鞘鞍醇激酶-2在转化生长因子-β1诱导心脏成纤维细胞增殖与活化过程中作用
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摘要
目的探讨鞘鞍醇激酶-2(SPHK2)在转化生长因子-β1(TGF-β1)诱导心脏成纤维细胞增殖与活化中的作用。方法分离乳鼠心脏成纤维细胞并体外培养。利用慢病毒分别将对照或SPHK2小分子干扰RNA(siRNA)转染心脏成纤维细胞。采用Western blot检测siRNA对SPHK2蛋白的干扰效率;采用酶联免疫吸附法检测心脏成纤维细胞1-磷酸鞘鞍醇(S1P)水平;采用TGF-β1处理诱导对照组和SPHK2 siRNA组心脏成纤维细胞增殖与活化后,CCK8法检测心脏成纤维细胞增殖能力,Western blot检测心脏成纤维细胞活化的标记蛋白α-平滑肌激动蛋白(α-SMA)的表达,q-PCR检测心脏成纤维细胞胶原蛋白Ⅰ(ColⅠ)和胶原蛋白Ⅲ(ColⅢ)的mRNA水平。结果慢病毒转染SPHK2 siRNA后,心脏成纤维细胞SPHK2蛋白表达水平显著低于对照组,组间比较,差异有统计学意义(P<0.05)。SPHK2 siRNA组心脏成纤维细胞S1P水平显著低于对照组,组间比较,差异有统计学意义(P<0.05)。给予TGF-β1处理后,SPHK2 siRNA组心脏成纤维细胞增殖能力、α-SMA蛋白和mRNA表达、ColⅠ和ColⅢ的mRNA表达显著低于对照组,组间比较,差异有统计学意义(P<0.05)。结论下调SPHK2表达可抑制TGF-β1诱导心脏成纤维细胞的增殖与活化。
Objective To investigate the role of sphingosine kinase-2(SPHK2) in transforming growth factor-β1(TGF-β1) induced proliferation and activation of cardiac fibroblasts.Methods Rat cardiac fibroblasts were isolated and cultured in vitro.Will compare respectively using slow virus or small molecules SPHK2 interference RNA(siRNA) transfection cardiac fibroblasts,using Western blot detection siRNA interference with SPHK2 protein efficiency,using enzyme-linked immunosorbent method to detect cardiac fibroblasts 1-phosphate sheath saddle alcohol(S1 P) level,the TGF-β1 treatment induced in the control group and SPHK2 siRNA group after cardiac fibroblasts proliferation and activation,CCK8 method to detect cardiac fibroblasts proliferation,Western blot detection of cardiac fibroblasts activation marker protein alpha smooth muscle actin(α-SMA) protein expression and the q-PCR detection of cardiac fibroblasts collagen Ⅰ(Col Ⅰ) and Ⅲ collagen(Col Ⅲ) mRNA level.Results After lentivirus transfection with SPHK2 siRNA,the expression level of SPHK2 protein in cardiac fibroblasts was significantly lower than that in the control group,with statistically significant difference between the two groups(P<0.05).The S1 P level of cardiac fibroblasts in the SPHK2 siRNA group was significantly lower than that in the control group(P<0.05).TGF-β1 was given after processing,cardiac fibroblasts proliferation,α-SMA protein and mRNA expression,Col Ⅰ and Col Ⅲ mRNA expression of SPHK2 siRNA group was significantly lower than that of the control group(P<0.05).Conclusion Down-regulation of SPHK2 expression inhibits TGF-β1 induced proliferation and activation of cardiac fibroblasts.
Objective To investigate the role of sphingosine kinase-2(SPHK2) in transforming growth factor-β1(TGF-β1) induced proliferation and activation of cardiac fibroblasts.Methods Rat cardiac fibroblasts were isolated and cultured in vitro.Will compare respectively using slow virus or small molecules SPHK2 interference RNA(siRNA) transfection cardiac fibroblasts,using Western blot detection siRNA interference with SPHK2 protein efficiency,using enzyme-linked immunosorbent method to detect cardiac fibroblasts 1-phosphate sheath saddle alcohol(S1 P) level,the TGF-β1 treatment induced in the control group and SPHK2 siRNA group after cardiac fibroblasts proliferation and activation,CCK8 method to detect cardiac fibroblasts proliferation,Western blot detection of cardiac fibroblasts activation marker protein alpha smooth muscle actin(α-SMA) protein expression and the q-PCR detection of cardiac fibroblasts collagen Ⅰ(Col Ⅰ) and Ⅲ collagen(Col Ⅲ) mRNA level.Results After lentivirus transfection with SPHK2 siRNA,the expression level of SPHK2 protein in cardiac fibroblasts was significantly lower than that in the control group,with statistically significant difference between the two groups(P<0.05).The S1 P level of cardiac fibroblasts in the SPHK2 siRNA group was significantly lower than that in the control group(P<0.05).TGF-β1 was given after processing,cardiac fibroblasts proliferation,α-SMA protein and mRNA expression,Col Ⅰ and Col Ⅲ mRNA expression of SPHK2 siRNA group was significantly lower than that of the control group(P<0.05).Conclusion Down-regulation of SPHK2 expression inhibits TGF-β1 induced proliferation and activation of cardiac fibroblasts.
引文
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[16] Liu X,Hong Q,Wang Z,et al.Transforming growth factor-β-sphingosine kinase 1/S1P signaling upregulates microRNA-21 to promote fibrosis in renal tubular epithelial cells[J].Exp Biol Med (Maywood),2016,241(3):265-272.
[2] Li L,Zhang Y,Li Y,et al.Mesenchymal stem cell transplantation attenuates cardiac fibrosis associated with isoproterenol-induced global heart failure[J].Transpl Int,2008,21(12):1181-1189.
[3] Chen TL,Liao JW,Chan WH,et al.Induction of cardiac fibrosis and transforming growth factor-β1 by motorcycle exhaust in rats[J].Inhal Toxicol,2013,25(9):525-535.
[4] Roubille F,Busseuil D,Merlet N,et al.Investigational drugs targeting cardiac fibrosis[J].Expert Rev Cardiovasc Ther,2014,12(1):111-125.
[5] Lei B,Hitomi H,Mori T,et al.Effect of efonidipine on TGF-β1-induced cardiac fibrosis through Smad2-dependent pathway in rat cardiac fibroblasts[J].J Pharmacol Sci,2011,117(2):98-105.
[6] Liu H,Sugiura M,Nava VE,et al.Molecular cloning and functional characterization of a novel mammalian sphingosine kinase type 2 isoform[J].J Biol Chem,2000,275(26):19513-19520.
[7] Xun C,Chen MB,Qi L,et al.Targeting sphingosine kinase 2 (SphK2) by ABC294640 inhibits colorectal cancer cell growth in vitro and in vivo[J].J Exp Clin Cancer Res,2015,34:94.
[8] Vessey DA,Li L,Imhof I,et al.FTY720 postconditions isolated perfused heart by a mechanism independent of sphingosine kinase 2 and different from S1P or ischemic postconditioning[J].Med Sci Monit Basic Res,2013,19:126-132.
[9] Jacoby JJ,Kalinowski A,Liu MG,et al.Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation,cardiac fibrosis,and heart failure with advanced age[J].Proc Natl Acad Sci USA,2003,100(22):12929-12934.
[10] Chen MM,Lam A,Abraham JA,et al.CTGF expression is induced by TGF- beta in cardiac fibroblasts and cardiac myocytes:a potential role in heart fibrosis[J].J Mol Cell Cardiol,2000,32(10):1805-1819.
[11] Schwab SR,Pereira JP,Matloubian M,et al.Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients[J].Science,2005,309(5741):1735-1739.
[12] Cao M,Ji C,Zhou Y,et al.Sphingosine kinase inhibitors:a patent review[J].Int J Mol Med,2018,41(5):2450-2460.
[13] Zhang R,Li L,Yuan L,et al.Hypoxic preconditioning protects cardiomyocytes against hypoxia/reoxygenation-induced cell apoptosis via sphingosine kinase 2 and FAK/AKT pathway[J].Exp Mol Pathol,2016,100(1):51-58.
[14] Gomez L,Paillard M,Price M,et al.A novel role for mitochondrial sphingosine-1-phosphate produced by sphingosine kinase-2 in PTP-mediated cell survival during cardioprotection[J].Basic Res Cardiol,2011,106(6):1341-1353.
[15] Santiago JJ,Dangerfield AL,Rattan SG,et al.Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro:expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts[J].Dev Dyn,2010,239(6):1573-1584.
[16] Liu X,Hong Q,Wang Z,et al.Transforming growth factor-β-sphingosine kinase 1/S1P signaling upregulates microRNA-21 to promote fibrosis in renal tubular epithelial cells[J].Exp Biol Med (Maywood),2016,241(3):265-272.