氧化苦参碱对人增生性瘢痕成纤维细胞增殖及凋亡的作用
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摘要
目的探讨氧化苦参碱对人增生性瘢痕成纤维细胞增殖及凋亡的影响。方法从人的增生性瘢痕组织中提取成纤维细胞进行培养,通过MTT实验观察不同浓度的氧化苦参碱对成纤维细胞增殖的影响。应用流式细胞术检测经氧化苦参碱处理后成纤维细胞的周期变化,并通过Annexin V-FITC/PI双染色法和Hoechst 33258染色观察细胞的凋亡情况。通过Western Blot和RT-PCR检测氧化苦参碱对细胞周期蛋白CDK4、CDK6,以及凋亡调节蛋白Bcl-2和Bax的作用。结果与对照组相比,氧化苦参碱的浓度为10μmol·L~(-1)时,24 h细胞增殖的抑制率为(18.92±3.77)%,成纤维细胞的早期和晚期凋亡率分别为(3.28±2.56)%和(2.98±1.78)%;氧化苦参碱的浓度为20μmol·L~(-1)时,24 h细胞增殖的抑制率为(32.93±2.68)%,成纤维细胞的早期和晚期凋亡率分别为(6.89±0.76)%和(1.47±2.57)%。氧化苦参碱能够通过抑制细胞周期蛋白CDK4、CDK6和抗凋亡蛋白Bcl-2的表达,诱导促凋亡蛋白Bax的表达来影响细胞周期进程,将细胞周期阻滞于G1期,使细胞增殖受到抑制,凋亡率增加。结论氧化苦参碱能够抑制人增生性瘢痕成纤维细胞的增殖并诱导其凋亡,可作为预防和治疗增生性瘢痕的潜在药物。
Objective To explore the effect of oxymatrine on proliferation and apoptosis of human hypertrophic scar fibroblasts.Methods Fibroblasts derived from human hypertrophic scar were isolated and cultured.The effect of different concentrations of oxymatrine on proliferation of fibroblasts was detected by MTT assay.After oxymatrine treated,the change of fibroblast cycle was examined by flow cytometry.The condition of fibroblasts apoptosis was observed through Annexin V-FITC/PI and Hochest 33258 staining.Effect of oxymatrine on cell cyclin protein of CDK4 and CDK6,and apoptosis-related protein of Bcl-2 and Bax was verified by Western Blot and RT-PCR assay.Results Compared with the control group,when oxymatrine concentration was 10 μmol·L~(-1),the inhibitory rate of 24 h cell proliferation was(18.92±3.77)%,and the early and late apoptosis rates were(3.28±2.56)% and(2.98±1.78)%,respectively.When the concentration of oxymatrine was 20 μmol·L~(-1),the inhibitory rate of 24 h cell proliferation was(32.93±2.68)%,and the early and late apoptosis rates were(6.89±0.76)% and(1.47±2.57)%,respectively.Oxymatrine can affect cell cycle progression by inhibiting the expression of cell cyclin protein of CDK4 and CDK6 and promoting the expression of apoptotic protein of Bcl-2 and Bax.Cell cycle was blocked in G1 phase and cell proliferation was inhibited.The apoptosis rate was increased.Conclusion Oxymatrine can inhibit the proliferation and promote the apoptosis of human hypertrophic scar fibroblasts,and it can be served as a potential drug for the prevention and treatment of hypertrophic scar.
Objective To explore the effect of oxymatrine on proliferation and apoptosis of human hypertrophic scar fibroblasts.Methods Fibroblasts derived from human hypertrophic scar were isolated and cultured.The effect of different concentrations of oxymatrine on proliferation of fibroblasts was detected by MTT assay.After oxymatrine treated,the change of fibroblast cycle was examined by flow cytometry.The condition of fibroblasts apoptosis was observed through Annexin V-FITC/PI and Hochest 33258 staining.Effect of oxymatrine on cell cyclin protein of CDK4 and CDK6,and apoptosis-related protein of Bcl-2 and Bax was verified by Western Blot and RT-PCR assay.Results Compared with the control group,when oxymatrine concentration was 10 μmol·L~(-1),the inhibitory rate of 24 h cell proliferation was(18.92±3.77)%,and the early and late apoptosis rates were(3.28±2.56)% and(2.98±1.78)%,respectively.When the concentration of oxymatrine was 20 μmol·L~(-1),the inhibitory rate of 24 h cell proliferation was(32.93±2.68)%,and the early and late apoptosis rates were(6.89±0.76)% and(1.47±2.57)%,respectively.Oxymatrine can affect cell cycle progression by inhibiting the expression of cell cyclin protein of CDK4 and CDK6 and promoting the expression of apoptotic protein of Bcl-2 and Bax.Cell cycle was blocked in G1 phase and cell proliferation was inhibited.The apoptosis rate was increased.Conclusion Oxymatrine can inhibit the proliferation and promote the apoptosis of human hypertrophic scar fibroblasts,and it can be served as a potential drug for the prevention and treatment of hypertrophic scar.
引文
[1] 高明月,蔺洁,张文显.增生性瘢痕的防治现状与展望[J].中国组织工程研究与临床康复,2010,14(20):3753-3756.
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[9] ZHANG Q,GUO B,HUI Q,et al.miR-137 inhibits proliferation and metastasis of hypertrophic scar fibroblasts via targeting pleiotrophin[J].Cellular Physiology and Biochemistry,2018,49(3):985-995.
[10] LI H,CAI H,DENG J,et al.TGF-β-mediated upregulation of Sox9 in fibroblast promotes renal fibrosis[J].Biochimica et Biophysica Acta-molecular Basis of Disease,2018,1864(2):520-532.
[11] LEE P H,CHU P M,HSIEH P L,et al.Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF-β/smad signaling[J].Environmental Toxicology,2018,33(2):248-255.
[12] BAI X,HE T,LIU J,et al.Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF-beta/Smad pathway[J].Experimental Dermatology,2015,24(5):355-360.
[13] DONG P,ZHANG C,PARKER B T,et al.Cyclin D/CDK4/6 activity controls G1 length in mammalian cells[J].PLoS One,2018,13(1):e0185637.
[14] SHERR C J,BEACH D,SHAPIRO G I.Targeting CDK4 and CDK6:From discovery to therapy[J].Cancer Discovery,2016,6(4):353-367.
[15] AZIMIAN H,DAYYANI M,TOOSSI M T B,et al.Bax/Bcl-2 expression ratio in prediction of response to breast cancer radiotherapy[J].Iranian Journal of Basic Medical Sciences,2018,21(3):325-332.
[16] 范东良,刘金超,郭澍,等.氧化苦参碱对瘢痕疙瘩成纤维细胞增殖及凋亡的影响[J].中国美容整形外科杂志,2012,23(3):177-179.
[2] 李大虎,吕大化.增生性瘢痕的发生机制及治疗进展[J].新乡医学院学报,2015,32(4):370-373.
[3] 吴琴,高云.氧化苦参碱药理作用的分子机制研究进展[J].中国药理学通报,2015,31(6):759-762.
[4] FAN D L,ZHAO W J,WANG Y X,et al.Oxymatrine inhibits collagen synthesis in keloid fibroblasts via inhibition of transforming growth factor-β1/Smad signaling pathway[J].International Journal of Dermatology,2012,51(4):463-472.
[5] ZHOU J,ZHAO Y,SIMONENKO V,et al.Simultaneous silencing of TGF-β1 and COX-2 reduces human skin hypertrophic scar through activation of fibroblast apoptosis[J].Oncotarget,2017,8(46):80651-80665.
[6] WANG X,ZHANG Y,JIANG B H,et al.Study on the role of Hsa-miR-31-5p in hypertrophic scar formation and the mechanism[J].Experimental Cell Research,2017,361(2):201-209.
[7] WANG H W,SHI L,XU Y P,et al.Oxymatrine inhibits renal fibrosis of obstructive nephropathy by downregulating the TGF-β1-Smad3 pathway[J].Renal Failure,2016,38(6):945-951.
[8] WU J,PAN L,JIN X,et al.The role of oxymatrine in regulating TGF-β1 in rats with hepatic fibrosis[J].Acta Cirurgica Brasileira,2018,33(3):207-215.
[9] ZHANG Q,GUO B,HUI Q,et al.miR-137 inhibits proliferation and metastasis of hypertrophic scar fibroblasts via targeting pleiotrophin[J].Cellular Physiology and Biochemistry,2018,49(3):985-995.
[10] LI H,CAI H,DENG J,et al.TGF-β-mediated upregulation of Sox9 in fibroblast promotes renal fibrosis[J].Biochimica et Biophysica Acta-molecular Basis of Disease,2018,1864(2):520-532.
[11] LEE P H,CHU P M,HSIEH P L,et al.Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF-β/smad signaling[J].Environmental Toxicology,2018,33(2):248-255.
[12] BAI X,HE T,LIU J,et al.Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF-beta/Smad pathway[J].Experimental Dermatology,2015,24(5):355-360.
[13] DONG P,ZHANG C,PARKER B T,et al.Cyclin D/CDK4/6 activity controls G1 length in mammalian cells[J].PLoS One,2018,13(1):e0185637.
[14] SHERR C J,BEACH D,SHAPIRO G I.Targeting CDK4 and CDK6:From discovery to therapy[J].Cancer Discovery,2016,6(4):353-367.
[15] AZIMIAN H,DAYYANI M,TOOSSI M T B,et al.Bax/Bcl-2 expression ratio in prediction of response to breast cancer radiotherapy[J].Iranian Journal of Basic Medical Sciences,2018,21(3):325-332.
[16] 范东良,刘金超,郭澍,等.氧化苦参碱对瘢痕疙瘩成纤维细胞增殖及凋亡的影响[J].中国美容整形外科杂志,2012,23(3):177-179.