白桦脂醇通过ER/MAPK信号通路对A375细胞黑素合成及机制
|
摘要
目的:研究植物雌激素成分白桦脂醇及加入受体阻断剂(ICI182780)后对A375细胞的黑素合成及机制的影响。方法:将1μmol·L~(-1)白桦脂醇作用于A375黑素细胞,实验分为空白组(DMEM完全培养液),雌二醇组(1×10~(-3)μmol·L~(-1)),白桦脂醇组(1μmol·L~(-1)),白桦脂醇+ICI182780组(1μmol·L~(-1)+1μmol·L~(-1))。用Na OH裂解法检测黑素含量、蛋白免疫印迹法(Western blot)和实时荧光定量PCR法(Real-time PCR)测定雌激素受体/丝裂原激活的蛋白激酶(ER/MAPK)通路中关键蛋白激酶p38 MAPK,ERβ,c-Jun氨基末端激酶(JNK),细胞外信号调节激酶2(ERK2)和小眼畸形相关转录因子(MITF),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)的蛋白及其mRNA表达。结果:与空白组比较,白桦脂醇组和白桦脂醇+ICI182780组对A375细胞黑素合成有显著抑制作用(P<0.01);1μmol·L~(-1)的白桦脂醇对A375细胞中上述蛋白表达有不同程度的抑制作用(P<0.05,P<0.01),对ERK2,MITF,TRP-1 mRNA的表达也有不同程度的抑制作用(P<0.05,P<0.01)。与白桦脂醇组比较,ICI182780可以逆转白桦脂醇对A375细胞黑素合成的抑制作用(P<0.01);ICI182780可以逆转白桦脂醇对A375细胞中上述蛋白表达的抑制作用及对ERK2,MITF,TRP-1 mRNA的表达的抑制作用(P<0.05,P<0.01)。结论:白桦脂醇能够通过ER/MAPK信号通路下调MITF,TYR,TRP-1及TRP-2蛋白表达量,从而减少黑素合成。
Objective: To study the effect of phytoestrogenic components of betulin and the addition of receptor blocker( ICI182780) on melanin synthesis and its mechanism on A375 cells. Method: 1 μmol·L~(-1) betulin was applied to A375 melanocytes and then the experimental cells were divided into blank group( DMEM complete culture fluid),estradiol group( 1 × 10~(-3)μmol·L~(-1)),betulin group( 1 μmol·L~(-1)),and betulin +ICI182780 group( 1 μmol·L~(-1)+ 1 μmol·L~(-1)). Melanin content was determined by Na OH lysis; Western blot,and real-time fluorescent quantitative polymerase chain reaction( Real-time PCR) were used to determine the protein content and mRNA expression of key protein kinase mitogen activated protein kinase( MAPK) p38 in estrogen receptor( ER)/mitogen activated protein kinase( MAPK) pathway,c-Jun N-terminal kinase( JNK),extracellular signal-regulated kinase 2( ERK2) and micrometamorphic-related transcription factor( MITF),dopa oxidation assay for tyrosinase( TYR),tyrosinase-related protein~(-1)( TRP-1),and tyrosinase-related protein-2( TRP-2). Result: As compared with the blank group,betulin group and betulin + ICI182780 group had significant inhibition on melanin synthesis in A375 cells( P < 0. 01); 1 μmol·L~(-1) betulin had different inhibitory effects on expression of the above proteins in A375 cells( P < 0. 05,P < 0. 01),as well as on ERK2,MITF,and TRP-1( P <0. 05,P < 0. 01) mRNA expression( P < 0. 05,P < 0. 01). As compared with the betulin group,ICI182780 can reverse the inhibitory effect of betulin on melanin synthesis of A375 cells( P < 0. 01); ICI182780 can reverse the inhibitory effect of betulin on the above protein expression in A375 cells as well as mRNA expression of ERK2,MITF,and TRP-1( P < 0. 05,P < 0. 01). Conclusion: Betulin can down-regulate the expression levels of MITF,TYR,TRP-1 and TRP-2 proteins through ER-MAPK signaling pathway,thus reducing melanin synthesis.
Objective: To study the effect of phytoestrogenic components of betulin and the addition of receptor blocker( ICI182780) on melanin synthesis and its mechanism on A375 cells. Method: 1 μmol·L~(-1) betulin was applied to A375 melanocytes and then the experimental cells were divided into blank group( DMEM complete culture fluid),estradiol group( 1 × 10~(-3)μmol·L~(-1)),betulin group( 1 μmol·L~(-1)),and betulin +ICI182780 group( 1 μmol·L~(-1)+ 1 μmol·L~(-1)). Melanin content was determined by Na OH lysis; Western blot,and real-time fluorescent quantitative polymerase chain reaction( Real-time PCR) were used to determine the protein content and mRNA expression of key protein kinase mitogen activated protein kinase( MAPK) p38 in estrogen receptor( ER)/mitogen activated protein kinase( MAPK) pathway,c-Jun N-terminal kinase( JNK),extracellular signal-regulated kinase 2( ERK2) and micrometamorphic-related transcription factor( MITF),dopa oxidation assay for tyrosinase( TYR),tyrosinase-related protein~(-1)( TRP-1),and tyrosinase-related protein-2( TRP-2). Result: As compared with the blank group,betulin group and betulin + ICI182780 group had significant inhibition on melanin synthesis in A375 cells( P < 0. 01); 1 μmol·L~(-1) betulin had different inhibitory effects on expression of the above proteins in A375 cells( P < 0. 05,P < 0. 01),as well as on ERK2,MITF,and TRP-1( P <0. 05,P < 0. 01) mRNA expression( P < 0. 05,P < 0. 01). As compared with the betulin group,ICI182780 can reverse the inhibitory effect of betulin on melanin synthesis of A375 cells( P < 0. 01); ICI182780 can reverse the inhibitory effect of betulin on the above protein expression in A375 cells as well as mRNA expression of ERK2,MITF,and TRP-1( P < 0. 05,P < 0. 01). Conclusion: Betulin can down-regulate the expression levels of MITF,TYR,TRP-1 and TRP-2 proteins through ER-MAPK signaling pathway,thus reducing melanin synthesis.
引文
[1]杨玮华.黄褐斑西医研究进展[J].中医临床研究,2016,8(12):68-74.
[2]王帅.三种植物雌激素成分A375细胞黑素合成的作用机制研究[D].哈尔滨:黑龙江中医药大学,2015.
[3]王银娟.黄褐斑相关发病机制的研究[D].昆明:昆明医科大学,2014.
[4]李志鸿,金娟.杨智荣教授祛斑养颜汤治疗女性黄褐斑临床疗效及对血清性激素水平影响的观察[J].中医药信息,2015,32(2):95-97.
[5]柏亚萍,薛至军.蔡瑞康教授治疗黄褐斑经验[J].世界中医西医结合杂志,2010,5(11):78-81.
[6]侯慧先,赵起.刺络放血拔罐疗法治疗黄褐斑(肝郁气滞型)的临床观察[J].中医药信息,2014,31(2):93-94.
[7]魏娜,贺海波,张长城,等.JNK信号通路与细胞凋亡关系的研究进展[J].中国临床药理学与治疗学,2013,18(7):807-812.
[8]Razandi M,Pedram A,Greene G L,et al.Cell membrane and nuclear estrogen receptprs(ERs)originate from a single transcript:studies of ERαand ERβexpressed in chinese hamster ovary cells[J].Mol Endocrinol,2009,13(2):307-319.
[9]Matsuoka H,Tsubaki M,Yamazoe Y,et al.Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways[J].Exp Cell Res,2009,315(12):2022-2032.
[10]唐建兵.雌激素在黄褐斑发病中的作用及黄褐斑的治疗研究[D].广州:南方医科大学,2011.
[11]李铁柱.杜仲果实和叶片转录组数据组及基因功能注释[J].中南林业科技大学学报,2012,32(11):122-130.
[12]袁天翔,方莲花,吕杨,等.杜仲叶的药理作用研究进展[J].中国中药杂志,2013,38(6):781-785.
[13]王志宏,彭胜,雷明盛,等.杜仲主要生物活性研究进展[J].天然产物研究与开发,2013,25:1302-1309.
[14]严颖,赵慧,邹立思,等.基于LC-QTOF MS/MS技术分析不同产地杜仲的差异化学成分[J].中国中药杂志,2017,42(14):2730-2737.
[15]张大维,李利.黄褐斑治疗的研究进展[J].现代临床医学,2008,34(5):391-396.
[16]王玉林,何锦凤,王维民,等.皮肤黑色素的产生及美白浅析[J].日用化学品科学,2013,36(2):33-36.
[17]Seong Z K,Lee S Y,Poudel A,et al.Constituents of cryptotaenia japonica inhibit melanogenesis via CREBand MAPK-assocuated signaling pathways in murine B16melanoma cells[J].Molecules,2016,21(10):1296.
[18]Ng L T,LIN L T,CHEN C L,et al.Anti-melanogenic effects ofδ-tocotrienol are associated with tyrosinaserelated proteins and MAPK signaling pathway in B16melanoma cells[J].Phytomedicine,2014,21(7):978-983.
[2]王帅.三种植物雌激素成分A375细胞黑素合成的作用机制研究[D].哈尔滨:黑龙江中医药大学,2015.
[3]王银娟.黄褐斑相关发病机制的研究[D].昆明:昆明医科大学,2014.
[4]李志鸿,金娟.杨智荣教授祛斑养颜汤治疗女性黄褐斑临床疗效及对血清性激素水平影响的观察[J].中医药信息,2015,32(2):95-97.
[5]柏亚萍,薛至军.蔡瑞康教授治疗黄褐斑经验[J].世界中医西医结合杂志,2010,5(11):78-81.
[6]侯慧先,赵起.刺络放血拔罐疗法治疗黄褐斑(肝郁气滞型)的临床观察[J].中医药信息,2014,31(2):93-94.
[7]魏娜,贺海波,张长城,等.JNK信号通路与细胞凋亡关系的研究进展[J].中国临床药理学与治疗学,2013,18(7):807-812.
[8]Razandi M,Pedram A,Greene G L,et al.Cell membrane and nuclear estrogen receptprs(ERs)originate from a single transcript:studies of ERαand ERβexpressed in chinese hamster ovary cells[J].Mol Endocrinol,2009,13(2):307-319.
[9]Matsuoka H,Tsubaki M,Yamazoe Y,et al.Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways[J].Exp Cell Res,2009,315(12):2022-2032.
[10]唐建兵.雌激素在黄褐斑发病中的作用及黄褐斑的治疗研究[D].广州:南方医科大学,2011.
[11]李铁柱.杜仲果实和叶片转录组数据组及基因功能注释[J].中南林业科技大学学报,2012,32(11):122-130.
[12]袁天翔,方莲花,吕杨,等.杜仲叶的药理作用研究进展[J].中国中药杂志,2013,38(6):781-785.
[13]王志宏,彭胜,雷明盛,等.杜仲主要生物活性研究进展[J].天然产物研究与开发,2013,25:1302-1309.
[14]严颖,赵慧,邹立思,等.基于LC-QTOF MS/MS技术分析不同产地杜仲的差异化学成分[J].中国中药杂志,2017,42(14):2730-2737.
[15]张大维,李利.黄褐斑治疗的研究进展[J].现代临床医学,2008,34(5):391-396.
[16]王玉林,何锦凤,王维民,等.皮肤黑色素的产生及美白浅析[J].日用化学品科学,2013,36(2):33-36.
[17]Seong Z K,Lee S Y,Poudel A,et al.Constituents of cryptotaenia japonica inhibit melanogenesis via CREBand MAPK-assocuated signaling pathways in murine B16melanoma cells[J].Molecules,2016,21(10):1296.
[18]Ng L T,LIN L T,CHEN C L,et al.Anti-melanogenic effects ofδ-tocotrienol are associated with tyrosinaserelated proteins and MAPK signaling pathway in B16melanoma cells[J].Phytomedicine,2014,21(7):978-983.