PPARα激动剂调控心脏糜酶介导大鼠心肌纤维化的作用及信号转导机制的研究
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摘要
研究背景及目的心肌纤维化是高血压左心室重构的重要病理改变之一,也是心脏舒张功能障碍的主要原因,更是心功能由代偿期向失代偿期转变的关键。因此,预防和逆转心肌纤维化、恢复心功能对高血压靶器官保护具有重要意义。大量研究表明,来源于心脏肥大细胞的糜酶参与心血管的病理性重构,在高血压、心肌梗死、心肌病、心力衰竭、冠脉支架植入术后再狭窄、动脉粥样硬化及动脉瘤等疾病的发生、发展中具有重要作用。文献报道,糜酶能抑制血管平滑肌细胞增殖,并促进心肌细胞肥大。心脏成纤维细胞(CFs)是心肌纤维化的主要效应细胞,其过度增殖及胶原合成增多是心肌纤维化的病理基础。但糜酶对CFs增殖与胶原合成有何影响及其信号转导机制目前尚不清楚。近年研究表明,糜酶能激活转化生长因子-β1(TGF-β1)的前体,但糜酶能否通过活化的TGF-β1诱导CFs增殖及胶原合成目前还不明确。Smads蛋白家族是近年发现的参与TGF-β1信号转导的细胞内效应分子之一,特异性地调节TGF-β1靶基因的表达。业已证实,TGF-β1/Smads信号通路的激活参与肝、肺、肾、腹膜和皮肤等器官和组织的纤维化病变,但该信号通路是否在高血压心肌纤维化的病理过程中发挥重要作用尚不清楚。过氧化物酶体增殖物激活受体(PPARs)是一类配体依赖的核转录因子,包括α、β/δ和γ三种亚型。近年来关于PPARγ及其激动剂噻唑烷二酮类药物在代谢综合征中的心血管保护作用已进行了广泛研究,但对于PPARα的研究相对较少。晚近研究表明,PPARα在心肌组织呈高水平表达,对心肌肥厚发挥负调控作用,这预示着PPARα信号通路将成为高血压左心室肥厚防治新策略的一个有效靶点。贝特类调脂药对原发性高甘油三脂血症有肯定的疗效,自从被确认为PPARα的特异性激动剂以来,非诺贝特调脂以外的心血管保护效应倍受关注。在体外培养的心肌细胞,非诺贝特可抑制多种炎症介质及细胞因子引起的心肌细胞肥大,但其对CFs增殖与胶原合成有无影响及其细胞内信号通路,目前尚不十分清楚。此外,新近研究表明,PPARγ激动剂可下调TGF-β1基因表达从而抑制肾间质纤维化,其机制可能与阻断TGF-β1/Smads信号通路有关。但是,在糜酶介导心肌纤维化的病理过程中有无TGF-β1/Smads及PPARα信号途径的参与,以及这两条信号转导通路之间是否存在“信息交流”(cross-talk),目前也不明确,有待探讨。因此,本研究在细胞和分子水平上观察心脏糜酶对大鼠CFs增殖、胶原合成的影响及其细胞内信号转导通路,探讨糜酶在心肌纤维化中的作用机制;研究PPARα及其激动剂非诺贝特对糜酶介导的TGF-β1/Smads信号途径的调控作用,阐明非诺贝特逆转高血压心肌纤维化的分子机制。旨在为临床防治高血压左室重构提供理论依据和治疗新思路。
研究方法本研究以体外培养的SD大鼠CFs为研究对象,采用MTT比色法、放射性核素掺入实验、流式细胞仪细胞周期分析、ELISA、RT-PCR及蛋白免疫印记等方法和技术,观察:(1)糜酶对大鼠CFs增殖和胶原合成的影响;(2)糜酶对大鼠CFs的TGF-β1、Smad2、Smad3、Smad7 mRNA和蛋白表达的影响;(3) PPARα激动剂非诺贝特对糜酶诱导的大鼠CFs增殖和胶原合成的影响:(4)非诺贝特干预对大鼠CFs的PPARα、TGF-β1mRNA和蛋白表达以及Smad2/3、p-Smad2/3、Smad7蛋白表达的影响。
研究结果(1)不同浓度的糜酶作用24 h,CFs的数目呈浓度依赖性增加,15、30和60 ng/ml组A_(490)值分别为0.263±0.033、0.348±0.031和0.387±0.026,均较对照组(0.201±0.019)显著增加(P＜0.01)。TGF-β1中和抗体预处理组和丝/苏氨酸激酶抑制剂预处理组的A_(490)值均明显低于30 ng/ml糜酶组(P＜0.05或P＜0.01),AT_1受体拮抗剂预处理组和AT_2受体拮抗剂预处理组的A_(490)值与糜酶组比较,均无显著性差异(P＞0.05)。(2) CFs的DNA合成随糜酶浓度的增加而增多,15、30和60 ng/ml组~3H-TdR掺入量分别为319±29、372±43和401±47(cpm/孔),较对照组(252±35 cpm/孔)明显升高(P＜0.01)。10μmol/L糜酶抑制剂预处理组的~3H-TdR掺入量显著低于30ng/ml糜酶组(P＜0.01)。(3)细胞周期分析结果表明,15、30和60 ng/ml糜酶作用24 h,CFs的G_0/G_1期细胞百分率均较对照组显著降低(P＜0.05或P＜0.01),S期细胞百分率和增殖指数(PI)则较对照组明显增高(P＜0.05或P＜0.01),G_2/M期细胞百分率与对照组比较无显著性差异(P＞0.05)。10μmol/L糜酶抑制剂预处理组G_0/G_1期细胞百分率显著高于30 ng/ml糜酶组(P＜0.01),S期细胞百分率和PI明显低于糜酶组(P＜0.01),G_2/M期细胞百分率与糜酶组及照组比较均无明显差异(P＞0.05)。(4)随着糜酶浓度的增高,CFs总胶原合成呈递增趋势,15、30和60 ng/ml组~3H-脯氨酸掺入量分别为520±75、684±62和769±58(cpm/孔),均较对照组(435±60 cpm/孔)显著增加(P＜0.05或P＜0.01)。TGF-β1中和抗体预处理组和丝/苏氨酸激酶抑制剂预处理组的~3H-脯氨酸掺入量均显著低于30 ng/ml糜酶组(P＜0.05或P＜0.01);AT_1受体拮抗剂预处理组和AT_2受体拮抗剂预处理组的~3H-脯氨酸掺入量与糜酶组比较无显著性差异(P＞0.05)。(5)不同浓度的糜酶作用24 h,Ⅰ、Ⅲ型胶原mRNA表达水平呈浓度依赖性增加,其中15、30和60 ng/ml组Ⅰ型和Ⅲ型胶原mRNA表达水平均较对照组显著升高(P＜0.01),但7.5 ng/ml组与对照组比较无显著性差异(P＞0.05)。(6)随着糜酶浓度的增高,CFs培养上清中Ⅰ、Ⅲ型胶原蛋白含量呈递增趋势,其中15、30和60 ng/ml组Ⅰ型和Ⅲ型胶原蛋白含量均较对照组明显增加(P＜0.05或P＜0.01),但7.5ng/ml组与对照组比较无显著性差异(P＞0.05)。(7) 15、30和60 ng/ml糜酶作用3h,TGF-β1 mRNA表达水平分别为0.698±0.051、1.096±0.078和1.242±0.065,均较对照组(0.299±0.035)明显增加(P＜0.01)。TGF-β1中和抗体预处理组的TGF-β1 mRNA表达水平明显低于30 ng/ml糜酶组(P＜0.05或P＜0.01),但AT_1受体拮抗剂和AT_2受体拮抗剂预处理组的TGF-β1 mRNA水平与糜酶组比较,均无显著性差异(P＞0.05)。(8) 15、30和60 ng/ml糜酶作用6 h,TGF-β1蛋白表达水平分别为0.968±0.069、1.782±0.058和2.656±0.085,均较对照组(0.333±0.023)明显升高(P＜0.05或P＜0.01)。TGF-β1中和抗体预处理组的TGF-β1蛋白表达水平较30 ng/ml糜酶组显著降低(P＜0.05或P＜0.01),但AT_1受体拮抗剂和AT_2受体拮抗剂预处理组的TGF-β1蛋白水平与糜酶组比较,均无明显差异(P＞0.05)。(9)不同浓度的糜酶作用6 h,Smad2和Smad3的mRNA表达水平与对照组比较,均无显著性差异(p＞0.05)。(10)在30 ng/mL糜酶作用下,3 h、6 h、12 h和24 h组p-Smad2/3表达水平均较对照组显著升高(P＜0.05或P＜0.01);Smad2/3蛋白表达水平与对照组比较均无显著性差异(P＞0.05)。3 h组Smad7蛋白表达水平较对照组显著升高(P＜0.05);6 h、12 h和24 h组Smad7蛋白水平均显著低于对照组(P＜0.05或P＜0.01)。(11)不同浓度的非诺贝特预处理后,CFs的数目呈递减趋势,其中50和100μmol/L组的A_(490)值均较糜酶组显著较少(P＜0.05或P＜0.01)。非诺贝特与其拮抗剂共同干预组的A_(490)值与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用对CFs的数目无明显影响(P＞0.05)。(12)不同浓度的非诺贝特预处理后,CFs的DNA合成呈浓度依赖性减少,其中50和100μmol/L组均较糜酶组明显减少(P＜0.01)。非诺贝特与其拮抗剂共同干预组的~3H-TdR掺入量与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用下,~3H-TdR掺入量无明显变化(P＞0.05)。(13)不同浓度的非诺贝特预处理后,CFs的G_0/G_1期细胞百分率呈递增趋势,S期细胞百分率和PI则呈递减趋势,其中50和100μmol/L组与糜酶组比较均有显著性差异(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用下,CFs的G_0/G_1期细胞百分率、S期细胞百分率和PI均无明显变化(P＞0.05)。(14) 10、50和100μmol/L非诺贝特预处理组CFs的总胶原合成呈浓度依赖性减少,其中50和100μmol/L组的~3H-脯氨酸掺入量均较糜酶组显著降低(P＜0.01)。非诺贝特与其拮抗剂共同干预组的~3H-脯氨酸掺入量与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用对~3H-脯氨酸掺入量无明显影响(P＞0.05)。(15)不同浓度的非诺贝特预处理后,CFs的Ⅰ、Ⅲ型胶原mRNA表达水平均呈递减趋势,其中50和100μmol/L组均较糜酶组显著减低(P＜0.01)。非诺贝特与其拮抗剂共同干预组的Ⅰ、Ⅲ型胶原mRNA表达水平与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用对Ⅰ、Ⅲ型胶原mRNA表达均无显著影响(P＞0.05)。(16)不同浓度的糜酶作用6 h,CFs的PPARαmRNA水平呈递减趋势,其中15、30和60 ng/ml组均较对照组明显减低(P＜0.01)。10、50和100μmol/L非诺贝特预处理后,CFs的PPARαmRNA表达水平呈浓度依赖性增加,其中50和100μmol/L组均较糜酶组显著增加(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用后,PPARαmRNA表达水平较对照组明显升高(P＜0.01)。(17)不同浓度的糜酶作用12 h,CFs的PPARα蛋白水平呈浓度依赖性下降,其中15、30和60 ng/ml组较对照组明显减少(P＜0.05或P＜0.01)。10、50和100μmol/L非诺贝特预处理后,CFs的PPARα蛋白表达水平呈递增趋势,其中50和100μmol/L组较糜酶组明显增加(P＜0.01)。100μmol/L非诺贝特单独作用组的PPARα蛋白表达水平较对照组显著升高(P＜0.01)。(18)不同浓度的非诺贝特预处理后,CFs的TGF-β1 mRNA表达水平呈递减趋势,其中50和100μmol/L组均较糜酶组明显减少(P＜0.01)。非诺贝特与其拮抗剂共同干预组的TGF-β1 mRNA水平与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用下,TGF-β1 mRNA水平无明显变化(P＞0.05)。(19)10、50和100μmol/L非诺贝特预处理组CFs的TGF-β1蛋白表达水平呈浓度依赖性减少,其中50和100μmol/L组均较糜酶组显著减少(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用对TGF-β1蛋白水平为无显著影响(P＞0.05)。(20)不同浓度的糜酶作用12h,CFs的Smad2/3蛋白表达水平与对照组比较均无明显差异(P＞0.05)。不同浓度的非诺贝特预处理后,Smad2/3蛋白表达水平亦无明显变化(P＞0.05)。(21) 15、30和60 ng/ml糜酶作用6h,CFs的p-Smad2/3蛋白表达水平均较对照组明显升高(P＜0.05或P＜0.01)。不同浓度的非诺贝特预处理后,p-Smad2/3蛋白表达水平呈递减趋势,其中50和100μmol/L组均较糜酶组显著下降(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用下,p-Smad2/3的蛋白表达水平无明显变化(P＞0.05)。(22) 15、30和60 ng/ml糜酶作用6 h,Smad7 mRNA表达水平均较对照组显著减少(P＜0.05或P＜0.01),不同浓度的非诺贝特预处理后,CFs的Smad7 mRNA表达水平呈浓度依赖性增加,其中50和100μmol/L组均较糜酶组明显增加(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用对Smad7 mRNA表达水平无显著影响(P＞0.05)。(23) 15、30和60 ng/ml糜酶作用12 h,CFs的Smad7蛋白表达水平均较对照组明显降低(P＜0.01)。不同浓度的非诺贝特干预后,Smad7蛋白表达水平呈浓度依赖性升高,其中50和100μmol/L组均较糜酶组明显升高(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用下,Smad7蛋白表达水平无明显变化(P＞0.05)。
研究结论(1)糜酶以浓度依赖的方式促进SD大鼠CFs增殖和胶原合成,提示心脏糜酶可能在心肌纤维化的发生、发展中具有重要的作用。(2)糜酶可上调TGF-β1表达,同时促进Smad2/3的磷酸化、下调Smad7表达,说明糜酶可激活TGF-β1/Smads信号通路。(3) TGF-β1中和抗体及丝/苏氨酸激酶抑制剂可抑制糜酶介导的CFs增殖和胶原合成作用,但血管紧张素Ⅱ受体阻滞剂(ARB)对糜酶诱导的CFs增殖、胶原合成及TGF-β1产生均无明显影响。表明TGF-β1/Smads信号通路参与糜酶致心肌纤维化作用,并且该作用与AngⅡ途径无关。(4) PPARα激动剂非诺贝特以浓度依赖的方式抑制糜酶诱导的大鼠CFs增殖和胶原合成,提示非诺贝特可能具有逆转心肌纤维化的作用。(5)非诺贝特通过激活PPARα抑制TGF-β1的生成;同时下调p-Smad2/3表达、上调Smad7表达。这可能是非诺贝特抑制CFs增殖和胶原合成,从而逆转心肌纤维化的分子生物学机制之一。(6) PPARα途径和TGF-β1/Smads信号通路可能存在信息交流:即活化的PPARα可下调TGF-β1的表达,进而抑制Smad2/3的磷酸化,并上调Smad7的表达,从而抑制TGF-β1的信号传导,对TGF-β1/Smads通路发挥负调控作用。
综上所述,不难看出心脏糜酶可诱导CFs增殖和胶原合成,因而具有促进心肌纤维化的作用,该作用的细胞内信号转导机制与TGF-β1表达上调、Smad2/3磷酸化增加以及Smad7表达下调有关。PPARα激动剂非诺贝特可抑制糜酶介导的心肌纤维化反应,从而发挥其调脂以外的心血管保护效应,其分子机制可能是通过激活PPARα抑制TGF-β1生成,从而下调p-Smad2/3表达并上调Smad7表达。因此,非诺贝特可能对高血压心肌纤维化发挥负调控作用,本研究结果有望为高血压左心室重构的发生提供理论依据和新的治疗靶点。
Background and objective Myocardial fibrosis is not only one of the important pathological bases of left ventricular remodeling induced by essential hypertension but also the main cause of cardiac diastolic dysfunction.Moreover, myocardial fibrosis is regarded as a major determinant leading to cardiac functional transition from compensation to decompensation.Therefore,it is very important to prevent and regress myocardial fibrosis,and to improve cardiac function for target organic protection of hypertension.A significant body of literature demonstrates that cardiac mast cell-derived chymase is involved in the cardivascular pathologic remodeling,which has been found to play a crucial role in the progression of many cardiovascular diseases,such as hypertension, myocardial infarction,cardiomyopathy,congestive heart failure,restenosis after percutaneous transluminal coronary angioplasty,atherosclerosis,aneurysm,and so on.Recent studies suggest that chymase could inhibit proliferation of vascular smooth muscle cells and elicit hypertrophic response of cardiac myocytes.Cardiac fibroblasts(CFs) have long been considered as major effectors of myocardial fibrosis.The excessive proliferation of CFs and enhanced collagen synthesis are thus regarded as pathological foundation of myocardial fibrosis.However,it remains unknown whether chymase can exert effects on CFs proliferation and collagen synthesis,and the underlying mechanisms of signal transduction are also obscure.Although it has been reported that chymase can activate latent transforming growth factor-β1 (TGF-β1) recently,the relative role of chymase-mediated TGF-β1 forming system in CFs proliferation has not been well established.In the last few years,a major advance in understanding TGF-β1 post-receptor signaling is the identification of Smad proteins,which may specifically modulate the transcriptions of TGF-β1 target genes.It has been well documented that the activation of TGF-β1/Smads signaling pathway may participate in the fibrotic progression of many organs,including liver,lung,kidney,peritoneum and skin. Little has been known about whether TGF-β1/Smads signaling pathway could contribute to myocardial fibrosis caused by hypertension.Peroxisome proliferator-activated receptors(PPARs) are ligand-dependent nuclear receptors, including PPARα,PPARβ/δand PPARγ,.Over the past few years,extensive studies have been focused on the protective effects of PPARγ,and its agonist, thiazolidinediones,on cardiovascular tissue which could be compromised in metabolic syndrome.In contrast,the knowledge about the possible role of PPARαin the prevention of cardiovascular disease is rather sparse.Recent evidence demonstrates that PPARαis highly expressed in myocardial tissue and exert an inhibitory effect on myocardial hypertrophy,indicating that further insight into the PPARαpathway may have important therapeutic implications on hypertensive left ventricular hypertrophy.It has been confirmed that fibrates,a sort of lipid-lowering drugs,have pronounced therapeutic effects on essential hypertriglyceridemia.Since being identified as PPARαactivator,fenofibrate has attracted a great deal of attention with regard to its beneficial effect on cardiovascular system beyond its contribution to lipid metabolism.In vitro, fenofibrate could inhibit the hypertrophy of cultured cardiac myocytes induced by many proinflammatory mediators and cytokines.However,whether fenofibrate can inhibit CFs proliferation and collagen synthesis,and the signaling pathway involved remain unclear so far.Currently,it has been observed that PPARγagonist can down-regulate gene expression of TGF-β1 and thus arrest the progression of renal interstitium fibrosis.The underlying mechanism may be related to the blockade of TGF-β1/Smad signaling pathway. However,it remains unclear whether TGF-β1/Smads and PPARαsignaling pathway participate in the chymase-induced myocardial fibrosis modulated by fenofibrate and whether there exist a cross-talk between the two pathways.This study was therefore designed to observe the effect of mast cell-derived chymase on cell proliferation and collagen synthesis of cultured CFs at cellular and molecular level,and to discuss the mechanisms of its effect on myocardial fibrosis.Moreover,we also investigated the role of PPARαand its agonist fenofibrate on TGF-β1/Smads signaling pathway activited by chymase in order to elucidate the mechanisms of preventive and therapeutic effects of fenofibrate on hypertensive myocardial fibrosis.Accordingly,the study was attempted to provide noval theoretical evidence and new strategy for the clinical treatment on hypertensive left ventricular hypertrophy.
Methods In this study,cultured CFs of neonatal SD rats were used as experimental models.Moreover,MTT assay,radionuclide incorporation method, flow cytometry technique,ELISA,RT-PCR and Western blot were applied to identify:(1) the effects of chymase on cell proliferation and collagen synthesis of cultured CFs in SD rats;(2) the effects of chymase on the mRNA and proteinic expression of TGF-β1,Smad2,Smad3 and Smad7 in CFs;(3) the effects of fenofibrate,a PPARαagonist,on CFs proliferation and collagen synthesis induced by chymase;(4) the intervention effects of fenofibrate on the mRNA and proteinic expression of PPARα,TGF-β1,and the proteinic expression of Smad2/3,p-Smad2/3 and Smad7 in CFs.
Results(1) Treatment with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 24 h significantly increased CFs number in a dose-dependent manner,and the A490 Value(0.263±0.033,0.348±0.031,and 0.387±0.026,respectively) were all significantly higher than that of control(0.201±0.019,P＜0.01).The A_(490) Value in 30 ng/ml chymase-treated CFs was markedly decreased by pretreatment with TGF-β1 neutralizing antibody or serine/threonine kinase inhibitor(P＜0.05 or P＜0.01).Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly altered the A_(490) Value of chymase-treated CFs(P＞0.05).(2) Administration with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 24 h significantly increased DNA synthesis in a dose-dependent manner,and the ~3H-TdR incorporation (319±29 cpm/well,372±43 cpm/well,and 401±47 cpm/well,respectively) were all remarkably higher than that of control(252±35 cpm/well,P＜0.01).The ~3H-TdR incorporation in 10μmol/L chymase inhibitor-pretreated group was notablely lower than that in 30 ng/ml chymase group(P＜0.01).(3) Cell cycle analysis revealed that treatment with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly decreased the percentages of cells in G_0/G_1 phase of the cell cycle(P＜0.05 or P＜0.01),and increased the percentages of cells in S phase and the proliferation index(PI) when compared with that of control(P＜0.05 or P＜0.01).Whereas The percentages of cells in G_2/M phase were not significantly changed by chymase(P＞0.05).Pretreatment with 10μmol/L chymase inhibitor markedly increased G_0/G_1 phase percentage(P＜0.01),and decreased S phase percentage and PI when compared with that of 30 ng/ml chymase group (P＜0.01).But pretreatment with 10μmol/L chymase inhibitor had no effect on G_2/M phase percentage when compared with chymase group or control group (P＞0.05).(4) Administration with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly increased total collagen synthesis in a dose-dependent manner,and the ~3H-Proline incorporation(520±75cpm/well,684±62 cpm/well and 769±58 cpm/well,respectively) were all remarkably higher than that of control(435±60 cpm/well,P＜0.05 or P＜0.01).The ~3H-proline incorporation in TGF-β1 neutralizing antibody or serine/threonine kinase inhibitor-pretreated group was significantly lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01).Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly changed the ~3H-proline incorporation of chymase-treated cells(P＞0.05).(5) Treatment with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h markedly increased the mRNA levels of collagen Type-Ⅰand -Ⅲin a dose-dependent manner when compared with control(P＜0.01),whereas chymase at 7.5 ng/ml had no significant effect on the mRNA expression of collagen Type-Ⅰand -Ⅲ(P＞0.05).(6) Administration with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly increased the protein content of collagen Type-Ⅰand -Ⅲin cultured supematant of CFs when compared with control (P＜0.01),whereas chymase at 7.5 ng/ml had no marked effect on the protein content of collagen Type-Ⅰand -Ⅲ(P＞0.05).(7) Treatment with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 3 h,the mRNA levels of TGF-β1 were 0.698±0.051,1.096±0.078,and 1.242±0.065,respectively,which were all notably higher than that of control(0.299±0.035,P＜0.01).The mRNA expression of TGF-β1 in TGF-β1 neutralizing antibody-pretreated group was remarkably lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01). Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly modified the mRNA expression of TGF-β1 induced by chymase(P＞0.05).(8) Administration with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 6 h,the protein levels of TGF-β1 were 0.968±0.069,1.782±0.058 and 2.656±0.085,respectively,which were all significantly higher than that of control(0.333±0.023,P＜0.05 or P＜0.01).The protein expression of TGF-β1 in TGF-β1 neutralizing antibody-pretreated group was markedly lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01).Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly altered the protein expression of TGF-β1 mediated by chymase(P＞0.05).(9) Treatment with chymase at different concentrations for 6 h had no significant effects on the mRNA expression of Smad2 and Smad3 (P＞0.05).(10) Administration with chymase at 30 ng/mL for 3 h,6 h,12 h and 24 h significantly increased the p-Smad2/3 when compared with control(P＜0.05 or P＜0.01),while the protein expressionof Smad2/3 was not notably altered (P＞0.05).The protein expression of Smad7 in 3 h group was higher than that in control group(P＜0.05).However,treatment with chymase for 6 h,12 h,and 24 h markedly decreased the protein expression of Smad7 when compared with control(P＜0.05 or P＜0.01).(11) Pretreatment with fenofibrate at different concentrations decreased the number of CFs in a concentration-dependent manner,and the A49o Value in 50μmol/L group and 100μmol/L group was significantly lower than that in chymase group(P＜0.05 or P＜0.01).The A_(490) Value was no significant difference between the chymase group and the co-pretreated with fenofibrate and its antagonist group(P＞0.05).100μmol/L Fenofbrate alone had no significant effect on the number of CFs(P＞0.05).(12) Pretreatment with fenofibrate at different concentrations decreased the DNA synthesis of CFs in a concentration-dependent manner,and the ~3H-TdR incorporation in 50μmol/L group and 100μmol/Lgroup was markedly lower than that in chymase group(P＜0.01).The ~3H-TdR incorporation was not changed by fenofibrate and its antagonist co-pretreatment when compared with that of chymase group(P＞0.05).100μmol/L fenofibrate alone had no significant effect on ~3H-TdR incorporation into CFs(P＞0.05).(13) Pretreatment with fenofibrate at different concentrations increased the percentages of cells in G_0/G_1 phase,and decreased those in S phase and the PI,which in 50μmol/L group and 100μmol/L group had signifcant difference when compared with that in chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no significant effect on PI,the percentages of cells in G_0/G_1 phase or S phase (P＞0.05).(14) Pretreatment with fenofibrate at 10μmoFL,50μmol/L and 100μmol/L remarkably decreased the ~3H-proline incorporation in a concentration-dependent manner,and the ~3H-proline incorporation at 50μmol/L and 100μmol/L was significantly lower than that of chymase group(P＜0.01). The ~3H-proline incorporation had no significant change in fenofibrate and its inhibitor-pretreated group when compared with that in chymase group(P＞0.05). 100μmol/L fenofibrate alone had no apparente effect on ~3H-proline incorporation into CFs(P＞0.05).(15) Pretreatment with fenofibrate at different concentrations decreased the mRNA expression of collagen Type-Ⅰand -Ⅲin a concentration-dependent manner,and the mRNA levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.01).The mRNA expression of collagen Type-Ⅰand -Ⅲhad no significant change in fenofibrate and its inhibitor-pretreated group when compared with that in chymase group(P＞0.05).100μmol/L fenofibrate alone had no marked effect on the mRNA expression of collagenⅠandⅢ(P＞0.05).(16) Administration with chymase at different concentrations for 6 h lowered the mRNA expression of PPARα,and the expression levels at 15 ng/ml,30 ng/ml and 60 ng/ml were significantly lower than that of control(P＜0.01).Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L increased mRNA expression of PPARαin a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group (P＜0.05 or P＜0.01).100μmol/L fenofibrate alone notably elevated the mRNA expression of PPARαwhen compared with control(P＜0.01).(17) Treatment with chymase at different concentrations for 12 h decreased the protein expression of PPARα,and the expression levels at 15 ng/ml,30 ng/ml and 60 ng/ml were significantly lower than that of control(P＜0.05 or P＜0.01). Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L up-regulated the protein expression of PPARαin a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.01).100μmol/L fenofibrate alone remarkably elevated the protein expression of PPARαwhen compared with control(P＜0.01).(18) Pretreatment with fenofibrate at different concentrations down-regulated the mRNA expression of TGF-β1,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.01).The expression level was not altered in fenofibrate and its antagonist co-pretreated group when compared with that in chymase group(P＞0.05).100μmol/L fenofibrate alone had no significant effect on the mRNA expression of TGF-β1(P＞0.05).(19) Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L down-regulated the protein expression of TGF-β1,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no marked effect on the protein expression of TGF-β1(P＞0.05).(20) Administration with chymase at different concentrations for 12 h had no significant effect on the protein expression of Smad2/3(P＞0.05) No changes of Smad2/3 in protein expression was observed when CFs were pretreated with different concentrations of fenofibrate(P＞0.05).(21) Treatment with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 6 h up-regulated the protein expression of p-Smad2/3 when compared with control(P＜0.05 or P＜0.01).Pretreatment with fenofibrate at different concentrations down-regulated the protein expression of p-Smad2/3,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no notable effect on the protein expression of p-Smad2/3(P＞0.05).(22) Administration with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 6 h down-regulated the mRNA expression of Smad7 when compared with control(P＜0.05 or P＜0.01). Pretreatment with fenofibrate at different concentrations up-regulated the mRNA expression of Smad7 in a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no significant effect on the mRNA expression of Smad7(P＞0.05).(23) Treatment with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 12 h down-regulated the protein expression of Smad7 when compared with control(P＜0.01). Pretreatment with fenofibrate at different concentrations up-regulated the protein expression of Smad7 in a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no remarkable effect on the protein expression of Smad7(P＞0.05).
Conclusion(1) Cardiac mast cell-derived chymase can promote cell proliferation and collagen synthesis of CFs in a dose-dependent manner, indicating that cardiac chymase may play a significant role in the formation and progression of myocardial fibrosis.(2) Chymase can up-regulate TGF-β1 expression,promote Smad2/3 phosphorylation and down-regulate Smad7 expression,suggesting that chymase can activate TGF-β1/Smads signaling pathway.(3) TGF-β1 neutralizing antibody and serine/threonine kinase inhibitor exert significant inhibitory effects on cell proliferation,collagen synthesis and TGF-β1 expression caused by chymase while angiotensinⅡreceptor blocker (ARB) valsartan and PD123319 fail to exhibit similar changes.The result further indicates that TGF-β1/Smads pathway is involved in myocardial fibrosis caused by chymase.(4) Fenofibrate,a PPARαagonist,can suppress cell proliferation and collagen synthesis of CFs induced by chymase.Thus,it may exert reverse effect on myocardial fibrosis.(5) Fenofibrate can not only activate PPARαand restrain TGF-β1 production,but also down-regulate p-Smad2/3 expression and up-regulate Smad7 expression.This may be one of the molecular and biological mechanisms involved in the inhibitory effects of fenofibrate on cell proliferation,collagen synthesis of CFs and then result in the regression of myocardial fibrosis.(6) There may exist cross-talk between PPARαand TGF-β1/Smads pathway.That is to say,activated PPARαcan exert negative modulation on TGF-β1/Smads pathway by down-regulating TGF-β1 expression, inhibiting Smad2/3 phosphorylation and up-regulating Smad7 expression.
In a summary,cardiac mast cell-derived chymase can induce cell proliferation and collagen synthesis of CFs and therefore,it can promote myocardial fibrosis.Up-regulation of TGF-β1 expression,phosphorylation of Smad2/3 and down-regulation of Smad7 expression are considered to be one of intra-cellular signal transduction mechariisms.Fenofibrate,a PPARαagonist, can reverse myocardial fibrotic response and thus exert protective effects on cardiovascular system apart from its effect on lipid metabolism.It may be one of the fenofibrate-mediated potential mechanisms that suppression of TGF-β1 expression caused by activating PPARαmay result in down-regulation of Smad2/3 phosphorylation and up-regulation of Smad7 expression.Therefore, this study may provide theoretical evidence and a novel therapeutic target for left ventricular hypertrophy caused by hypertension.
研究方法本研究以体外培养的SD大鼠CFs为研究对象,采用MTT比色法、放射性核素掺入实验、流式细胞仪细胞周期分析、ELISA、RT-PCR及蛋白免疫印记等方法和技术,观察:(1)糜酶对大鼠CFs增殖和胶原合成的影响;(2)糜酶对大鼠CFs的TGF-β1、Smad2、Smad3、Smad7 mRNA和蛋白表达的影响;(3) PPARα激动剂非诺贝特对糜酶诱导的大鼠CFs增殖和胶原合成的影响:(4)非诺贝特干预对大鼠CFs的PPARα、TGF-β1mRNA和蛋白表达以及Smad2/3、p-Smad2/3、Smad7蛋白表达的影响。
研究结果(1)不同浓度的糜酶作用24 h,CFs的数目呈浓度依赖性增加,15、30和60 ng/ml组A_(490)值分别为0.263±0.033、0.348±0.031和0.387±0.026,均较对照组(0.201±0.019)显著增加(P＜0.01)。TGF-β1中和抗体预处理组和丝/苏氨酸激酶抑制剂预处理组的A_(490)值均明显低于30 ng/ml糜酶组(P＜0.05或P＜0.01),AT_1受体拮抗剂预处理组和AT_2受体拮抗剂预处理组的A_(490)值与糜酶组比较,均无显著性差异(P＞0.05)。(2) CFs的DNA合成随糜酶浓度的增加而增多,15、30和60 ng/ml组~3H-TdR掺入量分别为319±29、372±43和401±47(cpm/孔),较对照组(252±35 cpm/孔)明显升高(P＜0.01)。10μmol/L糜酶抑制剂预处理组的~3H-TdR掺入量显著低于30ng/ml糜酶组(P＜0.01)。(3)细胞周期分析结果表明,15、30和60 ng/ml糜酶作用24 h,CFs的G_0/G_1期细胞百分率均较对照组显著降低(P＜0.05或P＜0.01),S期细胞百分率和增殖指数(PI)则较对照组明显增高(P＜0.05或P＜0.01),G_2/M期细胞百分率与对照组比较无显著性差异(P＞0.05)。10μmol/L糜酶抑制剂预处理组G_0/G_1期细胞百分率显著高于30 ng/ml糜酶组(P＜0.01),S期细胞百分率和PI明显低于糜酶组(P＜0.01),G_2/M期细胞百分率与糜酶组及照组比较均无明显差异(P＞0.05)。(4)随着糜酶浓度的增高,CFs总胶原合成呈递增趋势,15、30和60 ng/ml组~3H-脯氨酸掺入量分别为520±75、684±62和769±58(cpm/孔),均较对照组(435±60 cpm/孔)显著增加(P＜0.05或P＜0.01)。TGF-β1中和抗体预处理组和丝/苏氨酸激酶抑制剂预处理组的~3H-脯氨酸掺入量均显著低于30 ng/ml糜酶组(P＜0.05或P＜0.01);AT_1受体拮抗剂预处理组和AT_2受体拮抗剂预处理组的~3H-脯氨酸掺入量与糜酶组比较无显著性差异(P＞0.05)。(5)不同浓度的糜酶作用24 h,Ⅰ、Ⅲ型胶原mRNA表达水平呈浓度依赖性增加,其中15、30和60 ng/ml组Ⅰ型和Ⅲ型胶原mRNA表达水平均较对照组显著升高(P＜0.01),但7.5 ng/ml组与对照组比较无显著性差异(P＞0.05)。(6)随着糜酶浓度的增高,CFs培养上清中Ⅰ、Ⅲ型胶原蛋白含量呈递增趋势,其中15、30和60 ng/ml组Ⅰ型和Ⅲ型胶原蛋白含量均较对照组明显增加(P＜0.05或P＜0.01),但7.5ng/ml组与对照组比较无显著性差异(P＞0.05)。(7) 15、30和60 ng/ml糜酶作用3h,TGF-β1 mRNA表达水平分别为0.698±0.051、1.096±0.078和1.242±0.065,均较对照组(0.299±0.035)明显增加(P＜0.01)。TGF-β1中和抗体预处理组的TGF-β1 mRNA表达水平明显低于30 ng/ml糜酶组(P＜0.05或P＜0.01),但AT_1受体拮抗剂和AT_2受体拮抗剂预处理组的TGF-β1 mRNA水平与糜酶组比较,均无显著性差异(P＞0.05)。(8) 15、30和60 ng/ml糜酶作用6 h,TGF-β1蛋白表达水平分别为0.968±0.069、1.782±0.058和2.656±0.085,均较对照组(0.333±0.023)明显升高(P＜0.05或P＜0.01)。TGF-β1中和抗体预处理组的TGF-β1蛋白表达水平较30 ng/ml糜酶组显著降低(P＜0.05或P＜0.01),但AT_1受体拮抗剂和AT_2受体拮抗剂预处理组的TGF-β1蛋白水平与糜酶组比较,均无明显差异(P＞0.05)。(9)不同浓度的糜酶作用6 h,Smad2和Smad3的mRNA表达水平与对照组比较,均无显著性差异(p＞0.05)。(10)在30 ng/mL糜酶作用下,3 h、6 h、12 h和24 h组p-Smad2/3表达水平均较对照组显著升高(P＜0.05或P＜0.01);Smad2/3蛋白表达水平与对照组比较均无显著性差异(P＞0.05)。3 h组Smad7蛋白表达水平较对照组显著升高(P＜0.05);6 h、12 h和24 h组Smad7蛋白水平均显著低于对照组(P＜0.05或P＜0.01)。(11)不同浓度的非诺贝特预处理后,CFs的数目呈递减趋势,其中50和100μmol/L组的A_(490)值均较糜酶组显著较少(P＜0.05或P＜0.01)。非诺贝特与其拮抗剂共同干预组的A_(490)值与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用对CFs的数目无明显影响(P＞0.05)。(12)不同浓度的非诺贝特预处理后,CFs的DNA合成呈浓度依赖性减少,其中50和100μmol/L组均较糜酶组明显减少(P＜0.01)。非诺贝特与其拮抗剂共同干预组的~3H-TdR掺入量与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用下,~3H-TdR掺入量无明显变化(P＞0.05)。(13)不同浓度的非诺贝特预处理后,CFs的G_0/G_1期细胞百分率呈递增趋势,S期细胞百分率和PI则呈递减趋势,其中50和100μmol/L组与糜酶组比较均有显著性差异(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用下,CFs的G_0/G_1期细胞百分率、S期细胞百分率和PI均无明显变化(P＞0.05)。(14) 10、50和100μmol/L非诺贝特预处理组CFs的总胶原合成呈浓度依赖性减少,其中50和100μmol/L组的~3H-脯氨酸掺入量均较糜酶组显著降低(P＜0.01)。非诺贝特与其拮抗剂共同干预组的~3H-脯氨酸掺入量与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用对~3H-脯氨酸掺入量无明显影响(P＞0.05)。(15)不同浓度的非诺贝特预处理后,CFs的Ⅰ、Ⅲ型胶原mRNA表达水平均呈递减趋势,其中50和100μmol/L组均较糜酶组显著减低(P＜0.01)。非诺贝特与其拮抗剂共同干预组的Ⅰ、Ⅲ型胶原mRNA表达水平与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用对Ⅰ、Ⅲ型胶原mRNA表达均无显著影响(P＞0.05)。(16)不同浓度的糜酶作用6 h,CFs的PPARαmRNA水平呈递减趋势,其中15、30和60 ng/ml组均较对照组明显减低(P＜0.01)。10、50和100μmol/L非诺贝特预处理后,CFs的PPARαmRNA表达水平呈浓度依赖性增加,其中50和100μmol/L组均较糜酶组显著增加(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用后,PPARαmRNA表达水平较对照组明显升高(P＜0.01)。(17)不同浓度的糜酶作用12 h,CFs的PPARα蛋白水平呈浓度依赖性下降,其中15、30和60 ng/ml组较对照组明显减少(P＜0.05或P＜0.01)。10、50和100μmol/L非诺贝特预处理后,CFs的PPARα蛋白表达水平呈递增趋势,其中50和100μmol/L组较糜酶组明显增加(P＜0.01)。100μmol/L非诺贝特单独作用组的PPARα蛋白表达水平较对照组显著升高(P＜0.01)。(18)不同浓度的非诺贝特预处理后,CFs的TGF-β1 mRNA表达水平呈递减趋势,其中50和100μmol/L组均较糜酶组明显减少(P＜0.01)。非诺贝特与其拮抗剂共同干预组的TGF-β1 mRNA水平与糜酶组比较无显著性差异(P＞0.05)。100μmol/L非诺贝特单独作用下,TGF-β1 mRNA水平无明显变化(P＞0.05)。(19)10、50和100μmol/L非诺贝特预处理组CFs的TGF-β1蛋白表达水平呈浓度依赖性减少,其中50和100μmol/L组均较糜酶组显著减少(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用对TGF-β1蛋白水平为无显著影响(P＞0.05)。(20)不同浓度的糜酶作用12h,CFs的Smad2/3蛋白表达水平与对照组比较均无明显差异(P＞0.05)。不同浓度的非诺贝特预处理后,Smad2/3蛋白表达水平亦无明显变化(P＞0.05)。(21) 15、30和60 ng/ml糜酶作用6h,CFs的p-Smad2/3蛋白表达水平均较对照组明显升高(P＜0.05或P＜0.01)。不同浓度的非诺贝特预处理后,p-Smad2/3蛋白表达水平呈递减趋势,其中50和100μmol/L组均较糜酶组显著下降(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用下,p-Smad2/3的蛋白表达水平无明显变化(P＞0.05)。(22) 15、30和60 ng/ml糜酶作用6 h,Smad7 mRNA表达水平均较对照组显著减少(P＜0.05或P＜0.01),不同浓度的非诺贝特预处理后,CFs的Smad7 mRNA表达水平呈浓度依赖性增加,其中50和100μmol/L组均较糜酶组明显增加(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用对Smad7 mRNA表达水平无显著影响(P＞0.05)。(23) 15、30和60 ng/ml糜酶作用12 h,CFs的Smad7蛋白表达水平均较对照组明显降低(P＜0.01)。不同浓度的非诺贝特干预后,Smad7蛋白表达水平呈浓度依赖性升高,其中50和100μmol/L组均较糜酶组明显升高(P＜0.05或P＜0.01)。100μmol/L非诺贝特单独作用下,Smad7蛋白表达水平无明显变化(P＞0.05)。
研究结论(1)糜酶以浓度依赖的方式促进SD大鼠CFs增殖和胶原合成,提示心脏糜酶可能在心肌纤维化的发生、发展中具有重要的作用。(2)糜酶可上调TGF-β1表达,同时促进Smad2/3的磷酸化、下调Smad7表达,说明糜酶可激活TGF-β1/Smads信号通路。(3) TGF-β1中和抗体及丝/苏氨酸激酶抑制剂可抑制糜酶介导的CFs增殖和胶原合成作用,但血管紧张素Ⅱ受体阻滞剂(ARB)对糜酶诱导的CFs增殖、胶原合成及TGF-β1产生均无明显影响。表明TGF-β1/Smads信号通路参与糜酶致心肌纤维化作用,并且该作用与AngⅡ途径无关。(4) PPARα激动剂非诺贝特以浓度依赖的方式抑制糜酶诱导的大鼠CFs增殖和胶原合成,提示非诺贝特可能具有逆转心肌纤维化的作用。(5)非诺贝特通过激活PPARα抑制TGF-β1的生成;同时下调p-Smad2/3表达、上调Smad7表达。这可能是非诺贝特抑制CFs增殖和胶原合成,从而逆转心肌纤维化的分子生物学机制之一。(6) PPARα途径和TGF-β1/Smads信号通路可能存在信息交流:即活化的PPARα可下调TGF-β1的表达,进而抑制Smad2/3的磷酸化,并上调Smad7的表达,从而抑制TGF-β1的信号传导,对TGF-β1/Smads通路发挥负调控作用。
综上所述,不难看出心脏糜酶可诱导CFs增殖和胶原合成,因而具有促进心肌纤维化的作用,该作用的细胞内信号转导机制与TGF-β1表达上调、Smad2/3磷酸化增加以及Smad7表达下调有关。PPARα激动剂非诺贝特可抑制糜酶介导的心肌纤维化反应,从而发挥其调脂以外的心血管保护效应,其分子机制可能是通过激活PPARα抑制TGF-β1生成,从而下调p-Smad2/3表达并上调Smad7表达。因此,非诺贝特可能对高血压心肌纤维化发挥负调控作用,本研究结果有望为高血压左心室重构的发生提供理论依据和新的治疗靶点。
Background and objective Myocardial fibrosis is not only one of the important pathological bases of left ventricular remodeling induced by essential hypertension but also the main cause of cardiac diastolic dysfunction.Moreover, myocardial fibrosis is regarded as a major determinant leading to cardiac functional transition from compensation to decompensation.Therefore,it is very important to prevent and regress myocardial fibrosis,and to improve cardiac function for target organic protection of hypertension.A significant body of literature demonstrates that cardiac mast cell-derived chymase is involved in the cardivascular pathologic remodeling,which has been found to play a crucial role in the progression of many cardiovascular diseases,such as hypertension, myocardial infarction,cardiomyopathy,congestive heart failure,restenosis after percutaneous transluminal coronary angioplasty,atherosclerosis,aneurysm,and so on.Recent studies suggest that chymase could inhibit proliferation of vascular smooth muscle cells and elicit hypertrophic response of cardiac myocytes.Cardiac fibroblasts(CFs) have long been considered as major effectors of myocardial fibrosis.The excessive proliferation of CFs and enhanced collagen synthesis are thus regarded as pathological foundation of myocardial fibrosis.However,it remains unknown whether chymase can exert effects on CFs proliferation and collagen synthesis,and the underlying mechanisms of signal transduction are also obscure.Although it has been reported that chymase can activate latent transforming growth factor-β1 (TGF-β1) recently,the relative role of chymase-mediated TGF-β1 forming system in CFs proliferation has not been well established.In the last few years,a major advance in understanding TGF-β1 post-receptor signaling is the identification of Smad proteins,which may specifically modulate the transcriptions of TGF-β1 target genes.It has been well documented that the activation of TGF-β1/Smads signaling pathway may participate in the fibrotic progression of many organs,including liver,lung,kidney,peritoneum and skin. Little has been known about whether TGF-β1/Smads signaling pathway could contribute to myocardial fibrosis caused by hypertension.Peroxisome proliferator-activated receptors(PPARs) are ligand-dependent nuclear receptors, including PPARα,PPARβ/δand PPARγ,.Over the past few years,extensive studies have been focused on the protective effects of PPARγ,and its agonist, thiazolidinediones,on cardiovascular tissue which could be compromised in metabolic syndrome.In contrast,the knowledge about the possible role of PPARαin the prevention of cardiovascular disease is rather sparse.Recent evidence demonstrates that PPARαis highly expressed in myocardial tissue and exert an inhibitory effect on myocardial hypertrophy,indicating that further insight into the PPARαpathway may have important therapeutic implications on hypertensive left ventricular hypertrophy.It has been confirmed that fibrates,a sort of lipid-lowering drugs,have pronounced therapeutic effects on essential hypertriglyceridemia.Since being identified as PPARαactivator,fenofibrate has attracted a great deal of attention with regard to its beneficial effect on cardiovascular system beyond its contribution to lipid metabolism.In vitro, fenofibrate could inhibit the hypertrophy of cultured cardiac myocytes induced by many proinflammatory mediators and cytokines.However,whether fenofibrate can inhibit CFs proliferation and collagen synthesis,and the signaling pathway involved remain unclear so far.Currently,it has been observed that PPARγagonist can down-regulate gene expression of TGF-β1 and thus arrest the progression of renal interstitium fibrosis.The underlying mechanism may be related to the blockade of TGF-β1/Smad signaling pathway. However,it remains unclear whether TGF-β1/Smads and PPARαsignaling pathway participate in the chymase-induced myocardial fibrosis modulated by fenofibrate and whether there exist a cross-talk between the two pathways.This study was therefore designed to observe the effect of mast cell-derived chymase on cell proliferation and collagen synthesis of cultured CFs at cellular and molecular level,and to discuss the mechanisms of its effect on myocardial fibrosis.Moreover,we also investigated the role of PPARαand its agonist fenofibrate on TGF-β1/Smads signaling pathway activited by chymase in order to elucidate the mechanisms of preventive and therapeutic effects of fenofibrate on hypertensive myocardial fibrosis.Accordingly,the study was attempted to provide noval theoretical evidence and new strategy for the clinical treatment on hypertensive left ventricular hypertrophy.
Methods In this study,cultured CFs of neonatal SD rats were used as experimental models.Moreover,MTT assay,radionuclide incorporation method, flow cytometry technique,ELISA,RT-PCR and Western blot were applied to identify:(1) the effects of chymase on cell proliferation and collagen synthesis of cultured CFs in SD rats;(2) the effects of chymase on the mRNA and proteinic expression of TGF-β1,Smad2,Smad3 and Smad7 in CFs;(3) the effects of fenofibrate,a PPARαagonist,on CFs proliferation and collagen synthesis induced by chymase;(4) the intervention effects of fenofibrate on the mRNA and proteinic expression of PPARα,TGF-β1,and the proteinic expression of Smad2/3,p-Smad2/3 and Smad7 in CFs.
Results(1) Treatment with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 24 h significantly increased CFs number in a dose-dependent manner,and the A490 Value(0.263±0.033,0.348±0.031,and 0.387±0.026,respectively) were all significantly higher than that of control(0.201±0.019,P＜0.01).The A_(490) Value in 30 ng/ml chymase-treated CFs was markedly decreased by pretreatment with TGF-β1 neutralizing antibody or serine/threonine kinase inhibitor(P＜0.05 or P＜0.01).Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly altered the A_(490) Value of chymase-treated CFs(P＞0.05).(2) Administration with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 24 h significantly increased DNA synthesis in a dose-dependent manner,and the ~3H-TdR incorporation (319±29 cpm/well,372±43 cpm/well,and 401±47 cpm/well,respectively) were all remarkably higher than that of control(252±35 cpm/well,P＜0.01).The ~3H-TdR incorporation in 10μmol/L chymase inhibitor-pretreated group was notablely lower than that in 30 ng/ml chymase group(P＜0.01).(3) Cell cycle analysis revealed that treatment with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly decreased the percentages of cells in G_0/G_1 phase of the cell cycle(P＜0.05 or P＜0.01),and increased the percentages of cells in S phase and the proliferation index(PI) when compared with that of control(P＜0.05 or P＜0.01).Whereas The percentages of cells in G_2/M phase were not significantly changed by chymase(P＞0.05).Pretreatment with 10μmol/L chymase inhibitor markedly increased G_0/G_1 phase percentage(P＜0.01),and decreased S phase percentage and PI when compared with that of 30 ng/ml chymase group (P＜0.01).But pretreatment with 10μmol/L chymase inhibitor had no effect on G_2/M phase percentage when compared with chymase group or control group (P＞0.05).(4) Administration with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly increased total collagen synthesis in a dose-dependent manner,and the ~3H-Proline incorporation(520±75cpm/well,684±62 cpm/well and 769±58 cpm/well,respectively) were all remarkably higher than that of control(435±60 cpm/well,P＜0.05 or P＜0.01).The ~3H-proline incorporation in TGF-β1 neutralizing antibody or serine/threonine kinase inhibitor-pretreated group was significantly lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01).Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly changed the ~3H-proline incorporation of chymase-treated cells(P＞0.05).(5) Treatment with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h markedly increased the mRNA levels of collagen Type-Ⅰand -Ⅲin a dose-dependent manner when compared with control(P＜0.01),whereas chymase at 7.5 ng/ml had no significant effect on the mRNA expression of collagen Type-Ⅰand -Ⅲ(P＞0.05).(6) Administration with chymase(15 ng/ml,30 ng/ml and 60 ng/ml) for 24 h significantly increased the protein content of collagen Type-Ⅰand -Ⅲin cultured supematant of CFs when compared with control (P＜0.01),whereas chymase at 7.5 ng/ml had no marked effect on the protein content of collagen Type-Ⅰand -Ⅲ(P＞0.05).(7) Treatment with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 3 h,the mRNA levels of TGF-β1 were 0.698±0.051,1.096±0.078,and 1.242±0.065,respectively,which were all notably higher than that of control(0.299±0.035,P＜0.01).The mRNA expression of TGF-β1 in TGF-β1 neutralizing antibody-pretreated group was remarkably lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01). Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly modified the mRNA expression of TGF-β1 induced by chymase(P＞0.05).(8) Administration with 15 ng/ml,30 ng/ml and 60 ng/ml chymase for 6 h,the protein levels of TGF-β1 were 0.968±0.069,1.782±0.058 and 2.656±0.085,respectively,which were all significantly higher than that of control(0.333±0.023,P＜0.05 or P＜0.01).The protein expression of TGF-β1 in TGF-β1 neutralizing antibody-pretreated group was markedly lower than that in 30 ng/ml chymase group(P＜0.05 or P＜0.01).Neither AT_1 receptor blocker nor AT_2 receptor blocker significantly altered the protein expression of TGF-β1 mediated by chymase(P＞0.05).(9) Treatment with chymase at different concentrations for 6 h had no significant effects on the mRNA expression of Smad2 and Smad3 (P＞0.05).(10) Administration with chymase at 30 ng/mL for 3 h,6 h,12 h and 24 h significantly increased the p-Smad2/3 when compared with control(P＜0.05 or P＜0.01),while the protein expressionof Smad2/3 was not notably altered (P＞0.05).The protein expression of Smad7 in 3 h group was higher than that in control group(P＜0.05).However,treatment with chymase for 6 h,12 h,and 24 h markedly decreased the protein expression of Smad7 when compared with control(P＜0.05 or P＜0.01).(11) Pretreatment with fenofibrate at different concentrations decreased the number of CFs in a concentration-dependent manner,and the A49o Value in 50μmol/L group and 100μmol/L group was significantly lower than that in chymase group(P＜0.05 or P＜0.01).The A_(490) Value was no significant difference between the chymase group and the co-pretreated with fenofibrate and its antagonist group(P＞0.05).100μmol/L Fenofbrate alone had no significant effect on the number of CFs(P＞0.05).(12) Pretreatment with fenofibrate at different concentrations decreased the DNA synthesis of CFs in a concentration-dependent manner,and the ~3H-TdR incorporation in 50μmol/L group and 100μmol/Lgroup was markedly lower than that in chymase group(P＜0.01).The ~3H-TdR incorporation was not changed by fenofibrate and its antagonist co-pretreatment when compared with that of chymase group(P＞0.05).100μmol/L fenofibrate alone had no significant effect on ~3H-TdR incorporation into CFs(P＞0.05).(13) Pretreatment with fenofibrate at different concentrations increased the percentages of cells in G_0/G_1 phase,and decreased those in S phase and the PI,which in 50μmol/L group and 100μmol/L group had signifcant difference when compared with that in chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no significant effect on PI,the percentages of cells in G_0/G_1 phase or S phase (P＞0.05).(14) Pretreatment with fenofibrate at 10μmoFL,50μmol/L and 100μmol/L remarkably decreased the ~3H-proline incorporation in a concentration-dependent manner,and the ~3H-proline incorporation at 50μmol/L and 100μmol/L was significantly lower than that of chymase group(P＜0.01). The ~3H-proline incorporation had no significant change in fenofibrate and its inhibitor-pretreated group when compared with that in chymase group(P＞0.05). 100μmol/L fenofibrate alone had no apparente effect on ~3H-proline incorporation into CFs(P＞0.05).(15) Pretreatment with fenofibrate at different concentrations decreased the mRNA expression of collagen Type-Ⅰand -Ⅲin a concentration-dependent manner,and the mRNA levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.01).The mRNA expression of collagen Type-Ⅰand -Ⅲhad no significant change in fenofibrate and its inhibitor-pretreated group when compared with that in chymase group(P＞0.05).100μmol/L fenofibrate alone had no marked effect on the mRNA expression of collagenⅠandⅢ(P＞0.05).(16) Administration with chymase at different concentrations for 6 h lowered the mRNA expression of PPARα,and the expression levels at 15 ng/ml,30 ng/ml and 60 ng/ml were significantly lower than that of control(P＜0.01).Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L increased mRNA expression of PPARαin a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group (P＜0.05 or P＜0.01).100μmol/L fenofibrate alone notably elevated the mRNA expression of PPARαwhen compared with control(P＜0.01).(17) Treatment with chymase at different concentrations for 12 h decreased the protein expression of PPARα,and the expression levels at 15 ng/ml,30 ng/ml and 60 ng/ml were significantly lower than that of control(P＜0.05 or P＜0.01). Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L up-regulated the protein expression of PPARαin a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.01).100μmol/L fenofibrate alone remarkably elevated the protein expression of PPARαwhen compared with control(P＜0.01).(18) Pretreatment with fenofibrate at different concentrations down-regulated the mRNA expression of TGF-β1,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.01).The expression level was not altered in fenofibrate and its antagonist co-pretreated group when compared with that in chymase group(P＞0.05).100μmol/L fenofibrate alone had no significant effect on the mRNA expression of TGF-β1(P＞0.05).(19) Pretreatment with fenofibrate at 10μmol/L,50μmol/L and 100μmol/L down-regulated the protein expression of TGF-β1,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no marked effect on the protein expression of TGF-β1(P＞0.05).(20) Administration with chymase at different concentrations for 12 h had no significant effect on the protein expression of Smad2/3(P＞0.05) No changes of Smad2/3 in protein expression was observed when CFs were pretreated with different concentrations of fenofibrate(P＞0.05).(21) Treatment with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 6 h up-regulated the protein expression of p-Smad2/3 when compared with control(P＜0.05 or P＜0.01).Pretreatment with fenofibrate at different concentrations down-regulated the protein expression of p-Smad2/3,and the expression levels at 50μmol/L and 100μmol/L were significantly lower than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no notable effect on the protein expression of p-Smad2/3(P＞0.05).(22) Administration with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 6 h down-regulated the mRNA expression of Smad7 when compared with control(P＜0.05 or P＜0.01). Pretreatment with fenofibrate at different concentrations up-regulated the mRNA expression of Smad7 in a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no significant effect on the mRNA expression of Smad7(P＞0.05).(23) Treatment with chymase at 15 ng/ml,30 ng/ml and 60 ng/ml for 12 h down-regulated the protein expression of Smad7 when compared with control(P＜0.01). Pretreatment with fenofibrate at different concentrations up-regulated the protein expression of Smad7 in a concentration-dependent manner,and the expression levels at 50μmol/L and 100μmol/L were significantly higher than that of chymase group(P＜0.05 or P＜0.01).100μmol/L fenofibrate alone had no remarkable effect on the protein expression of Smad7(P＞0.05).
Conclusion(1) Cardiac mast cell-derived chymase can promote cell proliferation and collagen synthesis of CFs in a dose-dependent manner, indicating that cardiac chymase may play a significant role in the formation and progression of myocardial fibrosis.(2) Chymase can up-regulate TGF-β1 expression,promote Smad2/3 phosphorylation and down-regulate Smad7 expression,suggesting that chymase can activate TGF-β1/Smads signaling pathway.(3) TGF-β1 neutralizing antibody and serine/threonine kinase inhibitor exert significant inhibitory effects on cell proliferation,collagen synthesis and TGF-β1 expression caused by chymase while angiotensinⅡreceptor blocker (ARB) valsartan and PD123319 fail to exhibit similar changes.The result further indicates that TGF-β1/Smads pathway is involved in myocardial fibrosis caused by chymase.(4) Fenofibrate,a PPARαagonist,can suppress cell proliferation and collagen synthesis of CFs induced by chymase.Thus,it may exert reverse effect on myocardial fibrosis.(5) Fenofibrate can not only activate PPARαand restrain TGF-β1 production,but also down-regulate p-Smad2/3 expression and up-regulate Smad7 expression.This may be one of the molecular and biological mechanisms involved in the inhibitory effects of fenofibrate on cell proliferation,collagen synthesis of CFs and then result in the regression of myocardial fibrosis.(6) There may exist cross-talk between PPARαand TGF-β1/Smads pathway.That is to say,activated PPARαcan exert negative modulation on TGF-β1/Smads pathway by down-regulating TGF-β1 expression, inhibiting Smad2/3 phosphorylation and up-regulating Smad7 expression.
In a summary,cardiac mast cell-derived chymase can induce cell proliferation and collagen synthesis of CFs and therefore,it can promote myocardial fibrosis.Up-regulation of TGF-β1 expression,phosphorylation of Smad2/3 and down-regulation of Smad7 expression are considered to be one of intra-cellular signal transduction mechariisms.Fenofibrate,a PPARαagonist, can reverse myocardial fibrotic response and thus exert protective effects on cardiovascular system apart from its effect on lipid metabolism.It may be one of the fenofibrate-mediated potential mechanisms that suppression of TGF-β1 expression caused by activating PPARαmay result in down-regulation of Smad2/3 phosphorylation and up-regulation of Smad7 expression.Therefore, this study may provide theoretical evidence and a novel therapeutic target for left ventricular hypertrophy caused by hypertension.
引文
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