https://orcid.org/0000-0003-4743-2612
Abstract
To evaluate the protective effects of sodium butyrate (SB) on intestinal morphology, anti-oxidant status, inflammatory response and barrier integrity in male broilers under heat stress. A total of 180 21-d-old male Arbour Acres broilers with similar body weight were used for this 21-d experiment. Broilers were randomly allocated to three groups: CON group, basal diet and raised under 24 °C; HS group, basal diet and raised under heat stress condition (34 °C from 10:00 to 18:00 and 24 °C for the rest time); HS-SB group, basal diet with 1200 mg/kg SB and raised under heat stress condition. Compared to the HS group, SB supplementation increased average daily gain (ADG), villus height: crypt depth ratio (VH: CD), VH, glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity, and interleukin-10 (IL-10) level, decreased feed conversion ratio (FCR), CD, reactive oxygen species (ROS), IL-1β and tumour necrosis factor-alpha (TNF-α) level, up-regulated the mRNA expression of nuclear factor erythroid 2-related factor 2, haem oxygenase 1, GSH 1 (GPX1), SOD and CAT, down-regulated the mRNA expression of toll-like receptor 4 (TLR4), nuclear transcription factors, IL-1β, and TNF-α in jejunum of heat-stressed broilers. Furthermore, SB supplementation up-regulated the mRNA expression of occluding, claudin-1 and zona occludens-1 (ZO-1) in jejunum of heat-stressed broilers, accompanied with decreasing serum endotoxin and D-lactic acid concentration, and diamine oxidase (DAO) activity. There results suggested that SB supplementation could alleviate heat stress-induced jejunal tight junction structure dysfunction by up-regulated the expression of occluding, claudin-1 and ZO-1, presumably through alleviating oxidative stress and inflammatory response.
Sodium butyrate alleviated heat stress-induced jejunal morphology damage.
Sodium butyrate alleviated heat stress-induced jejunal oxidative damage.
Sodium butyrate alleviated heat stress-induced jejunal inflammatory response.
Sodium butyrate alleviated heat stress-induced tight junction structure dysfunction.
HIGHLIGHTS
Introduction
Heat stress is a heavy challenge for livestock production worldwide, especially for the fast-growing meat-type broilers in subtropical and tropical regions (Kpomasse et al. Citation2021). Heat stress induces numerous physiological disturbances, such as oxidative stress, electrolyte imbalance, immune dysregulation, endocrine disorders, which results in detrimental effects on feed intake, growth performance, mortality and welfare of broilers (Quinteiro-Filho et al. Citation2010; Varasteh et al. Citation2015; Lan, et al. Citation2020b). As one of the vital digestive organs, intestine plays a critical role in nutrient digestion and absorption, as well as maintain intestinal mucosal barrier function and immunity (Song, Cheng, et al. Citation2017). Intestinal mucosal barrier consists of a single layer of epithelial cells and their junctional intercellular complexes, and tight junction plays vital roles in maintaining intestinal barrier function (Otani and Furuse Citation2020). Intestine is highly sensitive to heat stress, which impairs intestinal function by inducing morphology damage, oxidative stress, inflammatory response and altering intestinal barrier function (Wu et al. Citation2018; Cheng et al. Citation2019; Lan, Li, Chang, Zhao Citation2020; Deng, Zheng, et al. Citation2023). Therefore, alleviating heat stress-induces intestinal dysfunction is vital for broilers’ health status and production. Currently, nutritional regulation is an effective and economic strategy to alleviate heat stress damage (Lan, Li, Chang, Zhao Citation2020; Deng, Zheng, et al. Citation2023). Former literature indicated that oxidative stress and inflammatory response increased intestinal permeability accompanied with down-regulated the mRNA expression of tight junction proteins-related genes (Song, Cheng, et al. Citation2017; Wu et al. Citation2018; Cheng et al. Citation2019; Chen et al. Citation2022). Therefore, feed additives with anti-oxidant and anti-inflammation effects can be used to alleviate heat stress in broilers (Lan, Li, Chang, Zhao Citation2020; Liu, Ou, et al. Citation2021). Increasing studies indicated that sodium butyrate (SB) possessed anti-oxidative, anti-inflammatory, anti-microbial and immunomodulatory capacity, which had beneficial effects on growth performance, intestinal morphology and microbiota, mucosal cells proliferation and differentiation, anti-oxidative, anti-inflammatory and intestinal barrier function of broilers (Zou et al. Citation2019; Elnesr et al. Citation2020; Wang et al. Citation2020; Wan et al. Citation2022; Deng, Tang, et al. Citation2023). The study in Nile tilapia reported that SB supplementation increased intestinal villus length and width (Dawood et al. Citation2020). The study in rats reported that SB supplementation alleviated lipopolysaccharides (LPS) induced jejunal villus injury and inflammatory infiltration by down-regulating the mRNA levels of toll-like receptor 4 (TLR4), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α), as well as maintain intestinal barrier function and microbiota balance (Dou et al. Citation2022). However, relatively few studies were conducted to evaluate the effects of SB supplementation on the intestinal health status in heat stressed-broilers. Our former studies indicated that SB supplementation alleviated heat stress induced oxidative damage in liver and breast muscle (Lan, Li, Chang, An Citation2020). Jiang et al. (Citation2015a, Citation2015b) reported that SB supplementation alleviated LPS induced inflammatory response by increasing duodenal and jejunal villus height: crypt depth ratio (VH: CD) and VH, as well as decreasing, nuclear transcription factors kappa-B (NF-κB) and TNF-α level. Jiang et al. (Citation2015a, Citation2015b) reported that SB supplementation alleviated corticosterone induced oxidative stress by increasing the duodenal and jejunal catalase activity and decreased the duodenal malondialdehyde (MDA) contend, as well as decreased the apoptosis index. Therefore, the aim of this study is to evaluate the effects of SB supplementation on intestinal morphology, anti-oxidant status, inflammatory response and barrier integrity of heat stressed-broilers.
Materials and methods
Animals, diets and management
A total of 180 21-d-old male Arbour Acres (AA) broilers with similar body weight (805.97 ± 39.93) were used in this 21-d experiment. Broilers were randomly allocated to three groups: CON group, basal diet and raised under 24 °C; HS group, basal diet and raised under heat stress condition (34 °C from 10:00 to 18:00 and 24 °C for the rest time); HS-SB group, basal diet with 1200 mg/kg SB (dose based on our former study) and raised under heat stress condition (Lan, Li, Chang, An, Zhao Citation2020; Lan, Li, Chang, An Citation2020). The temperature was regulated by air conditioner; the temperature and relative humidity were recorded at 10:00, 14:00 and 18:00 daily, and shown in Table S1. Each group had 6 replication cages and 10 broilers per cage. The broilers fed the corn-soybean based diet, and the feed composition was listed in Table S2. The SB used in this study contained 54% SB and protected by a physical and chemical matrix of buffer salts was provided by Beijing Shengtaiyuan Biotechnology Co, Ltd.
Growth performance
At the beginning and the end of the experiment, body weight and feed consumption were recorded on cage basis; average daily feed intake (ADFI), average daily gain (ADG) and feed conversion ratio (FCR) were calculated.
Sample collection
At the end day of the experiment, after 12-h fast, 1 broiler from each replication cage was randomly selected. Blood samples were collected from the brachial vein and centrifuged at 3500 g for 15 min at 4 °C to obtain serum and stored at −20 °C for later analysis. Then the broilers were sacrificed by cervical dislocation and exsanguinated. The jejunum sample (2 cm at the midpoint) was fixed in 10% buffered formalin for morphology examination. The remaining jejunum was opened longitudinally and flushed with ice-cold phosphate-buffered saline (PBS), the mucosa sample was collected using a sterile glass microscope slide, rapidly frozen in liquid nitrogen, then stored at −80 °C for later analysis.
Serum parameters
Test kits to determination corticosterone (H205-1-2), diamine oxidase (DAO, NO. A088-2-1) activity and D-lactate acid (D-LA, NO. A019-2-1) concentration were purchased from Nanjing Jiancheng Institute of Bioengineering, Nanjing, P. R. China, and endotoxin (NO. CB10535-Ch) was purchased from Shanghai Coibo Biotech Co., Ltd., the methods were according to the manufacturers’ instruction. The corticosterone content was detected to evaluate whether the broilers were undergoing stress, the result shown in Figure S1.
Intestinal morphology
The jejunum morphology analysis was followed our previous described methods (Lan, Li, Chang, An, Zhao Citation2020; Lan, Li, Chang, Zhao Citation2020). Briefly, formalin-fixed jejunum was dehydrated, cleared and embedded in paraffin, then cut into serial sections at 4 μm for staining with haematoxylin and eosin (H.E). VH and CD were measured using Image Pro Plus version 6.0 software (Media Cybernetics, Inc., Bethesda, MD), and the VH:CD was calculated.
Reactive oxygen species measurement
Reactive oxygen species (ROS) measurement was using an ROS-measurement kit (NO. E004-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) followed the methods described by the manufacturers’ instruction. ROS generation was quantified by the mean of the CON group.
Determination of jejunal mucosal oxidative status and inflammation cytokines
About 0.5 g of jejunum mucosa sample was homogenised at a ratio of 1: 9 (weight/volume) with ice-cold PBS, then centrifuged at 3000 g for 10 min at 4 °C to obtain supernatant. The protein concentration of the supernatant was determined by the Bradford method using bovine serum albumin as the standard. The activity of superoxide dismutase (SOD, NO. A001-1-2), glutathione peroxidase (GSH-Px, NO. A005-1-2), catalase (CAT, NO. A007-2-1), the content of MDA (NO. A003-4-1), IL-1β (NO. H002-1-2), IL-6 (NO. H007-1-2), IL-10 (NO. H009-1-2), and TNF-α (NO. H052-1-2) were measured with corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instruction.
Real-time quantitative PCR
Total mRNA extraction, cDNA reverse transcription, and quantitative real-time polymerase chain reaction (RT-PCR) analysis were followed the methods described in our previous research (Lan et al. Citation2014). The primers used were described in former literature (Song, Cheng, et al. Citation2017; Wu et al. Citation2018; Liu, Zhu, et al. Citation2021; Chang et al. Citation2022) and listed in . The PCR reactions were performed in triplicate and the results were normalised to β-actin mRNA expression. The relative mRNA expression was calculated by 2−△△Ct method.
Table 1. The primer sequences.
Statistical analysis
All data were analysed using one-way analysis of variance followed by Duncan’s multiple range test to analyse the difference among treatments. Difference was considered significant at p < 0.05.
Results
Growth performance
Compared to the CON group, broilers in the HS group had lower (p < 0.05) ADG, but higher FCR (). Compared to the HS group, SB supplementation increased (p < 0.05) ADG and decreased (p < 0.05) FCR.
Table 2. Effects of sodium butyrate on growth performance of male broilers under heat stress.
Intestinal morphology
Compared to the CON group, broilers in the HS group had lower (p < 0.05) VH and VH:CD, while higher (p < 0.05) CD (Figure ). Dietary SB supplementation increased (p < 0.05) the VH and VH:CD, while decreased (p < 0.05) CD in jejunum of heat-stressed broilers.
Figure 1. Effects of sodium butyrate on jejunal morphology of male broilers under heat stress. Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate.
Anti-oxidant capacity
The broilers in the HS group had higher (p < 0.05) jejunal ROS and MDA content, while had lower (p < 0.05) GSH-Px, SOD and CAT activity than the CON group (Figure ). Dietary SB supplementation reduced (p < 0.05) jejunal ROS level, increased (p < 0.05) GSH-Px and SOD activity in heat-stressed broilers. Compared to the CON group, heat stress down-regulated (p < 0.05) the mRNA expression of jejunal Nrf2, HO-1, GPX1, SOD and CAT (Figure ). Dietary SB supplementation up-regulated (p < 0.05) the mRNA expression of jejunal Nrf2, HO-1, GPX1, SOD and CAT in heat-stressed broilers.
Figure 2. Effects of sodium butyrate on jejunal anti-oxidative parameters in male broilers under heat stress Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate; ROS: reactive oxygen species; MDA: malondialdehyde; GSH-Px: glutathione peroxidase; SOD: superoxide dismutase; CAT: catalase.
Figure 3. Effects of sodium butyrate on the mRNA expression of anti-oxidant related genes expression in jejunum of male broilers under heat stress. Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate; Nrf2: nuclear factor erythroid 2-related factor 2; HO-1: haem oxygenase 1; GPX1: glutathione peroxidase; SOD: superoxide dismutase; CAT: catalase.
Intestinal permeability
Compared to the CON group, heat stress increased (p < 0.05) serum DAO activity and endotoxin and D-LA level (Figure ). Dietary SB supplementation decreased (p < 0.05) serum DAO activity, and endotoxin and D-LA level in heat-stressed broilers.
Figure 4. Effects of sodium butyrate on intestinal permeability in male broilers under heat stress. Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate; DAO: diamine oxidase; D-LA: D-lactic acid.
Inflammatory response
Compared to the CON group, heat stress increased jejunal IL-1β and TNF-α level, while decreased IL-10 level (Figure ). Dietary SB supplementation decreased (p < 0.05) jejunal IL-1β and TNF-α level, while increased (p < 0.05) IL-10 level in heat-stressed broilers. No significant difference was observed in IL-6 among the treatments. Compared with the CON group, heat stress up-regulated (p < 0.05) the mRNA expression of jejunal TLR4, NF-κB, IL-1β and TNF-α, whereas down-regulated (p < 0.05) the mRNA expression of IL-10 (Figure ). Dietary SB supplementation decreased (p < 0.05) the mRNA expression of jejunal TLR4, NF-κB, IL-1β and TNF-α in heat-stressed broilers.
Figure 5. Effects of sodium butyrate on jejunal inflammatory cytokines in male broilers under heat stress. Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate; IL: interleukin; TNF-α: tumour necrosis factor-alpha.
Figure 6. Effects of sodium butyrate on the mRNA expression of inflammation-related genes in the jejunum of male broilers under heat stress. Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate; TLR4: toll-like receptor 4; NF-κB: nuclear transcription factors; IL: interleukin; TNF-α: tumour necrosis factor-alpha.
The mRNA expression of tight junction proteins-related gene
Compared with the CON group, heat stress decreased (p < 0.05) the mRNA expression of jejunal ZO-1, occludin and claudin-1 (Figure ). Dietary SB supplementation up-regulated (p < 0.05) the mRNA expression jejunal ZO-1, occludin and claudin-1 in heat-stressed broilers.
Figure 7. Effects of sodium butyrate on the mRNA expression of tight junction proteins in the jejunum of male broilers under heat stress. Values are presented as mean ± standard error. Different letters marked on the bar graph mean significant difference (p < 0.05). CON: control group; HS: heat stress group; HS-SB: heat stress with 1200 mg/kg sodium butyrate; ZO-1: zona occludens-1.
Discussion
The main purpose of poultry production is to maintain animal health and enhance growth performance. However, high ambient temperature-induced heat stress had detrimental effects on the growth performance of broilers by decreasing feed efficiency and disordering intestinal function, resulting in enormous economic losses (Liu et al. Citation2020; Chaudhary et al. Citation2023; Khan et al. Citation2023). In this study, HS decreased ADG, while increased FCR. While, SB supplementation increased ADG and improve FCR. Consistent with our results, Deng, Tang, et al. (Citation2023) and Wan et al. (Citation2022) reported SB supplementation improved growth performance of broilers by improving final body weight, ADG and FCR. The possible explanation is that SB has anti-oxidant and anti-inflammation capacity, which maintains intestinal function, and enhance feed efficiency (Mallo et al. Citation2021; Zhao et al. Citation2022).
Heat stress is an inducer of oxidative stress, characterised by overproduction of ROS and impairing the balance between oxidant and anti-oxidant system (Lan, Li, Chang, Zhao Citation2020; Chang et al. Citation2022). Numerous studies indicated that either acute or chronic heat stress induced overproduction of ROS in broilers (Azad et al. Citation2010; Chang et al. Citation2022). In this study, the jejunal ROS level was significantly increased in the HS group than the CON group, which demonstrated that heat stress exposure induced overproduction of ROS. The increased ROS could lead to oxidative stress, which resulted in DAN, proteins and lipid oxidative damage (Akbarian et al. Citation2016). GSH-Px, SOD and CAT are vital enzymes of the anti-oxidant defence system, which plays important role in ROS elimination and against ROS-induced damage (Li et al. Citation2015; Hu et al. Citation2019). In this study, broilers in the HS group had higher MDA content, whereas lower GSH-Px, SOD and CAT activity than broilers in the CON group, which in consistent with the results reported by Lan, Li, Chang, Zhao (Citation2020) and Yousefi et al. (Citation2023). MDA is the end product of lipid peroxidation and acted as a biomarker of oxidative stress, the increased MDA content was companied with ROS accumulation, as well as decreased GSH-Px, SOD and CAT activity in heat-stressed broiler, indicating that heat stress could reduce ROS scavenging ability by lowering anti-oxidant enzyme activity and leading to ROS accumulation, and the higher ROS level induced lipid oxidation and MDA accumulation (Chen et al. Citation2022). The Nrf2 signalling pathway plays an important role in protecting against oxidative stress. Nrf2 specifically band to the anti-oxidant response element, which is located in the promoter region of phase II enzymes, regulating the anti-oxidant related genes expression, such as HO-1, GPX1, SOD and CAT (Shaw and Chattopadhyay Citation2020; Wang et al. Citation2021). Former studies indicated that heat stress inhibited the activation of the Nrf2 pathway by down-regulated the mRNA expression of Nrf2, HO-1, GPX1, SOD in jejunum, liver and breast muscle of broilers (Song et al. Citation2018; Wang et al. Citation2021). Similar with these results, in this study, heat stress down-regulated the mRNA expression of jejunal Nrf2, HO-1, GPX1, SOD and CAT. These results also corroborated with the decreasing jejunal GSH-Px, SOD and CAT activity. Former studies indicated that SB supplementation could improve intestinal health status through its anti-oxidant capacity in normal condition (Lan, Li, Chang, An Citation2020; Xiao et al. Citation2023). Meanwhile, SB supplementation could alleviate corticosterone exposure and heat stress induced oxidative damage in breast muscle and liver of broilers by increasing SOD, GSH-Px and CAT activity, and decreased MDA content (Zhang et al. Citation2011a; Lan, Zhao, et al. 2020). In this study, SB supplementation reduced the ROS level, increased the jejunal GSH-Px and SOD activity, as well as up-regulated the mRNA expression of Nrf2, GPX1, SOD and CAT. These results were supported by Zhou et al. (Citation2022), who reported that SB supplementation increased serum SOD and GSH-Px activity, decreased MDA content and up-regulated the mRNA expression of ileal HO-1 in diquat-challenged pullets. Zhao et al. (Citation2022) reported that SB supplementation increased jejunal and ileal SOD activity. Similar results also reported by Miao et al. (Citation2023), who reported SB supplementation increased GSH-Px activity and decreased MDA content in serum and liver, which accompanied with up-regulated the mRNA expression of Nrf2 and HO-1 in liver of old-aged laying hen. Additionally, the study in porcine intestinal epithelial cells also indicted that SB supplementation alleviated hydrogen peroxide-induced oxidative stress (Li et al. Citation2022). The intestinal oxidative status is associated with its morphology, permeability and barrier function (Wu et al. Citation2018; Cheng et al. Citation2019). Dietary SB supplementation enhanced the anti-oxidant capacity could explain the better intestinal morphology and the up-regulated tight junction protein related genes expression.
The small intestinal epithelium plays a vital role in nutrient digestion and absorption, therefore, maintain normal morphology of the small intestinal is crucial for growth performance and health status (Quinteiro-Filho et al. Citation2010). Former literature indicated that heat stress-induced intestinal morphology damage by leading to shorter VH, deeper CD and lower VH:CD ratio (Cheng et al. Citation2019; Lan, Li, Chang, Zhao Citation2020; Liu, Ou, et al. Citation2021). Similarly, in this study, heat stress induced shorter VH, lower VH:CD ratio and deeper CD in jejunum. Broilers subjected to heat stress, the peripherical circulation blood was increased to heat dissipation, which resulted in less blood circulation in the intestine and inducing intestinal morphology damage (Varasteh et al. Citation2015). Heat stress also decreased the feed intake and reduced the energy supply to the intestinal epithelial cells, thus lead to shorter VH and deeper CD (Wu et al. Citation2018). Butyrate was the primary energy source for intestinal epithelial cells and played vital roles in stimulating their proliferation and differentiation, as well as maintain gut homeostasis (Ahsan et al. Citation2016; Elnesr et al. Citation2020). Our former studies indicated that SB supplementation had beneficial effects on relative weight and length of intestine, and intestinal morphology in broilers (Lan, Li, Chang, An, Zhao Citation2020; Lan, Li, Chang, An Citation2020). Besides, SB or butyrate supplementation could alleviate the negative effects of heat stress induced poor growth performance and intestinal morphology damage (Abdelqader and Al-Fataftah Citation2016; Lan, Zhao, et al. 2020). In this study, SB supplementation increased VH and VH:CD ratio, whereas decreased CD in jejunum, these results suggested that dietary SB supplementation could alleviate heat stress induced intestinal morphology damage.
The increased serum DAO and endotoxin level and D-LA activity served as vital indicators of increased intestinal permeability (Chen et al. Citation2022). In this study, heat stress increased the serum DAO and endotoxin level, and D-LA activity, these results were in consistent with former literature (Deng, Zheng, et al. Citation2023), suggested that heat stress increased intestinal permeability. The increased intestinal permeability was associated with the destruction of tight junction structure, which was the main components of intestinal mucosal barrier and had vital roles in maintain intestinal barrier function (Guo et al. Citation2019). The tight junction was a multi-protein complex and mainly composed of the transmembrane proteins claudin-1 and occluding, and the cytoplasmic proteins ZO-1 (Otani and Furuse Citation2020). Therefore, the down-regulated the mRNA expression of claudin-1, occluding and ZO-1 would induce intestinal barrier dysfunction and finally increased intestinal permeability. Former literature illustrated that heat stress induced intestinal permeability increasing were associated with the down-regulated the mRNA expression of claudin-1, claudin-3, claudin-4, occluding and ZO-1 (Cheng et al. Citation2019; Alhotan et al. Citation2021; Deng, Zheng, et al. Citation2023; Du et al. Citation2023). Consistent with former literature, in this study, broilers in the HS group down-regulated the mRNA expression of claudin-1, occluding and ZO-1 in jejunum. These results suggested that the increased serum D-LA and endotoxin level and DAO activity in heat-stressed broilers was due to impaired intestinal morphology and the destruction of tight junction structure. Previous literature indicated that butyrate had beneficial effects on increasing intestinal barrier function and decreasing intestinal paracellular permeability by regulating tight junction proteins-related genes, SB supplementation up-regulated the mRNA expression of claudin-3 in ileum of broilers (Luo et al. Citation2021), up-regulated the mRNA expression of claudin-1, occluding, ZO-1 and ZO-2 in jejunum and ileum of laying hens (Miao et al. Citation2021). Besides, SB supplementation could alleviate necrotic enteritis-induced down-regulated expression of claudin-1, claudin-4, occluding and ZO-1 in jejunum of broilers (Song, Li, et al. Citation2017). In this study, our results indicated that SB supplementation up-regulated the mRNA expression of jejunal occluding, claudin-1 and ZO-1 in heat-stressed broilers, whereas decreased the intestinal permeability indicators (DAO, D-LA and endotoxin), these results suggested that SB supplementation could alleviate heat stress-induced intestinal barrier damage by maintain the tight junction structure.
Heat stress influenced the intestinal-immune barrier function, which allowed pathogenic bacteria migrated through the intestinal mucosa and activated the immune system, hence generating inflammatory infiltrate (Quinteiro-Filho et al. Citation2010). As expected, heat stress increased pro-inflammatory cytokines IL-1β and TNF-α level in the jejunum of broilers when compared to the CON group, whereas decreased anti-inflammatory cytokines IL-10 level, these results were in consistent with former literature (Wu et al. Citation2018; Lan, Li, Chang, Zhao, et al. 2020). The increased pro-inflammatory cytokines and decreased anti-inflammatory cytokines in the heat-stressed broilers indicated that heat stress regulated the activation of inflammatory response. TLR4/NF-κB is the prototypical pro-inflammatory signalling pathway, NF-κB plays a vital role in the expression of pro-inflammatory cytokines-related genes (Lawrence Citation2009; Mohyuddin et al. Citation2021). Former literature indicated that heat stress up-regulated the mRNA expression of TLR4, IL-1β, IL-6, IL-8, IL-18, IFN-γ and TNF-α in the ileum and/or jejunum of broilers, as well as down-regulated the mRNA expression of IL-10 (Varasteh et al. Citation2015; Song, Cheng, et al. Citation2017; Baxter et al. Citation2020). In consistent with former literature, in this study, heat stress up-regulated the mRNA expression of jejunal TLR4, NF-κB, IL-1β and TNF-α, whereas down-regulated the mRNA expression of IL-10 when compared to the CON group. The up-regulated TLR4 mRNA expression could be due to the disrupted intestinal barrier function hence pathogenic bacteria invasion or a direct response to heat stress, since TLR4 was a stress-related biosensor in initial injury response (Mollen et al. Citation2006; Chaussé et al. Citation2011). Heat stress up-regulated TLR4 mRNA expression level was accompanied with inflammatory response in jejunum by up-regulated the mRNA expression of IL-1β and TNF-α, and the higher IL-1β and TNF-α expression level were the results of NF-κB activation and transcription (Lawrence Citation2009). Former studies indicated that pro-inflammatory cytokines increased intestinal permeability by disrupting the intestinal tight junction barrier function (Al-Sadi et al. Citation2009; Kaminsky et al. Citation2021; Liu, Wang, et al. Citation2021), thus the increased IL-1β and TNF-α level and mRNA expression level may explain the down-regulated the mRNA expression level of occluding, claudin and ZO-1 in the jejunum of heat-stressed broilers. Former literature indicated that dietary SB supplementation could alleviate inflammatory response and intestinal barrier dysfunction by inhibiting the TLR4/NF-κB signalling pathway and reduced the pro-inflammatory cytokines under stress (Chen et al. Citation2018; Couto et al. Citation2020). Former literature also indicated that dietary SB supplementation decreased serum IL-6 and TNF-α level in LPS-challenged broilers (Zhang et al. Citation2011b). Our results were inconsistent with former studies, dietary SB supplementation inhibited the TLR4/NF-κB signalling pathway by down-regulated the mRNA expression of TLR4, NF-κB, IL-1β and TNF-α, as well as decreased the IL-1β and TNF-α level in the jejunum of heat-stressed broilers. These results were in consistent with the anti-inflammatory effects observed in humans, in which SB inhibited the NF-κB signalling pathway and reduced the mRNA expression of pro-inflammatory cytokines (Bedford and Gong Citation2018). The study in rats also indicated that SB alleviated LPS-induced intestinal inflammatory infiltration by down-regulating the mRNA expression of jejunal TLR4, TNF-α and IL-6 (Dou et al. Citation2022). In this study, dietary SB supplementation alleviated the inflammatory response in the jejunum of heat-stressed broiler, which might have beneficial effects on jejunum morphology and tight junction structure.
Conclusion
In conclusion, heat stress-induced morphology damage, oxidative stress, inflammatory response and impaired the tight junction structure in jejunum of heat-stressed broilers. Dietary SB supplementation could alleviate heat stress-induced tight junction structure dysfunction by up-regulated the expression of occluding, claudin-1 and ZO-1, presumably through alleviating oxidative stress and inflammatory response. Our findings indicated that dietary SB supplementation was an effective additive for alleviating heat stress induced intestinal damage in broilers.
Ethical approval
The experimental protocol used in this study was approved by the Animal Care and Use Committee of Guangdong Ocean University (SYXK-2018-0147).
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References
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