Chitosan Nanoparticles Attenuate Intestinal Damage and In ammatory Responses in LPS- challenged Weaned Piglets via Inhibition of the NF- κB Pathway

Yinglei Xu (  xuyl@zafu.edu.cn ) Zhejiang A&F University https://orcid.org/0000-0001-7375-7969 Pu Ge Chongqing University of Medical Science: Chongqing Medical University Qing Li Zhejiang Agriculture and Forestry University: Zhejiang A and F University Huiling Mao Zhejiang Agriculture and Forestry University: Zhejiang A and F University Caimei Yang Zhejiang Agriculture and Forestry University: Zhejiang A and F University


Introduction
An optimally functioning gastrointestinal tract (GIT) is important for the overall metabolism, physiology, disease status, and performance of animals. As the main site of transport and absorb nutrients, the GIT epithelium and underlying lamina propria are continually exposed to a harsh luminal environment, which includes massive amounts of toxins, antigens, and pathogens. Thus, the GIT has developed a barrier to prevent overwhelming immune activation and potentially sepsis, which is critical for host survival [1][2] .In weaning piglets, the abrupt change in diet from milk to cereal-based solid feed leads to not only marked structural and functional changes to the small intestine but also contributes to an intestinal in ammatory status that in turn compromises villous crypt architecture, GIT barrier function, and causes disruption of the microbiota [3][4][5] . Weaned piglets commonly suffer from gastroenteritis caused by enterotoxigenic Escherichia coli. Bacterial infections in weaned piglets are responsible for considerable economic losses and reduced animal welfare in the pork industry [6] .
Lipopolysaccharide (LPS), a component of the membrane of gram-negative bacteria, is one of the most effective stimulators of the immune system and has been widely applied in pigs as an experimental model for bacterial infection [7] .Toll-like receptor 4 (TLR4) is the major recognition receptor for LPS which interacts with the extracellular co-receptors, CD14 and MD2 [8][9] . After stimulation, two known intracellular signaling pathways are activated [10] : the MyD88-dependent and -independent pathways, which both end in the translocation of nuclear factor κB (NF-κB) into the nucleus, inducing transcription of multiple genes. This transcription results in the release of cytokines in a characteristic chronology [11] Chitosan is a natural polysaccharide and is considered the largest biomaterial after cellulose in terms of its application and distribution. It is produced from chitin, the most abundant natural amino polysaccharide obtained from the exoskeleton of crustaceans such as shrimps, lobsters, and crabs.
Chitosan has an abundance of hydroxyl (-OH) and amine (-NH 2 ) functional groups, which can be employed to react with cross-linking agents for in situ chemical cross-linking. Because of their unique chemical structures with desired biocompatibility and biodegradability, chitosan-based biomaterials have attracted a signi cant attention in the medical, food, and agricultural sectors [12] . Due to their small size and high zeta potential, chitosan-based particulate systems, particularly chitosan nanoparticles (CNP), exhibit higher oral absorption than chitosan, and have revealed great success as carriers for oral delivery of peptides, proteins, and nucleotides [13] .Our recent research found that CNP could protect Caco-2 cells, a model of human enterocyte, from LPS-induced cell membrane damage [14] , suggesting that CNP may relieve weaning-associated intestinal in ammation in various animal species. Therefore, in this research, LPS was applied on weaned piglets to investigate the protective effects of feeding CNP on the intestinal damage and the mechanism underline.

Materials And Methods
The experimental design and procedures in this study were reviewed and approved by the Animal Ethics Committee of Zhejiang A&F University, Hangzhou, Zhejiang, China Materials CNPs with a mean particle size of approximately 50 nm ( Fig. 1) were prepared in our laboratory using cationic chitosan (Chitosan Company of Pan'an, Zhejiang, China) with an average molecular weight of 220 kDa and a degree of deacetylation of 95%. LPS (Escherichia coli serotype O111:B4, Sigma Chemical, USA) was dissolved in sterile saline (0.9%).

Animals, housing, and treatments
A total of 24 piglets (21 ± 2 days, Duroc × Landrace × Yorkshire, initial mass: 8.58±0.59kg) were assigned to two dietary treatment groups, taking into consideration of principle of equal numbers of males and females and similar body weight in all groups. The piglets were acclimatized for 1 week before the study.
The dietary treatments included a corn-soybean meal-based control diet [15] and a control diet supplemented with 400 mg/kg CNP. On day 28 of the feeding trial, half of the piglets (male: female = 1:1) in each treatment group were injected via intraperitoneal injection (i.p.) with LPS at 100 μg/kg. The other piglets were injected with a sterile saline solution at an equal volume. Each group contained six piglets (male: female = 1:1) and each piglet was regarded as repeat. No antibiotics were administered to the piglets prior to or during the experimental period. The dosage of LPS was selected based on the results of previous studies [16] .

Sample collection
After 4 hours from the LPS challenge, the pigs were sacri ced by exsanguination. Samples of the duodenum, jejunum, and ileum (about 2 cm) were cut out and placed into prepared 4% formaldehyde for subsequent hematoxylinand eosin (H&E) staining, immunohistochemistry, and immuno uorescencestaining. Jejunum mucosa was collected for enzyme linked immunosorbent assay (ELISA) and western blot analysis.

Intestinal morphology
Intestinal tissues were embedded in para n. Sections of 5μm thickness weredepara nized in xylene, rehydrated in graded solutions of ethanol, and then stained with hematoxylin and eosin. Images were acquired using a Zeiss Axiovert microscope (Zeiss, Germany), and villus height and crypt depth were measured using Image Pro software (Media Cybernetics, MD, USA).

Immunohistochemistry staining to assess neutrophil in ltration
To analyze the in ltration status of neutrophilic granulocytes into the jejunum, Anti-CD177 antibody (Boster, China) was used. Brie y, the sections were depara nized in xylene and rehydrated in graded ethanol solutions. Antigen retrieval was performed at 95 °C for 40 min in0.01M sodium citrate buffer (pH 6.0). All sections were then immersed in 3% hydrogen peroxide for 20 min. Primary antibody was added at a dilution of 1:100 and incubated overnight at 4 °C. After washing three times with phosphate buffered saline, samples were incubated with rabbit anti-goat IgG (Boster, China) at a ratio of 1:200 at 4 °C for 1 h.
Then, DAB was added (DAKO, USA), and hematoxylin (H-3404; Vector, USA) was used to counter stain the slices. Finally, the samples were dehydrated with gradient alcohol, and xylene was used to increase the transparency of the slides, and a neutral balsam was applied for mounting. The integrated optical density from the immunohistochemistry stain was counted using the digital image software Image-Pro Plus 6.0 (Media Cybernetics company,MD,USA).
Markers of in ammation production in the jejunal mucosa The levels of three cytokines, interleukin (IL)-8, interferon (IFN)-γ and intercellular adhesion molecule-1 (ICAM-1) in the jejunum mucosa were determined through the double antibody sandwich method by using commercially available ELISA kits (Jiancheng Bioengineering, Nanjing, China).The absorbance (OD value) was determined at corresponding wave length with an FLx800 uorescence reader (BioTek, USA), and the concentrations of these cytokines in the samples were calculated using a standard curve.
IκB-α degradation in the cytoplasm and p65 accumulation in the nucleus Nuclear and cytoplasmic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Boster, China). Protein samples were quanti ed by the method of micro Bicinchoninic Acid protein assay (BCA), using a commercially available BCA kit (Boster, China).The absorbance (OD value) was determined at a 560 nm wavelength with an FLx800 uorescence reader (BioTek, USA), and the concentrations of protein in the samples were calculated using a standard curve. The expression of IκB-α (primary antibody from Abcam, USA) in the cytosol and NF-κB p65 (primary antibody from GeneTex, USA) in the nucleus were evaluated by western blot analysis. In brief, proteins from each sample were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes, 0.45μm (Boster, China). After blocking with 5% skimmed milk for 1 h at room temperature, membranes were incubated with appropriately diluted primary antibodies at 4 ℃ overnight. The next day, the membranes were washed with Tris Buffered Saline Tween (TBST) and immunoblotted with horseradish peroxide-conjugated secondary antibodies (Boster, China) for 1 h at room temperature, washed with TBST, and then cultured with ECL chemiluminescence reagent. Blots were visualized using an Odyssey CLx Infrared Imaging System (Li-Cor Biosciences,Nebraska, USA). β-actin (primary antibody from Boster, China) and Lamin-Bprotein (primary antibody from Boster, China) were used as the loading control for the cytoplasmic and nuclear fractions, respectively.
Nuclear translocation of the p65 subunit belonging to the NF-κB complex Sections of intestinal tissue were incubated with primary antibody NF-κB p65 (1:50 dilution) at 4 ℃ and then stained with goat anti-rabbit IgG-FITC (Boster, China) for 1 h at room temperature (RT) in the dark. After washing, nuclei were stained with DAPI for 10 min at RT, and the cover slips were mounted on slides with ProLong®Dia-mond Anti fade Mountant (Life Technology, NY, USA). Fluorescence was observed using a Zeiss Axiovert microscope (Zeiss, Germany).

Statistical analysis
Data were expressed as means ± standard error of mean (SEM) and examined for their statistical signi cance of their differences in an ordinary one-way ANOVA analysis followed by Tukey's multiple comparisons test, using GraphPad Prism 8.2.1.software (USA). P-values< 0.05 were considered to be statistically signi cant. The individual pigs were considered as the experimental unit.

Results
Effects of chitosan nanoparticleson intestinal morphology LPS induced the destruction of villus structure in the duodenum (Fig. 2D) and ileum (Fig. 2F) and atrophy in the jejunum (Fig. 2E). In contrast, administration of CNP alleviated the damage of LPS to intestinal villi ( Fig. 2J-L). Villus height and crypt depth in each section of the intestine were also measured ( Table 1). CNP administration tends to improve villus height and reduce crypt depth, which resulted in a signi cant improvement of V: C in the CNP group compared with the control (p<0.05) in the jejunum. Whereas, LPS treatment decreased villus height and increased crypt depth, which leaded to a signi cant decrease of V: C in LPS group compare with the CNP group in the jejunum (p<0.01) and Ileum (P<0.05). Effects of chitosan nanoparticles on the in ltration of neutrophils A signi cant (p<0.01) invasion of neutrophils in the jejunal mucosa was observed in the LPS group compared with the other groups, whereas the level of in ltration in the CNP+LPS group was attenuated (P<0.01) (Fig. 3).

Effects of chitosan nanoparticleson on the cytokine production
The ELISA (Fig.4) indicated that LPS treatment resulted in marked increases (p<0.01) in ICAM1, IL-8 and IFN-γ secretion in the jejunal mucosa, whereas CNP administration signi cantly reduced LPS-induced ICAM1 and IL-8 production (p<0.05).

Effect of chitosan nanoparticles on IκB-α degradation and p65 translocation
To further elucidate the protective mechanism of CNP on intestinal mucosa, the cytoplasmic level of IκBα and nuclear level of NF-κB p65 subunit were analyzed by western blotting. Immuno uorescence assays were performed to examine the location of p65 in the jejunum sections. Compared with the control group, LPS induced IκB-α degradation in the cytosol and translocation of NF-κB p65 subunit into the nucleus (p<0.01), whereas CNP administration inhibited the degradation of IκB-α (p<0.01) in the cytoplasm and p65 accumulation in the nucleus stimulated by LPS treatment (p<0.01) (Fig. 5). Immuno uorescence results further con rmed the results of the western blot assay. LPS stimulation induced the nuclear translocation of p65, whereas CNP exerted negligible effects on the translocation of p65 (Fig. 6). These results suggest that CNP can inhibit LPS-induced nuclear translocation of p65 by suppressing the degradation of IκB-α.

Discussion
In this study, LPS was used to simulate a bacterial infection in weaned piglets to investigate the potential anti-in ammatory effects of CNP and the underlying mechanism in vivo. Using H&E staining, we could clearly see that the integrity of the three parts of the small intestine was compromised in LPS treated animals. CNP administration improved the intestinal development in the piglets and alleviated the damage of the intestinal epithelium induced by LPS, which was indicated by the more intact intestinal epithelial structure and signi cantly higher V:C ratio, compared with the control and LPS group.
Along with disturbances in intestinal permeability induced by LPS, neutrophil in ltration of epithelial surfaces leads to injury and leakage of the mucosal barrier. Such barrier defects underlie the basis of a number of in ammatory disorders. Accumulation of neutrophil in epithelial intestinal crypts has been shown to be directly correlated with the severity of in ammation [17] . The current study showed that the level of neutrophil in ltration in the jejunum of LPS-treated piglets was signi cantly higher than that observed in the other three groups. This was accompanied by signi cantly increased secretion of markers of in ammation such as IL-8, ICAM-1, and IFNγ. IL-8 is mainly active on neutrophils, promoting their recruitment and strong activation, characterized by the activation of the leukotriene pathway [18] . The level of IL-8 expression correlates with the severity of the disease, particularly with neutrophilic in ammation and mucosal destruction [19][20] .ICAM-1 mainly mediates neutrophil adhesion. It was reported [21] that engagement of ICAM-1 exclusively on the luminal (apical) membrane of the intestinal epithelium would increase the accumulation of epithelial-associated neutrophils, thus contributing to mucosal injury. IFN-γ is considered to be one of the major drivers of the excessive immune response to commensal microbiota, leading to massive leukocyte in ltration and mucosal damage [22][23] . CNP administration signi cantly reduced the in ltration of neutrophils and secretion of IL-8, ICAM-1, and IFNγ, which in turn attenuated the damage of the intestinal epithelium as compared to the LPS group.
The activated NF-κB pathway stimulates the synthesis of markers of in ammation, including IL-8, ICAM-1, and IFNγ [24] . Thus, it will be interesting to explore whether NF-κB is involved in CNP-mediated regulation of the in ammatory response to LPS stimulationin in vivo. In non-in amed intestinal epithelial cells (IEC), NF-κB is kept inactive in the cytoplasm by binding to the inhibitory protein IκB-α. However, IκBα is phosphorylated and dissociated in response to stimulation, and thus the p65 subunit of NF-κB is liberated and translocated into the nucleus, where it activates the transcription of NF-κB-dependent genes. NF-κB activation in IECs results in increased levels of IEC-derived in ammatory cytokines [25][26] . Consistent with our previous in vitro study, LPS treatment led to the activation of NF-κB, as con rmed by IκB-α degradation and p65 nuclear translocation. CNP decreased the degradation of IκB-α and translocation of NF-κB p65 in the intestinal epithelium of LPS-in amed piglets. Therefore, it is likely that CNP alleviates the LPS-induced in ammatory response by weakening the activation of the NF-κB pathway.

Conclusions
In summary, CNP can suppress the activation of NF-κB signaling pathway in piglets by inhibiting the degradation of IκB. Impaired translocation of NF-κBp65 leads to reduced production of markers of in ammation and intestinal damage. It is speculated that CNP alleviates the in ammatory response to LPS stimulation via inhibition of the NF-κB signaling pathway in piglets. This underlying mechanism provides a theoretical basis for the application of CNP as a functional feed additive in piglets.

Consent for publication
All the authors read and agree to the content of this paper and its publication.   Western blot analysis of LPS activated IκB-α degradation in cytoplasm and p65 accumulation in nucleus.

Con ict of Interest
The density ratios were measured by densitometry. The average values of three male piglets of each group were calculated and analyzed by one-way ANOVA followed by Tukey's multiple comparisons test. * p<0.05, ** p<0.01 vs the control group. # p<0.05, ## p<0.01 vs the LPS group.