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

doi:10.3864/j.issn.0578-1752.2023.05.015

Abstract

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)

CUI HongJie, LU ChunTing, PAN LiQin, HU Hui, ZHONG PeiYun, ZHU JieYing, ZHANG KaiZhao, HUANG XiaoHong. Curcumin Alleviates Zearalenone-Induced Oxidative Damage in Porcine Renal Epithelial Cells via SIRT1/FOXO1 Pathway[J].Scientia Agricultura Sinica, 2023, 56(5): 1007-1018.

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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

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

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

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