Supplementation of Postbiotic RI11 Improves Antioxidant Enzymes Activity, Upregulated Gut Barrier Genes and Reduced Cytokines, Acute Phase Proteins and HSP70 Gene Expression Levels in Heat-Stressed Broilers

To alleviate the adverse impacts of stressful environmental conditions on poultry and promoting the animal's health and growth performance, antibiotics have been added to poultry diets as growth promoters. Nevertheless, improper and overuse of antibiotics as feed additives have resulted the emergence of antibiotic-resistant bacteria and increased the levels of antibiotic residues in animal products, which have disastrous effects on the health of both animals and humans. Postbiotics produced from probiotic Lactobacillus plantarum have been the recent research of interest as dietary additives for livestock and potential alternatives to antibiotics. However, there is very scarce of study has considered the effect of postbiotics on broilers under heat stress. The aim of this work was to evaluate the impacts of feeding different levels of postbiotic RI11 on antioxidant enzyme activity, physiological stress indicators, cytokines and gut barrier genes expression in broilers under heat stress. heat shock and cytokines in broiler Heat shock protein70; APPs: Acute phase proteins; α1-AGP: Alpha1-acid glycoprotein; CPN: Ceruloplasmin; M: Molar; mg: milligram; min: minute; mL: millilitre; mM: millimolar; ng: nanogram; nm: nanometre; IECs: Intestinal Epithelial Cells; x g: gravitational force; ROS: Reactive oxygen species.

dietary supplementation of postbiotics improved health and growth performance of broilers by promoting their immune status, growth genes expression and gut health as their supplementation signi cantly improved the intestinal villus, decreased the population of Enterobacteriaceae and faecal pH, and increased the population of lactic acid bacteria [25,[31][32][33]41]. Moreover, improvements in broiler meat quality and reduction in plasma cholesterol were observed with dietary supplementation of postbiotics in broilers [35,[41][42][43]. Our recent ndings from a companion study [44] revealed that dietary supplementation of postbiotics produced from L. plantarum increased body weight, body weight gain, FCR, intestinal villus height, immune response, IGF-1 and GHR mRNA expression, caecum non-pathogenic bacteria population, and reduced Enterobacteriaceae and E. coli population in heat-stressed broilers.
Aside from developing a healthy gut and promote growth performance, a preliminary study from this laboratory revealed that the postbiotics produced by L. plantarum have high antioxidant activities [40]. Similarly, bacterial cultures of L. plantarum were reported to exhibit high antioxidative activities [45,46]. In heat-stressed broilers, probiotics have been demonstrated to up-regulate the hepatic antioxidant capacity [47][48][49], inhibited the pro-in ammatory cytokines (TNF-α and IL-1β) and increased the antiin ammatory cytokines (IL-10) [50]. Another studies reported that chickens fed probiotics increased intestinal epithelial integrity by increased the mucin mRNA expression [51,52], and postbiotics produced from L. plantarum probiotic are expected to provide analogous bene ts to those from probiotic bacteria. Whilst considerable research has investigated the bene cial impacts of postbiotics on broiler chickens under normal temperature, there is still a scarcity of information on their impacts on heat-stressed broilers. Therefore, the purpose of this work was to investigate the impacts of feeding different inclusion levels of postbiotic RI11 on the antioxidant enzymes activity, and genes expression related to gut barrier function, acute phase proteins, heat shock protein70 and cytokines in broiler chickens under heat stress.

Postbiotic Production
The Lactobacillus plantarum RI11 strain was procured from the Industrial Biotechnology Laboratory, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia. The culture was preserved by the revival of culture following the procedure of Foo, et al. [53]. The culture was kept at −20°C in De Man, Rogosa and Sharpe (MRS) medium (Merck, Darmstadt, Germany) with 20% (v/v) glycerol.
A volume of 100 µL from stock culture was activated in 10 mL MRS broth, incubated at 30 °C for 48 h and sub-cultured in the same media for another 24 hours. The activated culture was spread onto a plate and incubated at 30 °C for 48 h. A single colony was picked from the plate, inoculated twice into MRS broth (10 mL) and incubated at 30 °C for 48 h and 24 h. Active cells of L. plantarum RI11 was rst washed using a 0.85% (w/v) NaCl (Merck, Darmstadt, Germany) sterile solution, then adjusted to 10 9 CFU/mL and used as an inoculum. For preparing the working culture of the L. plantarum RI11 strain, 10% (v/w) 10 9 CFU/mL bacterial cells were inoculated into MRS media, incubated for 10 hours at 30 °C, and centrifuged at 10,000 × g for 15 min at 4 °C. Cell-free supernatant (CFS) was ltered using a membrane of cellulose acetate (Sartorius Minisart, 0.22 µm, Gottingen, Germany) following the procedure described by Loh, et al. [54]. The harvested CFS (postbiotic RI11) was kept at 4 °C until applied in feed within 48 hours.
Ethical Note, Birds, Diets, Experimental Design and Housing The feeding trial was performed at the research facilities of the Institute of Tropical Agriculture and Food Security (ITAFoS), Universiti Putra Malaysia. The study was conducted following the guidelines approved by Animal Ethics Committee of Universiti Putra Malaysia (protocol no. UPM/ACUC/AUP-R085/2018), which ascertains that the use and care of research animals are ethical and humane. Two hundred and fty-two Cobb 500 male chicks (one-day-old) were supplied by a local hatchery. The chicks were housed in wire-oor cages placed in two identical rooms. The rooms were environmentally controlled with each of the two measuring 9.1 × 3.8 × 2.3 m, length × width × height, whereas measurement of each cage was 120 (length) × 120 (width) × 45 (height) cm. The birds were reared following the management recommendations of Cobb 500 from 1 to 21 days of age (starter period). The chickens in the two rooms were maintained at the recommended temperature of 32 ± 1 °C on the rst day, a gradually reduced to around 24 ± 1 °C by 21 days of age. During the nisher period (day 22 to day 42), the birds were divided into 7 treatment groups, 6 replicates per group with 6 chicks in each replicate. The birds were offered 1 of 7 diets: (1) A basal diet without any supplementation as negative control (NC) 0.0% RI11; (2) NC + 0.02% (w/w) oxytetracycline as positive control (OTC); (3) NC + 0.02% (w/w) ascorbic acid as antioxidant control (AA); or four further groups were NC + 0.2, 0.4, 0.6 and 0.8 % postbiotic RI11 (v/w) of the respective levels. The basal diets were formulated using FeedLIVE software [44]

Plasma Antioxidant Enzymes Biomarkers
Glutathione Peroxidase Activity Glutathione peroxidase (GPx) activity was analysed in plasma samples using the EnzyChrom TM Glutathione Peroxidase Assay Kit (EGPx-100, BioAssay Systems, Hayward, USA), which directly measured the consumption of NADPH in the enzyme-coupled reactions. The assay was carried out as recommended in the manufacturer's protocol. The detection range of the kit was 40-800 U/L GPx.
Approximately, 10 µL of the sample plus 90 µL of working reagent (80 µL assay buffer, 5 µL glutathione, 3 µL NADPH (35 mM), and 2 µL gr enzyme) were loaded into the microplate well and tap the plate to mix. A 100 µL of substrate solution was added to each sample and control wells. The optical density of the samples and standards were measured immediately at time zero (OD0), and again at 4 min (OD4). The absorbance of the GPx activity was recorded at 340 nm using microplate reader (Multiskan GO, Thermo Scienti c, Waltham, Massachusetts, USA). The NADPH standards were used to plot the standard curve.
The standard curve was used to calculate the GPx activity in the plasma samples.

Superoxide Dismutase Activity
Superoxide dismutase (SOD) assays were carried out using EnzyChrom™ Superoxide Dismutase Assay Kit (ESOD-100, BioAssay Systems, Hayward, USA) based on protocol provided from the manufacturer.
The detection range of the kit was 0.05 -3 U/mL SOD. The test depended on the addition of xanthine oxidase to the sample as a source of superoxide, and this superoxide reacted with a speci c dye to form a coloured product. Based on the activity of SOD in the sample, which acted as a superoxide scavenger, the superoxide was reduced, and then the intensity of colour decreased. The activity of SOD was determined by measuring the colour intensity at 440 nm using a microplate reader (Multiskan GO, Thermo Scienti c, Waltham, Massachusetts, USA). The standard curve was used to calculate the concentration of SOD in the samples.

Catalase Activity
Catalase (CAT) activity was measured from plasma using the EnzyChrom TM catalase assay kit (ECAT- the assay depended on the reaction of 5,5'-dithiobis 2-nitrobenzoic acid with reduced glutathione to form a yellow product. Brie y, 120 µL of 20-fold diluted sample was mixed with 120 µL of reagent A into 1.5 mL tube, centrifuged at 14000 rpm for 5 min and 200 µL of supernatant was transferred into the microplate well. A 100 µL of reagent B was added to each well of samples, tapped the plate for mixing, and incubated for 25 min at room temperature. A 400 µL of the calibrator was mixed in serial dilution with distilled water into separate wells as the standard. A 300 µL of distilled water was pipetted into a separate well as a blank. After incubation, the absorbance was read at 412 nm using a microplate reader (Multiskan GO, Thermo Scienti c, Waltham, Massachusetts, USA). The GSH concentration in the plasma was calculated using the standard curve of glutathione.

RNA extraction and RT-PCR of studied genes
The extraction of total RNA from liver and ileum tissue samples were conducted using an RNeasy ® Mini Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's recommendations and protocols. A thirty mg of liver and ileum tissue samples were homogenised with 600 μL of buffer RLT and centrifuged at 4 °C, 10000 x g for 2 min to obtain the supernatant. The collected supernatant was mixed with an equal volume of 70% (v/v) undenatured ethanol. Then, RNeasy spins column was used for RNA binding and series of buffer RW1 and buffer RPE were used for RNA puri cation. RNase-free water was used to elute the puri ed RNA from the spin column. The puri ed RNA was con rmed for its concentration and purity using a Nanodrop TM 2000 spectrophotometer (Thermo Scienti c, Wilmington, DE, USA) at 260/280 nm absorbance ratio. The complementary DNA (cDNA) was generated from puri ed RNA using a Quantitect ® reverse transcription kit (Qiagen, Hilden, Germany) for quantitative PCR.
Reverse transcription real-time PCR was performance on a Bio-Rad CFX96 PCR machine (Bio-Rad Laboratories, Hercules, CA, USA). The standardisation of target genes expression was determined by the GAPDH gene as housekeeping gene. The total of 20 μL PCR reaction mixture for every sample was prepared using QuantiNova™ SYBR Green PCR kit (Qiagen, Hilden, Germany) containing 10 μL of 2X SYBR Green Master Mix, 2 μL of sample cDNA, 1 μL each of 14 μM respective forward and reverse primers and 6 μL of RNase-free water. The sequence of forward and reverse primers of target and housekeeping genes is depicted in (Table 1). Table 1 The primer sequences of target genes used for RT-qPCR.

Antioxidant Enzymes Activities
Oxidative stress causes the production of ROS varieties, including hydroxyl free radical and superoxide anions. Various researches reported that over owed of ROS could damage the biological macromolecules such as proteins and nucleic acids, consequently leading to the development of diseases [57]. In chickens, the main antioxidant enzymes are glutathione peroxidase, superoxide dismutase, catalase and glutathione. These enzymes are important to transform reactive species into non-radical and non-toxic products [58].
In this study, the heat-stressed chickens supplemented with various levels of RI11 (excluding 0.2%) recorded increased GPx activity, but there was no difference between 0.2% RI11 and OTC. However, the result signi ed that higher levels of RI11 are required to improve GPx activity in broiler chickens under heat stress. There was no effect of the various treatments on SOD activity, whereas CAT and GSH activities were enhanced signi cantly following postbiotic supplementation with 0.4%, 0.6% and 0.8% RI11. This nding is consistent with Wang et al. [59], who indicated that the feed supplementation with probiotic enhanced CAT, GPx and SOD activities in broilers at day 21, which may be one of the mechanisms of its bene cial effects on health and growth performance of broilers. Another study found that feeding broiler on probiotic Bacillus subtilis increased the GPx, GSH and their mRNA expression level [60]. Likewise, two studies reported that broilers under heat challenge had increased activities of CAT, GPx, GSH and SOD [61,62]. Hence, the dietary postbiotic RI11 showed the capacity to improve antioxidant activities (concentrations of GPx, CAT and GSH) in the plasma of heat-stressed broilers.
Postbiotics are a natural source of antimicrobial and antioxidant that can safely alleviate the stress and improve the health of animals. As postbiotics possess the probiotic characteristics [34,39,41,44,63,64], probiotic studies can provide useful information to understand how postbiotics could improve the antioxidant capability and develop the oxidative resistance in the body under heat stress. Several studies reported that the supplementation of probiotics in the poultry diets reduced the adverse effects of oxidative stress and enhanced the antioxidant enzymes activities [57,65], which might reduce cell damages by inhibiting the production of ROS and nally improving the health of animals [66,67].
Consistent with our results, Shen, et al. [68] reported that blood antioxidant capacities were signi cantly enhanced by the inclusion of probiotic L. plantarum in the diets and promoted growth performance in broilers.
This study is the rst attempt to provide data on the effect of different levels of postbiotic RI11 on antioxidant activities in heat-stressed broilers. However, probiotics have been reported for their ROS removal capacity and promoting broiler health under normal [57] and high-temperature conditions [69].
Vitamin C has been equally reported to improve the activities of antioxidant enzymes including GPx and GSH in layers [70]. Yun et al. [71] showed that mRNA and activity of GPx were improved in heat-stressed broilers supplemented with vitamin C without affecting SOD activity.

Cytokines mRNA expression
Cytokines are small extracellular signaling protein produced by the host with crucial functions in immunity by enabling cell communication midst immunological development and immune response [72]. The pro and anti-in ammatory cytokines are produced by immune cells such as T lymphocytes, B lymphocytes, macrophages and natural killer cells [73]. T lymphocytes are divided into two types of cells which are Th1 and Th2. Generally, IL-2, IL-8, IFN-γ and TNF-α are known as a Th1 type cytokine which increases cellular immunity, whereas IL-6 and IL-10 are known as a Th2 type cytokine that acts in humoral immunity [74][75][76]. These small molecules proteins are released when the animal is exposed to infection, in ammation and shock as an immune response [77]. Heat stress affects intestinal integrity and increases intestinal permeability to endotoxin and antigens and in ammatory cytokines [78]. Heat stress has been shown to increase the expression of pro-in ammatory cytokines and suppressed antiin ammatory cytokines in broilers [79]. Heat stress leads to gut damage and induces commensal bacteria to release endotoxin that encourages the production of the pro-in ammatory cytokines [80]. The current study evident that the increase of the levels of pro-in ammatory cytokines in broilers under heat stress such as IL-8 and TNF-α could be alleviated by postbiotic dietary supplementation. Feeding postbiotic RI11 with various levels would modulate the in ammatory processes by restoring cytokine balance in order to reduce the potential in ammation-induced injury that occur following heat stress in broiler chickens.
In the present study, lower expression of IL-8 and TNF-α, and higher expression of IL-10 were found in postbiotic RI11 fed broilers compared to other treatments. The differential expression of IL-8 seen herein could be due to the interaction between the bene cial bacteria which enhanced by postbiotics and intestinal enterocytes and immune cells of the lamina propria [81]. These ndings were in line with that of Kareem et al. [31], who reported reduced cytokines expression in broiler chickens supplemented with various combinations of postbiotics and inulin. Wang [82] documented that supplemented probiotic B. subtilis in broiler diets under heat stress decreased the expression levels of IL-6 and TNF-α and increased IL-10 expression level. In ammatory cytokines, especially TNF-α, IL-2, IL-8 and IL-6 play important roles in the inducement and prolongation of in ammation caused by macrophages. The high levels of TNF-α have the capability to increase tissue damage or sepsis and death [83]. In this study, the TNF-α expression was down-regulated by supplementation with postbiotic RI11 in broilers diets under heat stress as compared to the negative control. Recently, supplementation with a polysaccharide-based bio occulant (PBB) extracted from B. subtilis F9 inhibited the expression of TNF-α and IL-1, whereas that of IL-10 was signi cantly increased as the anti-in ammatory potential of PBB [50]. Previous studies have demonstrated the effect of probiotics in reducing pro-in ammatory cytokine production [84,85]. In piglets, Yang et al. [86] posited that pre-treatment of porcine epithelial cells with L. reuteri led lowered the expression of TNF-α and IL-6. Moreover, the effects of feeding commensal bacteria such as LAB have been reported to have both pro-in ammatory and anti-in ammatory actions [87]. The high population of Lactobacillus and Bi dobacterium could play a role in anti-in ammatory cytokine expression, whereas the opposite reaction may be due to lowered pathogens load [88]. In piglets, increased lactobacilli population was associated with decreased expression of IL-8 [89]. This was exempli ed in this study based on the signi cant increment in Lactobacillus and Bi dobacterium count, reduced pathogenic load and down-regulation of IL-8. In other studies, involving probiotics, reduction in IL-8 secretion in intestinal epithelial cells (IECs) was suggested to occur through different pathways [79]. The IL-2, IL-6 and IFN were not affected by the inclusion of postbiotic in broiler diets under heat stress. However, IL-6 expression was up-regulated in broiler fed a combination of postbiotics and inulin [41] and lambs [39] fed postbiotic RG14.
The results of the current study allowed us to suggest that postbiotic RI11 in uences cytokine expression dynamics of broilers by the modulation of the balance between anti-in ammatory and pro-in ammatory cytokines under heat stress. Therefore, postbiotics could ameliorate heat tolerance by upregulation of cytokines expression to tissue stability and repairing mechanisms that are working during and after heat stress recovery.

Gut barrier genes expressions
The gut mucosal barrier is mainly formed by the intestinal epithelium and remains an essential part of the immune response in the intestine. Upon entry of foreign bodies such as pathogenic microbes, the intestinal epithelial cell (IEC) is the rst line of defense and they interact effectively with commensal bacteria and antimicrobial substances to protect intestinal barrier [90]. The IEC function is mediated by multiprotein complexes present at the apical end of the IECs and referred to as tight junctions (TJs). TJs play an immense role in regulating intestinal permeability by shutting the spaces between adjacent IECs [91]. Various factors affect TJ and mucosal barrier functions such as cytokines, probiotics, growth factors and pathogens by transcriptional regulation and post-translational modi cation of tight junction proteins [90][91][92]. OCLN, CLDN1 and ZO-1 are some of the major functional components of TJs [93,94]. The gene for mucin produced by goblet cells is often illustrated as MUC2 a vital component of the mucous layer covering the intestinal epithelium.
In this study, the expression of these TJs was investigated following the postbiotic feeding of broilers exposed to heat stress. The expression of ZO-1 and MUC2 were signi cantly higher in birds fed with postbiotic RI11 as compared with OTC and negative control, while 0.6% RI11 showed the highest effect among the postbiotic groups. The bene ts of inclusion postbiotic at level 0.6% may be attributed to the optimal environment provided for better bene cial bacteria growth, then improve the intestinal integrity, nutrient digestibility and increase the growth performance of broiler. These ndings showed that supplementation of postbiotics at various levels prevented the reduction in expression of ZO-1, OCLN and induced MUC2 gens by heat stress. These results were in agreement with the nding of Zhang et al. [95] who found that the addition of probiotics mixture in layer feed resulted in upregulation of ZO-1 mRNA expression under heat stress. Supplementation of probiotic B. subtilis in broiler diets signi cantly increased gene expression of intestinal MUC2 mRNA compared to those fed the control diet coincides with our nding [52]. Broilers fed L. fermentum 1.2029 strain showed signi cantly increased goblet cell density in the jejunum and the level of MUC2 mRNA in both the jejunum and ileum [96]. Inclusion of postbiotics did not affect the expression of CLDN1 gene in this study. These results corroborate the reports from studies conducted previously in mice and pigs, in which the expression of OCLN and ZO-1 were reduced in pulmonary cells in mice and porcine IECs respectively following LPS treatment [86,97]. A similar scenario regarding the reduced expression of ZO1, MUC2 and OCLN were reported in broilers fed with probiotic after LPS challenge [79].
To the best of our knowledge, there is a paucity of data relating to the expression of TJs related genes in broilers under heat stress. However, the expression of these TJs components illustrates the potential of RI11 to enhance barrier function and preventing antigen entry. The postbiotics used in this study could have improved barrier function, production of mucin and heat shock protein (HSP), thereby modulating signaling pathways and survival of IECs.
A recent study reported that inclusion of postbiotic in post-weaning lamb increased the expression level of TJs genes which is in line with our nding [39]. The improvement of TJs proteins is attributed to the postbiotics, which contain metabolites of probiotic bacteria with ability to affect the regulation of TJs integrity and mucosal barrier function [39]. The interactions of metabolites and bioactive molecules secreted by probiotics with intestinal immune cell receptors modulate epithelial cell function by increasing tight junction integrity and prevent its disruption [92,98].
Acute phase proteins and HSP70 mRNA expression Results from this study showed lower expression of AGP mRNA and HSP70 in heat-stressed broilers supplemented with postbiotics compared to the control. The parameters were also reduced in birds supplemented with ascorbic acid, but not as observed in the postbiotic treated groups.
HSP70 is a useful indicator of cellular insult and predicting the level of thermal stress in chickens [99]. The protein is highly preserved, and they are expressed under stress conditions such as transportation, feed restriction, unpleasant human contact and high temperature [100][101][102]. During acute heat stress, the level of HSP70 is increased through the synthesis of HSP70 mRNA either by increased amount of the protein or activity of the heat shock transcription factor [103,104]. Earlier studies have reported higher levels of HSP70 in various tissues of broilers following exposure to high ambient temperature [104,105].
In the present study, the down-regulation of HSP70 mRNA in the heat-stressed birds supplemented with postbiotics indicates the amelioration of the effect of the environmental stressor on birds' health status.
Moreover, for intestinal barrier to be improved, the expression of heat shock protein is important for the signaling pathways involved in the survival of IECs [106].
The ability of different levels of postbiotic RI11 to induce lower expression of HSP70 mRNA level as observed in this study could be a mechanism to defend the synthesised TJs proteins from the negative impact of heat stress. Accordingly, HSP70 acts as a chaperone by interacting with proteins to defend synthesized proteins against additional injury and reduce ROS production [107]. HSP70 induction guards against stresses such as hyperthermia, ischemia and in ammation [101,108].
This study showed that higher levels of RI11 and ascorbic acid supplementation induced similar positive effect in improving the HSP70 levels in heat-stressed broilers. Both postbiotics and ascorbic acid supplementation had greater positive effect compared to OTC treated birds. Previous studies have also shown the effect of ascorbic acid in the expression HSP70 mRNA [71,105,106,109]. Heat stressed rats supplemented with vitamin C had signi cantly lower hepatic HSP70 mRNA compared to those not given the dietary treatment; nevertheless, the thermic HSP70 was not affected by the treatment [71].
There is no scienti c report to date to investigate the effect of postbiotics on gene expression of HSP70 and APPs in heat-stressed broilers. However, the ndings are comparable to other studies that assessed the effect of other substances on the above-listed proteins. There was no effect of the various treatments on the mRNA expression of CPN. This could be related to the time variation and response kinetics of different APPs in avian species. For instance, hepatic α1-AGP was reported to be faster in reaction than CPN following exogenous administration of corticosterone [99]. Moreover, some authors have shown that variation of broiler breeds and examined organs could in uence the response of APPs to heat stress and targeted treatments [106,109].
Another likely mechanism for the positive impact of postbiotics on the HSP70 and α1-AGP mRNA expression level is the interaction with antioxidant activities. Gu et al. [105] posited a direct relationship between HSP70 level and antioxidant enzyme activities such as SOD, GPx and total antioxidant capacity. In this study, we observed improved antioxidant enzyme activity and reduced HSP70 and APPs mRNA expression in the heat-stressed broilers supplemented with various levels of postbiotic RI11. Roushdy et al. [109] reported that improved serum antioxidant enzymes level could explain the lower HSP70 expression in the different strains of broilers under heat stress. Such interaction with HSP70 mRNA expression could be vital in ameliorating the extent of mucosal oxidative injury.

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The results of the present study demonstrated that supplementation of postbiotic RI11 in different levels (particularly 0.6%) in the diet of broiler chickens under heat stress increased the plasma concentration of antioxidant enzymes activities (GPx, CAT and GSH) and reduced the heat stress biomarkers such as acute-phase proteins (α1-AGP and CPN) and HSP70 mRNA expression. It can be observed that higher mRNA expression level of IL-10, but lower expression IL-8 and TNF-α at the mucosal of ileum