
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00758-2
10.1016/j.psj.2024.104179
104179
IMMUNOLOGY, HEALTH AND DISEASE
Naringenin counteracts LPS-induced inflammation and immune deficits in chicken thymus by alleviating mtROS/ferroptosis levels
Yu Fei *
Shi Xu *
Li Ke *
Yin Yilin *
Xu Shiwen shiwenxu@neau.edu.cn
*†‡1
⁎ College of Veterinary Medicine, College of Animal Medicine, Northeast Agricultural University, Harbin 150030, China
† Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and Treatment, College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
‡ Laboratory of Embryo Biotechnology, College of Life Science, Department of Biotechnology, Northeast Agricultural University, Harbin, 150030, China
1 Corresponding author: shiwenxu@neau.edu.cn
06 8 2024
11 2024
06 8 2024
103 11 10417925 6 2024
1 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Naringenin is a flavonoid with significant anti-inflammatory and antioxidant properties. Mitochondrial dynamics, the mitochondrial respiratory chain, and mtROS are closely related to each other and regulate various biological processes. Ferroptosis is closely related to inflammatory responses and immune function in multiple tissues and organs. However, whether naringenin can alleviate LPS-induced inflammation and immune disorders in the chicken thymus via mtROS/ferroptosis has not been reported. Therefore, in this study, we constructed chicken thymus and MSB-1 cell models of LPS and naringenin based on screening for naringenin concentrations that have positive effects on inflammation and immune function to further investigate the anti-inflammatory, antiferroptosis, and maintenance of the immune function of naringenin. The results showed that 40 mg/kg naringenin alleviated LPS-induced tissue damage, elevated serum inflammatory factors, and decreased serum immune factors. The mechanism by which naringenin attenuates mtROS release by alleviating the imbalance of mitochondrial dynamics and the blockage of the respiratory chain. The effect of naringenin on alleviating LPS-induced lipid peroxidation, disruption of the GSH/GSSG system, iron overload, and GPx4 inactivation, thereby attenuating ferroptosis in thymus tissue, was inhibited by the addition of mtROS activators. In conclusion, naringenin alleviates LPS-induced ferroptosis in chicken thymus by attenuating mtROS release.

Key words

naringenin
mtROS/ferroptosis
inflammation and immune function
==== Body
pmcINTRODUCTION

Naringenin (4,5,7-trilethyl dihydroflavone, Nar) is a natural dihydroflavonoid found mainly in citrus fruits and can be widely used as the form of food additives in the food field (Li, Yao et al. 2024). Naringenin has a very wide range of pharmacological effects due to its remarkable antibacterial and anti-inflammatory properties, antioxidant damage, etc. There are many studies that have found naringenin to be capable of treating or preventing a variety of diseases such as obesity and diabetes mellitus (Gandhi, Vasconcelos et al. 2020, Li, Yao et al. 2024). Naringenin has the advantage of a comprehensive effect on multiple targets, organs and systems of the organism. The target organs of single oral administration in rats include stomach, intestines, liver, kidney, heart, spleen, fat and skeletal muscle (Zeng, Yao et al. 2020). In addition, naringenin reduces the toxicity of a number of common drugs, such as the antitumor drug cyclophosphamide (Akamo, Rotimi et al. 2021), cisplatin, and the antipyretic acetaminophen (Zhai, Dai et al. 2022), etc. 40 mg/kg naringenin prevented diabetic nephropathy against streptozotocin-induced diabetes mellitus in rats through the blockade of mitochondrial oxidative stress and attenuation of mitochondrial dysfunction . Naringenin's positive effects on anti-inflammatory and immune function are gradually being discovered by researchers (Tian, Wang et al. 2024). Naringenin can enhance antitumor immunity by activating the CD169-positive phenotype of lymph node macrophages (Fujiwara, Saito et al. 2018), suggesting that naringenin has a significant enhancing and activating effect on the immune system. Naringenin can alleviate the elevated serum IgE and IFN-γ levels caused by atopic dermatitis (Tian, Wang et al. 2024). For the immune cell aspect, oral naringenin limited T-cell proliferation by decreasing the level of anticollagen type II IgG (Li, Chen et al. 2015). Additionally, naringenin treatment leads to a significant decrease in serum total IgE and T-helper 2 cytokine levels in bronchoalveolar lavage fluid, which in turn may postpone the developments of airway remodeling (Shi, Tan et al. 2014).

Mitochondrial dynamics refers to the continuous division and fusion of mitochondria and regulates processes such as innate immunity, autophagy and apoptosis by providing energy (Liesa, Palacín et al. 2009). The process of mitochondrial fusion and division concerns the dynamic balance of mitochondria, which can directly affect the shape and function of mitochondria and has become a recognized trigger of cellular stress responses (Meyer, Leuthner et al. 2017). Another aspect, abnormal function of the mitochondrial respiratory chain complex (MRCC) limits adenosine triphosphate (ATP) synthesis and releases large amounts of mitochondrial ROS (mtROS) (Liu, Lin et al. 2023). One study found that 20 mg/kg BPA exposure disrupted the MRCC in the chicken brain, leading to mtROS burst as well as elevated the expression of mitochondrial division-related genes (Mff, Drp1 and Fis1) (Liu, Lin et al. 2023). Ferroptosis is an iron-dependent programmed cell death that is connected with cellular inflammation and immune responses (Shi, Xu et al. 2024). Iron overload, GPx4 inactivation and lipid peroxidation are 3 prerequisites for the occurrence of ferroptosis (Xu, Cui et al. 2023). 2.4 μg/mL emamectin benzoate induced ferroptosis in carp intestinal epithelial cells via lysosomes/ROS pathways (Shi, Xu et al. 2024). Polystyrene microplastics promote alveolar epithelial cell ferroptosis via cGAS/STING signaling, which induces lung fibrosis (Zhang, Du et al. 2024). In addition, a number of studies have found that drugs such as atractylodes macrocephala koidz (Zhang, Du et al. 2024), L-citrulline (Ba, Zhao et al. 2022), and quercetin (Zhang, Wang et al. 2024) are able to attenuate ferroptosis in thymus and kidney cells.

Naringenin as a flavonoid has significant anti-inflammatory and antioxidant effects (Fujiwara, Saito et al. 2018, Pérez, Mukdsi et al. 2023, Salama, Yassen et al. 2023). Mitochondrial dynamics, mitochondrial respiratory chain, and mtROS are closely related to each other and regulate a variety of biological processes (Meyer, Leuthner et al. 2017, Liu, Lin et al. 2023). Ferroptosis is connected with inflammatory responses and immune functions in multiple tissues, organs and cells (Shi, Xu et al. 2024). The thymus is an immune center with endocrine function and is the first line of immune defense. Thymus injury will directly affect the immune function crisis of the whole organism. But whether naringenin can alleviate chicken thymus inflammation and immune disorders has not been studied, and whether mitochondria-mediated ferroptosis is involved in this process is also unclear. Therefore, in this study, we constructed chicken thymus and MSB-1 cell models of biomolecule LPS and naringenin on the basis of screening for naringenin concentrations with positive effects on inflammation and immune function, and detected histopathological changes in the thymus, serum inflammatory factors, serum immune related factors, mitochondrial respiratory chain, mitochondrial fusion and fission, mtROS levels, lipid peroxidation, GSH/GSSG system, iron metabolism and GPx4 expression levels. Our results in this study provide a theoretical basis for naringenin pharmacology and a reference for comparative medicine.

MATERIALS AND METHODS

Animal Grouping and Model Establishment

Part I Animal Experiment

Eighty healthy 1-day-old white-feathered broilers were selected and randomly divided into 4 groups (n = 20): blank control group (C group), 40 mg/kg naringenin group (40 mg/kg group), 80 mg/kg naringenin group (80 mg/kg group) and 120 mg/kg naringenin group (120 mg/kg group) (Esan, Ebirim et al. 2024). Naringenin (S25634, purity > 98%, Yuanye) was administered by gavage from the first d and kept under the same environmental conditions. Chickens from each group were randomly selected and weighed on 7 d, 14 d, 21 d, and 28 d (n = 6). On 29st d, all broilers were euthanized, whole blood was collected from the heart and chicken thymus tissue was collected and stored in 4% paraformaldehyde and -80°C refrigerator.

Part II Animal Experiment

Eighty healthy 1-day-old white-feathered broilers were selected and randomly divided into 4 groups (n = 20): blank control group (C group), 40 mg/kg naringenin treatment group (Nar group), LPS exposure group (LPS group) and naringenin-antagonistic LPS group (Nar+LPS group). The Nar group and the Nar+LPS group were gavaged with 40 mg/kg naringenin from the first d; while C group and LPS group were gavaged with saline. On 28st d, the LPS group and Nar+LPS group were injected intraperitoneally with 1.5 mg/kg.BW LPS (BS904, potency≥ 500000 EU/mg, Biosharp) (Wang, Ding et al. 2023), whicle saline was injected intraperitoneally in the C group and Nar group. All broilers were executed 24 h after LPS exposure. Whole blood was collected from the heart, and chicken thymus tissues were taken and preserved in 4% paraformaldehyde (Solarbio, China) and -80°C refrigerator.

Thymus Index

When the broiler chicken thymus was collected, weighed and data recorded. The data was calculated using the following formula. Thymus index = thymus weight (mg)/body weight (g) × 100%.

Cell Culture and Grouping

Chicken lymphoma cells (MSB-1, laboratory preserved) were cultured with a mixture of RPMI1640 (Gibco) and 10% FBS (1705124, VivaCell, Shanghai, China) in a humidified incubator at 37°C, 5% CO2. According to the specific growth of the MSB-1 cells, the culture medium was changed and passaged. SMTIN-T140 (HY-147696, MCE) was used as an mtROS activator, the working solution concentration was 5 μM (Yang, Yoon et al. 2022).

CCK8 Cell Activity Assay

The influence of LPS on the MSB-1 cells viability and the screening of in vitro LPS concentration were determined using CCK-8 kit (Melun, China). 2×104 cells/well MSB-1 cells were inoculated into 96-well plates for culture, and the cells were treated with 0, 0.1, 0.5, 1, 5, 10, 50, and 100 µg/mL LPS and 0.05, 0.1, 0.5, 1, 10, 50, 100, and 200 µM Nar for 24 h. The viability of the MSB-1 cells was then detected by adding 200 µL CCK-8 solution to each 96-well. After 1.5 h, the absorbance (OD) was detected at 450 nm in a microplate reader (Thermo).

H&E Staining

H&E staining was used to visualize the effect of LPS and naringenin on chicken thymus morphology. The staining process was referred to the method reported by Shi et al (Shi, Xu et al. 2024). Finally, the samples were observed using a light microscope and images were taken.

Immunofluorescence

Immunofluorescence (IF) staining was used to assess the GPx4 level in chicken thymus and MSB-1 cells. Briefly, tissue sections were deparaffinized and rehydrated for antigen extraction. The sections were then incubated with GPx4 primary antibody (A1933, Abclonal) at 4°C overnight. After washing, the sections were incubated with secondary antibody (Dylight594, Abbkine) for 1 h. Finally, the nuclei were stained with DAPI (Leagene, China) to visualize the nuclei. For cultured MSB-1 cells, cells were fixed to permeabilize and incubated with GPx4 primary antibody (A1933, Abclonal) at 4°C overnight, followed by incubation with secondary antibody (Dylight594, Abbkine) for 1 h. Images were acquired using a fluorescence microscope and protein levels were quantified using image analysis software.

Detection of Fe2+ Levels

Thymus and MSB-1 cells were assayed for Fe2+ content using the Fe2+ Assay Kit (A039-2-12., NJJCBIO). 0.1 g chicken thymus tissue was homogenized in 0.9% NaCl, and processed MSB-1 cells were collected, lysed, and centrifuged (3500 r/min, 15 min) in 0.9% NaCl to obtain supernatants. Subsequent experiments were performed according to the manufacturer's instructions.

Detection of Intracellular mtROS

Mito-SOX stain was purchased from Thermo Fisher Scientific and was used to detect mitochondrial superoxide levels. MSB-1 cells from different treatment groups were treated accordingly and incubated in RPMI1640 culture medium containing 0.1 μM mitochondrial superoxide for 30 min. Then, fluorescence microscopy and Image J were used for observation and analysis.

Kit Testing

0.1 g chicken thymus tissue was homogenized in 0.9% NaCl, and processed MSB-1 cells were collected, lysed, and centrifuged (3500 r/min, 15 min) in 0.9% NaCl to obtain supernatants. Supernatants were used to assay MDA content (A039-2-1, NJJCBIO), Lipid peroxidation (LPO) content (A106-1-1, NJJCBIO), GSH and oxidized glutathione (GSSG) content (A061-2-1, NJJCBIO), GSH-Px activity (A005-1-2, NJJCBIO), and glutathione reductase (GR) activity (A062-1-1, NJJCBIO).

ELISA Kits

0.1 g chicken thymus tissue was homogenized in 0.9% NaCl, and processed MSB-1 cells were collected, lysed, and centrifuged (3500 r/min, 15 min) in 0.9% NaCl to obtain supernatants. We purchased TNF-α ELISA kits (ML002790, MLbio, China), IL-10 ELISA (ML059830), IL-1β ELISA (ML059835), IgA ELISA (ML002792), IgG ELISA (ML042771), and IFN-γ ELISA (ML023435) to assess the levels of inflammatory factors and immunoglobulins in chicken serums and MSB-1 cells.

Detection of Cellular Fe2+

FerroOrange stain was purchased from MX4559 of MKBio, which is a novel stain for detecting intracellular Fe2+ content; specifically, MSB-1 cells from different treatment groups were incubated in RPMI1640 medium with 1 μM FerroOrang for 30 min after corresponding treatment. Fluorescence microscopy and Image J were used for further analysis.

Quantitative Real-Time PCR

Quantitative real-time PCR (qRT-PCR) method was carried out according to the method of (Shi et al. 2024). Specifically, after treatment, 0.1 g chicken thymus tissue was weighed or 1×107 MSB-1 cells were collected. Total RNA was extracted by the TRIzol (Thermo) method. RNA reverse transcription was performed to obtain cDNA using the First-Strand cDNA Synthesis Kit purchased from Yugong Biotech Co., Ltd. 2×SYBR Green Mix was purchased from Bioer technology in Hangzhou, China. The qRT-PCR reaction was performed on a QuantStudio 6 instrument (Applied Biosystems). β-actin was used as an internal reference gene, and all primer sequences were shown in Supplementary Table S1, and the relative gene expression was analyzed by the 2−ΔΔCt method.

Western Blot Analysis

Western blot analysis was performed using the research method of (Sun et al. 2024). Specifically, 1×107 MSB-1 cells in C, Nar, LPS, and Nar+LPS groups were collected after the corresponding treatments. After ultrasonication and centrifugation (12000 r/min, 10 min), the cells were resuspended in 500 μL of RIPA lysis buffer (Solarbio, China), and then the supernatant of the cell lysate was retained for determination, followed by electrophoresis, membrane transfer, skimmed milk blocking, primary and secondary antibody incubation. Protein signals were detected using X-ray film. Primary antibodies, their sources and dilution concentrations were listed in Supplementary Table S2.

Statistical Analyses

Statistical analyses of all digital were reported as mean ± standard deviations (SD) using GraphPad Prism (version 8.0) and SPSS (version 28.0). The data was analyzed to determine the differences among the C, Nar, LPS, and Nar+LPS groups. Statistical analyses were conducted in triplicate and the results were expressed as the means ± SD. The same letter represents no significant difference (P > 0.05), completely different letters and “*” represent a significant difference (P < 0.05).

RESULTS

Positive effects of Naringenin on Inflammation and Immune Function in Chicken Thymus Tissue

In order to investigate the optimal concentration of naringenin added to chicken thymus tissue in 40 mg/kg, 80 mg/kg and 120 mg/kg Nar, we examined the appearance, tissue structure, body weight, thymus index, serum inflammatory factors and immunoglobulin levels. Compared with the C group, 80 and 120 mg/kg Nar caused thymus enlargement (Figure 1A), while no significant variations were found in the 40 mg/kg group. The results of body weight at 7 d, 14 d, 21 d and 28 d revealed that 40 mg/kg Nar (28 d) caused a significant ascend in chicken body weight (P < 0.05), while 120 mg/kg Nar caused a significant decrease in body weight (Figure 1B). In addition, 40 mg/kg Nar caused a decrease in thymus weight (P > 0.05) and thymus index (P < 0.05) compared to the C group (Figures 1C and 1D), with insignificant changes in the 80 mg/kg and 120 mg/kg groups. For the thymic structure (Figure 1E), compared with the control group, the corticomedullary demarcation was clear and the lymphocytes were intact and tightly arranged in the 40 mg/kg Nar group; the corticomedullary demarcation was clear and the arrangement of lymphocytes was a little loose or even partially missing, and there were many thymosomes in the medulla (yellow arrows) in the 80 mg/kg Nar group; the corticomedullary demarcation was blurred in the 120 mg/kg group, and was accompanied with congestion and disorganized lymphocyte arrangement.Figure 1 40 mg/kg naringenin has positive effects on inflammation and immunity (A) Photographs of the chicken thymus appearance in the control, 40 mg/kg, 80 mg/kg and 120 mg/kg groups. (B) Results of body weight (g) at 7th, 14th, 21st, and 28th d (n = 6). (C) Results of chicken thymus weight (mg), n = 6. (D) Results of chicken thymus index (mg/10g. BW), n = 6. (E) H&E staining results of chicken thymus in control, 40 mg/kg, 80 mg/kg and 120 mg/kg groups. The first and third rows were high magnification, scale = 100 μm; the second row was low magnification, scale = 20 μm. Yellow arrow: thymic vesicles. (F) Determination of TNF-α, IL-1β and IL-10 in chicken serum by ELISA method (n = 3). (G) Determination of IgA, IgG and IFN-γ in chicken serum by ELISA method (n = 3). (H) The mRNA levels of TNF-α and IL-1β in chicken thymus (n = 4). (I) The mRNA levels of IgG and IgA in chicken thymus (n = 4).

Figure 1

In Figure 1F, serum TNF-α and IL-1β levels were decreased and IL-10 levels were increased in the 40 mg/kg and 80 mg/kg Nar groups compared with the C group. Additionally, TNF-α was significantly enhanced in the 120 mg/kg group (P < 0.05), but changes in IL-1β and IL-10 were not significant. For the aspect of immune function (Figure 1G), the 40 mg/kg group caused an increase in the levels of serum IgG (P < 0.05), IgA (P > 0.05), and IFN-γ (P > 0.05) compared with the C group, and the variations of the above indexes were not significant in the 80 mg/kg and 120 mg/kg Nar groups. In addition, compared with the C group, the transcription levels of TNF-α and IL-1β showed a tendency to be declined, and IgA and IgG expression were significantly enhanced (P < 0.05) in the 40 mg/kg Nar group (Figures 1H and 1I). 80 Nar mg/kg group significantly induced a decrease in IL-1β and an increase in IgA. But the 120 mg/kg group induced an increase in TNF-α mRNA level (P < 0.05). The above results indicated that 40 mg/kg naringenin had a positive effect on the anti-inflammatory and immune functions of chicken thymus and did not cause damage to the tissue structure, so we chose 40 mg/kg naringenin for the subsequent experiments.

Naringenin Alleviates LPS-Induced Tissue Structure Damage in Chicken Thymus

First, we explored the role of naringenin in inflammation in terms of appearance, tissue structure, and thymus index. In Figure 2A, the LPS group induced bruising and swelling of the thymus compared with the control group, while the above conditions in the Nar+LPS group were alleviated. But there was no significant change in chicken body weight in the LPS and Nar+LPS groups (Figure 2B). The thymus weight (Figure 2C) and thymus index (Figure 2D) were higher in the LPS group compared to the C and Nar groups, and the 2 indices converged towards the control group in the Nar+LPS group (P < 0.05). In terms of thymic histology (Figure 2E), the LPS group caused a blurring of the thymic corticomedullary demarcation and a large aggregation of lymphocytes in the medulla (black arrows), accompanied by a large number of congestions and cavities, compared with the C group. Compared with the LPS group, all of the above conditions were restored in the Nar+LPS group.Figure 2 40 mg/kg naringenin alleviates LPS-induced thymus tissue damage and establishment of cellular models (A) Photographs of the chicken thymus appearance in the control, Nar, Nar+LPS and LPS groups. (B) Results of chicken weight (g) before execution, n = 6. (C) Results of chicken thymus weight (mg), n = 6. (D) Results of chicken thymus index (mg/10g. BW), n = 6. (E) H&E staining results of chicken thymus in control, Nar, Nar+LPS and LPS groups. The first and third rows were high magnification, scale = 100 μm; the second row was low magnification, scale = 20 μm. (F) Effect of 0.1, 0.5, 1, 5, 10, 50, 100 μg/mL LPS on the viability of MSB-1 cells (n = 3). (G) The IC50 of LPS was calculated by nonlinear equation. (H) Effect of 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 10 μM, 50 μM, 100 μM, 200 μM naringenin on MSB-1 cell viability when 10 μg/mL LPS.

Figure 2

In addition, we established the specific concentrations of LPS and Nar in vitro by CCK8 method on MSB-1 cells. It was found that the survival rate of MSB-1 cells gradually declined with the raising of LPS concentration (Figure 2F), and the cell viability with the addition of 10 μg/mL LPS was 70.88%. The LPS IC50 for MSB-1 cells was calculated to be 849.6 μg/mL by the nonlinear curve (Figure 2G). The addition of Nar in the 10 μg/mL LPS showed a tendency of increasing and then decreasing the cell survival rate with the increase of Nar concentration. Therefore, the LPS and Nar concentrations were selected as 10 μg/mL and 0.5 μM, respectively.

Naringenin Alleviates LPS-Induced Mitochondrial Damage

Given the important role of the mitochondrial respiratory chain in energy production, we detected the expression of mitochondrial complexes I-V. Compared with the C group, the LPS group induced an obvious decrease (P < 0.05) in the transcript (Figure 3A) and protein (Figure 3B) levels of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ, and ATP5A-V. Except for UQCRC2-III, the variations of the above genes in the Nar group were not significant. The transcript and protein levels of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ and ATP5A-V in the Nar+LPS group were obviously greater than LPS group, but did not reach the level of the C or Nar group. Additionally, the mitochondrial respiratory chain, mitochondrial dynamics (fusion and fission) are critical for mitochondrial homeostasis. Compared with the control group, the LPS group induced the obvious decreasing of mitochondrial fusion-related genes (OPA1, Mfn1, Mfn2) and the obvious increasing of mitochondrial division-related genes (Fis1, Mff, Drp1) (Figures 3C and 3D). In the Nar+LPS group, the fusion and division-related genes showed a tendency to recover toward the C or Nar group compared with the LPS group (P < 0.05). The above results suggest that Nar alleviated LPS-induced mitochondrial respiratory chain damage and mitochondrial dynamics imbalance in chicken thymus, resulting in mitochondrial damage.Figure 3 Naringenin alleviates LPS-induced mitochondrial damage in chicken thymus (A) The mRNA levels of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ, and ATP5A-V (n = 4). (B) The protein expression and quantitative of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ, and ATP5A-V (n = 3). (C) The mRNA levels of OPA1, Mfn1, Mfn2, Fis1, Mff, and Drp1 (n = 4). (D) The protein expression and quantitative of OPA1, Mfn1, Mfn2, Fis1, Mff, and Drp1 (n = 3).

Figure 3

We next verified the results obtained in vivo on MSB-1 cells. The transcript (Figure 4A) and protein (Figure 4B) levels of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ, and ATP5A-V were significantly decreased in the LPS (P < 0.05). Similar to the in vivo results, the changes in the levels of the above genes in the Nar group differed very little from the C group. The mRNA and protein levels of the mitochondrial respiratory chain complex in the Nar+LPS group showed different degrees of increase compared with the LPS group. Furthermore, mitochondrial fusion-related genes (OPA1, Mfn1, Mfn2) were obviously decreased and mitochondrial division-related genes (Fis1, Mff, Drp1) were increased in the LPS group compared with the C group (Figures 4C and 4D). The changes of the above genes in the Nar+LPS group, compared with the LPS group, converged toward the level of the C or Nar group. Based on the fact that mitochondrial damage releases mtROS, we detected the fluorescence level of mtROS in MSB-1 cells by mito-SOX staining (Figures 4E and 4F). It was found that the intensity of red fluorescence enhanced obviously in the LPS group compared with the C group (P < 0.05), and the change in the Nar group was not significant (P > 0.05), while the red mtROS fluorescence declined significantly in the Nar+LPS group (P < 0.05). The above results indicated that Nar alleviated LPS-induced mitochondrial respiratory chain damage and dynamics disorder, and reduced the release of mtROS.Figure 4 Naringin alleviates LPS-induced mitochondrial damage on MSB-1 cells (A) The mRNA levels of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ, and ATP5A-V (n = 4). (B) The protein expression and quantitative of NDUFB8-Ⅰ, SDHB-Ⅱ, UQCRC2-Ⅲ, MTCO1-Ⅳ, and ATP5A-V (n = 3). (C) The mRNA levels of OPA1, Mfn1, Mfn2, Fis1, Mff, and Drp1 (n = 4). (D) The protein expression and quantitative of OPA1, Mfn1, Mfn2, Fis1, Mff, and Drp1 (n = 3). (E–F) The detection and quantification of mtROS (Red) by Mito-SOX staining (Scale, 100 μm) (n = 3).

Figure 4

Naringenin Alleviates LPS-Induced Oxidative Stress, Lipid Peroxidation and GSH/GSSG System Disorder

In thymus tissues (Figures 5A and 5B), MDA and LPO were significantly elevated in the LPS group compared to the C group (P < 0.05), and there were no significant variations in the Nar group. Thymic MDA and LPO levels were obviously decreased in the Nar+LPS group compared to the LPS group (P > 0.05). In terms of glutamate transport (Figure 5C), the levels of thymic SLC3A2 and GCLC transcripts were significantly greater in the LPS group than in the C group, whereas the levels of SLC7A11 were significantly fewer than those in the C group. Because of the important role of lipid peroxidation products in ferroptosis, we discovered the mRNA levels of LPCAT3, PTGS2 and ACSL4 were significantly increased in the LPS group. The expression levels of SLC3A2, GCLC, LPCAT3, PTGS2 and ACSL4 were significantly declined and the SLC7A11 was raised in Nar+LPS group compared to LPS group. In Figure 5D, GSH content and GSH-Px activity were fewer and GSSG content and GR activity were greater in the LPS group than in the C group (P < 0.05). Compared with the LPS group, the GSH and GSSG contents and GSH-Px and GR activities in the Nar+LPS group converged toward the control or Nar group. The above results indicated that Nar alleviated LPS-induced thymic oxidative stress, lipid peroxidation, and GSH/GSSG system disorder.Figure 5 Naringenin alleviates LPS-induced oxidative stress, lipid peroxidation and GSH/GSSG system disorders (A) The contents of MDA (nmol/mgprot) and LPO (μmol/mgprot) in chicken thymus (n = 3, n = 6). (B) The mRNA levels of SLC3A2, SLC7A11, and GCLC in chicken thymus (n = 4). (C) The mRNA levels of LPCAT3, PTGS2, ACSL4 in chicken thymus (n = 4). (D) The activities of GSH-Px (U/mgprot) and GR (U/mgprot), the contents of GSH and GSSG (μmol/L) in chicken thymus (n = 3). (E) The contents of MDA (nmol/mgprot) and LPO (μmol/mgprot) on MSB-1 cell (n = 3, n = 6). (F) The mRNA levels of SLC3A2, SLC7A11, and GCLC on MSB-1 cell (n = 3). (G) The mRNA levels of LPCAT3, PTGS2, ACSL4 on MSB-1 cell (n = 4). (H) The activities of GSH-Px (U/mgprot) and GR (U/mgprot), the contents of GSH and GSSG (μmol/L) (n = 3) on MSB-1 cell.

Figure 5

On MSB-1 cells, we obtained similar results as in vivo. Compared with the C group, the contents of MDA and LPO (Fig. 5E), the expression levels of SLC3A2, GCLC, LPCAT3, PTGS2, and ACSL4 (Figures 5F and 5G) were enhanced and the expression level of SLC7A11 was declined in the LPS group, as well as GSH content and GSH-Px activity were decreased, and the content of GSSG and GR activity were increased (Figure 5H). Compared with the LPS group, the above indexes in the Nar+LPS group converged toward the control or Nar group. The addition of mtROS activator (SMTIN-T140) revealed that the Nar+LPS+SMTIN-T140 group showed an opposite trend to the Nar+LPS group. The above results indicated that Nar was able to alleviate LPS-induced oxidative stress, lipid peroxidation and GSH/GSSG system disorder in MSB-1 cells via mtROS.

Naringenin Alleviates LPS-Induced Thymic Iron Overload and Ferroptosis

Based on our finding of changes in lipid peroxidation, we subsequently examined changes in iron overload and ferroptosis related indicators. In Figure 6A and 6B, the transcript levels of Fe2+ content, iron transporter genes (TFR and TF), and iron utilization genes (FTH and FTL) were significantly enhanced in the LPS group compared to the C group. Compared with the LPS group, the expression changes of the above genes in the Nar+LPS group tended to be closer to those in the C or Nar group, suggesting that Nar alleviated the LPS-induced thymic iron overload and elevated Fe2+ content. In addition, the levels of transcript (Figure 6C), protein (Figure 6D) and fluorescence intensity (Figures 6E and 6F) of GPx4 in the LPS group were fewer than those in the control and Nar groups (P < 0.05). Compared with the LPS group, all the above indexes were obviously increased in the Nar+LPS group. And the protein levels of TFR and TF in the LPS group were greater than those in the Nar+LPS group, and also higher than the control and Nar groups. The above results suggested that Nar alleviated LPS-induced thymic iron overload and ferroptosis.Figure 6 Naringenin alleviates LPS-induced disorders of iron metabolism and GPx4 inactivation (A) The detection results of Fe2+ (mg/mgprot) content in chicken thymus (n = 3). (B) The mRNA levels of TFR, TF, FTH, and FTL in chicken thymus (n = 4). (C) The mRNA levels of GPx4 in chicken thymus (n = 4). (D) The protein expression and quantitative graphs of GPx4, TFR, and TF (n = 3). (E–F) IF results and quantification of GPx4 (Red) in chicken thymus. DAPI (Blue) stained nuclei (Scale, 50 μm, 20 μm). (G) The detection results of Fe2+ (mg/mgprot) content on MSB-1 cell (n = 3). (H) The mRNA levels of GPx4 on MSB-1 cell (n = 4). (I) The mRNA levels of TFR, TF, FTH, and FTL on MSB-1 cell (n = 4). (J) The protein expression and quantitative graphs of GPx4, TFR, and TF (n = 3). (K-L) IF results and quantification of GPx4 (Red) on MSB-1 cell. DAPI (Blue) stained MSB-1 cell nuclei (Scale, 100 μm). (M-N) The detection and quantification of Fe2+ content by FerroOrange staining (Scale, 100 μm) (n = 3).

Figure 6

On MSB-1 cells, we further validated the in vivo results. Compared with the C group, the Fe2+ content was obviously more in the LPS group and significantly higher than that in the Nar+LPS group (Figure 6G). The mRNA levels of GPx4 was significantly down-regulated in the LPS group, and the transcript levels of TFR, TF, FTH and FTL were significantly increased (Figures 6H and 6I), and the above indexes in the Nar+LPS group converged towards the C or Nar group. The protein levels of GPx4, TFR and TF had a similar trend with mRNA expression (Figure 6J). In addition, the red fluorescence intensity of GPx4 in the LPS group was notably lower than that in the Nar+LPS group, and both were lower than that in the C and Nar groups (Figures 6L and 6K). We also detected the Fe2+ content of MSB-1 cells by FerroOrange staining, and discovered that the intensity of Fe2+ fluorescence in the LPS group was notably higher than that in the Nar+LPS group, and both of them were more than that in the C group or Nar group (Figures 6M and 6N). The addition of mtROS activator found that the changes of the above indexes in the Nar+LPS+SMTIN-T140 group tended to be closer to those in the LPS group compared with the Nar+LPS group. Our results suggested that Nar alleviated LPS-induced iron overload and ferroptosis on MSB-1 cells by reducing mtROS.

Naringenin Alleviates LPS-Induced Thymic Inflammatory Response and Immune dysfunction

The thymus is an important portion of the immune system, we further detected inflammatory factors and immune-related indicators by ELISA and RT-PCR. The levels of TNF-α and IL-1β in the chicken serums (Figure 7A) and MSB-1 cells (Figure 7B) in the LPS group were enhanced, and the level of IL-10 was declined (P < 0.05). The pro-inflammatory factors in the Nar+LPS group were fewer than those in the LPS group, and the anti-inflammatory factors were higher than those in the LPS group. In addition, IgG, IgA and IFN-γ were decreased in chicken serums (Figure 7C) and MSB-1 cells (Figure 7D) in the LPS group (P < 0.05). The levels of IgG, IgA and IFN-γ in the Nar+LPS group, compared with the LPS group, converged toward the control and Nar groups. Finally, the transcription levels of TNF-α and IL-1β were also found to be significantly higher and the transcript levels of IgA and IgG were notably lower (P < 0.05) in the thymus (Figure 7E) and MSB-1 cells (Figure 7F) of the LPS group by RT-PCR. The above indicators in the Nar+LPS group converged toward the C and Nar groups compared with the LPS group. Compared with Nar+LPS group, the addition of mtROS activator found that the above indexes of Nar+LPS+SMTIN-T140 group converged toward LPS group. The above results suggested that Nar alleviated LPS-induced thymic inflammatory response and immune disorder by decreasing mtROS.Figure 7 Naringenin alleviates LPS-induced disorders of iron metabolism and GPx4 inactivation (A) Determination of TNF-α, IL-1β and IL-10 in chicken serum by ELISA method (n = 3). (B) Determination of TNF-α, IL-1β and IL-10 on MSB-1 cells by ELISA method (n = 3). (C) Determination of IgA, IgG and IFN-γ in chicken serum by ELISA method (n = 3). (D) Determination of IgA, IgG and IFN-γ on MSB-1 cells by ELISA method (n = 3). (E) The mRNA levels of TNF-α, IL-1β, IgG, and IgA in chicken thymus (n = 4). (F) The mRNA levels of TNF-α, IL-1β, IgG, and IgA on MSB-1 cells (n = 4).

Figure 7

DISCUSSION

Naringenin affects immune function through modulating oxidative stress and mitochondrial function (Fujiwara, Saito et al. 2018, Pérez, Mukdsi et al. 2023, Salama, Yassen et al. 2023). Ferroptosis is different from death forms such as autophagy and apoptosis and occurs with an inflammatory response that affects immune cells (Khan, Huo et al. 2024, Shi, Xu et al. 2024). Based on this, the present study investigated the positive effects of 40 mg/kg naringenin on inflammation and immune function in chicken thymus. The results revealed that naringenin alleviated LPS-induced elevated mitochondrial division and decreased fusion, mitochondrial respiratory chain damage, mtROS release, lipid peroxidation, disruption of the GSH/GSSG system, iron overload, and inactivation of GPx4 in the chicken thymus, thereby attenuating the ferroptosis, inflammation, and immune deficits.

Naringenin has significant growth-promoting, detoxifying and antiendotoxic effects, 80 mg/kg and 160 mg/kg naringenin were able to alleviate lead acetate-induced liver damage in egg-laying hen (Esan, Ajibade et al. 2023) and also had a positive effect on chicken appearances, inflammatory immune response and egg quality (Goliomytis, Simitzis et al. 2019). Supplementation with a mixture of phytoflavonoids resisted LPS challenge by increasing chicken immune organ indices (spleen, thymus and bursa) (Kamboh, Hang et al. 2016). In our study, we discovered that, compared to 80 mg/kg and 120 mg/kg groups, 40 mg/kg naringenin was effective in mitigating LPS-induced thymus structural damage and decrease in thymus index. The flavonoid Chinese medicine monomer quercetin attenuated LPS-induced release of IL-1β, IL-6 and ROS production in A549 cells (Lv, Han et al. 2024). 500 mg/kg baicalin alleviated LPS-induced reduction of lysozyme, IgG and IgA levels in the serum of broiler chickens (Lv, Li et al. 2023). In this study, we found that 40 mg/kg naringenin was able to maintain higher serum immunoglobulin and lower serum inflammatory factor levels in chicken thymus and significantly alleviated LPS-induced thymic inflammatory response and immune disorders. The above results indicate that 40 mg/kg naringenin alleviates LPS-induced inflammatory response and immune disorder in chicken thymus.

Mitochondrial homeostatic imbalance is strongly associated with increased mtROS release. One study found that α-ketoglutarate alleviated LPS-induced intestinal damage in piglets through mitochondrial kinetic dysfunction and endoplasmic reticulum stress, as evidenced by abnormal expression of OPA1, Drp1, and Fis1 (Lu, Liu et al. 2024). Fenoldopam blocked LPS-induced loss of mitochondrial NDUFB8 and ATP synthase in the mice lungs (Bone, Liu et al. 2017). Tanshinone IIA analog 15a reduced LPS-induced mtROS production, which inhibited the NLRP3 inflammatory vesicles to attenuate acute tubular necrosis in mice (Chen, Luo et al. 2023). In the present study, naringenin alleviated LPS-induced imbalance in mitochondrial dynamics in chicken thymus and MSB-1 cells, as evidenced by decreased fusion and elevated division. In addition, naringenin reduced LPS-induced mtROS production by rescuing the level of mitochondrial respiratory chain complex. The above results suggest that naringenin alleviates LPS-induced mitochondrial damage in chicken thymus, thereby reducing mtROS release.

The classical inflammation inducer LPS was able to cause a significant increase in lipid peroxidation and ferroptosis (Yang, Lin et al. 2024). Ginsenoside Rg1 was able to alleviate the LPS-increased lipid peroxidation product MDA and regulate ferroptosis-related proteins GPx4, FSP1, and TFR, which induced neuronal ferroptosis (Zhong, Qiao et al. 2024). 80 μM quercetin activated the SIRT1/p53/SLC7A11 signaling pathway to alleviate LPS-induced elevation of myocardial MDA, cytosolic Fe2+ levels and the increasing levels of GSH, GPx4 and ferritin in the rat heart (Tian, Wang et al. 2024). In the present study, naringenin alleviated LPS-induced lipid peroxidation (MDA, LPO), disruption of the GSH/GSSG system, and abnormalities in glutamate metabolism in chicken thymus and MSB-1 cells. Naringenin also rescued LPS-induced abnormal iron metabolism and GPx4 inactivation, which caused iron overload. The above results fulfilled the 3 conditions for ferroptosis, indicating that naringenin alleviated LPS-induced ferroptosis in chicken thymus tissue. We found similar results in Zhong et al.'s study (Zhong, Qiao et al. 2024) that mtROS does regulate the iron death process. And it has also been found that the occurrence of iron death further affects the inflammatory and immune responses of tissues and cells (Shi, Xu et al. 2024), which echoes the results of our study as well. In our study, the addition of the mtROS activator SMTIN-T140 to MSB-1 cells in vitro revealed that LPS-induced lipid peroxidation, disruption of the GSH/GSSG system, iron overload, and GPx4 inactivation attenuated by naringenin were re-activated when mtROS was activated. And mtROS activation reversed the effects of naringenin on LPS-induced serum inflammatory factors and immunoglobulin levels, suggesting that mtROS-mediated ferroptosis affects the inflammatory response and immune function in chicken thymus.

CONCLUSION

In conclusion, our results reveal the anti-inflammatory, antiferroptosis and immune-enhancing functions of naringenin in chicken thymus. 40 mg/kg naringenin alleviates LPS-induced imbalance in mitochondrial dynamics, blockage of mitochondrial respiratory chain, and mtROS release in chicken thymus. The excessive mtROS further induces iron metamorphosis pathway (iron overload, GSH/GSSG system and lipid peroxidation) and GPx4 inactivation which in turn caused ferroptosis, inflammation and immune dysfunction in thymic tissues. In short, naringenin alleviates LPS-induced ferroptosis, inflammation and immune dysfunction in chicken thymus tissue by releasing mtROS. Our results in this study are of great value for the pharmacology of enriched flavonoids and provide new possible therapeutic agents for inflammatory diseases.

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

Image, application 2

ACKNOWLEDGMENTS

The authors extend their sincere thanks to the members of the Veterinary Internal Medicine Laboratory, Key Laboratory of Experimental Animals and Embryo Biotechnology Laboratory of College of Life Sciences of Northeast Agricultural University, Northeast Agricultural University. This study was supported by the 10.13039/501100001809 National Natural Science Foundation of China (U22A20524 ), the 10.13039/501100005046 Natural Science Foundation of Heilongjiang Province (ZD2023C002 ), and the 10.13039/501100014219 National Science Fund for Distinguished Young Scholars (32202875 ).

Compliance with ethics requirements: All procedures used in this research were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (SRM-11).

Data availability statement: The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104179.
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