Gypenosides ameliorate heat stress-induced inflammatory injury in the bursa of Fabricius of broilers by activating the NF-κB signaling pathway
Description
Data Description and Interpretation Experimental Subjects and Design This study selected a total of 200 broilers at 28 days of age, randomly divided into five groups (40 broilers per group), with the specific groupings as follows: CON (Control Group): Basal diet + Normal temperature (24 ± 1 ℃) HS (Heat Stress Group): Basal diet + High temperature (33 ± 1 ℃, 8 hours per day) HSLGP (Low-Dose Gypenoside Group): Basal diet + 200 mg/kg GP + High temperature HSMGP (Medium-Dose Gypenoside Group): Basal diet + 300 mg/kg GP + High temperature HSHGP (High-Dose Gypenoside Group): Basal diet + 450 mg/kg GP + High temperature Data Content and Collection Method The data were derived from qPCR and Western Blot detection results of the bursa of Fabricius tissues from each group. Three samples were randomly selected from each group for analysis (reduced from the original plan of 8 samples to 3 for simplification). qPCR Data: The expression levels of genes related to inflammation and antioxidant defense (HO-1, ikba, nrf2, p65) were detected. WB Data: The expression levels of corresponding proteins (HO-1, IKBA, NRF2, P65) were measured. Significant Findings Gene Expression: Compared with the CON group, p65 gene expression was significantly elevated in the HS group, indicating that heat stress activated the NF-κB inflammatory pathway. After GP supplementation (in HSLGP, HSMGP, and HSHGP groups), p65 expression was downregulated, while antioxidant genes such as nrf2 were upregulated, with the most pronounced effect observed in the HSHGP group. Protein Expression: The WB results were consistent with the qPCR trends. P65 protein levels were elevated in the HS group and decreased after GP supplementation, while antioxidant proteins such as HO-1 showed increased expression. Data Interpretation Inflammation Regulation: GP alleviates heat stress-induced inflammatory responses by inhibiting p65 expression and blocking the NF-κB pathway. Antioxidant Effects: GP activates the Nrf2 pathway, promoting the expression of antioxidant proteins such as HO-1 and enhancing cellular defense against oxidative damage. Dose-Dependent Effects: High-dose GP (HSHGP) demonstrated the best regulatory effects on gene and protein expression, suggesting that GP's anti-inflammatory and antioxidant actions may exhibit dose dependency.
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Here's a refined overview of how the data was obtained, facilitating others to understand the data collection process and replicate the study: To obtain the experimental data, we employed the following methods and protocols: 1. Quantitative Real-Time PCR (qPCR): RNA Purification: Total RNA was purified from liver tissue using Trizol reagent, following the manufacturer's instructions. cDNA Synthesis: The purified RNA was reverse-transcribed into cDNA using a first-strand synthesis kit (ABclonal, China). qPCR Amplification: Real-time quantitative PCR was performed in a 20 μL reaction system using a qPCR kit (ABclonal, China). Gene-specific primers were used to amplify the target genes. Data Normalization: Gene expression levels were normalized using the expression of the housekeeping gene β-actin. Relative fold changes in gene expression were calculated using the 2−ΔΔCt method. 2. Western Blot Analysis: Protein Extraction: Approximately 20 mg of liver tissue was homogenized in RIPA lysate (PMSF: RIPA = 1:100) and centrifuged at 13,000 rpm at 4°C to obtain the protein extract. Protein Quantification: Protein levels in the extract were quantified using a BCA kit (Beyotime, China). Electrophoresis and Blotting: 10 μL of protein (1 μg/μL) was loaded onto a 10% SDS-PAGE gel for electrophoresis. Following electrophoresis, proteins were electroblotted onto a PVDF membrane. Blocking and Antibody Incubation: The membrane was blocked using 5% skim milk for 1 hour. It was then incubated with primary antibodies against Bak1 (1:800, Servicebio, China), Bcl-2 (1:2000, Proteintech Group, USA), and Caspase3 (1:1000, Proteintech Group, USA) at 4°C overnight (approximately 14 hours). After washing, the membrane was incubated with the secondary antibody for 1 hour. Visualization and Analysis: The ultrasensitive ECL chemiluminescence kit (Beyotime, China) was used to visualize the protein bands. Grayscale analysis of protein expression was performed using Image J software to quantify the band intensities. By detailing the instruments (e.g., centrifuge, electrophoresis apparatus), reagents (Trizol reagent, ABclonal qPCR kit, Beyotime BCA kit and ECL kit, etc.), antibodies, and software (Image J) used, along with the specific protocols, this overview provides a clear guide for others to replicate the study and understand the data collection process.
Institutions
- Anhui Science and Technology UniversityAnhui, Chuzhou