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

S0032-5791(24)00822-8
10.1016/j.psj.2024.104243
104243
METABOLISM AND NUTRITION
Early weaning damages the intestinal epithelial barrier of squabs through toll-like receptor signaling pathways
Xu Qianqian *†
Lv Mengqi *
Yuan Yiwei *
Ling Tianliang *
Zou Xiaoting †
Dong Xinyang sophiedxy@zju.edu.cn
†1
⁎ Key Laboratory of Characteristic Agricultural Product Quality and Hazardous Substance Control Technology of Zhejiang Province, Institute of Food Nutrition and Quality Safety, College of Life Science, China Jiliang University, Hangzhou, 310018, China
† Key laboratory for Molecular Animal Nutrition of Ministry of Education, Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Feed Science Institute, College of Animal Science, Zhejiang University (Zijingang Campus), Hangzhou, 310058, China
1 Corresponding author: sophiedxy@zju.edu.cn
22 8 2024
12 2024
22 8 2024
103 12 1042433 6 2024
18 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/).
Stress damage caused by early weaning and its possible mechanism have been studied mainly in young mammals, but rarely in altrices, especially in squabs. The study aimed to investigate the possible molecular mechanism of intestinal epithelial barrier damage caused by early weaning in squabs through determining the intestinal permeability, the ultrastructure of villous epithelium, the contents of ileal cytokines, and the protein relative expression of tight-junction proteins, TLRs and their mediated key factors in inflammatory signaling pathways. A total of 192 newly hatched squabs were randomly divided into 2 groups, 1 group was weaned and fed artificial pigeon milk from d 7, and the other group continued to be fed by the parent pigeons. The ileal mucosa and serum of 8 replicates were collected at 1, 4, 7, 10, and 14 d after weaning. The results indicated that early weaning could reduce the growth performance of squabs and damage the intestinal epithelial barrier, which is characterized by down-regulating the protein expression of claudin-1/3, up-regulating the protein expression of claudin-2, promoting the secretion of pro-inflammatory factors, inhibiting the secretion of anti-inflammatory factors, and increasing the permeability of the intestinal barrier. The specific mechanism of stress damage might be the activation of TLR2/4-MyD88-ERK/JNK inflammatory signaling pathway leading to the increase levels of IL-6 and TNF-α.

Key words

pigeon squab
intestinal barrier
early weaning
toll-like receptor
==== Body
pmcINTRODUCTION

Despite growing demand for pigeon squabs as a high-quality livestock product (Ji et al., 2020), the industry's productivity lags behind other poultry due to distinct growth characteristics (Gao et al., 2016; Jin et al., 2023). As altrices, the squabs are completely dependent on parental feeding and are usually weaned at 3 to 4 wk after hatching under natural conditions (Sales and Janssens, 2003; Mahdy, 2021; Ji et al., 2022). It means that during the period before the squabs are weaned, the breeding pigeons need to take care of the young all the time (Horseman and Buntin, 1995), and the next cycle of egg laying cannot be carried out as soon as possible. Obviously, the traditional breeding mode restricts the production efficiency of squabs, which has become a limiting factor in the pursuit of maximum economic benefits. If the squabs weaned early and left the nest, the parent pigeons could lay 10 to 20 d in advance, thus the annual egg production per each pair of parent pigeon could be increased from 6 pairs to 10 to 15 pairs (Xu et al., 2022). Therefore, artificial feeding of early weaned squabs is a necessary move to improve the production efficiency of squabs.

However, as with early weaned mammals, early weaned squabs also need to undergo a transition from parent nursing to artificial feeding, from a parent-young cohabitation environment to an independent life. This will inevitably cause severe physiological and psychological stress in the early stage of the young (Enríquez et al., 2011; Sun et al., 2016). There are evidences that early weaning stress leads to intestinal atrophy and dysfunction, intestinal flora imbalance, increased dyspepsia and diarrhea, mucosal barrier damage, etc., which are the main reasons for the retarded growth of the young mammals (Chen et al., 2017; Li et al., 2018). Our previous studies on squabs also found that weaning stress could lead to impairment of intestinal barrier function as well as disturbance of intestinal flora (Xu et al., 2022).

Due to the damage, bacterial penetration through the intestinal mucosa may increase. Translocation bacteria can be recognized by TLR, which can detect multiple pathogen-associated molecular patterns (PAMP) from bacteria. Our previous studies suggest that the TLR2 and TLR4 can be rapidly activated to identify bacterial PAMP in the intestine of weaned squabs, promoting the gene expression of inflammatory cytokines TNF-α and IL6 (Xu et al., 2022). The signaling pathways involved in TNF-α and IL6 production mediated by TLR2/4 mainly include NF-κB pathway and mitogen activated protein kinase (MAPK) signaling community (JNK, p38 MAPK, ERK) (Bioinformatics Center of Kyoto University, 1995). Therefore, the specific molecular mechanism needs to be further studied.

The purpose of this study was to explore the possible molecular mechanism of intestinal epithelial barrier damage caused by early weaning in squabs by determining the intestinal permeability, the ultrastructure of villous epithelium, the contents of ileal cytokines, and the protein relative expression of tight junction proteins, TLRs and their mediated key factors in inflammatory signaling pathways.

MATERIALS AND METHODS

All experimental protocols involving animals were approved by the Animal Care and Welfare Committee of Animal Science College and the Scientific Ethical Committee of Zhejiang University (Hangzhou, China).

Birds and Experimental Design

On the day of hatch, 192 newborn squabs (Columba livia) with similar body weight were selected. They were paired off and assigned to parent pigeons’ nests in place of the fake eggs used to satisfy the parent pigeons’ nesting characteristics. Each pair of parent pigeons raised 2 squabs. These squabs were fed pigeon milk beak-to-beak by their parents. On the d 7 after hatching, squabs were randomly divided into control group (CON) and early weaning group (EW). There were 8 replicates per group and 12 squabs per replicate. The squabs in CON continued to be fed by their parent pigeons, and those in EW began to be separated from the parent pigeons and fed artificial pigeon milk. The formula of artificial pigeon milk (contains 17.76% protein, 13.07 MJ/kg energy) was referred to the previous research of our group (Xu et al., 2022). The artificial pigeon milk powder was mixed with warm water at 37 °C and injected into the crop with a sterile syringe and a hose for artificial feeding. The environmental conditions of the 2 groups remained the same. During the whole study period, the ambient temperature was 18°C to 26°C, the relative humidity was 60-70%, and the light period was 12L:12D.

Sample Collection

At d 1 (D1), 4 (D4), 7 (D7), 10 (D10) and 14 (D14) after weaning, 8 squabs (1 per replicate) were randomly selected from the CON group and EW group, respectively, for sampling. All squabs were weighed and killed by cervical dislocation (squabs weighing more than 250 g were sedated before cervical dislocation). These squabs were slaughtered for serum and small intestine. The ileal segment was fixed with fixative solution for morphological analysis. Blood samples were collected and drawn into Eppendorf tubes (10 mL). After allowing the whole blood to clot, the serum was centrifuged for 10 min (3,000 g). Pure serum samples were aspirated by pipette, stored in 1.5-mL Eppendorf tubes at -80°C and thawed at 4°C before analyses of serum endotoxin, diamine oxidase and D-lactic acid. The ileal mucosa was collected for determining levels of inflammatory factors and the relative protein expression of key molecules in the inflammatory signaling pathway.

Intestinal Mucosal Permeability

The levels of endotoxin, diamine oxidase, and D-lactate in serum were determined by a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA) using commercial ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer's protocol.

Villus Morphology under Transmission Electron Microscope

Approximately 1×3 mm samples of ileum were collected. Samples were first fixed with 2.5% glutaraldehyde in phosphate buffer (0.1M, pH7.0) for more than 4h and washed 3 times in the phosphate buffer (0.1M, pH7.0) for 15 min at each step. Then these samples were postfixed with 1% OsO4 in phosphate buffer for 1 h and washed 3 times in the phosphate buffer (0.1M, pH7.0) for 15 min at each step. Next, the samples were first dehydrated by a graded series of ethanol (30%, 50%, 70%, 80%, 90% and 95%) for about 15 min at each step, dehydrated by alcohol for 20 min, and transferred to absolute acetone for 20 min. The samples were placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 h at room temperature, then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3h and to final Spurr resin mixture for overnight. These samples were placed in Eppendorf tubes contained Spurr resin and heated at 70°C for more than 9 h, after which the samples were sectioned in an ultramicrotome (LEICA EM UC7). The sections were stained by uranyl acetate and alkaline lead citrate for 5-10 min respectively and observed under transmission electron microscope (Hitachi Model H-7650). The measurement of microvilli length was in line with Karcher and Applegate (2008).

Cytokine Concentration Analysis

The homogenates of ileal mucosa were prepared with PBS for cytokine concentration analysis. The concentrations of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1β, IL-4, IL-10, and interferon-γ (IFN-γ) were determined with commercial ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China). The standard samples and diluent solution were added at 100 μL per well in duplicate. The plate was incubated for 2 h at 37°C, and washed by washing solution for 3 times. The biotin-antibody was added at 100 μL per well, with incubation at 37°C for 1 h and washing for 5 times. The HRP conjugate was added at 100 μL per well, with incubation at 37°C for 1 h and washing for 5 times. Next, 100 μL color solution was added into each well and develop color at 37°C for 15 min. Finally, 50 μL stop solution was added into each well to stop the reaction. The absorbance changes at a wavelength of 450 nm were immediately determined with a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). The final cytokine concentration was expressed as pg per mg of protein.

Protein Extraction and Western Blotting

RIPA lysate (strong) was supplemented with 50*cooktail, PMSF (100 mM) and phosphorylase inhibitors before use according to instruction. The ileal mucosa was cut into small pieces and placed in Eppendorf tubes, and the lysate was added at a ratio of 50 mg tissue to 1 mL lysate. The samples were homogenized at 4°C. After centrifugation at 12,000 g for 5 min, the supernatant was collected, which was the total protein solution. The protein concentration was determined by BCA protein quantitative detection kit. Pregel (4–20%) (Genscript Biotechnology Co., LTD, Nanjing, China) were obtained. The protein sample was added to 1/4 sample volume of 5× protein loading buffer and heated in a metal bath at 95°C for 10 min to fully denature the protein. After the protein sample was cooled to room temperature, the sample is directly fed into the Pregel filling hole. Electrophoresis was carried out by connecting the electrophoresis apparatus, and the voltage was set to 180V. Until bromophenol blue is about 1 cm away from the bottom, electrophoresis could be terminated, and membrane transfer operation was ready. Six pieces of 7 × 9 cm filter paper and 5 × 8 cm PVDF (0.45 μm) membrane were prepared. The PVDF membrane should be activated by methanol for 1 min before use. In the basin with transfer buffer, the clip for transfer film, 2 sponge pads, filter paper and activated PVDF membrane were placed. The clip was opened with the red side on the left and the black side on the right. A sponge and 3 layers of filter paper were added on each side. The separation glue was carefully peeled off and placed on the filter paper. The PVDF membrane was put on the glue and there was no bubble during operation. Then 3 pieces of filter paper and another sponge were covered on the membrane in turn. The whole was put into the transfer electrophoresis apparatus and the transfer condition was set to 300 mA for 30 min. After the transfer, the PVDF membrane was obtained and immersed in blocking solution, shielded from light, and blocked on a shaker for 1h. The PVDF membrane was washed 3 times with TBST buffer for 5 min each time. After washing, the PVDF membrane was immersed in primary antibody (diluted according to the instructions), shielded from light, and incubated at 4°C overnight. The antibody was recycled, and the PVDF membrane was washed in TBST buffer for 3 times. The secondary antibody (diluted according to the instructions) was added and the PVDF membrane was incubated for 1 h at room temperature in darkness. After incubation, the secondary antibody was recycled, and the PVDF membrane was washed 3 times with TBST buffer for 5 min each time. The luminescence solution was prepared according to the ECL chemiluminescence kit (Wuhan Servicebio Technology Co. Ltd., Wuhan, China), and the membrane with protein was immersed in the ECL luminescence solution. The reaction was carried out for 2 min at room temperature, and the chemiluminescence detector was used for imaging observation. Analysis of protein bands were performed on Image J.

Statistical Analysis

The data obtained from this experiment were subjected to an independent-sample t-test in SPSS 24.0 (SPSS Inc., Chicago) for Windows. The level of significance was chosen at P < 0.05. The level of highly significance was chosen at P < 0.01. Plotting was performed with GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, USA). Values were presented as means with their standard errors of 8 squabs.

RESULTS

Body Weight

The effects of early weaning on body weight (BW) and average daily gain (ADG) of squabs are shown in Figure 1. The BW of early weaned squabs at various time points (D1, D4, D7, D10 and D14 after weaning) were significantly decreased (P < 0.01) compared with that of CON group. The ADG of squabs in EW group were significantly reduced (P < 0.01) compared with that of CON group.Figure 1 Effects of early weaning on body weight (BW) and average daily gain (ADG) of squabs. (A) BW, (B) ADG. D1, D4, D7, D10, and D14 means 1, 4, 7, 10, and 14 d after weaning, respectively; CON = control group; EW = early weaning group (the same below). Values are means with their standard errors of 8 squabs. ** means P < 0.01, respectively, within each time points after weaning.

Figure 1

Intestinal Permeability

The effects of early weaning on the intestinal mucosal permeability of squabs are shown in Table 1. The intestinal mucosal permeability of squabs was reflected by levels of serum endotoxin, diamine oxidase and D-lactic acid. Early weaning resulted in significant increase of serum endotoxin levels in squabs at D1, D4, D7, D10 and D14 after weaning (P < 0.01, P < 0.01, P = 0.01, P = 0.04, and P < 0.01, respectively). And the serum diamine oxidase level was significantly higher (P < 0.01) in early weaned squabs at D4 after weaning compared with CON group. There was no significance (P > 0.05) in serum D-lactic acid level between these 2 groups.Table 1 Effects of early weaning on intestinal mucosal permeability of squabs.1

Table 1Items	Group	Weaning time	
D1	D4	D7	D10	D14	
Endotoxin (EU/mL)	CON	83.82 ± 1.68	87.04 ± 1.83	92.79 ± 2.76	92.11 ± 1.97	83.73 ± 1.58	
EW	101.81 ± 1.77	106.17 ± 1.16	105.12 ± 3.09	100.51 ± 3.23	96.18 ± 1.74	
P-value	<0.01	<0.01	0.01	0.04	<0.01	
Diamine oxidase (ng/mL)	CON	17.88 ± 0.43	17.82 ± 0.32	20.91 ± 0.36	20.20 ± 0.63	19.34 ± 0.42	
EW	18.57 ± 0.36	20.66 ± 0.46	20.15 ± 0.71	19.54 ± 0.38	19.04 ± 0.58	
P-value	0.21	<0.01	0.36	0.39	0.68	
D-lactic acid (nmol/L)	CON	105.13 ± 7.66	106.64 ± 12.95	105.13 ± 9.16	114.50 ± 12.32	114.84 ± 7.26	
EW	97.17 ± 8.74	119.05 ± 6.77	122.00 ± 4.93	116.32 ± 5.76	109.43 ± 4.90	
P-value	0.51	0.41	0.12	0.90	0.55	
1 Values are means with their standard errors of 8 squabs. P < 0.05 indicates significant difference between the 2 groups at each time points after weaning. D1, D4, D7, D10, and D14 means 1, 4, 7, 10, and 14 d after weaning, respectively. CON = control group; EW = early weaning group.

Ultrastructure of Villous Epithelium

The effects of early weaning on the ileal villi ultrastructure of squabs are shown in Figure 2. The junctions between epithelial cells in ileum of early weaned pigeon were damaged. The cytoplasm vacuolated, and the mitochondrial ridge was broken. Besides, the microvilli length in ileum of early weaned pigeon in EW group at D7, D10 and D14 after weaning was significantly lower (P < 0.01, P < 0.01, and P < 0.01, respectively) than that in CON group.Figure 2 Effects of early weaning on the ileal villi ultrastructure of squabs. D1, D4, D7, D10, and D14 means 1, 4, 7, 10, and 14 d after weaning, respectively; CON = control group; EW = early weaning group. (A) Ileal villi ultrastructure under transmission electron microscope. Bar = 2 μm. (B) Microvillus length of ileal villi. Values are means with their standard errors of 8 squabs. ** means P < 0.01, respectively, within each time points after weaning.

Figure 2

Intestinal Cytokine Levels

The effects of early weaning on the concentrations of cytokines in ileum of squabs are shown in Figure 3. Compared with CON group, the intestinal TNF-α level in EW group was significantly increased (P < 0.05 or P < 0.01) at D7, D10, D14 after weaning. The intestinal IL-6 level of squabs in EW group was significantly higher than that in CON group at various time points after weaning. Compared with CON group, the levels of intestinal IL-4 and IL-10 in EW group were significantly decreased (P < 0.01 or P < 0.05) at various time points after weaning. However, there was no significant difference (P > 0.05) in the levels of IL-1β and IFN-γ between these 2 groups at any time points.Figure 3 Effects of early weaning on the concentrations of cytokines in ileum of squabs. CON = control group, EW = early weaning group. ** means P < 0.01, respectively, within each time points after weaning.

Figure 3

Relative Protein Expression of Tight Junction Proteins

The effects of early weaning on the relative protein expression of tight junction proteins in ileum of squabs are shown in Figure 4. Early weaning significantly increased the relative protein expression of claudin-2 in ileum of squabs (P < 0.01), while significantly decreased the relative protein expression of claudin-1 and claudin-3 in ileum of squabs (P < 0.01).Figure 4 Effects of early weaning on the relative protein expression of tight junction proteins in ileum of squabs. CON = control group, EW = weaning group. CLDN = claudin. **Represents P < 0.01 between the 2 groups.

Figure 4

Relative Protein Expression of Key Signaling Molecules

The effects of early weaning on the relative protein expression of TLRs in ileum of squabs are shown in Figure 5. Early weaning significantly increased the relative protein expression of both TLR2 and TLR4 in ileum of squabs (P < 0.01). The effects of early weaning on the relative protein expression of MyD88 in ileum of squabs are shown in Figure 6. The relative expression of adaptor protein MyD88 in ileum of EW group was significantly higher than that of CON group (P < 0.01).Figure 5 Effects of early weaning on the relative protein expression of toll-like receptors in ileum of squabs. CON = control group, EW = weaning group. **Represents P < 0.01 between the 2 groups.

Figure 5

Figure 6 Effects of early weaning on the relative protein expression of MyD88 in ileum of squabs. CON = control group, EW = weaning group. **Represents P < 0.01 between the 2 groups.

Figure 6

The effects of early weaning on the relative expression of key proteins of MAPK pathway in ileum of squabs are shown in Figure 7. Early weaning significantly increased the expression levels of JNK, phosphorylated JNK and phosphorylated ERK proteins (all P < 0.01). Compared with CON group, there was no significant change in ERK, p38 and phosphorylated p38 protein levels in EW group (P > 0.05).Figure 7 Effects of early weaning on the relative expression of key proteins of MAPK pathway in ileum of squabs. CON = control group, EW = early weaning group. **Represents P < 0.01 between the 2 groups.

Figure 7

The effects of early weaning on the relative expression of key molecular proteins of NF-κB pathway in ileum of squabs are shown in Figure 8. Early weaning had no significant effects on the relative protein expression of IκBα, phosphorylated IκBα and p65NFκB in ileum of squabs (P > 0.05).Figure 8 Effects of early weaning on the relative expression of key proteins of NF-κB pathway in ileum of squabs. CON = control group, EW = early weaning group. No ‘*’ represents P > 0.05 between the 2 groups.

Figure 8

DISCUSSION

The results of our experiments showed that early weaning caused a rapid and continuous decline in growth performance of pigeon squabs over the first 14 d after weaning. The impaired intestinal function associated with abrupt weaning may be the main reason for the poor growth rate after weaning.

The integrity of the intestinal barrier is fundamental to maintaining epithelial permeability and preventing the entry of intestinal pathogens and endotoxins. Increased intestinal permeability has been shown to be closely related to villi atrophy (Jeurissen et al., 2002). The levels of serum endotoxin, diamine oxidase and D-lactic acid, which were positively correlated with intestinal permeability, were used to reflect the degree of intestinal mucosal injury. It was found that the levels of endotoxin and diamine oxidase increased significantly at one or more time points after weaning. In addition, the endotoxin level in the EW group was still high at D14 after weaning, indicating that early weaning resulted in a continuous increase in intestinal permeability. The permeability of the intestine is mainly regulated by an orderly system called tight junctions. Based on gene expression results, previous study showed that weaning regulated the expression of the claudin family of proteins (namely claudin-1, claudin-2, and claudin-3), leading to impairment of the intestinal barrier and increased permeability (Xu et al., 2022). The claudin-1 and claudin-3 are known to be barrier-forming tight junction proteins, while the expression of claudin-2 is associated with decreased epithelial barrier tightness and increased intestinal permeability, therefore claudin-2 is called a pore-forming tight junction protein (Suzuki et al., 2011; Ariyadi et al., 2013). In the current study, the protein expression levels of claudin-1 and claudin-3 were down-regulated in the EW group, whereas the the protein expression level of claudin-2 was up-regulated. These results were consistent with an increase in intestinal permeability in early weaned squabs, which means the above conclusion that weaning stress impaired intestinal barrier function by weakening tight junctions was also confirmed. The observed damaged connections between villous epithelial cells, cytoplasmic vacuolation, mitochondrial ridge breakage, and microvilli length reduction in ultrastructure of villi also suggested the intestinal barrier injury in early weaned squabs.

Intestinal barrier damage means that the bacteria or pathogens are more likely to invade the body. Once the foreign invaders breached the intestinal mucosal barrier, the contact of TLR on mucosal cells and the subsequent activation of signaling cascades would promote the production of pro-inflammatory cytokines such as TNF-α, IL1-β, IL6, and IL12 (Lavelle et al., 2010). Many studies have shown that early weaning is associated with changes in the expression of pro-inflammatory cytokines in the mammalian intestine (Hu et al., 2013; Mclamb et al., 2013). In the current study, early weaning significantly increased the TNF-α level in ileum of squabs at D7∼D14 after weaning and the IL6 level in ileum of squabs within 14 d after weaning, but had no effect on the levels of IL1β and INF-γ. The results suggest that the intestinal mucosal immune system is activated under early weaning stress, and the inflammation is mainly caused by the increase of TNF-α and IL6. Besides, in this study, the levels of IL4 and IL10 were decreased in early weaned squabs. As anti-inflammatory cytokines, IL4 and IL10 play important roles in the weakening of inflammatory processes. In contrast to that, Hu et al. (2013) found that there was no significant change in anti-inflammatory cytokines in piglets at the first 2 wk after weaning. In our study, the decrease of anti-inflammatory cytokines is likely due to the synergistic role of cytokines in the inflammatory process. In other words, high levels of pro-inflammatory cytokines might inhibit the production of anti-inflammatory cytokines (Gao et al., 2013). Moreover, overproduction of pro-inflammatory cytokines negatively regulated the integrity of intestinal barrier (Bruewer et al., 2003; Al-Sadi et al., 2009).

The secretory stimulating of inflammatory factors TNF-α and IL-6 may be mediated by the activation of TLR pathway (Hochdörfer et al., 2011; Hong et al., 2014). However, TLR signaling pathway network is complex. How does TLR2/4 induced by weaning stress regulate downstream factors and ultimately promote the secretion of inflammatory factors TNF-α and IL-6? Based on this question, the expression of key factors related to the pathway was concerned. MyD88 is a key adaptor molecule in the TLR signaling pathway and plays an important role in upstream signaling and immune response mediating (Verstak et al., 2009). Depending on whether MyD88 is connected or not, the downstream of TLR can be divided into MyD88 dependent signal pathway and MyD88 independent signal pathway (Wieland et al., 2005). The most important adaptor protein in TLR2/4 pathway that ultimately induces TNF-α and IL-6 is MyD88. In our current study, early weaning significantly increased the relative protein expression of MyD88 in ileum of squabs, suggesting that TLR2/4 did indeed activate downstream signaling in a MyD88-dependent pathway. Then the key molecules of the NF-κB pathway and MAPK pathway community were determined. JNK belongs to the MAPK family, which regulates signaling mechanisms of cell proliferation, differentiation, survival, death, and inflammation (Catherine et al., 2013). Li et al. (2020) found that JNK phosphorylation levels in jejunum of weaned piglets and porcine jejunum epithelial cell line J2 cells induced by toxic carotene in vitro were significantly increased. In the current experiment, early weaning also significantly increased the relative protein expression of JNK and phosphorylated JNK in the ileum of squabs, suggesting that early weaning stress not only induced the expression of JNK protein, but also played a role in MAPK (JNK) signaling pathway. The MAPK family also includes extracellular signal-regulated kinases ERK and p38 MAPK. The 3 MAPK signal cascades are considered parallel pathways, although crosstalk may exist (Xiao et al., 2002; Shifflett et al., 2004). The results of current study showed that the protein expression level of phosphorylated ERK also significantly increased under weaning stress, indicating that MAPK (ERK) signaling pathway is also involved in inducing the release of pro-inflammatory factors TNF-α and IL-6, causing damage to the intestinal epithelial barrier of weaned squabs. While, the determination of p38 protein expression level showed that TLR2/4 signaling pathway induced by early weaning does not promote cytokine secretion via MAPK (p38) pathway. In contrast to our results, previous studies showed that weaning in 21-day-old piglets induced the p38 MAPK signaling pathway without altering ERK protein phosphorylation levels (Li et al., 2020). And Hu et al. (2013) found that 3 kinds of MAPK signaling cascades were all activated in piglets weaned at 3 d and 7 d. Several previous studies have shown that early weaning damaged intestinal barrier function in young animals (Wang et al., 2015; Xiong et al., 2015), resulting in a large number of intestinal bacteria and endotoxin lipopolysaccharides invading intestinal tissues and circulation, leading to bacterial translocation and intestinal endotoxemia (Dong et al., 2020). In porcine jejunum epithelial cell line J2 cells, LPS not only induces inflammation through MAPK signaling cascade, but also induces pro-inflammatory response through NF-κB signaling pathway (Dong et al., 2020). However, in this study, the expression and phosphorylation level of IκBα protein, a key molecule of NF-κB pathway, and the expression level of p65 NFκB protein were analyzed, and it was found that early weaning did not activate NF-κB pathway. It is speculated that the molecular mechanism of weaning stress might be different in different species, weaning age and the time after weaning.

In conclusion, early weaning could reduce the growth performance of squabs and damage the intestinal epithelial barrier, which is characterized by down-regulating the protein expression of barrier-forming claudins, up-regulating the protein expression of pore-forming claudin, promoting the secretion of pro-inflammatory factors, inhibiting the secretion of anti-inflammatory factors, and increasing the permeability of the intestinal barrier. The specific mechanism of stress damage might be the activation of TLR2/4-MyD88-ERK/JNK inflammatory signaling pathway leading to the increase levels of IL-6 and TNF-α.

DISCLOSURES

The authors declare that they have no competing interests.

ACKNOWLEDGMENTS

This work was supported by Zhejiang Provincial Natural Science Foundation of China [ZCLQ24C1701 ], the Fundamental Research Funds for the Provincial Universities of Zhejiang [2023YW61 ], the Open project of Key Laboratory of Animal Molecular Nutrition of Ministry of Education (Zhejiang University)[KLMAN202301 ] and Hangzhou Science and Technology Commissioner Project [20231122t48 ].
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