中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (11): 797-801.doi: 10.3760/cma.j.issn.0412-4030.2018.11.004

• 论著 • 上一篇    下一篇

京尼平苷通过PI3K-Akt途径拮抗黑素细胞氧化损伤

鹿文静 赵煜 王克玉   

  1. 山东大学齐鲁医院(青岛)皮肤科(鹿文静);山东大学齐鲁医院皮肤科(赵煜、王克玉)
  • 收稿日期:2018-01-26 修回日期:2018-08-20 出版日期:2018-11-16 发布日期:2018-10-31
  • 通讯作者: 王克玉 E-mail:qlyywky@163.com
  • 基金资助:
    山东省科技发展计划项目(2015GSF119006)

Geniposide protects melanocytes from oxidative damage through the PI3K-Akt pathway

Lu Wenjing, Zhao Yu, Wang Keyu   

  1. Department of Dermatology, Qilu Hospital of Shandong University (Qingdao), Qingdao 266035, China (Lu WJ); Department of Dermatology, Qilu Hospital of Shandong University, Jinan 250012, China (Zhao Y, Wang KY)
  • Received:2018-01-26 Revised:2018-08-20 Online:2018-11-16 Published:2018-10-31
  • Contact: Wang Keyu E-mail:qlyywky@163.com
  • Supported by:
    Shandong Science and Technology Development Project(2015GSF119006)

摘要: 目的 探讨京尼平苷对体外培养的人黑素细胞氧化损伤的拮抗作用,以及PI3K-Akt途径在其中的作用。方法 取健康青少年环切术后包皮,分离并培养表皮黑素细胞。取第2 ~ 3代黑素细胞,分为对照组(不做任何处理)、京尼平苷组(125 μmol/L京尼平苷处理)、LY294002组(5 μmol/L LY294002处理)、H2O2组(250 μmol/L H2O2处理)、京尼平苷 + H2O2组(125 μmol/L京尼平苷处理24 h后,加入250 μmol/L H2O2作用4 h)、京尼平苷 + LY294002 + H2O2组(125 μmol/L京尼平苷处理24 h后,加入5 μmol/L LY294002作用1 h,然后用 250 μmol/L H2O2作用4 h)。作用完成后,用噻唑蓝(MTT)法检测黑素细胞增殖活性,Western印迹检测Akt、磷酸化Akt(p-Akt)、血红素加氧酶1(HO-1)、谷胱甘肽过氧化物酶1(GPx-1)蛋白的表达,生物化学方法检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,流式细胞仪检测细胞内活性氧(ROS)含量。通过单因素方差分析(ANOVA)对各组间数据进行比较,采用SNK-q检验进行组间两两比较。结果 H2O2组细胞增殖率较对照组明显降低(P < 0.01),p-Akt(P < 0.01)、HO-1(P < 0.01)、GPx-1(P < 0.05)蛋白表达水平下降,SOD活性、CAT活性降低(均P < 0.01),ROS含量显著升高(P < 0.01)。与H2O2组相比,京尼平苷+ H2O2组黑素细胞增殖率上升(72.98% ± 8.92%比50.53% ± 10.85%,P < 0.05),p-Akt(P < 0.05)、HO-1(P < 0.01)、GPx-1(P < 0.01)蛋白表达上调,SOD[(6.82 ± 1.03) U/mg比(1.29 ± 0.43 ) U/mg,P < 0.05]和CAT[(46.08 ± 4.16) U/mg比(18.71 ± 3.09) U/mg,P < 0.05]酶活性增高,ROS含量(1 284.33 ± 110.64比2 158 ± 222.75,P < 0.01)减少;京尼平苷 + LY294002 + H2O2组黑素细胞增殖率(44.35% ± 14.85%)较京尼平苷 + H2O2组降低(P < 0.05),p-Akt(P < 0.01)、HO-1(P < 0.05)、GPx-1(P < 0.01)蛋白表达下调,SOD活性[(1.31 ± 0.65) U/mg,P < 0.05]和CAT活性[(23.25 ± 5.56) U/mg,P < 0.05]减弱,ROS含量增加(1 668 ± 62.03,P < 0.05)。结论 京尼平苷可通过PI3K-Akt途径促进黑素细胞HO-1和GPx-1表达,增强SOD和CAT活性,拮抗黑素细胞氧化应激损伤。

关键词: 黑素细胞, 白癜风, 氧化性应激, 磷酸肌醇3-激酶类, 癌基因蛋白质v?akt, 超氧化物歧化酶, 过氧化氢酶, 京尼平苷

Abstract: Lu Wenjing, Zhao Yu, Wang Keyu Department of Dermatology, Qilu Hospital of Shandong University (Qingdao), Qingdao 266035, China (Lu WJ); Department of Dermatology, Qilu Hospital of Shandong University, Jinan 250012, China (Zhao Y, Wang KY) Corresponding author: Wang Keyu, Email: qlyywky@163.com 【Abstract】 Objective To evaluate the inhibitory effect of geniposide on the oxidative damage to in vitro cultured human melanocytes, and to explore the role of PI3K-Akt signaling pathway in this inhibitory effect. Methods Epidermal melanocytes were isolated from the circumcised foreskin of healthy adolescent males, and then subjected to culture. Melanocytes at passage 2 - 3 were divided to six groups: control group receiving no treatment, geniposide group treated with 125 μmol/L geniposide alone, LY294002 group treated with 5 μmol/L LY294002 alone, H2O2 group treated with 250 μmol/L H2O2 alone, geniposide + H2O2 group firstly treated with 125 μmol/L geniposide for 24 hours followed by 4-hour treatment with 250 μmol/L H2O2, and geniposide + LY294002 + H2O2 group treated with 125 μmol/L geniposide for 24 hours, then with 5 μmol/L LY294002 for 1 hour, followed by 4-hour treatment with 250 μmol/L H2O2. After the above treatment, methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cellular proliferative activity, Western blot analysis to determine the protein expression of Akt, phosphorylated Akt (p-Akt), heme oxygenase1 (HO-1) and glutathione peroxidase (GPx-1), and flow cytometry to detect the level of cellular reactive oxygen species (ROS), and biochemical methods were used to evaluate the activity of superoxide dismutase (SOD) and catalase (CAT). Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparison, and Student-Newman-Keuls-q (SNK-q) test for multiple com-parisons. Results Compared with the control group, the H2O2 group showed significantly decreased cellular proliferative activity (P < 0.01), protein expression of p-Akt (P < 0.01), HO-1 (P < 0.01) and GPx-1 (P < 0.05), SOD activity (P < 0.01) and CAT activity (P < 0.01), but significantly increased ROS level (P < 0.01). Compared with the H2O2 group, the geniposide + H2O2 group showed significantly increased cellular proliferative activity (72.98% ± 8.92% vs. 50.53% ± 10.85%, P < 0.05), up-regulated protein expression of p-Akt (P < 0.05), HO-1 (P < 0.01) and GPx-1 (P < 0.01), increased SOD activity (6.82 ± 1.03 U/mg vs. 1.29 ± 0.43 U/mg, P < 0.05) and CAT activity (46.08 ± 4.16 U/mg vs. 18.71 ± 3.09 U/mg, P < 0.05), but decreased ROS level (1 284.33 ± 110.64 vs. 2 158.00 ± 222.75, P < 0.01). The proliferative activity of melanocytes was significantly lower in the geniposide + LY294002 + H2O2 group (44.35% ± 14.85%) than in the geniposide + H2O2 group (P < 0.05). In addition, the geniposide + LY294002 + H2O2 group showed significantly decreased protein expression of p-Akt (P < 0.01), HO-1 (P < 0.05) and GPx-1 (P < 0.01), SOD (1.31 ± 0.65 U/mg, P < 0.05) and CAT activity (23.25 ± 5.56 U/mg, P < 0.05), but significantly increased ROS level (1 668.00 ± 62.03, P < 0.05)compared with the geniposide + H2O2 group. Conclusion Through the PI3K-Akt pathway, geniposide can promote the protein expression of HO-1 and GPx-1 in melanocytes, enhance SOD and CAT activity, and antagonize the oxidative damage of melanocytes.

Key words: Melanocytes, Vitiligo, Oxidative stress, Phosphatidylinositol 3?kinases, Oncogene protein v?akt, Superoxide dismutase, Catalase, Geniposide