表儿茶素对增生性瘢痕成纤维细胞增殖及ERK1/2 MAPK信号通路影响的研究
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摘要
目的:研究表儿茶素对增生性瘢痕成纤维细胞增殖及ERK1/2 MAPK信号通路的影响。方法:取4例增生性腹部瘢痕组织,分离成纤维细胞并鉴定和培养,根据表儿茶素对成纤维细胞增殖抑制的半数抑制浓度(The median inhibition concentration,IC50)确定表儿茶素的浓度,用表儿茶素分别作用24 h、48 h、72 h,用MTT法检测细胞抑制情况,Western Blot法检测细胞外信号调节激酶1/2 (Extracellular-signal-regulated kinase1/2,ERK1/2)、磷酸化细胞外信号调节激酶1/2 (p-ERK1/2)和CyclinD1蛋白表达水平,流式细胞术检测细胞周期。结果:表儿茶素对成纤维细胞的24 h IC50为20.47 mg/mL,故选择浓度为20.00 mg/mL进行后续实验。随着表儿茶素作用时间的增加,抑制情况增加越显著(P <0.05);表儿茶素对成纤维细胞ERK1/2蛋白表达影响不显著;与表儿茶素作用0 h比较,作用24 h、48 h和72 h后成纤维细胞p-ERK1/2、CyclinD1蛋白表达和G0/G1期降低,成纤维细胞G2/M期增加(P <0.05);随着作用时间的增加,成纤维细胞p-ERK1/2、CyclinD1蛋白表达和G0/G1期降低越显著,G2/M期增加越显著(P <0.05)。结论:表儿茶素对增生性瘢痕成纤维细胞增殖有抑制作用,其机制可能与抑制ERK1/2磷酸化,阻滞细胞周期有关。
Objective: To study the effect of epicatechin on proliferation and ERK1/2 MAPK signaling pathways of hypertrophic scar fibroblasts. Methods:Fibroblasts were isolated from four cases of hypertrophic abdominal scar tissues,and were identified and cultured;the concentration of epicatechin was determined according to the median inhibition concentration(IC50) of epicatechin inhibiting the fibroblast proliferation; fibroblasts were exposed to epicatechin for 24, 48 and 72 hours respectively; the inhibition of cells was detected through MTT assay; extracellular-signal-regulated kinase1/2(ERK1/2),phosphorylated extracellular signal-regulated kinase1/2(p-ERK1/2) and the protein expression level of cyclinD1 were detected through western blot method;the cell cycle was detected through flow cytometry. Results:The 24-hour IC50 of epicatechin in fibroblasts was 20.47 mg/m L, therefore, IC50 of epicatechin at the concentration of 20.00 mg/m L was selected for the subsequent experiments. With the increase of action time of epicatechin, the inhibition increased more significantly(P <0.05); there was no significant effect of epicatechin on ERK1/2 protein expression in fibroblasts; after 24 h, 48 h and 72 h respectively,compared with those when the action time of epicatechin was 0 h, the protein expressions of p-ERK1/2 and cyclinD1 as well as G0/G1 in fibroblasts were decreased, while G2/M in fibroblasts was increased(P < 0.05); with the increase of the action time,the protein expressions of p-ERK1/2 and cyclinD1 as well as G0/G1 in fibroblasts were decreased more significantly,while G2/M was increased more significantly(P < 0.05). Conclusion:Epicatechin can inhibit the proliferation of hypertrophic scar fibroblasts, whose mechanism may be related to its inhibition of phosphorylation of ERK1/2 and its blockage of cell cycle.
Objective: To study the effect of epicatechin on proliferation and ERK1/2 MAPK signaling pathways of hypertrophic scar fibroblasts. Methods:Fibroblasts were isolated from four cases of hypertrophic abdominal scar tissues,and were identified and cultured;the concentration of epicatechin was determined according to the median inhibition concentration(IC50) of epicatechin inhibiting the fibroblast proliferation; fibroblasts were exposed to epicatechin for 24, 48 and 72 hours respectively; the inhibition of cells was detected through MTT assay; extracellular-signal-regulated kinase1/2(ERK1/2),phosphorylated extracellular signal-regulated kinase1/2(p-ERK1/2) and the protein expression level of cyclinD1 were detected through western blot method;the cell cycle was detected through flow cytometry. Results:The 24-hour IC50 of epicatechin in fibroblasts was 20.47 mg/m L, therefore, IC50 of epicatechin at the concentration of 20.00 mg/m L was selected for the subsequent experiments. With the increase of action time of epicatechin, the inhibition increased more significantly(P <0.05); there was no significant effect of epicatechin on ERK1/2 protein expression in fibroblasts; after 24 h, 48 h and 72 h respectively,compared with those when the action time of epicatechin was 0 h, the protein expressions of p-ERK1/2 and cyclinD1 as well as G0/G1 in fibroblasts were decreased, while G2/M in fibroblasts was increased(P < 0.05); with the increase of the action time,the protein expressions of p-ERK1/2 and cyclinD1 as well as G0/G1 in fibroblasts were decreased more significantly,while G2/M was increased more significantly(P < 0.05). Conclusion:Epicatechin can inhibit the proliferation of hypertrophic scar fibroblasts, whose mechanism may be related to its inhibition of phosphorylation of ERK1/2 and its blockage of cell cycle.
引文
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[2]KWON S H,GURTNER G C.Is early inflammation good or bad?Linking early immune changes to hypertrophic scarring[J].Experimental Dermatology,2017,26(2):133-139.
[3]TAMAR E,KOLER M,VAKNIN A.The role of motility and chemotaxis in the bacterial colonization of protected surfaces[J].Scientific Reports,2016,6(4):19616-19622.
[4]CHUN Q,ZHIYONG W,FEI S,et al.Dynamic biological changes in fibroblasts during hypertrophic scar formation and regression[J].International Wound Journal,2016,13(2):257-262.
[5]ZHANG Y F,ZHOU S Z,CHENG X Y,et al.Baicalein attenuates hypertrophic scar formation via inhibition of the transforming growth factor-β/Smad2/3 signalling pathway[J].British Journal of Dermatology,2016,174(1):120-130.
[6]LAKE D,CORR魭A S A L,M譈LLER J.Negative feedback regulation of the ERK1/2 MAPK pathway[J].Cellular and Molecular Life Sciences,2016,73(23):4397-4413.
[7]LAI J M,ZHANG X,LIU F F,et al.Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation:a dual role of ERK1/2[J].Oncotarget,2016,7(28):43731-43745.
[8]徐莲,符旭东,熊蕊,等.3种中药提取物对人真皮成纤维细胞增殖及胶原合成的影响[J].中国药师,2016,19(3):448-452.
[9]ANITUA E,PINO A,ORIVE G.Plasma rich in growth factors promotes dermal fibroblast proliferation,migration and biosynthetic activity[J].Journal of Wound Care,2016,25(11):680-689.
[10]李周娜,陈香儒,金哲虎.4-羟苯基维胺在不同载体中对人瘢痕疙瘩成纤维细胞增殖及凋亡影响[J].中华皮肤科杂志,2017,50(3):195-198.
[11]TAO H,CAO W,YANG J J,et al.Long noncoding RNAH19 controls DUSP5/ERK1/2 axis in cardiac fibroblast proliferation and fibrosis[J].Cardiovascular Pathology,2016,25(5):381-389.
[12]SCHROETER H,HEISS C,BALZER J,et al.(-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(4):1024-1029.
[13]刘清,张尔婷,李华平,等.茶多酚对人皮肤成纤维细胞Nrf2/Bach1m RNA及核蛋白的影响[J].中华皮肤科杂志,2017,50(3):199-203.
[14]LATIMER H R,VEAL E A.Peroxiredoxins in regulation of MAPK signalling pathways;sensors and barriers to signal transduction[J].Molecules&Cells,2016,39(1):40-44.
[15]ASHIDA Y,NAKAJIMA-KOYAMA M,HIROTA A,et al.Negative feedback regulation of the ERK1/2 MAPK pathway[J].Cellular and Molecular Life Sciences,2016,73(23):4397-4413.
[16]李佳,吴松,唐宏图,等.电针内关穴对心肌肥厚大鼠Raf、ERK1/2及p-ERK1/2表达的影响[J].中国中西医结合杂志,2016,36(11):1335-1339.
[17]NAGPAL H,FUKAGAWA T.Kinetochore assembly and function through the cell cycle[J].Chromosoma,2016,125(4):645-659.
[18]PAUKLIN S,MADRIGAL P,BERTERO A,et al.Initiation of stem cell differentiation involves cell cycle-dependent regulation of developmental genes by Cyclin D[J].Genes&Development,2016,30(4):421-429.
[19]MENS M,GHANBARI M.Cell cycle regulation of stem cells by micro RNAs[J].Stem Cell Reviews&Reports,2018,14(3):1-14.