坏死因子-α(TNF-α)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的水平,采用Western blotting检测小鼠心肌组织中collagenⅠ、collagenⅢ、Bcl-2、Bax、caspase-3、TGF-β1、Smad3和p-Smad3水平。采用RT-qPCR检测心肌组织中collagenⅠ和collagenⅢ的表达,通过免疫荧光染色检测TGF-β1和p-Smad3的表达。研究显示,EGCG可有效减弱心力衰竭所致细胞坏死、组织肿胀、心肌纤维断裂等病理变化,并且显著降低心力衰竭诊断标志物BNP和NT-proBNP的水平。EGCG可显著降低小鼠心肌组织中的collagenⅠ和collagenⅢ的表达水平。EGCG处理可显著降低TNF-α、IL-1β和IL-6的水平。EGCG处理显著降低了心力衰竭小鼠血清MDA水平,而升高了SOD和GSH-Px水平。EGCG可显著上调心力衰竭小鼠心肌组织中Bcl2的表达,而下调Bax和caspase-3的表达。TGF-β信号通路激活剂可显著上调心肌组织中的TGF-β表达,而EGCG处理可显著抑制TGF-β1和p-Smad3蛋白的表达。表明EGCG可抑制心力衰竭小鼠的心肌纤维化、炎症反应、氧化应激和细胞凋亡,从而减轻心肌损伤。EGCG对心肌的保护作用可能与抑制TGF-β1/smad3信号通路有关。     

黄芪甲苷激活TGF-β1/Smad2信号通路诱导骨髓间充质干细胞向周细胞分化的作用研究

目的:观察黄芪甲苷促进大鼠骨髓间充质干细胞(Mesenchymal stem cell,MSCs)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法:本研究以大鼠骨髓间充质干细胞为研究对象,采用全骨髓培养法,从SD大鼠乳鼠(5d-7d)股骨提取原代细胞,体外传代纯化,P4-P5代用于实验。实验分阴性对照组,大鼠BMSCs 15%胎牛血清(Fetal Bovine Serum,FBS)向周细胞分化的作用,揭示中药黄芪治疗缺血性心脏病的意义。方法:本研究以大鼠骨髓间培养基培养,不给予药物干预;阳性对照组,给予转化生长因子β1(TGF-β1)5 ng/mL干预3天;黄芪甲苷(Astragaloside,Ast)组,给予黄芪甲苷4μg/mL分别干预1 d、2 d、3 d、5 d、7d;阻断剂组,TGF-β1受体阻断剂sb431542预处理后,再加入黄芪甲苷4μg/mL分别诱导1 d、2 d、3 d。实时定量PCR(RT-PCR)检测神经元-胶质细胞抗原2(Neuron-glial antigen 2,NG2)、α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)信使核糖核酸(mRNA)表达;印迹法(Western blot)检测NG2、α-SMA、Smad2/3、p-Smad2蛋白表达。结果:经黄芪甲苷诱导刺激后,大鼠骨髓间充质干细胞在mRNA和蛋白水平上,NG2、α-SMA的表达量均升高;Ast 3d组,与阴性对照组和阳性对照组相比,NG2、α-SMA蛋白和mRNA的表达量均显著升高(p0.05)。黄芪甲苷刺激细胞后,TGF-β1/Smad2信号传导通路被激活,从第一天开始,p-Smad2蛋白表达量升高,第3天到达顶峰,随后降低;其中第三天,p-Smad2蛋白表达量显著高于Ast 0d组(p0.01)。加入阻断剂后,黄芪甲苷受到sb431542影响,对细胞的作用减弱,NG2、α-SMA蛋白和mRNA表达量均下调;sb431542+Ast 3d组,与Ast 3d组相比,NG2、α-SMA蛋白和mRNA的表达量显著降低(p0.001)。受阻断剂的影响,黄芪甲苷对TGF-β1/Smad2信号传导通路的作用减弱,前三天p-Smad2蛋白的表达量均降低,sb431542+Ast 3d组,与Ast 3d组相比,p-Smad2蛋白的表达量显著降低(p0.001)。结论:黄芪甲苷具有促进大鼠BMSCs向周细胞分化的作用,其机制与激活TGF-β1/Smad2信号传导通路相关。     

异钩藤碱调控ERK/p27^(Kip1)信号通路改善博莱霉素诱导的小鼠肺纤维化CSCD

基于细胞外调节蛋白激酶1/2(ERK1/2)、p27^(Kip1)信号通路探究异钩藤碱(isorhynchophylline,IRN)对博莱霉素(bleomycin,BLM)诱导的小鼠肺纤维化(pulmonary fibrosis,PF)的作用及机制。C57BL/6J小鼠48只,随机正常组、BLM组、BLM+IRN(10、20 mg/kg)两个剂量组,每组12只。气管注射BLM(5000 U/kg)诱导PF小鼠模型,造模后连续灌胃给药21天。HE和Masson染色观察肺组织病理变化及胶原沉积情况。免疫组化检测肺组织α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)的表达。体外培养小鼠原代肺成纤维细胞,实验设对照组、转化生长因子-β1(transforming growth factor-beta 1,TGF-β1)(10 ng/mL)组和TGF-β1+IRN(5、10、20μmol/L)三个剂量组。EdU掺入法和流式细胞术检测细胞增殖,Transwell观察细胞的迁移能力。RT-qPCR检测肺组织或肺成纤维细胞TGF-β1、collagen I和α-SMA mRNA的表达。Western blot检测肺组织和(或)肺成纤维细胞TGF-β1、collagen I、α-SMA、p-ERK1/2,p27^(Kip1)、CDK2和Cyclin E1的蛋白水平。动物实验结果显示,与BLM组相比,不同剂量IRN均能明显减轻肺组织结构的损伤、降低炎症细胞的浸润和胶原的沉积;此外,IRN不同程度地降低肺组织TGF-β1、collagen I和α-SMA mRNA和蛋白的表达;同时,IRN还抑制了肺组织ERK1/2的磷酸化、上调p27^(Kip1)和下调CDK2和Cyclin E1的蛋白表达。细胞实验结果显示,与TGF-β1组相比,不同剂量IRN能够明显抑制TGF-β1诱导的肺成纤维细胞增殖、显著降低细胞迁移能力;明显降低TGF-β1诱导的collagen I和α-SMA mRNA和蛋白的表达,同时降低ERK1/2的磷酸化水平、上调p27^(Kip1)和下调CDK2和Cyclin E1的蛋白表达。以上结果表明IRN可能通过抑制ERK1/2信号通路、上调p27^(Kip1)的表达而抑制了肺成纤维细胞向肌成纤维细胞的转化,从而减轻了BLM诱导的PF。     

大黄素对TGF-β1诱导的肾小管上皮细胞间质转分化的影响

目的:探讨大黄素对TGF-β1诱导的人肾小管上皮细胞(HK-2)间质转分化的影响。方法:不同浓度大黄素分别作用于TGF-β1诱导HK-2细胞24 h和48 h,通过细胞增殖实验确定最佳大黄素最佳给药浓度。TGF-β1诱导HK-2细胞24 h后收集细胞用于免疫印迹Western blot和实时荧光定量PCR(RT-PCR)分析。Western印迹法分别检测纤维化相关蛋白Collagen IV的表达,和肾小管上皮细胞向间充质细胞转分化关键蛋白α-SMA和E-Cadherin的表达;RT-PCR法检测肾小管上皮细胞向间充质细胞转分化关键蛋白α-SMA的表达。结果:由细胞增殖实验结果表明40μM大黄素是最佳给药浓度。Western结果表明,与模型组相比,大黄素组下调纤维化相关蛋白Collagen IV的表达,大黄素组与模型组蛋白差异有统计学意义(P0.05)。与模型组相比,大黄素组下调α-SMA蛋白表达水平,而上调E-Cadherin蛋白表达,差异有统计学意义(P0.05)。RT-PCR结果表明,与模型组相比,大黄素组降低α-SMA mRNA的含量,大黄素组与模型组α-SMA mRNA含量差异有统计学意义(P0.05)。结论:大黄素可通过抑制TGF-β1诱导的HK-2细胞间质转分化,从而发挥延缓肾间质纤维化的过程。     

人脐带间充质干细胞缓解马兜铃酸诱导小鼠肾纤维化的作用及机制

该实验探究了人脐带间充质干细胞(human umbilical cord mesenchymal stem cell, hucMSC)缓解马兜铃酸(aristolochic acid, AA)诱导小鼠肾纤维化的作用及可能机制。我们将huc-MSC经尾静脉注射干预AA诱导的肾纤维化小鼠模型。HE、PAS和Masson染色观察肾脏形态变化, Western blot和免疫组化检测上皮间质转化相关标志物E-cadherin、N-cadherin和TGF-β/Smad信号通路蛋白TGF-β1和p-Smad2/3表达水平。组织形态学染色结果显示, AA可诱导小鼠出现肾小管扩张、结构破坏,肾间质区胶原纤维沉积,呈纤维化改变; Western blot和免疫组化结果显示,其E-cadherin表达降低,N-cadherin、TGF-β1及p-Smad2/3表达增高。huc-MSC干预后,肾脏形态明显改善,胶原纤维沉积减少,E-cadherin表达增高, N-cadherin、TGF-β1及p-Smad2/3表达受到降低。研究结果表明, huc-MSC能够通过抑制TGF-β/Smad信号通路减轻肾脏上皮间质转化,从而缓解马兜铃酸诱导的小鼠肾纤维化。     

间充质干细胞来源的外泌体通过TGF-β1/Smad2/3信号通路抑制高糖诱导的成纤维细胞转分化

心脏纤维化是糖尿病患者心肌功能障碍的主要原因。成纤维细胞转分化为成肌纤维细胞是心脏纤维化过程中的一个关键性事件。该研究的目的是探究高糖诱导成纤维细胞转分化的分子机制,并找寻抑制成纤维细胞转分化的方法。结果显示,经高糖处理的BJ细胞(人皮肤成纤维细胞系)与正常BJ细胞相比,α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达明显上调。通过使用SB525334或转化生长因子-β1(transforming growth factor-β1,TGF-β1)si RNA抑制TGF-β1/Smad2/3信号通路的活化,发现α-SMA和胶原I的蛋白质水平及Smad2/3的磷酸化水平均降低。同时,SB525334也抑制了高糖诱导的BJ细胞增殖。大鼠骨髓间充质干细胞来源的外泌体(mesenchymal stem cell-derived exosome,MSC-Exo)通过降低Smad2/3磷酸化水平,抑制高糖诱导的α-SMA表达。综上所述,高糖通过激活TGF-β1信号通路导致BJ细胞的转分化,而MSC-Exo通过抑制该通路防止BJ细胞的转分化。     

AcSDKP对TGF-β1诱导大鼠MSCs向肌成纤维细胞分化的抑制作用

目的:研究N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartly-lysyl-proline,Ac SDKP)对转化生长因子β1(Transforming growth factor beta 1,TGF-β1)诱导大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)向肌成纤维细胞(myofibroblast,MF)分化的影响,探讨Ac SDKP抗纤维化作用的可能机制。方法:全骨髓贴壁法分离培养大鼠骨髓MSCs。使用免疫组化,Western blotting技术分析α-SMA蛋白的表达以及Smad2/3,ERK1/2蛋白磷酸化的变化情况。结果:和对照组相比,TGF-β1诱导的MSC中α-SMA、磷酸化-Smad2/3及磷酸化-ERK1/2的表达大大增强,使用Ac SDKP干预细胞则三者的表达量明显下降且呈一定的剂量依赖性。结论:Ac SDKP可以显著抑制TGF-β1诱导的大鼠MSCs向MF分化,可能通过抑制TGF-β/Smad/ERK1/2信号通路的激活,从而发挥其抗器官纤维化作用。     

TGF-β1 induces EMT reprogramming of porcine bladder urothelial cells into collagen producing fibroblasts-like cells in a Smad2/Smad3-dependent manner

Activation of fibroblasts and their differentiation into myofibroblasts, excessive collagen production and fibrosis occurs in a number of bladder diseases. Similarly, conversion of epithelial cells into mesenchymal cells (EMT) has been shown to increase fibroblasts like cells. TGF-β1 can induce the EMT and the role of TGF-β1-induced EMT during bladder injury leading to fibrosis and possible organ failure is gaining increasing interest. Here we show that EMT and fibrosis in porcine bladder urothelial (UC) cells are Smad dependent. Fresh normal porcine bladder urothelial cells were grown in culture with or without TGF-β1 and EMT markers were assessed. TGF-β1 treatment induced changes in cellular morphology as depicted by a significant decrease in the expression of E-cadherin and corresponding increase in N-cadherin and α-SMA. We knocked down Smad2 and Smad3 by Smad specific siRNA. Downregulation of E-cadherin expression by TGF-β1 was Smad3-dependent, whereas N-cadherin and α-SMA were dependent on both Smad2 and Smad3. Connective tissue growth factor (CTGF/CCN2), matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) has been shown to play important roles in the pathogenesis of fibrosis. Induction of these genes by TGF-β1 was found to be time dependent. Upregulation of CTGF/CCN2 by TGF-β1 was Smad3 dependent; whereas MMP-2 was Smad2 dependent. Smad2 and Smad3 both participated in MMP-9 expression. TGF-β1 reprogrammed mesenchymal fibroblast like cells robustly expressed collagen I and III and these was inhibited by SB-431542, a TGF-β receptor inhibitor. Our results indicate that EMT of porcine bladder UC cells is TGF-β1 dependent and is mediated through Smad2 and Smad3. TGF-β1 may be an important factor in the development of bladder fibrosis via an EMT mechanism. This identifies a potential amenable therapeutic target.     

Effects of rhDecorin on TGF-β1 induced human hepatic stellate cells LX-2 activation

Decorin is a small leucine-rich extracellular matrix proteoglycan composed of a core protein with a single glycosaminoglycan (GAG) chain near the N-terminus and N-glycosylated at three potential sites. Decorin is involved in the regulation of formation and organization of collagen fibrils, modulation of the activity of growth factors such as transforming growth factor β (TGF-β), and exerts other effects on cell proliferation and behavior. Increasing evidences show that decorin plays an important role in fibrogenesis by regulating TGF-β, a key stimulator of fibrosis, and by directly modulating the degradation of extracellular matrix (ECM) from activated hepatic stellate cells (HSCs). In this study, the core protein of human decorin was cloned and expressed in Escherichia coli. The purified recombinant human decorin (rhDecorin) significantly inhibited the proliferation of LX-2 cells, a human HSC cell line, stimulated by TGF-β1. RT-PCR result showed that the expression of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were reduced by rhDecorin in LX-2 cells stimulated by TGF-β1. Furthermore, the protein expression of smooth muscle-α-actin (α-SMA), collagen type III and phosphorylated Smad2 (p-Smad2) was significantly decreased in the presence of rhDecorin. rhDecorin also reduced fibrillogenesis of collagen type I in a dose-dependent manner. Gene expression profiles of LX-2 cells stimulated by TGF-β1 in the presence and the absence of rhDecorin were obtained by using cDNA microarray technique and differentially expressed genes were identified to provide further insight into the molecular action mechanism of decorin on LX-2 cells.     

Apigenin suppresses TGF-β1-induced cardiac fibroblast differentiation and collagen synthesis through the downregulation of HIF-1α expression by miR-122-5p

BackgroundApigenin can reduce cardiomyocyte hypertrophy by downregulating hypoxia inducible factor-1 alpha (HIF-1α) expression. However, its effects on cardiac fibroblasts (CFs) and its exact inhibitory molecular mechanisms on HIF-1α remain unclear.PurposeThis study aims to examine the effects of apigenin on cell proliferation and differentiation, microRNA-122-5p (miR-122-5p) expression, and HIF-1α-mediated Smad signaling pathway in transforming growth factor beta 1 (TGF-β1)-stimulated CFs and cardiac fibrosis and to investigate the relationship between miR-122-5p and HIF-1α.MethodsThe TGF-β1-stimulated CFs, the combination of TGF-β1-stimulated and miR-122-5p mimic-transfected CFs, the combination of TGF-β1-stimulated and miR-122-5p inhibitor-transfected CFs, and the isoproterenol-induced cardiac fibrotic mice were used and treated with or without apigenin. The recombinant lentiviruses overexpressing HIF-1α vector and miR-122-5p mimic were co-transfected to observe their interaction. Related mRNA and protein expressions and myocardial collagen were determined. The luciferase reporter gene that contains HIF-1α wild type or mutant type 3’-UTR was used, and the luciferase activity was determined to verify the direct link between miR-122-5p and HIF-1α.ResultsIn the TGF-β1-stimulated CFs, apigenin treatment increased the miR-122-5p and Smad7 expressions and decreased the HIF-1α, α-smooth muscle actin, collagen Ⅰ/Ⅲ, Smad2/3, and p-Smad2/3 expressions. Similar and inverse results were observed in the miR-122-5p mimic- and inhibitor-transfected CFs, respectively. Moreover, the miR-122-5p mimic could antagonize the effects of TGF-β1 in the TGF-β1 and miR-122-5p mimic-combined CFs, and the miR-122-5p inhibitor could enhance the effects of TGF-β1 in the TGF-β1 and miR-122-5p inhibitor-combined CFs. In the two aforementioned cell models, the addition of apigenin could further enhance the effects of miR-122-5p mimic and partially reverse the effects of miR-122-5p inhibitor. After treatment of HIF-1α-transfected CFs with miR-122-5p mimic, the HIF-1α expression decreased. Further study confirmed that HIF-1α was a direct target of miR-122-5p. Apigenin also decreased the myocardial collagen accumulation in cardiac fibrotic mice.ConclusionApigenin could suppress the differentiation and collagen synthesis of TGF-β1-stimulated CFs and mouse cardiac fibrosis, and its mechanisms were related to the increment of miR-122-5p expression and subsequent downregulation of HIF-1α expression via direct interaction, which might finally result in the decrements of Smad2/3 and p-Smad2/3 expressions and increment of Smad7 expression.     

TGF-β2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF

Transforming growth factor (TGF)-β2, gremlin and connective tissue growth factor (CTGF) are known to play important roles in the induction of epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis. However, the complex functional relationship among gremlin, CTGF and TGF-β2 in the induction of EMT and ECM synthesis in human lens epithelial cells (HLECs) has not been reported. In this study, we found that TGF-β2, CTGF and gremlin can individually induce the expression of α-smooth muscle actin (α-SMA), fibronectin (Fn), collagen type I (COL-I), Smad2 and Smad3 in HLECs. Blockade of CTGF and gremlin effectively inhibited TGF-β2-induced expression of α-SMA, Fn, COL-I, Smad2, and Smad3 in HLECs. Furthermore blockade of Smad2 and Smad3 effectively inhibited CTGF and gremlin induced expression of α-SMA, Fn, COL-I in HLECs. In conclusion, TGF-β2, CTGF and gremlin are all involved in EMT and ECM synthesis via activation of Smad signaling pathway in HLECs. Specifically silencing CTGF and gremlin can effectively block the TGF-β2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.     

Inflammatory microenvironment and tumor necrosis factor alpha as modulators of periostin and CCN2 expression in human non-healing skin wounds and dermal fibroblasts

Non-healing skin wounds remain a significant clinical burden, and in recent years, the regulatory role of matricellular proteins in skin healing has received significant attention. Periostin and CCN2 are both upregulated at day 3 post-wounding in murine skin, where they regulate aspects of the proliferative phase of repair including mesenchymal cell infiltration and myofibroblast differentiation. In this study, we examined 1) the wound phenotype and expression patterns of periostin and CCN2 in non-healing skin wounds in humans and 2) the regulation of their expression in wound fibroblasts by tumor necrosis factor α (TNFα) and transforming growth factor-β1 (TGF-β1). Chronic skin wounds had a pro-inflammatory phenotype, characterized by macrophage infiltration, TNFα immunoreactivity, and neutrophil infiltration. Periostin, but not CCN2, was significantly suppressed in non-healing wound edge tissue at the mRNA and protein level compared with non-involved skin. In vitro, human wound edge fibroblasts populations were still able to proliferate and contract collagen gels. Compared to cells from non-involved skin, periostin and α-SMA mRNA levels increased significantly in the presence of TGF-β1 in wound cells and were significantly decreased by TNFα, but not those of Col1A2 or CCN2. In the presence of both TGF-β1 and TNFα, periostin and α-SMA mRNA levels were significantly reduced compared to TGF-β1 treated wound cells. Effects of TGF-β1 and TNFα on gene expression were also more pronounced in wound edge cells compared to non-involved fibroblasts. We conclude that variations in the expression of periostin and CCN2, are related to an inflammatory microenvironment and the presence of TNFα in human chronic wounds.     

Atrial natriuretic peptide: A novel mediator for TGF-β1-induced epithelial-mesenchymal transition in 16HBE-14o and A549 cells

Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-β1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA expressions were increased after induction by TGF-β1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and α-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased α-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with α-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-β1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-β1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.     

The inhibitory effect of ginsan on TGF-β mediated fibrotic process

Transforming growth factor-beta (TGF-β) plays a central role in the development of fibrosis by stimulating extracellular matrix accumulation, and signals either directly or indirectly through types I, II, and III (TβRI, II, and III) TGF-β receptor complexes. Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. Here, we examine whether ginsan regulates the fibrogenic process by interfering with TGF-β signaling pathways. TGF-β treatment of murine or human normal lung fibroblasts enhanced the levels of several fibrotic markers, including smooth muscle alpha actin (α-SMA), collagen-1, and fibronectin. Interestingly, ginsan treatment either before or after TGF-β administration led to significant reductions in all of α-SMA, collagen-1, and fibronectin expression levels. Ginsan not only inhibited phosphorylation of Smad2 and Smad3, but also attenuated pERK and pAKT signaling induced by TGF-β. Moreover, ginsan restored TβRIII protein expression, which was significantly downregulated by TGF-β, but reduced TβRI and TβRII protein levels. In a murine model of bleomycin (BLM)-induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α-SMA, and TGF-β. These data collectively suggest that ginsan acts as an effective anti-fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF-β signaling pathways.     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

Mesenchymal stem cells ameliorate silica-induced pulmonary fibrosis by inhibition of inflammation and epithelial-mesenchymal transition

Silicosis is a devastating occupational disease caused by long-term inhalation of silica particles, inducing irreversible lung damage and affecting lung function, without effective treatment. Mesenchymal stem cells (MSCs) are a heterogeneous subset of adult stem cells that exhibit excellent self-renewal capacity, multi-lineage differentiation potential and immunomodulatory properties. The aim of this study was to explore the effect of bone marrow-derived mesenchymal stem cells (BMSCs) in a silica-induced rat model of pulmonary fibrosis. The rats were treated with BMSCs on days 14, 28 and 42 after perfusion with silica. Histological examination and hydroxyproline assays showed that BMSCs alleviated silica-induced pulmonary fibrosis in rats. Results from ELISA and qRT-PCR indicated that BMSCs inhibited the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 in lung tissues and bronchoalveolar lavage fluid of rats exposed to silica particles. We also performed qRT-PCR, Western blot and immunohistochemistry to examine epithelial-mesenchymal transition (EMT)–related indicators and demonstrated that BMSCs up-regulate E-cadherin and down-regulate vimentin and extracellular matrix (ECM) components such as fibronectin and collagen Ⅰ. Additionally, BMSCs inhibited the silica-induced increase in TGF-β1, p-Smad2 and p-Smad3 and decrease in Smad7. These results suggested that BMSCs can inhibit inflammation and reverse EMT through the inhibition of the TGF-β/Smad signalling pathway to exhibit an anti-fibrotic effect in the rat silicosis model. Our study provides a new and meaningful perspective for silicosis treatment strategies.