Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (5): 1007-1018.doi: 10.3864/j.issn.0578-1752.2023.05.015

• RESEARCH NOTES • Previous Articles    

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 Online:2023-03-01 Published:2023-03-13

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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)

Table 1

Primer sequences for Real-time PCR"

基因 Target gene 登录号 Accession No. 引物序列Primer sequence (5′→3′) 片段长度 Product length (bp)
SIRT1 NM_001145750.2 F:CGGCAGGAGAAGGAAACAATGGG
R:TCGTCGTCGTCGTCGTAGAAGTC		 88
FOXO1 NM_214014.3 F:TGTCCTACGCCGACCTCATCAC
R:GCACGCTCTTGACCATCCACTC		 96
CAT NM_214301.2 F:CGCCTATTTGCCTATCCTGACACTC
R:GCACGGAAGGGACAGTTCACAG		 83
Mn-SOD NM_214127.2 F:TTTCTGGACAAATCTGAGCCCTAACG
R:CGACGGATACAGCGGTCAACTTC		 122
β-Actin NC_010445.4 F:CAAGGACCTCTACGCCAACAC
R: TGGAGGCGCGATGATCTT		 130

Table 2

Real-time PCR reaction system"

试剂
Reagent		 体积
Volume (μL)
TB Green Premix Ex Taq П 10
上游引物 Upstream Primer 0.8
下游引物 Travelling Primer 0.8
Rox Reference Dye П (50×) 0.4
DNA模板 DNA template 2
灭菌水 Sterile water 6
总体系 Overall system 20

Table 3

PCR reaction procedure"

步骤 Step 循环数 Number of cycles 温度Temperature (℃) 时间Time (s)
预变性 Pre-denaturation 1 95 30
PCR反应PCR reaction 40 95 5
40 60 34

Fig. 1

Inhibitory effect of ZEA on PK-15 cell viability"

Fig. 2

Effect of curcumin on the viability of PK-15 cells Compared with control group, * means significant difference (P<0.05), ** means extremely significant difference (P<0.01)"

Fig. 3

Effect of different concentration of Cur on PK-15 cell morphology (Scale =100 μm)"

Fig. 4

Effect of different concentrations of Cur on ZEA-induced morphological changes of PK-15 cells (Scale =100 μm)"

Fig. 5

Effects of different concentrations of Cur on cell viability of PK-15 cells induced by ZEA Compared with control group, * means significant difference (P<0.05), and ** means extremely significant difference (P<0.01); compared with control group, # means significant difference (P<0.05), and ## means extremely significant difference (P<0.01). The same as below"

Fig. 6

Effect of curcumin on ZEA-induced ROS in PK-15 cells（Scale = 100 μm）"

Fig. 7

Relative fluorescence intensity of reactive oxygen species"

Fig. 8

Effects of Cur and ZEA on oxidative stress index in PK-15 cells A: Sod activity B: Cat activity C: MDA content"

Fig. 9

Cur and Zea effect on the expression of oxidative stress-related gene mRNA in PK-15 cells A: SIRT1 mRNA expression; b: FOXO1 mRNA expression; C: Mn-SOD mRNA expression; D: CAT mRNA expression"

Fig. 10

Cur and Zea effect on the expression of oxidative stress related gene protein in PK-15 cells A: SIRT1, Acetyl-FOXO1 and FOXO1 protein band; B: SIRT1 protein expression; C: Acetyl-FOXO1 protein expression"

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