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. 2023 Mar 3;102(6):102622. doi: 10.1016/j.psj.2023.102622

The anti-inflammatory effect of lutein in broilers is mediated by regulating Toll-like receptor 4/myeloid-differentiation-factor 88 signaling pathway

Zhi-Xin Lin ⁎, Min Zhang *, Rui Yang *, Yao Min *, Ping-Ting Guo *, Jing Zhang *, Chang-Kang Wang *, Ling Jin †,1, Yu-Yun Gao ⁎
PMCID: PMC10122034  PMID: 37019074

Abstract

The anti-inflammatory role of lutein has been widely recognized, however, the underlying mechanism is still not fully elucidated. Hence, the effects of lutein on the intestinal health and growth performance of broilers and the action of mechanism were investigated. 288 male yellow-feathered broilers (1-day old) were randomly allocated to 3 treatment groups with 8 replicates of 12 birds each, and the control group was fed a broken rice-soybean basal diet, while the test groups were fed a basal diet added with 20 mg/kg and 40 mg/kg of lutein (LU20, LU40), respectively. The feeding trial lasted for 21 d. The results showed that 40 mg/kg lutein supplementation tended to increase ADFI (P = 0.10) and ADG (P = 0.08) of broilers. Moreover, the addition of lutein caused a decreasing trend of gene expression and concentration of proinflammatory cytokines IL-1β (P = 0.08, P = 0.10, respectively) and IL-6 (P = 0.06, P = 0.06, respectively) and also tended to decrease the gene expression of TLR4 (P = 0.09) and MyD88 (P = 0.07) while increasing gene expression and concentration of anti-inflammatory cytokines IL-4 and IL-10 (P < 0.05) in the jejunum mucosa of broilers. Additionally, lutein supplementation increased the jejunal villi height of broilers (P < 0.05) and reduced villi damage. The experiment in vitro showed that lutein treatment reduced the gene expression of IL-1β, IL-6, and IFN-γ in chicken intestinal epithelial cells (P < 0.05). However, this effect was diminished after knock-down of TLR4 or MyD88 genes using RNAi technology. In conclusion, lutein can inhibit the expression and secretion of proinflammatory cytokines in the jejunum mucosa and promote intestinal development of broilers, and the anti-inflammatory effect may be achieved by regulating TLR4/MyD88 signaling pathway.

Key words: lutein, TLR4/MyD88 signaling pathway, yellow-feathered broilers, intestinal health, cytokines

INTRODUCTION

Lutein, also known as phytoxanthin, is a dihydroxy carotenoid containing 2 ionone rings, which is widely found in all kinds of dark green leafy vegetables, among which marigold is the richest in lutein (Craft and Soares, 1992). The hydroxyl groups in its structure make lutein more polar and hydrophilic than other carotenoids, which can better react with oxygen in serum and effectively scavenge reactive oxygen species (ROS) (including superoxide anions, peroxyl radicals, and hydroxyl radicals, etc.), thus preventing the lone pair of electrons of free radicals from damaging the lipid molecular layer of cell membranes, proteins, and DNA (Krinsky and Johnson, 2005). In addition, superoxide radicals can be converted to hydrogen peroxide and singlet oxygen through nonenzymatic reactions. Like other free radicals, singlet oxygen can also inflict cellular damage through oxidation reactions. Lutein can prevent this damage by quenching singlet oxygen through a triplet-state excited energy transfer process (Foote and Denny, 1968). Several studies have indicated that oxidative stress and inflammation are interactive, as free radical oxidation leads to elevated levels of inflammation, while inflammation, in turn, promotes the production of free radicals (Biswas, 2016; Agita and Alsagaff, 2017), so the anti-inflammatory activity of lutein, which has superb antioxidant capacity, should not be underestimated. In clinical studies, lutein has shown its ability to prevent a variety of inflammatory diseases, including neurodegenerative diseases (Shimazu et al., 2019), osteoporosis (Qiao et al., 2018), vascular diseases (Han et al., 2015), skin diseases (Oh et al., 2013), liver injury (Kim et al., 2012), etc. However, despite numerous studies, the underlying mechanism of the anti-inflammatory activity of lutein remains unclear.

Toll-like receptor 4 (TLR4), a type I transmembrane protein expressed in a variety of tissue cells, recognizes lipopolysaccharide (LPS) from gram-negative bacteria and promotes the secretion of proinflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interferon-γ (IFN-γ) (Kawai and Akira, 2010; Płóciennikowska et al., 2015), through either the myeloid-differentiation-factor 88 (MyD88, an important junction protein in the TLR4 pathway) dependent pathway or the MyD88 independent pathway, ultimately leading to a severe inflammatory response (Khan et al., 2006; Deguine and Barton, 2014). Gram-negative bacilli such as Salmonella (Barrow, 2000), Escherichia coli (Ateya et al., 2019), and Campylobacter (Nooreh et al., 2021) often cause intestinal health problems in broiler production and lead to reduced growth performance. Therefore, exploring TLR4 signaling pathway regulators would be a key target to overcome the problem of bacterial enteritis in broilers. The objectives of this study were to investigate the effects of lutein on growth performance, anti-inflammatory capacity, and intestinal development of yellow-feathered broilers, and then to further investigate the possible mechanisms of lutein through in vitro experiments.

MATERIALS AND METHODS

Experimental Design, Animals, and Diets

A total of 288 one-day-old male yellow-feathered broilers (average weight 36 g, purchased from Fujian Wenshi Poultry Co., Ltd., Fujian, China) were randomly divided into 3 treatments with 8 replicates each and 12 birds per replicate. To reduce the content of other oxygenated carotenoids in the feed, a broken rice-soybean basal diet (Jinhualong Feed Factory, Fujian, China) with a measured carotenoid value of 3.13 mg/kg was used in this experiment. The control group was fed a basal diet, and the test groups were fed a basal diet supplemented with 20 mg/kg and 40 mg/kg of lutein (purity ≥2%, Juyuan Biochemical Co. Ltd., Guangzhou, China) respectively for 21 d. As we previously reported, the addition of lutein in this experiment was chosen to be similar to the pigment content in the commercial diets (Gao et al., 2013). All birds had ad libitum access to feed and water. The diets were prepared according to the Chinese Feeding Standard of Chicken (2004). The composition and nutrition level of the basal diet is shown in Table 1. All the experimental procedures applied in this study were reviewed and approved by the Committee of Animal Experiments of Fujian Agriculture and Forestry University (Fuzhou, Fujian, China, approval ID 202003016). All efforts were made to minimize suffering.

Table 1.

Ingredient composition and nutrient levels of the basal diets (as-fed basis, %).

Items 1–21 d
Ingredients
 Broken rice 61.86
 Soybean meal 32.55
 Expanded soybean 1.00
 Limestone 1.21
 Calcium hydro phosphate 1.87
 DL-Methionine 0.21
 Premix1) 1.00
 Salt 0.30
 Total 100.00
Calculated nutrient levels 2)
 Apparent metabolizable energy (MJ/kg) 12.12
 Crude protein 21.00
 Calcium 1.00
 Total phosphorus 0.73
 Available phosphorus 0.45
 Lysine 1.13
 Methionine + cysteine 0.85
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1

Premix provided per kilogram diet: vitamin A, 8,000 IU; vitamin D3, 2,000 IU; vitamin E, 20 IU; vitamin K3, 1 mg; thiamin, 2.6 mg; riboflavin, 5.4 mg; vitamin B6, 6 mg; vitamin B12, 0.02 mg; niacin, 40 mg; folic acid, 0.8 mg; biotin, 0.2 mg; choline, 1,200 mg; pantothenic acid, 20 mg; Zn, 60 mg; Mn, 80 mg; Fe, 80 mg; Cu, 8 mg; I, 0.35 mg; Se, 0.15 mg.

2

Nutrient levels were calculated values.

Growth Performance Measurement

The daily feed intake was recorded in replicates, and the total feed consumption of each replicate was counted at 18:00 on the day before the end of the experiment, and the total weight of broilers in each replicate was weighed at 6:00 on the following day. The average daily feed intake (ADFI), average daily weight gain (ADG), feed-to-gain ratio (F/G), and survival rate (SR) was calculated for each group of 21-day-old broilers.

Sample Collection

At 21 d, 8 medium-weight broilers (1 bird from each replication) were randomly selected from each treatment. Then, birds were individually weighed, euthanized by cervical dislocation, and collected for jejunum tissue samples. The mid-jejunum tissues were collected and gently rinsed with 4°C phosphate buffered saline (PBS) to remove the contents, then 2 cm intestine segments were rapidly cut with ophthalmic scissors. Intestinal tissues used for histomorphology observation were processed as following. The remaining intestinal tissues were quickly stored in liquid nitrogen, and then frozen at −80°C for further analysis.

Jejunum Histomorphology Observation

Obtained jejunum tissues were fixed in a 4% paraformaldehyde solution, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Paraffin sections of jejunum tissue were observed under a light microscope (Nikon Corporation, Tokyo, Japan) and photographed, and intestinal villi height (VH) and crypt depth (CD) were measured using Image Pro Plus 6.0 software (Media Cybernetics, MD), and then the ratio of villi height to crypt depth (VH/CD) was calculated.

Jejunum Ultrastructure Observation

Scanning electron microscopy sections were made as described in our previous study (Wang et al., 2022). The obtained 2-cm-long mid-jejunum was dissected longitudinally to expose the mucosal portion and rinsed 3 to 4 times with 0.1 mol/L of PBS, then cut it into thin strips (2 mm × 2 mm). The jejunum tissue was fixed in a 2.5% glutaraldehyde solution for 48 h at 4°C then rinsed with PBS. After rinsing well, specimens were fixed with 1% osmium acid for 1.5 h, dehydrated in gradient alcohol, transitioned with isoamyl acetate, and critical point dried with a K850 point dryer (Quorum, Edinburgh, UK) to make scanning electron microscopy (SEM) sections. Gold was sputtered with MC1000 sputter coater (Hitachi, Tokyo, Japan) and then observed and photographed using SU8010 scanning electron microscope (Hitachi, Tokyo, Japan). The SEM images were measured and analyzed using the Image J software program (Media Cybernetics, MD).

Cell Isolation and Culture

The whole process of cell isolation from chicken intestine was carried out in a sterile environment using sterile solutions and materials. The isolation procedure is a modification of the method used by Dimier-Poisson et al. (2004). Chicken intestinal embryonic tissue was obtained from 16-day-old embryo SPF egg (Spafax Biotechnology, Shandong, China). The mesenteric fat was removed, and the intestine was longitudinally opened and thoroughly rinsed 5 times with 37°C PBS. The intestine was then cut into small pieces (approximately 1 mm), suspended with a small amount of DMEM (DMEM; Hyclone Corporation, Logan, UT) and left to stand for 3 min at room temperature. The digestion protocol was a modification of previous studies (Perreault and Beaulieu, 1996; Golaz et al., 2007). A mixture of 300 U/mL of type Ⅺ collagenase and 0.1 mg/mL of type I neutral protease (Nordmark Pharma GmbH, Heidelberg, Germany) was added and codigested by shaking for 25 min at 37°C in a water bath. The tissue blocks were repeatedly blown with a pipette for 3 to 5 min and then centrifuged at 100 × g for 5 min. After the digestion, the digestive medium was removed and 5 mL DMEM was used to float the tissue pieces. Then the pieces were centrifuged at 100 × g for 10 min and the above process was repeated twice. The supernatant was discarded, and the cell precipitate was added to DMEM (containing 10% (v/v) fetal bovine serum (FBS; Cellmax, Beijing, China), 10 g/L penicillin and streptomycin), which was carefully blown and mixed with a pipette and then seeded into cell culture plates. The cells were incubated in a 37°C, 5% CO2 incubator. After 3 h, the cell culture medium together with the unadhered cells were collected into a centrifuge tube and centrifuged at 100 × g for 10 min. The supernatant was discarded and DMEM was added again to the cell precipitate, after which it was seeded into cell culture plates and placed in an incubator at 37°C with 5% CO2. The purity of the isolated cells was assessed to be 90%.

Cell Transfection

According to the manufacturer's instructions, well-grown chicken intestinal epithelial cells were seeded into 6-well cell plate and cultured for 24 h. Transfection was performed when cells reached 70% confluence using Lipofectamine 2000 (Thermo Fisher Scientific, Shanghai, China). Firstly, the siRNA was prepared with DEPC water to a concentration of 20 μM solution, and then 1.5 μL siRNA was diluted with 50 μL Opti-MEM and mixed well to make siRNA dilution solution. Subsequently, 50 μL Opti-MEM was added to 1 μL Lipofectamine 2000, mixed by gently blowing 3 to 5 times, and left at room temperature for 3 to 5 min to form transfection reagent. Finally, the transfection reagent and siRNA dilution were gently mixed to form the cell transfection complex. The solution is left at room temperature for 20 min before starting transfection. The original culture medium was discarded, then added 100 μL of the prepared transfection solution and 400 μL Opti-MEM to the 6-well cell plate, and shook the cell plate gently to mix well (the control group used 100 μL Opti-MEM instead of transfection solution). The chicken intestinal epithelial cells were incubated in a 37°C, 5% CO2 cell culture incubator for 24 h and then replaced with a complete medium for another 24 h. The siRNA sequences are as follows: TLR4 siRNA sense: 5′-GCAACUCUUUGAGAACAATT-3′ and antisense 5′-UUGUCUCAAAGGAGUGCTT-3′; and MyD88 siRNA sense 5′-GCCAAAGACUUCAGAGCUGTT-3′ and antisense: 5′-CAGCUCUGAAGUCUUUGGCTT-3′.

Cells Treatments

To find the optimal concentration of lutein treatment, the isolated chicken intestinal epithelial cells were first seeded into 6-well plates and then incubated with different concentrations of lutein (0, 10, 20, 40, 80, 160 μmol/L) (Product Number: X6250, purity more than 75%, Sigma Aldrich Trading, Shanghai, China) for 24 h. After determining the concentration of lutein treatment, cell transfection experiments were then performed as described above. Control group, cells were transfected with scrambled siRNA (si-NC) and incubated for 24 h; LPS group, cells were transfected with si-NC for 24 h, then stimulated with 20 μg/mL LPS (Product Number: L2880, Sigma Aldrich Trading, Shanghai, China) for another 24 h; Lutein group, cells were transfected with si-NC for 24 h, then treated with 80 μmol/L lutein for another 24 h. siTLR4 and siMyD88 groups, cells were transfected with corresponding siRNA for 24 h; siRNA + LPS group, cells were transfected with siRNA for 24 h and then stimulated with 20 μg/mL LPS for another 24 h; siRNA + Lutein group, cells were transfected with siRNA for 24 h and treated with 80 μmol/L lutein for another 24 h; siRNA + Lutein + LPS group, cells were transfected with siRNA for 24 h, and then treated cells with 80 μmol/L lutein for 12 h, and then stimulated cells with 20 μg/mL LPS, and cocultured for 24 h.

qRT-PCR

The method to determine gene expression was generally the same as we previously reported (Gao et al., 2012). Total RNA was first extracted from jejunum mucosa and chicken intestinal epithelial cell using TRIzol reagent (Promega Biotechnology, Beijing, China) according to the manufacturer's instructions. Then, the quantity and quality of total RNA was assessed using Nanodrop 2000 (Thermo Fisher Scientific, MA). The integrity of RNA was checked by electrophoretic analysis. Reverse transcription was performed using the PrimeScript RT kit (Promega Biotechnology Co., Ltd., Beijing, China). qRT-PCR amplification conditions were as follows: initial denaturation at 94°C for 30 s, followed by 40 cycles of denaturation at 94°C for 5 s, annealing at 56°C for 15 s, and extension at 72°C for 10 s. β-Actin was used as an internal control for normalizing gene, and gene expression levels were analyzed using the 2−ΔΔCT method. The primer sequences used to detect the mRNA levels of the relevant genes in the jejunum mucosa are shown in Table 2.

Table 2.

Primer sequences.

Gene Primer sequence
β-Actin F: 5′-CAAAAGCCAACAGAGAGAAGAT-3′
R: 5′-CATCACCAGAGTCCATCACAAT-3′
MyD88 F: 5′-GGGATGTCTTGCCAGGAACG -3′
R: 5′-TGCACTTGACCGGAATCAGC-3′
TLR4 F: 5′- TTCGGTTGGTGGACCTGAATCTTG-3′
R: 5′-ACAGCTTCTCAGCAGGCAATTCC-3′
IL-1β F: 5′-GGAGCAGGGACTTTGCTGAC-3′
R: 5′-AAGGACTGTGAGCGGGTGTA-3′
IL-6 F: 5′-AAATCCCTCCTCGCCAATCT-3′
R: 5′-CCCTCACGGTCTTCTCCATAAA-3ʹ
IFN-γ F: 5′-CTTCCTGATGGCGTGAAGA-3′
R: 5′-GAGGATCCACCAGCTTCTGT-3′
IL-4 F: 5′-GGGAGGAGTGGCAGAAGTAG-3′
R: 5′-GCTAAACGAGGTCCAGCATT-3′
IL-10 F: 5′-CATCCATAGCCAGAAGAGGAA-3′
R: 5′-GGCGAAATCCATCAGGAA-3′
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ELISA Analyses

The levels of IL-1β, interleukin-4 (IL-4), IL-6, interleukin-10 (IL-10), and IFN-γ in the jejunum mucosa were determined using chicken-specific ELISA kits purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturer's instructions. Total protein was measured by assay kits A045 (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Data Analysis

Statistical analysis was performed using SPSS, version 20.0 (SPSS, Inc., Chicago, IL). The data were analyzed using 1-way analysis of variance (ANOVA) and Tukey's multiple range tests for multiple comparisons. Results are presented as mean ± standard deviation. P < 0.05 were considered statistically significant, and 0.05 < P ≤ 0.10 were taken to indicate a statistical tendency.

RESULTS

Growth Performance

The effects of lutein on growth performance are presented in Table 3. Compared with the control group, the ADFI (P = 0.10) and ADG (P = 0.07) of broilers in the group supplemented with 40 mg/kg of lutein tended to increase, but there was no significant change in F/G and SR (P > 0.05).

Table 3.

Effect of dietary supplementation of lutein on the growth performance of yellow-feathered broilers1.

Groups1
Items2 Control LU20 LU40 P value
ADFI (g) 24.36 ± 0.67 24.67 ± 0.79 25.23 ± 0.86 0.10
ADG (g) 14.41 ± 0.61 14.69 ± 0.61 15.15 ± 0.65 0.08
F/G 1.69 ± 0.09 1.68 ± 0.03 1.67 ± 0.07 0.72
SR (%) 99.0 ± 2.95 100 ± 0.00 100 ± 0.00 0.39
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Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; F/G, feed to gain ratio; SR, survival rate.

1

Control group, basal diet; LU20 group, basal diet added with 20 mg/kg lutein; LU40 group, basal diet added with 40 mg/kg lutein.

2

Values are mean ± SD, n = 8.

Jejunum Histomorphology of Broilers

As shown in Table 4, dietary supplementation with 40 mg/kg of lutein increased the jejunum villi of broilers (P < 0.05), but had no significant effect on VH/CD (P > 0.05).

Table 4.

Effect of dietary supplementation of lutein on jejunum intestinal morphology in yellow-feathered broilers1.

Groups1
Items2 Control LU20 LU40 P value
Villus length (μm) 852.7 ± 81.54b 922.8 ± 110.71ab 976.9 ± 94.29a 0.04
Crypt depth (μm) 106.6 ± 10.68 109.4 ± 15.99 108.7 ± 17.72 0.91
VH/CD3 8.08 ± 1.20 8.51 ± 0.91 9.16 ± 1.56 0.17
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1

Control group, basal diet; LU20 group, basal diet added with 20 mg/kg lutein; LU40 group, basal diet added with 40 mg/kg lutein.

2

Values are mean ± SD, n = 8. Mean values within a row with different superscripts letters (a, b) denote statistical significantly difference, P < 0.05.

3

VH/CD represents the ratio of intestinal villi height to crypt depth.

Jejunum Ultrastructure of Broilers

Under the low magnification SEM, the intestinal villi of the control broiler group were atrophied (Figure 1A), and the microvilli on the surface of the intestinal villi were damaged and lost in many places, forming microvillous pits (Figure 1A'). In contrast, the intestinal microvilli of broilers in the lutein-added group were more intact (Figure 1B, C). The microvilli were much denser and the microvillous pits were smaller (Figure 1B'), and some of the small microvillous pits were in a state of imminent healing (Figure 1C').

Figure 1.

Figure 1

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Scanning electron micrograph of the ultrastructure of the jejunum (low magnification). Red arrow, the villi damage. Control group, basal diet; LU20 group, basal diet added with 20 mg/kg lutein; LU40 group, basal diet added with 40 mg/kg lutein. Scale bar of Figure A, B, C is 500 μm. Scale bar of Figure A’, B’, C’ is 50 μm.

Under high magnification SEM, the control broilers had the sparsest microvilli (Figure 2A), the most spongy pores on the surface of microvilli, and the length of microvilli was about 14 μm (the small grid in the figure is 2 μm) (Figure 2A'). Broilers in the lutein-added group had denser and longer microvilli with fewer spongy pores (Figure 2B, C). The length of microvilli in the LU20 group was about 16 μm (Figure 2B'), and the length of microvilli in the LU40 group was about 17 to 18 μm (Figure 2C').

Figure 2.

Figure 2

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Scanning electron micrograph of the ultrastructure of the jejunum (high magnification). Control group, basal diet; LU20 group, basal diet added with 20 mg/kg lutein; LU40 group, basal diet added with 40 mg/kg lutein. Scale bar = 2 μm. Figure A, B, C shows the cross-section of jejunum, and Figure A', B', C' shows the longitudinal section of jejunum.

Gene Expression and Concentration of Cytokines in the Jejunum Mucosa

The effects of lutein on cytokine gene expression and concentration in jejunum mucosa are shown in Figure 3. Compared with the control group, dietary supplementation of 40 mg/kg lutein caused a decreasing trend of gene expression and concentration of proinflammatory cytokines IL-1β (P = 0.08, P = 0.10) and IL-6 (P = 0.06, P = 0.06) but had no significant effect on IFN-γ (P > 0.05) in the jejunum mucosa of broilers. However, the gene expression and concentration of anti-inflammatory cytokines IL-4 and IL-10 (P < 0.05) in the jejunum mucosa of broilers in the LU40 group was remarkably increased compared with the control group.

Figure 3.

Figure 3

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Effect of lutein on gene expression (A) and concentration (B) of cytokines in jejunum mucosa of yellow-feathered broilers. Control group, basal diet; LU20 group, basal diet added with 20 mg/kg lutein; LU40 group, basal diet added with 40 mg/kg lutein. Values are mean ± SD. Labeled means without a common letter differ, P < 0.05.

Gene Expression of TLR4 and MyD88 in the Jejunum Mucosa

As shown in Figure 4, there was a trend of decreasing gene expression of TLR4 (P = 0.09) and MyD88 (P = 0.07) in the jejunal mucosa of broilers in the lutein-added group compared with the control group.

Figure 4.

Figure 4

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Effect of lutein on gene expression of TLR4 and MyD88 in jejunum mucosa of yellow-feathered broilers. Control group, basal diet; LU20 group, basal diet added with 20 mg/kg lutein; LU40 group, basal diet added with 40 mg/kg lutein. Values are mean ± SD. Labeled means without a common letter differ, P < 0.05.

Gene Expression Levels of Cytokines in Chicken Intestinal Epithelial Cells

Compared with the control group, 10 μmol/L lutein decreased the gene expression of IL-1β and IL-6 (P < 0.05) in chicken intestinal epithelial cells, but had no significant effect on IFN-γ (P > 0.05), and 20, 40, 80, and 160 μmol/L lutein downregulated the gene expression level of IL-1β, IL-6, and IFN-γ (P < 0.05), and 80 μmol/L lutein had the best down-regulation effect (Figure 5A–C).

Figure 5.

Figure 5

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Effects of different concentrations of lutein on the relative mRNA expression of IL-1β (A), Il-6 (B), IFN-γ (C), TLR4 (D), MyD88 (E) in chicken intestinal epithelial cells. Values are mean ± SD. Labeled means without a common letter differ, P < 0.05. In the control group, 10% FBS was added to the complete medium, while in the test group, medium containing lutein concentrations of 10 μmol/L, 20 μmol/L, 40 μmol/L, 80 μmol/L, and 160 μmol/L were added and the gene expression levels of cytokines were measured after 24 h incubation in a CO2 cell incubator.

The Expression Levels of TLR4 and MyD88 Genes in Chicken Intestinal Epithelial Cells

As shown in Figure 5D and E, all lutein-treated groups downregulated the gene expression level of TLR4 and MyD88 (P < 0.05) in chicken intestinal epithelial cells compared with the control group, and the addition of 80 μmol/L of lutein still achieved the best down-regulation effect.

Cytokine Gene Expression Levels After Interfering With TLR4 and MyD88 in Chicken Intestinal Epithelial Cells

As shown in Figure 6, lutein decreased the gene expression levels of IL-1β, IL-6, and IFN-γ compared with the control group (P < 0.05). However, in the presence of siRNA, there was no significant change in IL-1β, IL-6, and IFN-γ gene expression in the siTLR4 + Lutein group compared with the siTLR4 group (P > 0.05). And the gene expressions of IL-1β, IL-6, and IFN-γ were also not significantly changed in the siMyD88+lutein group compared with the siMyD88 group (P > 0.05). The above results suggested that the knock-down of TLR4 or MyD88 genes greatly diminished the inhibitory effect of lutein on proinflammatory cytokines, and it was inferred that lutein is likely to act through the TLR4/MyD88 pathway.

Figure 6.

Figure 6

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Effect of lutein on the relative mRNA expression of IL-1β (A), IL-6 (B), IFN-γ (C) in chicken intestinal epithelial cells after knock-down of TLR4 or MyD88 genes. Values are mean ± SD. *P < 0.05.

DISCUSSION

In this study, dietary supplementation with 40 mg/kg lutein resulted in a trend toward increased ADFI and ADG in yellow-feathered broilers. However, F/G and SR were not affected by lutein addition. This is in general agreement with the results obtained by Zhao et al. who showed that the addition of Barranca yajiagengensis powder (rich in lutein) to the diet of fish had no significant effect on feed conversion rate, but could improve the body weight gain of the test fish to some extent (Zhao et al., 2022).

Villi are thin, hair-like structures arranged inside the small intestine. Villi expand the surface area of the intestine and help the body absorb nutrients from food as it passes through the digestive tract (Dailey, 2014). The intestinal villi length depends on the balance between proliferation and apoptosis of intestinal epithelial cells (Kai, 2021). In addition, the intestinal epithelial cells and intercellular junction complexes together form a mechanical barrier to the intestinal tract to protect it from external pathogens, thus maintaining intestinal health (Groschwitz and Hogan, 2009). Therefore, the survival of intestinal cells not only affects the length of the intestinal villi but also plays an important role in the integrity of the intestinal barrier. In the present study, dietary supplemented with lutein promoted the growth of jejunum villi and maintained the integrity of intestinal villi in 21-day-old yellow-feathered broilers. Our previous study (Gao et al., 2016) showed that lutein upregulated the gene expression of B cell lymphoma/leukemia-2 protein while suppressing the apoptosis-related protein caspase-3, thereby inhibiting intestinal apoptosis. Given the role of intestinal epithelial cells in the length of the intestinal villi and in the intestinal barrier, we suggest that lutein is likely to promote cell survival and thus the growth of intestinal villi in broilers by inhibiting the expression of apoptosis-related proteins, reducing intestinal villi damage, and maintaining the integrity of the intestinal barrier.

Cytokines, which are mainly secreted by helper T cells, can be classified as pro- and anti-inflammatory cytokines and play a key role in the initiation, enhancement, and persistence of inflammation (Bamias et al., 2012). IL-1β has broad immunomodulatory effects and may mediate inflammation or be directly involved in inflammatory processes (Lopez-Castejon and Brough, 2011). IL-6 is a cytokine with multiple immunomodulatory functions that stimulates the proliferation of B cells, T cells, and stem cells, promotes B cell production of immunoglobulins, and promotes cytotoxic lymphocyte and stem cell differentiation (Tanaka et al., 2014). IFN-γ is a soluble dimeric cytokine, the only member of type II interferons, with an important role in intrinsic and adaptive immunity against viruses, certain bacteria, and protozoan infections (Mendoza et al., 2019), and abnormal expression of IFN-γ is associated with many auto-inflammatory and autoimmune diseases (Ikeda et al., 2002). Thus, IL-1β, IL-6, and IFN-γ are proinflammatory cytokines that play an important role in promoting inflammation. In contrast, IL-4 and IL-10 are typical anti-inflammatory cytokines that can antagonize the proinflammatory effects of other cytokines, thereby controlling inflammation (Rutz and Ouyang, 2016; Brown and Hural, 2017). Meurer et al. (2019) showed that aqueous extract of marigold (rich in lutein) reduced the content of IL-6 and TNF-α in the colon of mice. Another study (Shanmugasundaram and Selvaraj, 2011) reported that dietary supplementation of lutein downregulated the mRNA level of IL-1β in turkey spleen. Our previous research (Gao et al., 2012) also found that lutein had a modulatory effect on inflammatory response indicators in chickens, reduced the expression of the proinflammatory cytokines IFN-γ, IL-1β, and IL-6, while increasing the expression of anti-inflammatory cytokines IL-4 and IL-10 in the liver and jejunum of breeding hens and chicks. Similarly, the results of this experiment showed that 40 mg/kg lutein caused a tendency to reduce the gene expression of IL-1β, IL-6, and IFN-γ and increase the gene expression of IL-4 and IL-10 in the jejunum mucosa of broiler and the same effect was shown for lutein in vitro experiments. Therefore, we suggested that lutein suppresses inflammation by inhibiting the secretion of proinflammatory factors and increasing the secretion of anti-inflammatory factors.

Previous studies have shown that when inflammation occurs in the animal body, it leads to a redistribution of nutrients, with a large number of nutrients being used for immunity and inflammation, as well as inducing anorexia and a reduction in ADFI (Dinarello et al., 1990). Lutein can inhibit inflammation and thus avoid nutrient wastage. In addition, lutein increases the length of intestinal villi and reduces intestinal villi damage in broilers, which helps to improve nutrient absorption by the organism (Walton et al., 2018), which would also positively affect growth performance in broilers. Thus, lutein may affect growth performance in yellow-feathered broilers through a combination of both inhibition of inflammation and increase in intestinal villi length.

Subsequently, we explored the potential mechanisms underlying the anti-inflammatory effects of lutein. TLR4/MyD88 is a classical inflammatory signaling pathway that plays a key role in regulating the transcription of the proinflammatory cytokines IL-1β, IL-6, and IFN-γ (Li et al., 2019). We found that lutein significantly inhibited the expression of TLR4 and MyD88 genes in chicken intestinal epithelial cells. To further confirm the effect of lutein on the TLR4/MyD88 signaling pathway, an immune stress model of chicken intestinal epithelial cells was established using LPS, and our previous study showed that the optimal concentration of LPS is 20 μg/mL. Then the effects of lutein on the expression levels of IL-1β, IL-6, and IFN-γ genes under different conditions were investigated. The data indicated that the inhibitory effect of lutein on proinflammatory cytokines became insignificant after knock-down of TLR4 or MyD88 gene. Therefore, we suggest that lutein is likely to achieve its anti-inflammatory effect through the TLR4/MyD88 signaling pathway. In addition, the present experiment revealed that lutein alleviated the elevated expression of LPS-induced proinflammatory cytokines both before and after knock-down of TLR4 or MyD88 gene. As mentioned previously, lutein has a strong antioxidant capacity and can effectively scavenge intracellular ROS, which can lead to decreased secretion of proinflammatory cytokines through multiple pathways (Fang and Richardson, 2005). Thus, lutein may achieve its anti-inflammatory function primarily by affecting the TLR4/MyD88 signaling pathway, but it may also achieve this effect by directly scavenging ROS.

CONCLUSIONS

Lutein promoted intestinal villi growth, and reduced villi damage. In addition, lutein inhibited the expression of proinflammatory cytokines IL-1β, IL-6, and IFN-γ both in vivo and in vitro, this effect was mainly achieved through regulating the TLR4/MyD88 signaling pathway.

ACKNOWLEDGMENTS

This study was supported by National Natural Science Foundation of China (31802079), Natural Science Foundation of Fujian Province (2022J01587), Fujian Specialist Funds of Chicken Industrial System in China (K83139297, 2019-2022), and Science and Technology Development Projects Funded by Chinese Central and Local Governments (20022L3085).

DISCLOSURES

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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