安五脂素对α-黑色素细胞刺激素诱导的小鼠黑色素瘤B16F10细胞中黑色素生成的影响及机制  

Effect and mechanism of anwulignan on melanocyte-stimulating hormone-induced melanin production in B16F10 melanoma cells

作　　者：庄文越 苏小明 赵鸣瑶 李贺[3] 王春梅[3] 陈建光[3] 李正祎 邱旭东[5] 杜兴旭[5] ZHUANG Wen-yue;SU Xiao-ming;ZHAO Ming-yao;LI He;WANG Chun-mei;CHEN Jian-guang;LI Zheng-yi;QIU Xu-dong;DU Xing-xu(College of Medical Technology,Jilin 132013,China;Clinical Lab,Jilin Provincial Cancer Hospital,Changchun 130000,China;College of Pharmacy,Jilin 132013,China;Laboratory Academy,Jilin Medical University,Jilin 132013,China;Affiliated Hospital,Beihua University,Jilin 132013,China)

机构地区：[1]北华大学医学技术学院,吉林吉林132013 [2]吉林省肿瘤医院检验科,吉林长春130000 [3]北华大学药学院,吉林吉林132013 [4]吉林医药学院检验学院,吉林吉林132013 [5]北华大学附属医院,吉林吉林132013

出　　处：《中国药理学与毒理学杂志》2022年第5期364-369,共6页Chinese Journal of Pharmacology and Toxicology

基　　金：吉林省科技发展计划项目(20200404053YY);吉林省科技发展计划项目(20160101176JC)。

摘　　要：目的研究安五脂素对小鼠黑色素瘤B16F10细胞黑色素生成的影响及机制。方法体外培养B16F10细胞,用MTT法检测B16F10细胞存活率。随后实验分为细胞对照组、α-黑色素细胞刺激素(α-MSH)诱导模型组和模型+安五脂素5,10和20μmol·L^(-1)组,培养48 h。NaOH裂解法和多巴氧化速率法检测细胞内黑色素含量和酪氨酸酶活性,WST-1法检测细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,化学荧光法检测活性氧(ROS)水平,实时-定量PCR检测血红素加氧酶1(HO-1)mRNA表达水平,Western印迹法检测核因子E2相关因子2(Nrf-2)和HO-1蛋白表达水平。结果安五脂素5,10和20μmol·L^(-1)对B16F10细胞存活无影响。与细胞对照组相比,模型组黑色素含量和酪氨酸酶活性显著升高(P<0.01);SOD活性降低(P<0.01),MDA和ROS水平升高(P<0.01);Nrf-2蛋白及下游HO-1 mRNA和蛋白表达显著降低(P<0.01)。与模型组相比,模型+安五脂素10和20μmol·L^(-1)组B16F10细胞黑色素含量和酪氨酸酶活性降低(P<0.05,P<0.01);SOD活性升高(P<0.01),MDA和ROS含量明显降低(P<0.05,P<0.01);Nrf-2蛋白及下游HO-1 mRNA和蛋白表达水平升高(P<0.01)。结论安五脂素可能通过抑制B16F10细胞氧化应激反应而抑制其黑色素合成,且可能与抑制Nrf-2/HO-1信号通路有关。OBJECTIVE To investigate the effect and mechanism of anwulignan on melanin production in melanoma cells.METHODS B16F10 cells were cultured in vitro and MTT was performed to assess cell viability.The experiment was divided into the control group,α-melanocyte-stimulating hormone(α-MSH)model group and model+anwulignan 5,10 and 20μmol·L^(-1) groups.Melanin content was detected using the NaOH decomposition method.Tyrosinase activity was determined by measuring the rate of dopachrome formation of levodopa.The activity of superoxide dismutase(SOD)and content of malondialdehyde(MDA)were determined by WST-1 assay.Chemical fluorescence assay was used to detect the content of reactive oxygen species(ROS).The mRNA expression of heme oxygenase-1(HO-1)was measured by real time-quantitative reverse transcription polymerase chain reaction.The protein expressions of nuclear factor erythroid 2-related factor 2(Nrf-2)and HO-1 were measured by Western blotting.RESULTS Anwulignan 5,10 and 20μmol·L^(-1) had no significant effect on the viability of B16F10 cells.Compared with the cell control group,the melanin content and tyrosinase activity were significantly enhanced(P<0.01),the intracellular SOD activity was decreased(P<0.01),the MDA and ROS contents were increased(P<0.01),the expressions of Nrf-2 protein and its downstream target HO-1 mRNA and protein were significantly reduced(P<0.01)in the model group.Compared with the model group,the melanin content was reduced(P<0.05,P<0.01),the activity of tyrosinase was inhibited(P<0.05,P<0.01),the intracellular SOD activity was increased(P<0.01),the MDA and ROS contents were significantly decreased(P<0.05,P<0.01),and the expressions of Nrf-2 protein and its downstream target HO-1 mRNA and protein were enhanced(P<0.01)in model+anwulignan 10 and 20μmol·L^(-1) groups.CONCLUSION Anwulignan may inhibit melanin production by suppressing oxidative stress.The mechanism may be related to its inhibition of Nrf-2/HO-1 signal pathway.

关 键 词：安五脂素 黑色素 核因子-E2相关因子2 血红素加氧酶1 

分 类 号：R285.5[医药卫生—中药学]