醛糖还原酶（AR）对TGF-β1诱导人系膜细胞（HMC）表达Fibronectin的影响及其机制的研究
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摘要
前言
肾小球硬化是各种原因所致肾小球损伤和病变的共同结局,是由多种生物活性物质和多种细胞成分参与的一个复杂病理过程,其主要的病理特征之一是肾小球内细胞外基质(Extracellular Matrix,ECM)的异常沉积。研究认为ECM合成和(或)降解失衡是肾小球硬化发生的重要机制之一,而系膜细胞(Mesangial Cell,MsC)增生则是许多肾小球疾病发生发展过程中的中心环节,并且在肾小球内ECM代谢及调控过程中起重要作用,体内外各种生长因子和细胞因子均可刺激MsC增殖。因此,研究肾小球系膜细胞与肾小球硬化的发生、发展之间的相互关系也是近年来器官纤维化研究领域的重要研究内容之一。
MsC是肾小球内固有的一种血管外周细胞,大量研究证实,该细胞可自分泌多种细胞因子:如血小板源生长因子、IL-6、TGF-β1、内皮素1等,后者可促进MsC自身分裂、增生及细胞外基质的合成和分泌,在肾小球硬化过程中发挥重要作用。研究已证实,肾脏MsC高表达转化生长因子β1(transforming growth factorβ1,TGF-β1),TGF-β1是一个多功能细胞因子,具有调节细胞增殖、分化以及细胞凋亡等功能。TGF-β1具有调节ECM合成、代谢过程的作用,可以直接促进ECM成分的合成或影响ECM合成和(或)降解主要调节酶系统的活性而促进ECM合成增加和沉积,从而在肾小球硬化发生与发展的病理过程中具有重要的作用。
醛糖还原酶(Aldose Reductase,AR)为NADPH依赖性醛-酮还原酶家族成员之一,是多元醇代谢通路中的限速酶,可催化己糖或戊糖的NADPH依赖性还原反应,使之变成相应的糖醇或多元醇。AR广泛分布于神经、肌肉、脑、肾脏、胎盘、血管、视网膜等组织中,在不同组织中的分子构成、免疫原性及其他理化性质有差别。对AR的研究,以往都集中在糖尿病及其并发症中,但新近的研究发现,AR的某些功能和高糖环境并不相关。我们课题组前期通过SSH-PCR研究发现,AR是大鼠MsC中TGF-β1相关反应性基因之一,而TGF-β1在组织纤维化,特别是肾脏纤维化中起着重要作用,因此AR是否参与了TGF-β1在非高糖状态下所导致的肾小球硬化、肾脏纤维化及其病理过程值得深入研究。
纤连蛋白(Fibronectin,FN)是构成细胞外基质的一个重要组成部分,是一种大分子糖蛋白,在正常组织中分布广泛,具有促进细胞移动、分化、调理吞噬等功能,在细胞与细胞、细胞与基质的相互作用中起着重要作用。FN可诱导成纤维细胞增生,产生胶原纤维,促进纤维化的形成,研究发现FN的合成及降解的异常在肾纤维化的发生、发展中起很大的作用。
本课题采用细胞培养、基因转染、免疫荧光、RNA干扰、RT-PCR、Real-Time PCR、Western Blot等实验方法,观察AR在人系膜细胞中的表达及其与TGF-β1的相互关系,观察AR在TGF-β1诱导Fibronectin表达增高过程中的作用,并初步探讨这种作用所涉及的细胞信号传导通路等作用机制,以明确AR在肾小球硬化等病理过程中的作用及意义,进一步认识和了解AR在非糖尿病性肾脏疾病(主要为肾小球肾炎、纤维化和硬化)中的新作用,以期能够为肾小球硬化发生发展的病理机制的认识提供新的实验依据。
第一部分TGF-β1对人系膜细胞表达AR、FN以及MAPKs信号通路的影响
目的探讨外源性TGF-β1对体外培养人系膜细胞(HMC)表达AR、FN以及对MAPKs信号通路的影响。
方法培养HMC,在培养液中添加外源性TGF-β1因子,采用免疫荧光和WesternBlot方法观察HMC内AR、FN和MAPKs表达的变化。
结果以不同浓度TGF-β1(1-10ng/ml)刺激HMC 24h,结果发现AR和FN表达均增高,其中以1ng/ml TGF-β1作用浓度下AR、FN表达升高最为明显(P＜0.05);再以1ng/ml TGF-β1以不同的作用时间(0-4h)刺激HMC,可见AR、FN在15min时表达即开始增高,其中AR表达在TGF-β1作用后1h升高最明显(P＜0.05),FN在2h升高最明显(P＜0.05)。与此同时,TGF-β1激活了MAPKs信号通路,TGF-β1作用15min后可见磷酸化的ERK、JNK和p38表达升高,其中磷酸化的ERK、JNK在作用后30min达高峰,磷酸化的p38在1h后达高峰(P＜0.05),然后逐渐下降;磷酸化的ERK表达量和持续时间明显多于磷酸化JNK和p38。
小结外源性TGF-β1可上调HMC内AR、FN的表达;TGF-β1可以活化MAPKs信号通路,其中以ERK信号通路活化效果最明显,提示TGF-β1上调AR、FN的表达可能与激活MAPKs信号通路有关,ERK信号通路在这过程中可能起着主要的作用。TGF-β1可调节AR表达的实验结果也证实了在HMC内,AR也是TGF-β1的反应性基因。
第二部分抑制AR活性,阻断MAPKs和PKC信号通路对TGF-β1诱导FN表达的影响
目的探讨AR抑制剂Zopolrestat和Sorbinil,MAPKs信号通路抑制剂U0126(ERK抑制剂)、SB203580(p38抑制剂)、SP600125(JNK抑制剂)和G(o|¨)6983(PKC抑制剂)对体外培养的HMC使用TGF-β1诱导FN表达的影响。
方法培养HMC,通过预先在培养液中分别添加AR抑制剂Zopolrestat和Sorbinil,MAPKs信号通路抑制剂U0126、SB203580、SP600125和PKC信号通路抑制剂G(o|¨)6983作用后,再用外源性TGF-β1因子刺激,采用Western Blot方法观察HMC内FN表达的变化。
结果仅使用AR抑制剂Zopolrestat和Sorbinil作用于HMC,与未使用AR抑制剂的HMC相比,FN的表达未见减少;但在使用Zopolrestat和Sorbinil预孵育HMC后,再用TGF-β1刺激,则可发现FN的表达明显减少(P＜0.05),使用Zopolrestat预作用组FN的表达比单独使用TGF-β1刺激时减少了2.9倍,使用Sorbinil预作用组的FN的表达减少了2.5倍;分别以不同浓度的ERK、p38、JNK和PKC信号通路抑制剂作用于HMC,与正常对照组相比,FN的表达未见减少。进一步研究发现,在预先使用ERK、p38、JNK和PKC抑制剂作用于细胞后,再用外源性TGF-β1刺激,则发现FN的表达明显减少(P＜0.05),ERK、p38、JNK和PKC抑制剂预作用HMC组中的FN表达分别减少了2倍、1.6倍、1.8倍和2.4倍;预先使用Zopolrestat孵育,再用ERK、p38、JNK和PKC通路抑制剂作用,最后使用TGF-β1刺激,结果发现ERK、JNK和PKC抑制剂使FN的表达分别减少2.3倍、2.4倍和2.1倍(P＜0.05)。但AR抑制剂和p38通路抑制剂合用后,与对照组比较,FN的表达未减少。
小结在无TGF-β1刺激时,单独使用AR抑制剂(Zopolrestat和Sorbinil),或者单独使用MAPKs和PKC信号通路抑制剂并不能减少HMC内FN的表达,但在使用AR抑制剂,或者ERK、JNK、p38和PKC信号通路抑制剂预作用HMC后,再以TGF-β1刺激时,则能明显减少细胞内FN的表达。说明AR、ERK、JNK、p38和PKC信号通路参与了TGF-β1诱导的FN表达过程的调控。以AR抑制剂和ERK、JNK和PKC通路抑制剂共同预作用HMC后,FN的表达明显减少,尤其是ERK和JNK通路更加明显,证明AR,ERK和JNK通路对TGF-β1诱导FN的表达过程可能存在叠加作用,也初步证明AR对TGF-β1诱导FN的表达过程的调控作用可能通过ERK、JNK和PKC通路实现的。AR抑制剂和p38信号通路抑制剂共同作用后,TGF-β1诱导FN的表达并未减少,推测其原因可能为AR对HMC的作用与p38信号通路的作用相互拮抗,但具体机制不清楚,有待进一步研究。提示在HMC内TGF-β1诱导的MAPKs信号通路活化过程中,ERK、JNK与p38信号通路的作用存在着一定的差异。同时AR抑制剂必须在TGF-β1刺激的情况下才能发挥对FN表达的调控作用,也更进一步的证明了在HMC中AR为TGF-β1的反应性基因。
第三部分醛糖还原酶基因对TGF-β1诱导FN的表达及对MAPKs信号通路的影响
目的进一步证明醛糖还原酶基因在TGF-β1诱导的FN表达过程中的作用及对MAPKs信号通路的影响。
方法培养HMC,转染PcDNA3.0-AR至HMC内及用RNA干扰技术干扰HMC内AR基因表达,观察HMC表达AR、FN以及MAPKs通路变化情况;转染和干扰AR基因后的细胞预先使用MAPKs、PKC信号通路抑制剂作用,再用外源性TGF-β1刺激;采用免疫荧光、Western Blot、RT-PCR、Real-Time PCR方法观察HMC内AR、FN和MAPKs信号通路的表达情况。
结果成功转染和干扰HMC内AR基因,并经多种方法验证转染和干扰效果明显。转染AR基因后,与正常对照相比,HMC内AR蛋白表达增高了4.3倍,FN蛋白表达增高2.5倍(P＜0.05);转染AR基因的HMC再经TGF-β1刺激后,FN表达增加更为明显(为对照组的3.6倍,P＜0.05);RNA干扰AR基因后,HMC中AR和FN表达明显减少(P＜0.05),干扰后AR表达在蛋白水平减少了6.8倍,FN表达减少了16.2倍(P＜0.05);RNA干扰后的HMC再用TGF-β1刺激,与对照组比较,FN的表达减少8.1倍(P＜0.05),和单纯干扰组比较有所上升,但却明显低于未干扰组;转染PcDNA3.0-AR后,使用免疫荧光方法检测,与正常对照组比较,系膜细胞内AR和FN荧光表达明显增强;通过RT-PCR、Real-Time PCR验证,AR在mRNA水平增多与对照组相比超过10倍以上。在AR转染组HMC中使用MAPKs、PKC信号通路抑制剂预孵育后,再用TGF-β1刺激,发现FN表达减少(P＜0.05),但与未转染AR基因的细胞相比,FN减少的程度明显降低,说明AR活性增高对MAPKs、PKC信号通路活性有一定的影响。在使用RNA干扰AR基因后的系膜细胞用免疫荧光检测,与正常对照组比较,细胞内AR和FN荧光表达明显减弱,通过RT-PCR、Real-Time PCR验证,AR在mRNA水平与对照组相比减少超过了80%;进一步实验在RNA干扰AR基因的细胞,预先使用MAPKs、PKC通路抑制剂孵育后,再用TGF-β1刺激,结果发现FN表达明显减少(P＜0.05),与未受RNA干扰的细胞相比,FN表达减少的程度明显加重,且干扰后MAPKs信号通路的磷酸化水平明显受到抑制,包括磷酸化的ERK、p38和JNK表达均明显减少,更进一步的证明了AR基因参与了TGF-β1诱导的MAPKs通路的活化过程。
小结转染PcDNA3.0-AR后,即使在无TGF-β1刺激的情况下,FN的表达也明显增多,TGF-β1刺激后FN的表达增多更加明显。用RNA干扰技术干扰AR基因后,即使无TGF-β1的刺激,FN的表达也明显的减少,说明改变HMC内AR基因表达的变化本身可以影响FN表达。在TGF-β1刺激时,转染和干扰AR基因对FN表达具有明显的影响,进一步说明了AR参与了TGF-β1诱导的FN的调控过程,转染AR基因后用MAPKs、PKC信号通路抑制剂孵育,再用TGF-β1刺激,与未转染AR基因的细胞相比,FN减少的程度明显降低;在RNA干扰AR基因的细胞,预先使用MAPKs、PKC通路抑制剂,再用TGF-β1刺激,与未受RNA干扰的细胞相比,FN表达减少程度明显加重,进一步的说明AR对MAPKs,PKC信号通路的活性存在一定的影响;干扰AR基因后,MAPKs信号通路的活性也明显受到抑制,证明了TGF-β1诱导的MAPKs信号通路的活化过程与AR的活性状态有关。
结论
1.外源性TGF-β1可上调HMC内AR、FN的表达,提示在HMC内,AR仍然是TGF-β1的下游反应性基因之一,这也进一步证明了课题组前期研究结果。TGF-β1在增加AR、FN表达的同时激活了包括ERK、p38和JNK信号通路,其中ERK信号通路活化效果最明显,说明TGF-β1对人系膜细胞内FN的表达的调控作用,可能与TGF-β1刺激活化了ERK、p38和JNK信号通路有关,ERK信号通路可能起着重要作用。
2.在无外源性TGF-β1刺激的情况下,单独使用AR抑制剂或者单独使用MAPKs和PKC信号通路抑制剂并不能减少HMC内FN的表达。但在TGF-β1刺激下,AR抑制剂、MAPKs和PKC信号通路抑制剂能明显的减少HMC内FN的表达,说明AR参与了TGF-β1诱导FN高表达的调控过程,且这一过程可能与MAPKs和PKC信号通路有关。AR抑制剂必须在TGF-β1刺激的情况下才能发挥对FN表达的调控作用,也更进一步的证明了AR为TGF-β1的反应性基因。
3.转染PcDNA3.0-AR后,即使在无TGF-β1刺激的情况下,FN的表达也明显增多,添加TGF-β1刺激后FN的表达增多更加明显。用RNA干扰AR基因后,在无TGF-β1刺激的情况下,FN的表达也明显的减少,说明AR本身也参与了HMC内FN的表达调控作用。在TGF-β1刺激时,转染和干扰AR基因对FN表达有更加明显的影响,转染后使FN的表达增多更明显,干扰后FN的表达减少也更显著,进一步说明了AR参与了TGF-β1诱导的FN的调控过程。在转染AR基因后的细胞,用MAPKs和PKC信号通路抑制剂作用,FN的减少程度不如未转染的细胞,进一步的说明AR对MAPKs,PKC信号通路的活性存在一定的影响,高表达AR能够削弱通路抑制剂对信号通路的抑制作用;干扰AR基因后,MAPKs信号通路的活性也明显受到抑制,证明了TGF-β1诱导的MAPKs信号通路的活化过程与AR有关。
Glomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular diseases,characterized by extracellular matrix(ECM) proteins accumulation,represents a common pathway of different renal diseases.There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis.Glomerular MsC plays an important role in the regulation of ECM metabolism.Recent research points are focused on the MsC and regulation ECM metabolism,as well as its correlation with the development of glomerulosclerosis.
As resident cells in glomerulus,the mesangial cells play an important role not only in maintaining the normal structure and functions of glomerulus,but also in acute glomerulonephritis and progressive glomerulosclerosis.Several reports have implicated that MsC excretes various cellular factors,PDGF,TGF-β1 and IL6.Recent researches have demonstrated that transforming growth factor-β1(TGF-β1) in kidney diseases,including glomerulosclerosis,since the MsC high expression of TGF-β1 can result in ECM deposition by inducing ECM proteins expression.
Aldose reductase,a member of the aldo-keto reductase superfamily,can catalyse the reduction of a wide range of aldehydes including glucose.This enzyme represents the first and the rate-limiting step of the polyol pathway.Increased polyol pathway activity has been linked to the aetiology of hyperglycaemic injury and inhibition of AR in preventing or delaying diabetes-related changes in the lens,retina,peripheral nerve and kidney.Our group has found that AR gene was one of TGF-β1-responsive genes by SSH-PCR.As one of TGF-β1-responsive genes,whether AR has any relations to glomerulosclerosis just like TGF-β1 under normal glucose concentration remains unclear.
Fibronectin(FN) is a heterodimeric extracellular matrix glycoprotein implicated in a number of physiological events during embryogenesis,angiogenesis,thrombosis,as well as inflammation.It is a major component of the ECM.FN plays an important role during development and wound healing by promoting cell adhesion,migration and cytoskeletal organization.However,FN is often observed following oncogenic transformation,leading to decreased adhesion and increased metastatic potential.Overproduction of matrix components including FN is the main pathological finding in tissue fibrosis.
In this study we used molecular biologic technologies and cell culture to examine the expression of TGF-β1 and AR in HMC and the relations between them,and to observe the effect of AR in TGF-β1-induced expression of ECM components,fibronectin,and to analyze the effect related signaling pathways.The purpose of our research was to elucidate the function and significance of AR in glomerulosclerosis,and provide new experiments data for enriching the pathologic mechanism of glomerulosclerosis.
PartⅠTGF-β1 induced expression of AR,FN and MAPKs signaling pathway phosphorylation in cultured human mesangial cells
Objective To explore the effect of TGF-β1 induced expression of AR,FN and MAPKs phosphorylation in cultured human mesangial cells.
Methods Cultured HMCs was treated with recombinant human TGF-β1 in different concentrations or for different time.The expression of AR,FN and MAPKs were examined by Western Blot analysis,respectively.
Results The cultured HMC treated with TGF-β1 showed increased expression of AR,FN and phosphorylation MAPKs.When using the single concentration,1ng/ml TGF-β1,the high expression were observed when incubated for 15min,60min is the most effective in AR and 120min in FN.When using different concentrations for 24h incubation,the high expression was observed when used from1ng/ml to 8ng/ml TGF-β1,and reached a peak at 1ng/ml,TGF-β1 activated the three MAPK cascades p38,ERK and JNK pathway.The maximum activation of p38 was observed after 60 min of stimulation,ERK and JNK was observed after 30 min of stimulation.The activation of JNK and p38 by TGF-β1 was sustained up to 120 min,ERK activation was more sustained a longer time.
Conclusions The recombinant human cytokine TGF-β1 can up-regulate the expression of AR, FN and MAPKs in dose and time-dependent manners in cultured HMC,which provide new proof for our former conclusion,namely AR gene was one of TGF-β1-responsive genes.
PartⅡThe effect of inhibitors of AR,MAPKs and PKC on Expression of FN by TGF-β1 induced
Objective To elucidate the role of AR and signal pathway in TGF-β1-induced expression of FN and explore the expression of FN and AR by inhibit the activation of AR,MAPKs and PKC.
Methods Cultured HMCs were treated with inhibitors of AR(sorbinil and zopolrestat), inhibitors of p38,JNK and ERK(SB203580,SP600125 and U0126) in HMC,then the different groups of cells were treated with TGF-β1(1ng/ml) for 1h.The expression of AR and FN were examined by Western Blot analysis,respectively.
Results The normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR and MAPKs.Pre-incubation of cells with sorbinil or zopolrestat, then the different groups of cells were treated with TGF-β1(1ng/ml).More than 2.9 folds decrease in the protein level of FN was observed in cells treated with zopolrestat.Similar results were observed in cells treated with sorbinil,which showed that ARI could reduce the expression of FN.The results showed that TGF-β1-induced expression of FN protein levels was suppressed by G_(O丨¨)6983,SB203580,SP600125 and U0126,indicating that the PKC, ERK,JNK and p38-mediated signaling pathways are involved in the TGF-β1-induced up-regulation of FN protein expression.TGF-β1-induced up-regulation of FN was significantly suppressed in together with inhibitors of AR,ERK,JNK and PKC.In contrast, co-incubated inhibitor of AR,p38 did not suppressed FN expression.
Conclusions These results indicate that TGF-β1-induced FN expressing could be attributed to AR activation,which is regulated,at least in part,by PKC,ERK,JNK and p38 signaling pathways.
PartⅢThe effect of AR on TGF-β1-induced ECM components and the related signaling pathway
Objective To further clarify the role of AR in TGF-β1 induced FN expression,explore the effect of AR and AR on TGF-β1-induced expression of FN and the related signaling pathway.
Methods Restriction endonucleases digestion and ligation was used to reconstructing eukaryotic expression plasmid pcDNA3-AR and siRNA.Lipofectin2000 was used to transfect AR vectors into HMC.RT-PCR,Real-Time PCR,Western Blot and immunofluorescence analysis were performed to verify the transfection and RNAi.Western Blot was used to analyse the expression of fibronectin and MAPKs proteins with or without the stimulation of TGF-β1.
Results The HMC transfected with AR showed increased expression of AR and FN (P＜0.05).After stimulation of TGF-β1,the HMC transfected with AR showed increased expression of FN.The HMC knock-down of AR gene with RNAi showed decreased expression of AR and FN(P＜0.05).After stimulation of TGF-β1,the results showed decreased expression of FN.The HMC transfected with AR and knocked down AR gene showed obviously increased or reduced expression of FN(P＜0.05).There was no change in expression of total MAPKs in different groups of HMC with or without stimulation of TGF-β1.The normal HMC showed increased expression of phospho-ERK,phospho-JNK, and phospho-p38 after stimulation of TGF-β1,and the HMC knocked down AR gene with RNAi showed reduced expression of phospho-JNK,phospho-p38 and phospho-ERK (p＜0.05).
Conclusions AR can regulates the expression of FN with the stimulation of TGF-β1 as one of the responsive genes of TGF-β1,which may have relations with the activation of JNK-MAPK,ERK-MAPK and p38-MAPK signaling pathways.These data indicate that AR may probably play a role in the pathogenesis of glomerulosclersis.
1.TGF-β1 can induce the expression of AR and FN in time and dose-dependent manners, which show the controllability of AR gene as one of TGF-β1-responsive genes.
2.These results showed that ARI could reduce the expression of FN,indicating that the PKC,ERK,JNK and p38-mediated signaling pathways are involved in the TGF-β1-induced up-regulation of FN protein expression.
3.AR may regulate the TGF-β1-induced expression of FN,one of ECM components, probably via MAPKs and PKC signaling pathway,which can be considered one of new function of AR under normal glucose concentration in glomerulosclerosis.
肾小球硬化是各种原因所致肾小球损伤和病变的共同结局,是由多种生物活性物质和多种细胞成分参与的一个复杂病理过程,其主要的病理特征之一是肾小球内细胞外基质(Extracellular Matrix,ECM)的异常沉积。研究认为ECM合成和(或)降解失衡是肾小球硬化发生的重要机制之一,而系膜细胞(Mesangial Cell,MsC)增生则是许多肾小球疾病发生发展过程中的中心环节,并且在肾小球内ECM代谢及调控过程中起重要作用,体内外各种生长因子和细胞因子均可刺激MsC增殖。因此,研究肾小球系膜细胞与肾小球硬化的发生、发展之间的相互关系也是近年来器官纤维化研究领域的重要研究内容之一。
MsC是肾小球内固有的一种血管外周细胞,大量研究证实,该细胞可自分泌多种细胞因子:如血小板源生长因子、IL-6、TGF-β1、内皮素1等,后者可促进MsC自身分裂、增生及细胞外基质的合成和分泌,在肾小球硬化过程中发挥重要作用。研究已证实,肾脏MsC高表达转化生长因子β1(transforming growth factorβ1,TGF-β1),TGF-β1是一个多功能细胞因子,具有调节细胞增殖、分化以及细胞凋亡等功能。TGF-β1具有调节ECM合成、代谢过程的作用,可以直接促进ECM成分的合成或影响ECM合成和(或)降解主要调节酶系统的活性而促进ECM合成增加和沉积,从而在肾小球硬化发生与发展的病理过程中具有重要的作用。
醛糖还原酶(Aldose Reductase,AR)为NADPH依赖性醛-酮还原酶家族成员之一,是多元醇代谢通路中的限速酶,可催化己糖或戊糖的NADPH依赖性还原反应,使之变成相应的糖醇或多元醇。AR广泛分布于神经、肌肉、脑、肾脏、胎盘、血管、视网膜等组织中,在不同组织中的分子构成、免疫原性及其他理化性质有差别。对AR的研究,以往都集中在糖尿病及其并发症中,但新近的研究发现,AR的某些功能和高糖环境并不相关。我们课题组前期通过SSH-PCR研究发现,AR是大鼠MsC中TGF-β1相关反应性基因之一,而TGF-β1在组织纤维化,特别是肾脏纤维化中起着重要作用,因此AR是否参与了TGF-β1在非高糖状态下所导致的肾小球硬化、肾脏纤维化及其病理过程值得深入研究。
纤连蛋白(Fibronectin,FN)是构成细胞外基质的一个重要组成部分,是一种大分子糖蛋白,在正常组织中分布广泛,具有促进细胞移动、分化、调理吞噬等功能,在细胞与细胞、细胞与基质的相互作用中起着重要作用。FN可诱导成纤维细胞增生,产生胶原纤维,促进纤维化的形成,研究发现FN的合成及降解的异常在肾纤维化的发生、发展中起很大的作用。
本课题采用细胞培养、基因转染、免疫荧光、RNA干扰、RT-PCR、Real-Time PCR、Western Blot等实验方法,观察AR在人系膜细胞中的表达及其与TGF-β1的相互关系,观察AR在TGF-β1诱导Fibronectin表达增高过程中的作用,并初步探讨这种作用所涉及的细胞信号传导通路等作用机制,以明确AR在肾小球硬化等病理过程中的作用及意义,进一步认识和了解AR在非糖尿病性肾脏疾病(主要为肾小球肾炎、纤维化和硬化)中的新作用,以期能够为肾小球硬化发生发展的病理机制的认识提供新的实验依据。
第一部分TGF-β1对人系膜细胞表达AR、FN以及MAPKs信号通路的影响
目的探讨外源性TGF-β1对体外培养人系膜细胞(HMC)表达AR、FN以及对MAPKs信号通路的影响。
方法培养HMC,在培养液中添加外源性TGF-β1因子,采用免疫荧光和WesternBlot方法观察HMC内AR、FN和MAPKs表达的变化。
结果以不同浓度TGF-β1(1-10ng/ml)刺激HMC 24h,结果发现AR和FN表达均增高,其中以1ng/ml TGF-β1作用浓度下AR、FN表达升高最为明显(P＜0.05);再以1ng/ml TGF-β1以不同的作用时间(0-4h)刺激HMC,可见AR、FN在15min时表达即开始增高,其中AR表达在TGF-β1作用后1h升高最明显(P＜0.05),FN在2h升高最明显(P＜0.05)。与此同时,TGF-β1激活了MAPKs信号通路,TGF-β1作用15min后可见磷酸化的ERK、JNK和p38表达升高,其中磷酸化的ERK、JNK在作用后30min达高峰,磷酸化的p38在1h后达高峰(P＜0.05),然后逐渐下降;磷酸化的ERK表达量和持续时间明显多于磷酸化JNK和p38。
小结外源性TGF-β1可上调HMC内AR、FN的表达;TGF-β1可以活化MAPKs信号通路,其中以ERK信号通路活化效果最明显,提示TGF-β1上调AR、FN的表达可能与激活MAPKs信号通路有关,ERK信号通路在这过程中可能起着主要的作用。TGF-β1可调节AR表达的实验结果也证实了在HMC内,AR也是TGF-β1的反应性基因。
第二部分抑制AR活性,阻断MAPKs和PKC信号通路对TGF-β1诱导FN表达的影响
目的探讨AR抑制剂Zopolrestat和Sorbinil,MAPKs信号通路抑制剂U0126(ERK抑制剂)、SB203580(p38抑制剂)、SP600125(JNK抑制剂)和G(o|¨)6983(PKC抑制剂)对体外培养的HMC使用TGF-β1诱导FN表达的影响。
方法培养HMC,通过预先在培养液中分别添加AR抑制剂Zopolrestat和Sorbinil,MAPKs信号通路抑制剂U0126、SB203580、SP600125和PKC信号通路抑制剂G(o|¨)6983作用后,再用外源性TGF-β1因子刺激,采用Western Blot方法观察HMC内FN表达的变化。
结果仅使用AR抑制剂Zopolrestat和Sorbinil作用于HMC,与未使用AR抑制剂的HMC相比,FN的表达未见减少;但在使用Zopolrestat和Sorbinil预孵育HMC后,再用TGF-β1刺激,则可发现FN的表达明显减少(P＜0.05),使用Zopolrestat预作用组FN的表达比单独使用TGF-β1刺激时减少了2.9倍,使用Sorbinil预作用组的FN的表达减少了2.5倍;分别以不同浓度的ERK、p38、JNK和PKC信号通路抑制剂作用于HMC,与正常对照组相比,FN的表达未见减少。进一步研究发现,在预先使用ERK、p38、JNK和PKC抑制剂作用于细胞后,再用外源性TGF-β1刺激,则发现FN的表达明显减少(P＜0.05),ERK、p38、JNK和PKC抑制剂预作用HMC组中的FN表达分别减少了2倍、1.6倍、1.8倍和2.4倍;预先使用Zopolrestat孵育,再用ERK、p38、JNK和PKC通路抑制剂作用,最后使用TGF-β1刺激,结果发现ERK、JNK和PKC抑制剂使FN的表达分别减少2.3倍、2.4倍和2.1倍(P＜0.05)。但AR抑制剂和p38通路抑制剂合用后,与对照组比较,FN的表达未减少。
小结在无TGF-β1刺激时,单独使用AR抑制剂(Zopolrestat和Sorbinil),或者单独使用MAPKs和PKC信号通路抑制剂并不能减少HMC内FN的表达,但在使用AR抑制剂,或者ERK、JNK、p38和PKC信号通路抑制剂预作用HMC后,再以TGF-β1刺激时,则能明显减少细胞内FN的表达。说明AR、ERK、JNK、p38和PKC信号通路参与了TGF-β1诱导的FN表达过程的调控。以AR抑制剂和ERK、JNK和PKC通路抑制剂共同预作用HMC后,FN的表达明显减少,尤其是ERK和JNK通路更加明显,证明AR,ERK和JNK通路对TGF-β1诱导FN的表达过程可能存在叠加作用,也初步证明AR对TGF-β1诱导FN的表达过程的调控作用可能通过ERK、JNK和PKC通路实现的。AR抑制剂和p38信号通路抑制剂共同作用后,TGF-β1诱导FN的表达并未减少,推测其原因可能为AR对HMC的作用与p38信号通路的作用相互拮抗,但具体机制不清楚,有待进一步研究。提示在HMC内TGF-β1诱导的MAPKs信号通路活化过程中,ERK、JNK与p38信号通路的作用存在着一定的差异。同时AR抑制剂必须在TGF-β1刺激的情况下才能发挥对FN表达的调控作用,也更进一步的证明了在HMC中AR为TGF-β1的反应性基因。
第三部分醛糖还原酶基因对TGF-β1诱导FN的表达及对MAPKs信号通路的影响
目的进一步证明醛糖还原酶基因在TGF-β1诱导的FN表达过程中的作用及对MAPKs信号通路的影响。
方法培养HMC,转染PcDNA3.0-AR至HMC内及用RNA干扰技术干扰HMC内AR基因表达,观察HMC表达AR、FN以及MAPKs通路变化情况;转染和干扰AR基因后的细胞预先使用MAPKs、PKC信号通路抑制剂作用,再用外源性TGF-β1刺激;采用免疫荧光、Western Blot、RT-PCR、Real-Time PCR方法观察HMC内AR、FN和MAPKs信号通路的表达情况。
结果成功转染和干扰HMC内AR基因,并经多种方法验证转染和干扰效果明显。转染AR基因后,与正常对照相比,HMC内AR蛋白表达增高了4.3倍,FN蛋白表达增高2.5倍(P＜0.05);转染AR基因的HMC再经TGF-β1刺激后,FN表达增加更为明显(为对照组的3.6倍,P＜0.05);RNA干扰AR基因后,HMC中AR和FN表达明显减少(P＜0.05),干扰后AR表达在蛋白水平减少了6.8倍,FN表达减少了16.2倍(P＜0.05);RNA干扰后的HMC再用TGF-β1刺激,与对照组比较,FN的表达减少8.1倍(P＜0.05),和单纯干扰组比较有所上升,但却明显低于未干扰组;转染PcDNA3.0-AR后,使用免疫荧光方法检测,与正常对照组比较,系膜细胞内AR和FN荧光表达明显增强;通过RT-PCR、Real-Time PCR验证,AR在mRNA水平增多与对照组相比超过10倍以上。在AR转染组HMC中使用MAPKs、PKC信号通路抑制剂预孵育后,再用TGF-β1刺激,发现FN表达减少(P＜0.05),但与未转染AR基因的细胞相比,FN减少的程度明显降低,说明AR活性增高对MAPKs、PKC信号通路活性有一定的影响。在使用RNA干扰AR基因后的系膜细胞用免疫荧光检测,与正常对照组比较,细胞内AR和FN荧光表达明显减弱,通过RT-PCR、Real-Time PCR验证,AR在mRNA水平与对照组相比减少超过了80%;进一步实验在RNA干扰AR基因的细胞,预先使用MAPKs、PKC通路抑制剂孵育后,再用TGF-β1刺激,结果发现FN表达明显减少(P＜0.05),与未受RNA干扰的细胞相比,FN表达减少的程度明显加重,且干扰后MAPKs信号通路的磷酸化水平明显受到抑制,包括磷酸化的ERK、p38和JNK表达均明显减少,更进一步的证明了AR基因参与了TGF-β1诱导的MAPKs通路的活化过程。
小结转染PcDNA3.0-AR后,即使在无TGF-β1刺激的情况下,FN的表达也明显增多,TGF-β1刺激后FN的表达增多更加明显。用RNA干扰技术干扰AR基因后,即使无TGF-β1的刺激,FN的表达也明显的减少,说明改变HMC内AR基因表达的变化本身可以影响FN表达。在TGF-β1刺激时,转染和干扰AR基因对FN表达具有明显的影响,进一步说明了AR参与了TGF-β1诱导的FN的调控过程,转染AR基因后用MAPKs、PKC信号通路抑制剂孵育,再用TGF-β1刺激,与未转染AR基因的细胞相比,FN减少的程度明显降低;在RNA干扰AR基因的细胞,预先使用MAPKs、PKC通路抑制剂,再用TGF-β1刺激,与未受RNA干扰的细胞相比,FN表达减少程度明显加重,进一步的说明AR对MAPKs,PKC信号通路的活性存在一定的影响;干扰AR基因后,MAPKs信号通路的活性也明显受到抑制,证明了TGF-β1诱导的MAPKs信号通路的活化过程与AR的活性状态有关。
结论
1.外源性TGF-β1可上调HMC内AR、FN的表达,提示在HMC内,AR仍然是TGF-β1的下游反应性基因之一,这也进一步证明了课题组前期研究结果。TGF-β1在增加AR、FN表达的同时激活了包括ERK、p38和JNK信号通路,其中ERK信号通路活化效果最明显,说明TGF-β1对人系膜细胞内FN的表达的调控作用,可能与TGF-β1刺激活化了ERK、p38和JNK信号通路有关,ERK信号通路可能起着重要作用。
2.在无外源性TGF-β1刺激的情况下,单独使用AR抑制剂或者单独使用MAPKs和PKC信号通路抑制剂并不能减少HMC内FN的表达。但在TGF-β1刺激下,AR抑制剂、MAPKs和PKC信号通路抑制剂能明显的减少HMC内FN的表达,说明AR参与了TGF-β1诱导FN高表达的调控过程,且这一过程可能与MAPKs和PKC信号通路有关。AR抑制剂必须在TGF-β1刺激的情况下才能发挥对FN表达的调控作用,也更进一步的证明了AR为TGF-β1的反应性基因。
3.转染PcDNA3.0-AR后,即使在无TGF-β1刺激的情况下,FN的表达也明显增多,添加TGF-β1刺激后FN的表达增多更加明显。用RNA干扰AR基因后,在无TGF-β1刺激的情况下,FN的表达也明显的减少,说明AR本身也参与了HMC内FN的表达调控作用。在TGF-β1刺激时,转染和干扰AR基因对FN表达有更加明显的影响,转染后使FN的表达增多更明显,干扰后FN的表达减少也更显著,进一步说明了AR参与了TGF-β1诱导的FN的调控过程。在转染AR基因后的细胞,用MAPKs和PKC信号通路抑制剂作用,FN的减少程度不如未转染的细胞,进一步的说明AR对MAPKs,PKC信号通路的活性存在一定的影响,高表达AR能够削弱通路抑制剂对信号通路的抑制作用;干扰AR基因后,MAPKs信号通路的活性也明显受到抑制,证明了TGF-β1诱导的MAPKs信号通路的活化过程与AR有关。
Glomerulosclerosis is a final common outcome of glomerular injury in various types of human glomerular diseases,characterized by extracellular matrix(ECM) proteins accumulation,represents a common pathway of different renal diseases.There is increasing evidence that the imbalance of ECM synthesis and degradation may be an important mechanism of glomerulosclerosis.Glomerular MsC plays an important role in the regulation of ECM metabolism.Recent research points are focused on the MsC and regulation ECM metabolism,as well as its correlation with the development of glomerulosclerosis.
As resident cells in glomerulus,the mesangial cells play an important role not only in maintaining the normal structure and functions of glomerulus,but also in acute glomerulonephritis and progressive glomerulosclerosis.Several reports have implicated that MsC excretes various cellular factors,PDGF,TGF-β1 and IL6.Recent researches have demonstrated that transforming growth factor-β1(TGF-β1) in kidney diseases,including glomerulosclerosis,since the MsC high expression of TGF-β1 can result in ECM deposition by inducing ECM proteins expression.
Aldose reductase,a member of the aldo-keto reductase superfamily,can catalyse the reduction of a wide range of aldehydes including glucose.This enzyme represents the first and the rate-limiting step of the polyol pathway.Increased polyol pathway activity has been linked to the aetiology of hyperglycaemic injury and inhibition of AR in preventing or delaying diabetes-related changes in the lens,retina,peripheral nerve and kidney.Our group has found that AR gene was one of TGF-β1-responsive genes by SSH-PCR.As one of TGF-β1-responsive genes,whether AR has any relations to glomerulosclerosis just like TGF-β1 under normal glucose concentration remains unclear.
Fibronectin(FN) is a heterodimeric extracellular matrix glycoprotein implicated in a number of physiological events during embryogenesis,angiogenesis,thrombosis,as well as inflammation.It is a major component of the ECM.FN plays an important role during development and wound healing by promoting cell adhesion,migration and cytoskeletal organization.However,FN is often observed following oncogenic transformation,leading to decreased adhesion and increased metastatic potential.Overproduction of matrix components including FN is the main pathological finding in tissue fibrosis.
In this study we used molecular biologic technologies and cell culture to examine the expression of TGF-β1 and AR in HMC and the relations between them,and to observe the effect of AR in TGF-β1-induced expression of ECM components,fibronectin,and to analyze the effect related signaling pathways.The purpose of our research was to elucidate the function and significance of AR in glomerulosclerosis,and provide new experiments data for enriching the pathologic mechanism of glomerulosclerosis.
PartⅠTGF-β1 induced expression of AR,FN and MAPKs signaling pathway phosphorylation in cultured human mesangial cells
Objective To explore the effect of TGF-β1 induced expression of AR,FN and MAPKs phosphorylation in cultured human mesangial cells.
Methods Cultured HMCs was treated with recombinant human TGF-β1 in different concentrations or for different time.The expression of AR,FN and MAPKs were examined by Western Blot analysis,respectively.
Results The cultured HMC treated with TGF-β1 showed increased expression of AR,FN and phosphorylation MAPKs.When using the single concentration,1ng/ml TGF-β1,the high expression were observed when incubated for 15min,60min is the most effective in AR and 120min in FN.When using different concentrations for 24h incubation,the high expression was observed when used from1ng/ml to 8ng/ml TGF-β1,and reached a peak at 1ng/ml,TGF-β1 activated the three MAPK cascades p38,ERK and JNK pathway.The maximum activation of p38 was observed after 60 min of stimulation,ERK and JNK was observed after 30 min of stimulation.The activation of JNK and p38 by TGF-β1 was sustained up to 120 min,ERK activation was more sustained a longer time.
Conclusions The recombinant human cytokine TGF-β1 can up-regulate the expression of AR, FN and MAPKs in dose and time-dependent manners in cultured HMC,which provide new proof for our former conclusion,namely AR gene was one of TGF-β1-responsive genes.
PartⅡThe effect of inhibitors of AR,MAPKs and PKC on Expression of FN by TGF-β1 induced
Objective To elucidate the role of AR and signal pathway in TGF-β1-induced expression of FN and explore the expression of FN and AR by inhibit the activation of AR,MAPKs and PKC.
Methods Cultured HMCs were treated with inhibitors of AR(sorbinil and zopolrestat), inhibitors of p38,JNK and ERK(SB203580,SP600125 and U0126) in HMC,then the different groups of cells were treated with TGF-β1(1ng/ml) for 1h.The expression of AR and FN were examined by Western Blot analysis,respectively.
Results The normal HMC showed not reduced expression of FN after incubation with single inhibitors of AR and MAPKs.Pre-incubation of cells with sorbinil or zopolrestat, then the different groups of cells were treated with TGF-β1(1ng/ml).More than 2.9 folds decrease in the protein level of FN was observed in cells treated with zopolrestat.Similar results were observed in cells treated with sorbinil,which showed that ARI could reduce the expression of FN.The results showed that TGF-β1-induced expression of FN protein levels was suppressed by G_(O丨¨)6983,SB203580,SP600125 and U0126,indicating that the PKC, ERK,JNK and p38-mediated signaling pathways are involved in the TGF-β1-induced up-regulation of FN protein expression.TGF-β1-induced up-regulation of FN was significantly suppressed in together with inhibitors of AR,ERK,JNK and PKC.In contrast, co-incubated inhibitor of AR,p38 did not suppressed FN expression.
Conclusions These results indicate that TGF-β1-induced FN expressing could be attributed to AR activation,which is regulated,at least in part,by PKC,ERK,JNK and p38 signaling pathways.
PartⅢThe effect of AR on TGF-β1-induced ECM components and the related signaling pathway
Objective To further clarify the role of AR in TGF-β1 induced FN expression,explore the effect of AR and AR on TGF-β1-induced expression of FN and the related signaling pathway.
Methods Restriction endonucleases digestion and ligation was used to reconstructing eukaryotic expression plasmid pcDNA3-AR and siRNA.Lipofectin2000 was used to transfect AR vectors into HMC.RT-PCR,Real-Time PCR,Western Blot and immunofluorescence analysis were performed to verify the transfection and RNAi.Western Blot was used to analyse the expression of fibronectin and MAPKs proteins with or without the stimulation of TGF-β1.
Results The HMC transfected with AR showed increased expression of AR and FN (P＜0.05).After stimulation of TGF-β1,the HMC transfected with AR showed increased expression of FN.The HMC knock-down of AR gene with RNAi showed decreased expression of AR and FN(P＜0.05).After stimulation of TGF-β1,the results showed decreased expression of FN.The HMC transfected with AR and knocked down AR gene showed obviously increased or reduced expression of FN(P＜0.05).There was no change in expression of total MAPKs in different groups of HMC with or without stimulation of TGF-β1.The normal HMC showed increased expression of phospho-ERK,phospho-JNK, and phospho-p38 after stimulation of TGF-β1,and the HMC knocked down AR gene with RNAi showed reduced expression of phospho-JNK,phospho-p38 and phospho-ERK (p＜0.05).
Conclusions AR can regulates the expression of FN with the stimulation of TGF-β1 as one of the responsive genes of TGF-β1,which may have relations with the activation of JNK-MAPK,ERK-MAPK and p38-MAPK signaling pathways.These data indicate that AR may probably play a role in the pathogenesis of glomerulosclersis.
1.TGF-β1 can induce the expression of AR and FN in time and dose-dependent manners, which show the controllability of AR gene as one of TGF-β1-responsive genes.
2.These results showed that ARI could reduce the expression of FN,indicating that the PKC,ERK,JNK and p38-mediated signaling pathways are involved in the TGF-β1-induced up-regulation of FN protein expression.
3.AR may regulate the TGF-β1-induced expression of FN,one of ECM components, probably via MAPKs and PKC signaling pathway,which can be considered one of new function of AR under normal glucose concentration in glomerulosclerosis.
引文
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5、 WS.Lin,N.Zhang.Upregulation of MMP-2 by all-trans retinoics acid is mediated by TGF-β1 in cultured rat mesangial cell[J].Fibrinosis and Proteolysis,2000,14(4):235-241.
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13、Kapor Drezgic J,Zhou X,Babazono T,et al.Effect of high glucose onmesangial cell protein kinase C-delta and epsilon is polyol pathway dependent.J Am Soc Nephrol,,1999;10(6):1193-1203.
14、林文生,张农,秦蓉,等.转化生长因子β1对大鼠系膜细胞醛糖还原酶表达的影响.中华医学杂志,2001,81:744-747.
15、蒋涛,刘国元,赵仲华,等。TGF-β1对其醛糖还原酶在大鼠系膜细胞及人肾小球肾炎中表达的影响。复旦学报医学科学版,2004,31:565-569.
16、 Bessho K,Mizuno S,Matsumoto K,et al.Counter active effects of HGF on PDGF-induced mesangial cell p roliferation in a rat model of glomerulonephritis.Am J Physiol Renal Physiol,2003,284(6):F1171 - F1180.
17、 Schnaper HW,Hayashida T,Hubchak SC,et al.TGF -beta signal transduction and mesangial cell fibrogenesis.Am J Physiol Renal Physiol,2003,284(2):F243 - F252.
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28、 Hayashida T, Schnaper HW.High ambient glucose enhances sensitivity to TGF-beta1 via extracellular signal—regulated kinase and protein kinase Cdelta activities in human mesangial cells. J Am Soc Nephrol. 2004;15(8):2032-2041.
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30、 Yoshioka K,Takemura T,Murakami K,et al.Transforming growth factor-beta protein and mRNA in glomeruli in normal and diseased human kidneys[J].Lab Invest,1993,68:154～163.
31、 Wahab NA, Weston BS, Mason RM.Modulation of the TGFbeta/Smad signaling pathway in mesangial cells by CTGF/CCN2. Exp Cell Res. 2005;307(2):305-314.
32、 Poncelet AC, de Caestecker MP, Schnaper HW. The transforming growth factor-beta /SMAD signaling pathway is p resent and functional in human mesangial cells1 Kidney Int, 1999, 56 (4): 1354-1365.
33、 Runyan CE, Schnaper HW, PonceletAC. Smad3 and PKC delta mediate TGF-beta1-induced collagen I exp ression in human mesangial cells1 Am J Physiol Renal Physiol, 2003, 85 ( 3 ) :413-422.
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