Effects of Kushenol F on oxidative stress and autophagy in melanocytes

SHI Jiachen; LI Xiaorui; WU Ping; ZHAO Pengfei; LI Yongmei

doi:10.16306/j.1008-861x.2021.04.011

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Effects of Kushenol F on oxidative stress and autophagy in melanocytes

Experiment Research | 更新时间：2022-09-09

- Effects of Kushenol F on oxidative stress and autophagy in melanocytes
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 35, Issue 4, Pages: 73-78(2021)
- 作者机构：
  
  1.上海中医药大学附属龙华医院皮肤科（上海　200032）
  2.上海中医药大学（上海　201203）
  3.南京中医药大学附属苏州市中医医院（江苏　苏州　215000）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2021.04.011
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SHI Jiachen, LI Xiaorui, WU Ping, et al. Effects of Kushenol F on oxidative stress and autophagy in melanocytes. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(4):73-78(2021)
DOI：

SHI Jiachen, LI Xiaorui, WU Ping, et al. Effects of Kushenol F on oxidative stress and autophagy in melanocytes. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(4):73-78(2021) DOI： 10.16306/j.1008-861x.2021.04.011.

摘要

目的：,2,探究苦参新醇F对黑素细胞氧化应激及自噬的影响。,方法：,2,以PIG1细胞株为研究对象，将细胞分为对照组、模型组、阳性对照（小檗碱，5 μmol/L）组以及苦参新醇F低、中、高剂量（26、52、78 μmol/L）组。除对照组外，其他各组加入1.0 mmol/L过氧化氢（H,2,O,2,）干预24 h，以诱导氧化应激损伤。H,2,O,2,刺激后，各药物组给予相应药物干预24 h。CCK8法检测细胞活力；ELISA检测活性氧（ROS）、丙二醛（MDA）、谷胱甘肽（GSH）水平；透射电镜观察自噬小体；免疫荧光检测E-钙黏素蛋白表达；Western blot检测氧化应激及自噬相关蛋白的表达，包括：Beclin-1、P62、微管相关蛋白1轻链3（LC3）、核转录因子E2相关因子2（Nrf2）。,结果：,2,①与模型组相比，苦参新醇F不同剂量组的细胞活力均显著升高（,P,<,0.01），且高剂量组细胞活力明显高于低剂量组、阳性对照组（,P,<,0.05）。②与模型组相比，苦参新醇F不同剂量组的ROS、MDA水平明显降低（,P,<,0.05，,P,<,0.01），GSH水平明显升高（,P,<,0.05，,P,<,0.01）；且高剂量组的ROS、MDA水平低于低剂量组、阳性对照组（,P,<,0.05，,P,<,0.01），GSH水平高于低剂量组（,P,<,0.05）。③与模型组相比，苦参新醇F不同剂量组的自噬小体数目均显著增多（,P,<,0.05，,P,<,0.01）；且中、高剂量组的自噬小体数目明显多于低剂量组（,P,<,0.05，,P,<,0.01）。④苦参新醇F中、高剂量组E-钙黏素表达较模型组显著升高（,P,<,0.05，,P,<,0.01），且明显高于低剂量组（,P,<,0.05，,P,<,0.01）。⑤与模型组相比，不同剂量苦参新醇F干预后，Beclin-1、LC3Ⅱ/LC3Ⅰ、Nrf2、P62蛋白表达水平均显著升高（,P,<,0.05，,P,<,0.01）。,结论：,2,苦参新醇F可能通过上调Nrf2-P62信号通路，促进黑素细胞自噬，从而抵抗氧化应激损伤。

Abstract

Objective:,2,To explore the effects of Kushenol F on oxidative stress and autophagy in melanocytes.,Methods:,2,PIG1 cell line was used as the research object.The cells were divided into the control group, model group, positive control(berberine, 5 μmol/L) group and Kushenol F groups with low-(26 μmol/L), middle-(52 μmol/L) and high-(78 μmol/L) dose.Except the control group, the other groups were treated with 1.0 mmol/L hydrogen peroxide(H,2,O,2,) for 24 hours to induce oxidative stress injury.After H,2,O,2, stimulation, each drug group was treated with the corresponding drug for 24 hours.The cell viability was detected by CCK8 assay.The levels of reactive oxygen species(ROS), malondialdehyde(MDA) and glutathione(GSH) were detected by ELISA.The autophagosomes were observed by transmission electron microscope.The expression of E-cadherin was detected by immunofluorescence.The expressions of oxidative stress and autophagy related proteins were detected by Western blot, including Beclin-1, P62, microtubule-associated protein 1 light chain 3(LC3) and nuclear factor erythroid 2-related factor 2(Nrf2) .,Results:,2,①Compared with the model group, the cell viability of Kushenol F groups with different doses was significantly increased(,P,<,0.01), and the cell viability of high-dose group was significantly higher than that of low-dose group and positive control group(,P,<,0.05) .②Compared with the model group, the levels of ROS and MDA were significantly decreased(,P,<,0.05,P,<,0.01) and the level of GSH was significantly increased(,P,<,0.05,P,<,0.01) in the Kushenol F groups with different doses.The levels of ROS and MDA in the high-dose group were lower than those in the low-dose group and positive control group((,P,<,0.05,P,<,0.01), and the level of GSH in the high-dose group was higher than that in the low-dose group(,P,<,0.05) .③Compared with the model group, the number of autophagosomes in the Kushenol F groups with different doses was significantly increased(,P,<,0.05,P,<,0.01) ; and the number of autophagosomes in the middle-and high-dose groups was significantly more than that in the low-dose group(,P,<,0.05,P,<,0.01) .④The expression of E-cadherin in the Kushenol F groups with middle-and high-dose was significantly higher than that in the model group(,P,<,0.05,P,<,0.01), and was significantly higher than that in the low-dose group(,P,<,0.05,P,<,0.01) .⑤Compared with the model group, after treatment with Kushenol F at different doses, the protein expression levels of Beclin-1, LC3Ⅱ/LC3Ⅰ, Nrf2 and P62 were significantly increased(,P,<,0.05,P,<,0.01) .,Conclusion:,2,Kushenol F may up-regulate Nrf2-P62 signaling pathway, promote melanocyte autophagy, and thus resist oxidative stress injury.

关键词

苦参新醇F白癜风黑素细胞氧化应激自噬

Keywords

Kushenol Fvitiligomelanocyteoxidative stressautophagy

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