Alleviation Effect of Phloretin on Oxidative Stress in Cattle Adipose Stem Cells Induced by Hydrogen Peroxide

JIANG Enhui; LIU Yuan; LI Jie; MA Sijia; LI Xiangchen; LAN Xianyong

doi:10.3969/j.issn.1006-267x.2022.01.055

Chinese Journal of Animal Nutrition >

2022 , Vol. 34 >Issue 1: 600 - 611

DOI: https://doi.org/10.3969/j.issn.1006-267x.2022.01.055

RESEARCH PAPER

Alleviation Effect of Phloretin on Oxidative Stress in Cattle Adipose Stem Cells Induced by Hydrogen Peroxide

JIANG Enhui ,
LIU Yuan ,
LI Jie ,
MA Sijia ,
LI Xiangchen ,
LAN Xianyong

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1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;
2. College of Animal Science and Technology·College of Veterinary Medicine, Zhejiang A&F University, Hangzhou 311300, China;
3. Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, Hangzhou 311300, China

Received date: 2021-06-17

Online published: 2022-01-18

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Abstract

The aim of this study was to investigate the effects of phloretin (PT) on oxidative stress and apoptosis of cattle adipose-derived stem cells (ADSCs) induced by hydrogen peroxide (H₂O₂). Bovine adipocytes were selected and collagenase was used to isolate cattle ADSCs. After subculture, the surface marker proteins CD29, CD44, CD73 and Vimentin of cattle ADSCs were analyzed by immunofluorescence technique. Different concentrations (0, 100, 200, 500 and 1 000 μmol/L) of H₂O₂ were set to treat cattle ADSCs for 24 h, the cell viability was determined by CCK-8 method to screen the optimal concentration of H₂O₂ to build oxidative stress in cattle ADSCs. Different concentrations (0, 10, 50 and 100 μmol/L) of PT were set to treat cattle ADSCs for 4 h. The cell viability was determined by CCK-8 method to determine the optimal concentration of PT to protect cattle ADSCs. Cattle ADSCs were pretreated with different concentrations (0, 10 and 50 μmol/L) of PT for 4 h, and then treated with 500 μmol/L H₂O₂ for 24 h. The oxidative stress related indices such as malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in cattle ADSCs were detected by corresponding kit. The intracellular reactive oxygen species (ROS) content was determined by fluorescence probe 2',7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA). The mRNA relative expression levels of apoptosis related genes B-cell lymphoma/leukaemia-2-associated X protein (Bax), B-cell lymphoma/leukaemia-2 (Bcl-2) and cysteinyl aspartate specific proteinase-3 (Caspase-3) were detected by quantitative real-time PCR (qRT-PCR). The results showed as follows:500 μmol/L H₂O₂ extremely significantly decreased cattle ADSCs viability (P<0.01), which could induce oxidative stress in cattle ADSCs. 10 and 50 μmol/L PT had no significant difference in cattle ADSCs viability (P>0.05), but 100 μmol/L PT significantly decreased cattle ADSCs viability (P<0.05). Therefore, subsequent experiment was selected 10 and 50 μmol/L PT to treat cattle ADSCs for 4 h. Immunofluorescence detection results showed that the expression of CD29, CD44, CD73 and Vimentin were positive in cattle ADSCs. Oxidative stress related indices detection results showed that 500 μmol/L H₂O₂ extremely significantly increased MDA content (P<0.01), and extremely significantly decreased SOD content in cattle ADSCs compared with the blank group (0 μmol/L PT, 0 μmol/L H₂O₂) (P<0.01), this demonstrated that 500 μmol/L H₂O₂ treatment caused oxidative stress in cattle ADSCs. Compared with the oxidative stress group (0 μmol/L PT, 500 μmol/L H₂O₂), the co-treatment of 10 or 50 μmol/L PT and 500 μmol/L H₂O₂ could significantly reduce the MDA content in cattle ADSCs (P<0.05), and the co-treatment of 50 μmol/L PT and 500 μmol/L H₂O₂ could significantly increase the SOD activity in cattle ADSCs (P<0.05), this showed that optimal concentration of PT could relieve the oxidative stress in cattle ADSCs induced by H₂O₂. ROS staining results showed that the co-treatment of 10 or 50 μmol/L PT and 500 μmol/L H₂O₂ could extremely significantly reduce the ROS content in cattle ADSCs, that also showed that optimal concentration of PT could relieve the oxidative stress in cattle ADSCs induced by H₂O₂. In addition, qPCR detection results showed that compared with the blank group, 500 μmol/L H₂O₂ significantly increased the mRNA relative expression levels of pro-apoptotic gene Bax and Caspase-3, and extremely significantly decreased the mRNA relative expression level of anti-apoptotic gene Bcl-2 (P<0.05). this showed that 500 μmol/L H₂O₂ treatment could cause the apoptosis of cattle ADSCs. However, compared with 500 μmol/L H₂O₂ alone treatment, the co-treatment of 10 or 50 μmol/L PT and 500 μmol/L H₂O₂ had no significant difference in the mRNA relative expression levels of apoptosis-related genes in cattle ADSCs (P>0.05), which showed PT could not relieve the apoptosis of cattle ADSCs induced by H₂O₂ under this experiment condition. In conclusion, the optimal amount of PT can effectively alleviate the oxidative stress in cattle ADSCs induced by H₂O₂.

Key words： cattle; adipose derived stem cells; phloretin; oxidative stress; cell apoptosis

Cite this article

JIANG Enhui , LIU Yuan , LI Jie , MA Sijia , LI Xiangchen , LAN Xianyong . Alleviation Effect of Phloretin on Oxidative Stress in Cattle Adipose Stem Cells Induced by Hydrogen Peroxide[J]. Chinese Journal of Animal Nutrition, 2022 , 34(1) : 600 -611 . DOI: 10.3969/j.issn.1006-267x.2022.01.055

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