桑白皮多酚对B16细胞内黑色素生成的影响及其机制
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摘要
目的研究桑白皮多酚(polyphenol from Cortex Mori,CMP)对小鼠B16细胞内黑色素生成的影响,并探究其作用机制。方法体外培养小鼠B16细胞,构建α-黑素细胞刺激素(α-MSH)诱导的黑色素高表达细胞模型。CMP干预B16细胞,MTT法测定细胞活性;分别利用NaOH裂解法和L-Dopa氧化法,分析细胞内黑色素生成含量和酪氨酸酶活性的变化;Western blot和实时荧光定量PCR法分别测定B16细胞中酪氨酸酶(TYR)、酪氨酸酶相关蛋白-1(TRP-1)、酪氨酸酶相关蛋白-2(TRP-2)、小眼畸形相关转录因子(MITF)的蛋白质和mRNA水平。结果 CMP对α-MSH诱导的B16细胞内黑色素生成及酪氨酸酶活力均具有明显的抑制作用(P<0.05),且呈量效关系。当CMP浓度为20 mg·L~(-1)时,对细胞内黑色素生成及酪氨酸酶活性抑制率分别为52.95%、32.85%,阳性对照熊果苷(100 mg·L~(-1))的抑制率分别为17.29%、16.75%,表明CMP对黑色素生成的抑制效果强于熊果苷。与α-MSH模型组相比,CMP干预后细胞内TYR、TRP-1、TRP-2、MITF的mRNA和蛋白表达被明显抑制(P<0.05)。结论 CMP明显抑制α-MSH诱导黑色素的生成,其机制可能是通过调控TYR、TRP-1、TRP-2、MITF mRNA和蛋白表达,进而抑制酪氨酸酶活性实现的。
Aim To investigate the inhibitory effect of polyphenol from Cortex Mori (CMP) on melanogenesis in mouse melanoma B16 cells and its possible mechanism. Methods Melanoma B16 cells with high expression melanin were induced by α-melanocyte-stimulating hormone (α-MSH) to establish cell model. Cell viability was detected by MTT assay. The melanin synthesis and tyrosinase activity were measured by NaOH and L-Dopa assays, respectively. The tyrosinase (TYR),tyrosinase-related protein-1 (TRP-1),tyrosinase-related protein-2 (TRP-2) and microphthalmia associated transcription factor (MITF) protein and mRNA levels were measured by Western blot and qRTPCR,respectively. Results CMP could inhibit the melanin synthesis and tyrosinase activity in α-MSH stimulated B16 cells in a dose-dependent manner (P <0. 05). The melanin content and tyrosinase activity significantly decreased by 52. 95%,32. 85% at 20 mg·L~(-1) of CMP,respectively. Treatment of 100 mg ·L~(-1) of arbutin reduced the melanin content and tyrosinase activity by 17. 29%, 16. 75%, respectively.Based on the results of this study,CMP showed a stronger anti-melanogenesis activity than that of positive control arbutin. After treated by CMP,the protein and mRNA levels of TYR,TRP-1,TRP-2 and MITF were significantly inhibited compared to the α-MSH group (P < 0. 05). Conclusions CMP could suppress the melanogenesis in α-MSH stimulated B16 cells,and its mechanism may be related to its regulation of the protein and mRNA expressions of TYR,TRP-1,TRP-2 and MITF,and the inhibition of tyrosinase activity.
Aim To investigate the inhibitory effect of polyphenol from Cortex Mori (CMP) on melanogenesis in mouse melanoma B16 cells and its possible mechanism. Methods Melanoma B16 cells with high expression melanin were induced by α-melanocyte-stimulating hormone (α-MSH) to establish cell model. Cell viability was detected by MTT assay. The melanin synthesis and tyrosinase activity were measured by NaOH and L-Dopa assays, respectively. The tyrosinase (TYR),tyrosinase-related protein-1 (TRP-1),tyrosinase-related protein-2 (TRP-2) and microphthalmia associated transcription factor (MITF) protein and mRNA levels were measured by Western blot and qRTPCR,respectively. Results CMP could inhibit the melanin synthesis and tyrosinase activity in α-MSH stimulated B16 cells in a dose-dependent manner (P <0. 05). The melanin content and tyrosinase activity significantly decreased by 52. 95%,32. 85% at 20 mg·L~(-1) of CMP,respectively. Treatment of 100 mg ·L~(-1) of arbutin reduced the melanin content and tyrosinase activity by 17. 29%, 16. 75%, respectively.Based on the results of this study,CMP showed a stronger anti-melanogenesis activity than that of positive control arbutin. After treated by CMP,the protein and mRNA levels of TYR,TRP-1,TRP-2 and MITF were significantly inhibited compared to the α-MSH group (P < 0. 05). Conclusions CMP could suppress the melanogenesis in α-MSH stimulated B16 cells,and its mechanism may be related to its regulation of the protein and mRNA expressions of TYR,TRP-1,TRP-2 and MITF,and the inhibition of tyrosinase activity.
引文
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[15]Sim M O,Ham J R,Lee M K.Young leaves of reed(Phragmites communis)suppress melanogenesis and oxidative stress in B16F10melanoma cells[J].Biomed Pharmacother,2017,93:165-71.
[2]Mustapha N,Bzéouich I M,Ghedira K,et al.Compounds isolated from the aerial part of Crataegus azarolus inhibit growth of B16F10 melanoma cells and exert a potent inhibition of the melanin synthesis[J].Biomed Pharmacother,2015,69:139-44.
[3]Kim B H,Park K C,Park J H,et al.Inhibition of tyrosinase activity and melanin production by the chalcone derivative 1-(2-cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone[J].Biochem Biophys Res Commun,2016,480(4):648-54.
[4]张向娜,林勇,黄建安,等.茶叶提取物EGCG和GCG及ECG对B16细胞内黑色素生成的抑制作用[J].湖南农业大学(自然科学版),2017,43(4):405-10.[4]Zhang X N,Lin Y,Huang J A,et al.Inhibitory effects of tea extracts EGCG,GCG and ECG on the melanogenesis in melanoma cell B16[J].J Hunan Agri Univ(Nat Sci),2017,43(4):405-10.
[5]王鹏,李素霞,韩福森,等.葡萄籽提取物对B16细胞内黑色素合成的抑制作用[J].日用化学工业,2013,43(4):280-4.[5]Wang P,Li S X,Han F S,et al.Inhibition effect of grape seed extract on melanin formation in B16 murine melanoma cells[J].China Surf Deter Cosme,2017,43(4):280-4.
[6]李墨灵,张晗,夏庆梅.桑白皮的化学、药理与药代动力学研究进展[J].西部中医药,2017,30(2):137-9.[6]Li M L,Zhang H,Xia Q M,et al.Research progress of chemistry,pharmacology,pharmacokinetics of Sang Bai Pi[J].West J Tradit Chin Med,2017,30(2):137-9.
[7]Butt M S,Nazir A,Sultan M T,et al.Morus alba L.nature’s functional tonic[J].Trends Food Sci Tech,2008,19(10):505-12.
[8]Dai S J,Wu Y,Wang Y H,et al.New diels-alder type adducts from Morus macroura and their antioxidant activities[J].Chem Pharm Bull,2004,52(10):1190-3.
[9]杨利红,赵费敏,张特,等.桑白皮抗炎活性成分的分离及抗炎机制研究[J].中华中医药学刊,2016,34(12):3008-12.[9]Yang L H,Zhao F M,Zhang T,et al.Isolation of active compounds from Cortex Mori and its mechanism on anti-inflammatory[J].Chin Arch Trad Chin Med,2016,34(12):3008-12.
[10]Wu Y X,Kim Y J,Li S,et al.Anti-obese effects of mulberry(Morus alba L.)root bark through the inhibition of digestive enzymes and 3T3-L1 adipocyte differentiation[J].Korean J Food Preser,2015,22(1):27-35.
[11]Singleton V L,Orthofer R,Lamuela-Raventos R M.Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent[J].Method Enzymol,1999,299:152-78.
[12]马艳霞,吴勉华,姜泽群,等.山茱萸环烯醚萜苷对D-Gal N/TNF-α损伤肝细胞的保护作用研究[J].中国药理学通报,2018,34(1):118-22.[12]Ma Y X,Wu M H,Jiang Z Q,et al.Protective effect of cornel iridoid glycoside on hepatocytes injured by D-galactosamine/tumor necrosis factor-α[J].Chin Pharmacol Bull,2018,34(1):118-22.
[13]王艳明,刘瑛,阎新燕,等.甘草查尔酮A抑制小鼠黑色素瘤B16F10细胞增殖机制研究[J].中国药理学通报,2015,31(7):967-72.[13]Wang Y M,Liu Y,Yan X Y,et al.Inhibitory effects of licochalcone A on proliferation of melanoma B16F10 cells[J].Chin Pharmacol Bull,2015,31(7):967-72.
[14]Maack A,Pegard A.Populus nigra(Salicaceae)absolute rich in phenolic acids,phenylpropano6ds and flavonoids as a new potent tyrosinase inhibitor[J].Fitoterapia,2016,111:95-101.
[15]Sim M O,Ham J R,Lee M K.Young leaves of reed(Phragmites communis)suppress melanogenesis and oxidative stress in B16F10melanoma cells[J].Biomed Pharmacother,2017,93:165-71.