Hesperidin alleviates terbuthylazine-induced ferroptosis via maintenance of mitochondria-associated endoplasmic reticulum membrane integrity in chicken hepatocytes

Description

This data recorded the body weight and liver weight of the chicken in this paper. We tested the protein levels of ER stress by Western Blot, and tested the gene levels of ER stress by RT-qPCR to investigate the effect of hesperidin on ER stress induced by TBA. In addition, to investigate whether hesperidin alleviates the MAM disorder caused by TBA, Western Blot was used to detect the expression level of MAM proteins, as well as used Immunohistochemistry to test the expression level of GRP75. Furthermore, we used Western Blot and RT-qPCR to investigate the effect of hesperidin on ferroptosis induced by TBA. Immunohistochemistry was used to test the expression level of TF and XCT proteins.

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A total 240 of chicken (1-day-old) were adaptively fed for 1 week and the ingredients and nutritional diet for the control groups are shown in Table S1. Then, these chickens were random into four groups (60 broilers per group), including control group (C group), H group (4 mg/kg TBA), H+G group (4 mg/kg TBA+ 150mg/kg HSP), and G group (150mg/kg HSP). After 28 days of treatment, pentobarbital sodium was used for euthanasia, and liver tissue samples were collected. All the experiments involving animals have been received approval by the Ethics Committee of South China Agricultural University. Immunohistochemical staining was performed according to previous methods (Li et al., 2022). The anti-transferrin (TF) (1:200) (A19130, ABclonal, Wuhan, China), cystine/glutamate transporter (XCT) (1:200) (A13685, ABclonal, Wuhan, China), glucose-regulated protein 75 (GRP75) (1:200) (A0241, ABclonal, Wuhan, China) primary antibodies were used. Total RNA was extracted from liver tissue using Trizol reagent. Confirm RNA concentration and quality using a spectrophotometer, followed by reverse transcription (Vazyme, Nanjing) of total RNA of liver tissue. The primer sequences for all mRNAs are shown in Table S2. Housekeeping genes (GAPDH) are used as standardized reference genes. The results were calculated by 2-ΔΔCt method. The total protein was extracted from liver tissue with RIPA buffer, Protein concentration is determined using the BCA assay (Vazyme, Nanjing). The protein samples were separated by SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane, blocked with 5% skim milk for 1 h, then incubated with primary antibodies overnight at 4 °C. The primary antibody used are shown in Table S3. The proteins blot was visualized by using Enhanced Chemiluminescence (ECL) Assay Kit (P10200, New Cell & Molecular Biotech, China), and results were analyzed by using image J software (NIH, USA). The data were calculated as mean ± SD value. All results were performed by using GraphPad Prism 7.0 (GraphPad Inc., USA). Differences were analyzed by t-test between two groups. The standard was considered statistically significant at P<0.05.

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