雷帕霉素调控光老化成纤维细胞MMPs和COL-1表达
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摘要
目的探讨雷帕霉素(RAPA)对光老化皮肤成纤维细胞(FBs)基质金属蛋白酶(MMPs)表达和Ⅰ型胶原蛋白(collagen-Ⅰ)合成的影响。方法采用cck-8法检测RAPA对FBs活性的影响,甄选最佳应用浓度,利用UVB体外反复照射构建成纤维细胞的老化模型,RAPA预处理细胞,造模结束24 h后,实时荧光定量检测MMPs的表达;造模结束48 h,蛋白质印迹法检测Ⅰ型胶原的表达。结果低剂量RAPA对FBs的细胞活性无影响,5μm/L的RAPA作为后续细胞处理的药物浓度为佳。UVB组的光老化FBs中,MMP-1、2、9的表达均显著升高;而RAPA预处理后能明显降低UVB引起的MMPs过度分泌;同时使Ⅰ型胶原的表达量增多。结论 RAPA可降低光老化FBs中MMPs的表达,增加Ⅰ型胶原的合成,维持光老化皮肤ECM成分的稳定,在一定程度上缓解细胞光老化程度。
Objective To investigate the influence of rapamycin(RAPA) on the expression of matrix metalloproteinases(MMPs)and the synthesis of type I collagen(collagen-I) in photo-aging skin fibroblasts(FBs). Methods The best concentration was selected by the effect of RAPA on the proliferative activity of FBs tested by cell counting kit-8(CCK-8) assay. The photo-aging model of FBs was structured by repeated UVB radiation. The cells were pretreated with RAPA. The influence of RAPA on the expression of MMPs of photo-aging skin FBs was detected by real-time fluorescence quantitative PCR at 24 hours after modeling and the synthesis of collagen I was detected by western blotting at 48 h after modeling. Results The proliferative activity of fibroblasts was not affected by RAPA in a low dose manner, 5 μm/L was the best concentration for the subsequent treatment. The UVB irradiation increased the expressions of MMP-1, 2, 9 of FBs. RAPA pretreatment significantly decreased expression of MMPs caused by UVB and increased the expression of collagen I.Conclusion RAPA can maintain the metabolic balance of dermal ECM by decreasing the expression of MMPs and increasing the synthesis of collagen I in photo-aging FBs, and to a certain extent, alleviate the degree of cell photoaging.
Objective To investigate the influence of rapamycin(RAPA) on the expression of matrix metalloproteinases(MMPs)and the synthesis of type I collagen(collagen-I) in photo-aging skin fibroblasts(FBs). Methods The best concentration was selected by the effect of RAPA on the proliferative activity of FBs tested by cell counting kit-8(CCK-8) assay. The photo-aging model of FBs was structured by repeated UVB radiation. The cells were pretreated with RAPA. The influence of RAPA on the expression of MMPs of photo-aging skin FBs was detected by real-time fluorescence quantitative PCR at 24 hours after modeling and the synthesis of collagen I was detected by western blotting at 48 h after modeling. Results The proliferative activity of fibroblasts was not affected by RAPA in a low dose manner, 5 μm/L was the best concentration for the subsequent treatment. The UVB irradiation increased the expressions of MMP-1, 2, 9 of FBs. RAPA pretreatment significantly decreased expression of MMPs caused by UVB and increased the expression of collagen I.Conclusion RAPA can maintain the metabolic balance of dermal ECM by decreasing the expression of MMPs and increasing the synthesis of collagen I in photo-aging FBs, and to a certain extent, alleviate the degree of cell photoaging.
引文
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[2]Zeng JP,Bi B,Chen L,et al.Repeated exposure of mouse dermal fibroblasts at a sub-cytotoxic dose of UVB leads to premature senescence:a robust model of cellular photoaging[J].J Dermatol Sci,2014,73(1):49-56.
[3]Chen L,Bi B,Zeng J,et al.Rosiglitazone ameliorates senescence-like phenotypes in a cellular photoaging model[J].J Dermatol Sci,2015,77(3):173-181.
[4]Demidenko ZN,Zubova SG,Bukreeva EI,et al.Rapamycin decelerates cellular senescence[J].Cell Cycle,2009,8(12):1888-1895.
[5]Kohl E,Steinbauer J,Landthaler M,et al.Skin ageing[J].J Eur Acad Dermatol Venereol,2011,25(8):873-884.
[6]陈亮,毕波,曾继平,等.罗格列酮对UVB诱导的小鼠光老化皮肤成纤维细胞MMPs表达和细胞外基质蛋白合成的影响[J].中国美容整形外科杂志,2015,26(8):500-503.
[7]Kim EJ,Kim YK,Kim M,et al.UV-induced inhibition of adipokine production in subcutaneous fat aggravates dermal matrix degradation in human skin[J].Scientific Reports,2016,6:25616.
[8]Sun Z,Hwang E,Park SY,et al.Angelica archangelia prevented collagen degradation by blocking production of matrix metalloproteinases in UVB-exposed dermal fibroblasts[J].Photochem Photobiol,2016,92(4):604-610.
[9]Karthikeyan R,Kanimozhi G,Prasad NR,et al.7-Hydroxycoumarin prevents UVB-induced activation of NF-kappa B and subsequent overexpression of matrix metalloproteinases and inflammatory markers in human dermal fibroblast cells[J].J Photochem Photobiol B,2016,161:170-176.
[10]Pittayapruek P,Meephansan J,Prapapan O,et al.Role of matrix metalloproteinases in photoaging and photocarcinogenesis[J].Int J Mol Sci,2016,17(6)[2016].
[11]Baek B,Lee SH,Kim K,et al.Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts[J].Korean J Physiol Pharmacol,2016,20(3):269-277.
[12]Lu J,Guo JH,Tu XL,et al.Tiron inhibits UVB-induced AP-1binding sites transcriptional activation on MMP-1 and MMP-3promoters by MAPK signaling pathway in Human dermal fibroblasts[J].PLo S One,2016,11(8):e159998.
[13]Bosch R,Philips N,Suárez-Pérez JA,et al.Mechanisms of photoaging and cutaneous photocarcinogenesis,and photoprotective strategies with phytochemicals[J].Antioxidants(Basel),2015,4(2):248-268.
[14]Bravo K,Duque L,Ferreres F,et al.Passiflora tarminiana fruits reduce UVB-induced photoaging in human skin fibroblasts[J].J Photochem Photobiol B,2017,168:78-88.
[15]Bae JS,Han M,Shin HS,et al.Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin[J].J Ethnopharmacol,2017,195:334-342.
[16]Rubinsztein DC,Marino G,Kroemer G.Autophagy and aging[J].Cell,2011,146(5):682-695.
[17]Giordano S,Darley-Usmar V,Zhang J.Autophagy as an essential cellular antioxidant pathway in neurodegenerative disease[J].Redox Biol,2013,2:82-90.
[18]Nacarelli T,Azar A,Sell C.Inhibition of m TOR prevents ROS production initiated by ethidium bromide-induced mitochondrial DNA depletion[J].Front Endocrinol,2014,5:122.
[19]Kang HT,Lee KB,Kim SY,et al.Autophagy impairment induces premature senescence in primary human fibroblasts[J].PLo S One,2011,6(8):e23367.
[20]Huang J,Manning BD.A complex interplay between Akt,TSC2 and the twom TOR complexes[J].Biochem Soc Trans,2009,37(Pt 1):217.
[21]Finkel T.Relief with rapamycin:m TOR inhibition protects against radiation-induced mucositis[J].Cell Stem Cell,2012,11(3):287-288.