中国农业科学 ›› 2023, Vol. 56 ›› Issue (5): 1007-1018.doi: 10.3864/j.issn.0578-1752.2023.05.015

• 研究简报 • 上一篇    

姜黄素通过SIRT1/FOXO1通路缓解玉米赤霉烯酮诱导的猪肾上皮细胞氧化损伤

崔红杰1,2(), 卢春亭1, 潘丽琴1, 胡会1, 钟佩云1, 朱洁莹1, 张凯照1,2, 黄小红1,2()   

  1. 1 福建农林大学动物科学学院,福州 350002
    2 中西兽医结合与动物保健福建省高校重点实验室/福建省兽医中药与动物保健重点实验室,福建农林大学,福州 350002
  • 收稿日期:2021-10-21 接受日期:2022-05-17 出版日期:2023-03-01 发布日期:2023-03-13
  • 通信作者: 黄小红,Tel:13774550003;E-mail:984158392@qq.com
  • 联系方式: 崔红杰,Tel:13696892917;E-mail:tsuisearcher@163.com。
  • 基金资助:
    福建省科技厅重大专项(2021NZ029008); 福建省自然科学基金(2021J01080); 福建省中青年教师教育科研项目(JAT210066); 横向课题项目(KH190363A)

Curcumin Alleviates Zearalenone-Induced Oxidative Damage in Porcine Renal Epithelial Cells via SIRT1/FOXO1 Pathway

CUI HongJie1,2(), LU ChunTing1, PAN LiQin1, HU Hui1, ZHONG PeiYun1, ZHU JieYing1, ZHANG KaiZhao1,2, HUANG XiaoHong1,2()   

  1. 1 College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002
    2 University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province/Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou 350002
  • Received:2021-10-21 Accepted:2022-05-17 Published:2023-03-01 Online:2023-03-13

摘要:

【目的】探究姜黄素(Cur)对玉米赤霉烯酮(ZEA)诱导猪肾上皮细胞(PK-15)氧化损伤的保护作用,并基于SIRT1/FOXO1信号通路阐明其作用机制,为姜黄素的兽医临床应用提供依据。【方法】试验分为5组:对照组、ZEA组(36.55 μg·mL-1 ZEA)、Cur 6.25组(36.55 μg·mL-1 ZEA+6.25 μmol·L-1 Cur)、Cur 12.5组(36.55 μg·mL-1 ZEA+12.5 μmol·L-1 Cur)、Cur 25组(36.55 μg·mL-1 ZEA +25 μmol·L-1 Cur);通过MTT法测定ZEA的半数抑制浓度和Cur对PK-15细胞的最大安全浓度;使用倒置显微镜观察PK-15细胞的形态变化;采用试剂盒检测细胞内活性氧(ROS)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)以及丙二醛(MDA)的水平;qRT-PCR检测细胞SIRT1、FOXO1、CAT、Mn-SOD的mRNA水平;Western Blot检测SIRT1、FOXO1和Acetyl-FOXO1蛋白的表达量。【结果】ZEA的IC50为36.55 μg·mL-1,Cur的最大安全浓度为25 μmol·L-1;与对照组相比,ZEA极显著降低PK-15细胞活力(P<0.01),极显著提高ROS和MDA水平(P<0.01),极显著降低SOD、CAT活性(P<0.01);与ZEA组对比,不同浓度的Cur(6.25、12.5、25 μmol·L-1)显著提高PK-15细胞的存活率(P<0.05),改善细胞形态;极显著(P<0.01)降低ZEA诱导的ROS和MDA水平;极显著升高细胞内SOD和CAT活性(P<0.01)。qRT-PCR结果显示,与对照组相比,ZEA降低SIRT1 mRNA表达量,极显著升高FOXO1 mRNA表达量(P<0.01),升高Mn-SOD mRNA表达量,极显著降低CAT mRNA表达量(P<0.01)。与ZEA组对比,各Cur组不同程度的升高SIRT1和CAT的mRNA表达量,极显著的降低FOXO1的mRNA表达量(P<0.01);极显著升高Mn-SOD的mRNA表达量(P<0.01)。Western Blot结果表明,与对照组对比,ZEA显著降低SIRT1蛋白表达(P<0.05),极显著升高Acetyl-FOXO1蛋白表达(P<0.01);与ZEA组对比,各Cur组均极显著升高SIRT1蛋白表达(P<0.01),极显著降低Acetyl-FOXO1蛋白表达(P<0.01)。【结论】Cur可以上调SIRT1的表达,降低FOXO1的乙酰化水平,诱导抗氧化酶Mn-SOD和CAT的表达,从而清除ROS,降低MDA水平,减轻ZEA对PK-15细胞的氧化损伤作用。

关键词: 姜黄素, 玉米赤霉烯酮, 氧化应激, 沉默信息调节因子1 (SIRT1)

Abstract:

【Objective】 The purpose of this study was to investigate the protective effect of curcumin (Cur) on zearalenone (ZEA)-induced oxidative damage in porcine renal epithelial cells (PK-15), and to elucidate the protective mechanism based on SIRT1/FOXO1 signaling pathway. 【Method】 The experiment was divided into 5 groups: control group, ZEA group (36.55 μg·mL-1 ZEA), Cur6.25 group (36.55 μg·mL-1 ZEA+6.25 μmol·L-1 Cur), Cur12.5 group (36.55 μg·mL-1 ZEA+12.5 μmol·L-1 Cur), and Cur25 group (36.55 μg·mL-1 ZEA +25 μmol·L-1 Cur). MTT assay was used to determine the half inhibitory concentration of ZEA and the maximum safe concentration of Cur on PK-15 cells. The morphological changes were observed by inverted microscope. The levels of intracellular reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were detected by the reagent kits. Real-time quantitative PCR was used to detect the mRNA levels of SIRT1, FOXO1, CAT and Mn-SOD. The expression levels of SIRT1, FOXO1 and Acetyl-FOXO1 proteins were detected by Western Blot. 【Result】 The IC50 of ZEA was 36.55 μg·mL-1, and the maximum safe concentration of Cur was 25 μmol·L-1. Compared with the control group, ZEA significantly decreased the cell viability of PK-15 cells (P<0.01), significantly increased the levels of ROS and MDA (P<0.01), and significantly decreased the activities of SOD and CAT (P<0.01). Compared with ZEA group, the different concentrations of Cur (6.25, 12.5, 25 μmol·L-1) significantly increased the cell viability of PK-15 cells (P<0.05) and improved the cell morphology. ROS and MDA levels induced by ZEA were also significantly reduced by Cur (P<0.01). Moreover, SOD and CAT activities in cells were significantly increased (P<0.01). qRT-PCR results showed that, compared with the control group, ZEA decreased SIRT1 mRNA expression, significantly increased FOXO1 mRNA expression (P<0.01), increased Mn-SOD mRNA expression, and significantly decreased CAT mRNA expression (P<0.01). Compared with ZEA group, mRNA expression levels of SIRT1 and CAT were increased in different degrees, FOXO1 mRNA expression levels were significantly decreased (P<0.01), and Mn-SOD mRNA expression levels were significantly increased (P<0.01) in all Cur groups. Western Blot results showed that ZEA significantly reduced SIRT1 protein expression (P<0.05), and significantly increased Acetyl-FOXO1 protein expression (P<0.01). Compared with ZEA group, SIRT1 protein expression was significantly increased (P<0.01), while Acetyl-FOXO1 protein expression was significantly decreased (P<0.01) in all Cur groups.【Conclusion】 Cur could up-regulate the expression of SIRT1, reduce the acetylation level of FOXO1, and induce the expression of antioxidant enzymes Mn-SOD and CAT, thereby eliminating ROS, reducing the level of MDA, and alleviating the oxidative damage of ZEA on PK-15 cells.

Key words: curcumin, zearalenone, oxidative stress, silencing information regulator 1 (SIRT1)