Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (8): 1787-1794.doi: 10.3864/j.issn.0578-1752.2021.08.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Curcumin Alleviates H2O2-Induced Oxidative Stress in Bovine Mammary Epithelial Cells Via the Nrf2 Signaling Pathway

JIANG ChunHui(),SUN XuDong(),TANG Yan,LUO ShengBin,XU Chuang(),CHEN YuanYuan()   

  1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang
  • Received:2020-05-06 Accepted:2021-01-25 Online:2021-04-16 Published:2021-04-25
  • Contact: Chuang XU,YuanYuan CHEN E-mail:jiangchunh0606@163.com;sunxudong0323@163.com;xuchuang7175@163.com;18249636785@163.com

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Abstract:

【Objective】The aim of this study was to investigate whether curcumin alleviated oxidative stress in bovine mammary epithelial cells induced by H2O2 via the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. 【Method】Bovine mammary epithelial cells MAC-T cells were treated with H2O2 (500 μmol·L-1) for 24 h, followed by incubation of curcumin (0, 5, 15 or 30 μmol·L -1) for an additional 3 h; the MAC-T cells were transfected with Nrf2 siRNA for 48 h, followed by incubation of H2O2 (500 μmol·L-1) for 24 h and then treated with curcumin (30 μmol·L -1) for an additional 3 h. Real-time quantitative PCR (qPCR), Western blot (WB) were used to detect the protein abundance of Nrf2 and the mRNA and protein abundance of NAD(P)H quinone oxidoreductase 1 (NQO1) and Heme oxygenase 1 (HO-1), the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and the content of malondialdehyde (MDA). 【Result】 (1) Compared with the control group, H2O2 treatment significantly increased MDA content (P<0.01), while it decreased the activity of SOD, GSH-Px, and CAT (P<0.01). Compared with the H2O2 group, the content of MDA in MAC-T cells in the 15 μmol·L-1 or 30 μmol·L -1 curcumin with H2O2 treatment groups was significantly decreased (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.05, P<0.01). (2) Compared with the control group, H2O2 treatment significantly decreased Nrf2 protein abundance (P<0.01) and decreased HO-1 and NQO1 their mRNA and protein abundance (P<0.01). However, compared with the control group, curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). Compared with the H2O2 group, H2O2+curcumin treatment significantly increased Nrf2 protein abundance (P<0.01) and increased HO-1 and NQO1 mRNA and protein abundance (P<0.01). (3) Compared with the control group, si-Nrf2 treatment group significantly decreased Nrf2 mRNA abundance (P<0.01). Compared with si-Control+H2O group, the content of MDA was significantly decreased in si-Control+H2O2+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly increased (P<0.01). However, compared with si-Control+H2O2+curcum group, the content of MDA was significantly increased in si-Nrf2+H2O2+curcumin treatment group (P<0.01), while the activity of SOD, GSH-Px, and CAT were significantly decreased (P<0.01). 【Conclusion】These results suggested that curcumin could alleviate the oxidative stress of bovine mammary epithelial cells induced by H2O2 through increasing the expression of Nrf2 and inducing the transcription of downstream antioxidant molecules. This study provided a theoretical basis for the prevention and treatment of oxidative damage of mammary epithelial cells caused by metabolic disorderd in perinatal dairy cows.

Key words: curcumin, bovine, mammary epithelial cells, oxidative stress, Nrf2

Table 1

Gene-specific primers of Real-time PCR"

基因
Gene		 引物序列
Primer sequences (5′-3′)		 产物大小
Length (bp)
Nrf2 F GCCCTCACTGGATAAAGAA 202
R CATGCCGTTGCTGGTAC
NQO1 F AACCAACAGACCAGCCAATC 154
R CACAGTGACCTCCCATCCTT
HO-1 F CAAGGAGAACCCCGTCTACA 225
R CCAGACAGGTCTCCCAGGTA
β-Actin F GCCCTGAGGCTCTCTTCCA 101
R GCGGATGTCGACGTCACA
GAPDH F TCTTCACTACCATGGAGAAGG 197
R TCATGGATGACCTTGGCCAG

Fig. 1

Curcumin activates the antioxidant enzyme system and reduces toxicity markers A: MDA content; B: CAT activity; C: SOD activity; D: GSH-PX activity. * Means differ significantly (P<0.05), ** Means differ highly significantly (P<0.01). The same as below"

Fig. 2

Curcumin induced Nrf2 expression A: Western blot analysis of Nrf2; B: Relative protein level of Nrf2"

Fig. 3

Curcumin induced Nrf2 target genes HO-1 and NQO1 mRNA and protein expression A: HO-1 mRNA level; B: NQO1 mRNA level; C: Western blot analysis of HO-1 and NQO1; D: Relative protein level of HO-1; E: Relative protein level of NQO1"

Fig. 4

Silence of Nrf2 attenuated the beneficial effects of curcumin on H2O2-induced oxidative stress A: Nrf2 mRNA level; B: MDA content; C: CAT activity; D: SOD activity; E: GSH-PX activity"

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