丹酚酸B-TAT脂质体对HSF细胞增殖、侵袭和细胞周期的影响
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摘要
制备载丹酚酸B的穿膜肽TAT修饰脂质体(SAB-TAT-LIP),研究其对人皮肤成纤维细胞(HSF)生长增殖、迁移、侵袭和细胞周期的影响,初步评价其用于防治增生性瘢痕的效果。以体外培养的HSF细胞为研究对象,采用四甲基偶氮唑盐MTT法检测SAB-TAT-LIP对细胞生长增殖的抑制作用;采用Transwell小室法和划痕法检测SAB-TAT-LIP对细胞侵袭和迁移能力的影响;采用流式细胞术检测细胞周期变化。空白脂质体对HSF细胞基本无毒副作用,不同浓度的SAB-TAT-LIP干预HSF细胞不同时间后,均具有一定的增殖抑制作用,且呈现剂量和时间依赖性,同时细胞迁移和侵袭能力明显降低; SAB-TATLIP干预HSF细胞48 h后,可使细胞周期阻滞于G0/G1期,与空白对照组相比,P<0. 01。SAB-TAT-LIP对HSF细胞的增殖、侵袭和迁移有显著的抑制作用,对细胞周期G0/G1期有阻滞作用。
Hypertrophic scar( HS) is a very common skin fibrosis disorder after human skin injury and wound healing. The objective of this study was to investigate the efficacy of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B( SAB-TAT-LIP) on proliferation,migration and cell cycle of human skin fibroblasts( HSF),and preliminarily evaluate its effect on prevention and treatment of HS. HSF were cultured in vitro,and MTT assay was used to detect the inhibitory effect of SAB-TAT-LIP on cell proliferation. Cell migration was assessed by Transwell chamber method and scratch method; and cell cycle change was detected by flow cytometry. In vitro cell studies showed that blank liposome basically had no toxic effect on HSF. Different concentrations of SABTAT-LIP inhibited proliferation on HSF in varying degrees after intervention for different periods in a dose and time dependent manner;meanwhile,SAB-TAT-LIP significantly inhibited the migration and invasion of HSF. At the same time,SAB-TAT-LIP could block the cell cycle at G0/G1 phase after intervention for 48 h,P<0.01 as compared with the blank control group. Conclusively,our experimental data quantitatively demonstrate that SAB-TAT-LIP has significant inhibitory effect on cells proliferation,invasion and migration,with blocking effect on G0/G1 phase. This may offer a promising therapeutic strategy for transdermal delivery in prevention and treatment of HS.
Hypertrophic scar( HS) is a very common skin fibrosis disorder after human skin injury and wound healing. The objective of this study was to investigate the efficacy of cell penetrating peptide TAT-modified liposomes loaded with salvianolic acid B( SAB-TAT-LIP) on proliferation,migration and cell cycle of human skin fibroblasts( HSF),and preliminarily evaluate its effect on prevention and treatment of HS. HSF were cultured in vitro,and MTT assay was used to detect the inhibitory effect of SAB-TAT-LIP on cell proliferation. Cell migration was assessed by Transwell chamber method and scratch method; and cell cycle change was detected by flow cytometry. In vitro cell studies showed that blank liposome basically had no toxic effect on HSF. Different concentrations of SABTAT-LIP inhibited proliferation on HSF in varying degrees after intervention for different periods in a dose and time dependent manner;meanwhile,SAB-TAT-LIP significantly inhibited the migration and invasion of HSF. At the same time,SAB-TAT-LIP could block the cell cycle at G0/G1 phase after intervention for 48 h,P<0.01 as compared with the blank control group. Conclusively,our experimental data quantitatively demonstrate that SAB-TAT-LIP has significant inhibitory effect on cells proliferation,invasion and migration,with blocking effect on G0/G1 phase. This may offer a promising therapeutic strategy for transdermal delivery in prevention and treatment of HS.
引文
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[22] Li Y,Shi S,Gao J,et al. Cryptotanshinone downregulates the profibrotic activities of hypertrophic scar fibroblasts and accelerates wound healing:a potential therapy for the reduction of skin scarring[J]. Biomed Pharmacother,2016,80(5):80.
[23] Xu L,Shen P,Bi Y,et al. Danshen injection ameliorates STZinduced diabetic nephropathy in association with suppression of oxidative stress,pro-inflammatory factors and fibrosis[J]. Int Immunopharmacol,2016,38(90):385.
[24]王丽娜,陶艳艳,李书,等.丹酚酸B盐对大鼠纤维化肝组织脂质过氧化及其MMP-2活性的影响[J].中国中药杂志,2010,35(1):71.
[25]梁文仪,陈文静,杨光辉,等.丹参酚酸类成分研究进展[J].中国中药杂志,2016,41(5):806.
[26] Wang Y,Xu F,Chen J,et al. Matrix metalloproteinase-9 induces cardiac fibroblast migration,collagen and cytokine secretion:Inhibition by salvianolic acid B from Salvia miltiorrhiza[J].Phytomedicine,2012,19(1):13.
[27] Wang Q L,Tao Y Y,Yuan J L,et al. Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-β1 signal transduction pathway in vivo and in vitro[J]. BMC Cell Biol,2010,11(1):1.
[28] Ledon J A,Savas J,Franca K,et al. Intralesional treatment for keloids and hypertrophic scars:a review[J]. Dermatol Surg,2013,39(12):1745.
[2] Liu Q,Chu H,Ma Y,et al. Salvianolic acid B attenuates experimental pulmonary fibrosis through inhibition of the TGF-βsignaling pathway[J]. Sci Rep,2016,6(9):27610.
[3] Hou J,Tian J,Jiang W,et al. Therapeutic effects of SMND-309,a new metabolite of salvianolic acid B,on experimental liver fibrosis[J]. Eur J Pharmacol,2011,650(1):390.
[4] Liu M,Xu H,Zhang L,et al. Salvianolic acid B inhibits myofibroblast transdifferentiation in experimental pulmonary fibrosis via the up-regulation of Nrf2[J]. Biochem Biophys Res Commun,2018,495(1):325.
[5]罗红,王春花,赵玲璐,等.丹酚酸B抗心肌纤维化的机制研究[J].中国药房,2017,28(28):3900.
[6] Finnerty C C,Jeschke M G,Branski L K,et al. Hypertrophic scarring:the greatest unmet challenge following burn injury[J].Lancet,2016,388(10052):1427.
[7] Chawla S,Ghosh S. Regulation of fibrotic changes by the synergistic effects of cytokines,dimensionality and matrix:towards the development of an in vitro human dermal hypertrophic scar model[J]. Acta Biomaterialia,2018,69(15):131.
[8] Curran T A,Ghahary A. Evidence of a role for fibrocyte and keratinocyte-like cells in the formation of hypertrophic scars[J]. J Burn Care Res,2013,34(2):227.
[9] Li G,Zhou R,Zhang Q,et al. Fibroproliferative effect of microRNA-21 in hypertrophic scar derived fibroblasts[J]. Exp Cell Res,2016,345(1):93.
[10] Chun Q,Zhiyong W,Fei S,et al. Dynamic biological changes in fibroblasts during hypertrophic scar formation and regression[J].Int Wound J,2016,13(2):257.
[11] Kwon S S,Kim S Y,Kong B J,et al. Cell penetrating peptide conjugated liposomes as transdermal delivery system of Polygonum aviculare L. extract[J]. Int J Pharm,2015,483(1/2):26.
[12] Fu H,Hu G L,He Q. Preparation of cell penetrating peptide TAT and cleavable PEGco-modified liposomes loaded with paclitaxel and its in vitro apoptosis assay[J]. Acta Pharm Sin B,2014,49(7):1054.
[13] Patlolla R R,Desai P R,Belay K,et al. Translocation of cell penetrating peptide engrafted nanoparticles across skin layers[J].Biomaterials,2010,31(21):5598.
[14] Zhang L,Han L,Sun X,et al. The use of PEGylated liposomes to prolong the circulation lifetime of salvianolic acid B[J]. Fitoterapia,2012,83(4):678.
[15]林珈好.丹参酮ⅡA、丹酚酸B与甘草次酸复方脂质体构建及体内外评价研究[D].北京:北京中医药大学,2014.
[16] Gong Y F,Zhang X M,Yu J,et al. Effect of recombinant human endostatin on hypertrophic scar fibroblast apoptosis in a rabbit ear model[J]. Biomed Pharmacother,2017,91(7):680.
[17] Wang J,Dodd C,Shankowsky H A,et al. Deep dermal fibroblasts contribute to hypertrophic scarring[J]. Lab Invest,2008,88(12):1278.
[18] Bai X,He T,Liu J,et al. Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF-β/Smad pathway[J]. Exp Dermatol,2015,24(5):355.
[19]罗宏宾.紫草素对皮肤成纤维细胞增殖、凋亡的调控及抑制增生性瘢痕形成的机制研究[D].杭州:浙江中医药大学,2017.
[20]时军,张小灵,郭波红,等.双丹脂质体凝胶抑制兔耳瘢痕增生的作用及机制研究[J].广东药科大学学报,2017,33(1):84.
[21]袁媛,吴芹,石京山,等.丹参及其主要成分保肝作用的研究进展[J].中国中药杂志,2015,40(4):588.
[22] Li Y,Shi S,Gao J,et al. Cryptotanshinone downregulates the profibrotic activities of hypertrophic scar fibroblasts and accelerates wound healing:a potential therapy for the reduction of skin scarring[J]. Biomed Pharmacother,2016,80(5):80.
[23] Xu L,Shen P,Bi Y,et al. Danshen injection ameliorates STZinduced diabetic nephropathy in association with suppression of oxidative stress,pro-inflammatory factors and fibrosis[J]. Int Immunopharmacol,2016,38(90):385.
[24]王丽娜,陶艳艳,李书,等.丹酚酸B盐对大鼠纤维化肝组织脂质过氧化及其MMP-2活性的影响[J].中国中药杂志,2010,35(1):71.
[25]梁文仪,陈文静,杨光辉,等.丹参酚酸类成分研究进展[J].中国中药杂志,2016,41(5):806.
[26] Wang Y,Xu F,Chen J,et al. Matrix metalloproteinase-9 induces cardiac fibroblast migration,collagen and cytokine secretion:Inhibition by salvianolic acid B from Salvia miltiorrhiza[J].Phytomedicine,2012,19(1):13.
[27] Wang Q L,Tao Y Y,Yuan J L,et al. Salvianolic acid B prevents epithelial-to-mesenchymal transition through the TGF-β1 signal transduction pathway in vivo and in vitro[J]. BMC Cell Biol,2010,11(1):1.
[28] Ledon J A,Savas J,Franca K,et al. Intralesional treatment for keloids and hypertrophic scars:a review[J]. Dermatol Surg,2013,39(12):1745.