Establishment of Oxidative Injury Model of Grass Carp Hepatocytes Induced by Hydrogen Peroxide and Its Application in Antioxidant Ability Evaluation of Natural Plants

SHI Yaoyao; YE Yuantu; JIANG Rong; CAI Chunfang; WU Ping

doi:10.3969/j.issn.1006-267x.2021.04.047

2021 , Vol. 33 >Issue 4: 2290 - 2301

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.04.047

Establishment of Oxidative Injury Model of Grass Carp Hepatocytes Induced by Hydrogen Peroxide and Its Application in Antioxidant Ability Evaluation of Natural Plants

SHI Yaoyao ,
YE Yuantu ,
JIANG Rong ,
CAI Chunfang ,
WU Ping

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1. Key Laboratory of Aquatic Nutrition of Jiangsu Province, School of Biology and Basic Medical Sciences, Soochow University, Suzhou 215123, China;
2. Wuxi Sanzhi Bio-Technology Co., Ltd., Wuxi 214135, China

Received date: 2020-09-22

Online published: 2021-04-15

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Abstract

This experiment was to explore the conditions for the establishment of in vitro oxidative injury model of grass carp primary hepatocytes induced by hydrogen peroxide (H₂O₂), and to evaluate the antioxidant ability of 8 kinds of natural plants using this model. Methyl thiazolyl tetrazolium (MTT) staining method was used to detect the survival rate of cells treated with different concentrations (0, 100, 200, 400 and 600 μmol/L) of H₂O₂ for 0.5, 1.0, 2.0 and 4.0 h, and the lactate dehydrogenase (LDH) activity in culture fluid was measured by microplate method. The hepatocytes were treated with different concentrations (100, 200, 400 and 600 μmol/L) of H₂O₂ for 1.0 h. Cell morphology was observed by microscope. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in cells were detected by spectrophotometry. And cell apoptosis rate and the level of reactive oxygen species (ROS) in cells were detected by flow cytometry. The antioxidant ability of 8 kinds of natural plants such as Terminalia chebula Retz, Polygoni Cuspidati Rhizoma et Radix, Folium nelumbinis, Plantaginis herb, Platycladi cacumen, Glycyrrhizae Radix et Rhizoma, Rhei Radix et Rhizoma and Cinnamomi cortex was evaluated by determining the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging capacity and the survival rate of H₂O₂ injured cells. The results showed as follows:compared with the control group, after treated with 200 μmol/L H₂O₂ for 1.0 h, the cell survival rate was reduced, which dropped to (64.85±0.38)%; the content of LDH and MDA was significantly or extremely significantly increased (P<0.05 or P<0.01), and the activity of SOD was extremely significantly decreased (P<0.01); the level of ROS and cell apoptosis rate were extremely significantly increased (P<0.01). The cell survival rate of Terminalia chebula Retz group was the highest, which was 80.17% to 150.68% higher than that of model group. The order of the DPPH free radical scavenging capacity was Terminalia chebula Retz ≥ Polygoni Cuspidati Rhizoma et Radix ≥ Folium nelumbinis ≥ Rhei Radix et Rhizoma ≥ Cinnamomi cortex ≥ Platycladi cacumen ≥ Plantaginis herba ≥ Glycyrrhizae Radix et Rhizoma, and the strong-to-weak sequence of oxidative injury repair ability was basically the same as the DPPH free radical scavenging capacity. The results indicate that 200 μmol/L H₂O₂ treatment for 1.0 h is the best experimental condition for the establishment of oxidative injury model of grass carp primary hepatocytes. In 8 kinds of natural plants, Terminalia chebula Retz has the best antioxidant activity and its oxidative injury repair ability also is the strongest.

Key words： grass carp; primary hepatocytes; hydrogen peroxide; oxidative stress; natural plants

Cite this article

SHI Yaoyao , YE Yuantu , JIANG Rong , CAI Chunfang , WU Ping . Establishment of Oxidative Injury Model of Grass Carp Hepatocytes Induced by Hydrogen Peroxide and Its Application in Antioxidant Ability Evaluation of Natural Plants[J]. Chinese Journal of Animal Nutrition, 2021 , 33(4) : 2290 -2301 . DOI: 10.3969/j.issn.1006-267x.2021.04.047

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