Icariin Enhances Intestinal Barrier Function by Inhibiting NF-Κb

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Icariin enhances intestinal barrier function by inhibiting NF-kB signaling pathways and Cite this: RSC Adv.,2019,9, 37947 modulating gut microbiota in a piglet model†

Wen Xiong, a Haoyue Ma,b Zhu Zhang,a Meilan Jin,a Jian Wang,a Yuwei Xua and Zili Wang*a

This study investigated the effects of icariin on intestinal barrier function and its underlying mechanisms. The icariin diet improved the growth rate and reduced the diarrhea rate in piglets. The icariin diet also reduced the levels of plasma and colonic IL-1b, -6, -8, TNF-a, and MDA but increased the plasma and colonic activity of SOD, GPx, and CAT. Besides, the levels of plasma and colonic endotoxin, DAO, D- lactate, and zonulin were markedly reduced in icariin groups. Meanwhile, dietary intake icariin significantly increased the gene and protein expression of ZO-1, Occludin, and Claudin-1 in the colon. Furthermore, the gene and protein expressions of TLR4, MyD88, and NF-kB were significantly inhibited Received 7th September 2019 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. in the colon of icariin fed piglets. The intestinal microbiota composition and function was changed by Accepted 6th November 2019 the icariin diet. Collectively, these findings increase our understanding of the mechanisms by which ICA DOI: 10.1039/c9ra07176h enhances the intestinal barrier function and promotes the development of nutritional intervention rsc.li/rsc-advances strategies.

Introduction inammatory bowel disease and tissue damage.6 In addition, intestinal oxidative stress disrupts the intestinal barrier by The intestine is not only the main digestive and absorbing reducing redox sensitivity, resulting in increased intestinal 7 This article is licensed under a organ of nutrients but is also an effective barrier against various permeability. Microorganisms that inhabit the gut and their pathogenic infections.1,2 The main function of the intestinal benecial metabolites can regulate intestinal health.8 Increasing epithelium is to isolate the digesta, microorganisms, and its evidences suggest that gut microbiota disorder is closely related metabolites from the intestinal epithelial cells, preventing to inammation and oxidative stress. A previous study has shown Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. harmful substances in the intestinal lumen from damaging the that gut microbiota disorder redisposed to inammatory bowel intestinal epithelial cells and then entering the lymph or blood diseases such as ulcerative colitis, Crohn disease, and indeter- circulation.3 Under normal physiological conditions, the intes- minate colitis.9 Besides, it has been reported that change in the tinal tract can selectively penetrate nutrients. However, under gut microbiota may lead to apoptosis and further affect the pathological conditions, harmful metabolic substances in the intestinal barrier by affecting oxidative stress.10 Therefore, the intestinal lumen may escape into the blood circulation due to inhibition of pathogenic proliferation by means of nutritional the destruction of intestinal integrity, causing local or systemic intervention is necessary to alleviate inammation and oxidative diseases.4 Thus, to prevent harmful metabolites from damaging stress. Microbial metabolites such as short-chain fatty acids the intestinal or distant organs, the structural and functional (SCFAs) could serve as an energy source for intestinal epithelial integrity of the mucosal epithelium needs to be maintained. cells, and could also act as signaling molecules to regulate Impaired intestinal integrity increases intestinal permeability, metabolic and immune functions.11,12 NF-kB is a nuclear tran- inammatory response, and oxidative stress, leading to bacterial scription factor that has been extensively studied, and many translocation and subsequent intestinal disease.5 Excessive pro- reports have demonstrated that NF-kB mediates inammatory inammatory cytokines in the intestinal mucosa can cause responses and oxidative stress.13,14 Therefore, maintaining the homeostasis of the gut microbiota and limiting the expression of NF-kB is critical for protecting intestinal health. aCollege of Animal Science and Technology, Southwest University, Chongqing, China. Evidence in rodents and humans has revealed that natural E-mail: [email protected]; [email protected]; [email protected]; product medicines have good health effects, such as alleviation [email protected]; [email protected]; [email protected] of type 2 diabetes, chronic liver damage, and irritable bowel bCollege of Parmaceutical Sciences, Southwest University, Chongqing, China. E-mail: syndrome.15–17 As a common natural product, the academic [email protected] † Electronic supplementary information (ESI) available. See DOI: community has conducted a comprehensive study on the 10.1039/c9ra07176h chemical constituents of epimedium. Among them, icariin

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(ICA) is a representative active ingredient of epimedium.18 the diarrhea rate was signicantly lower than that of the CON Emerging data suggest that ICA exerts its benecial effects by group (P < 0.05). regulating some biological processes, such as inhibiting inammatory responses and oxidative stress.19,20 However, it is unclear whether ICA contributes to maintaining or enhancing Effect of diet supplemented with ICA on plasma and colonic the intestinal barrier function. cytokines and oxidative status in weaned piglets Pigs are omnivores with similar organ functions and meta- We detected biomarkers related to the inammation and bolic pathways as humans. Importantly, the gut microbiota of oxidative stress in the piglets. These biomarkers included IL-1b, these two species shares a comparatively broad range of gut IL-6, IL-8, and TNF-a as markers for inammation and MDA, 21,22 microbiota. Therefore, pigs are generally considered to be SOD, GSH-px, and CAT as markers for oxidative stress. The excellent model species for studying human digestion and levels of plasma and colonic IL-1b, IL-6, IL-8, and TNF-a were 23,24 nutrition. In the current study, we employed piglets as markedly reduced (P < 0.05) in the piglets from the ICA groups ff a model to investigate the e ects of ICA on intestinal barrier (Fig. 1). The levels of plasma and colonic MDA were dramati- function. In addition, the potential mechanisms of ICA for cally decreased and the enzymatic activity of SOD, GSH-px, and intestinal barrier function were elucidated by studying gut CAT were signicantly increased (P < 0.05) in the piglets from k microbiota and NF- B signaling pathways. the ICA groups (Fig. 2).

Effect of diet supplemented with ICA on intestinal Results permeability in weaned piglets Effect of diet supplemented with ICA on growth performance We further detected biomarkers related to the intestinal in weaned piglets permeability of piglets. These biomarkers included endotoxin, Creative Commons Attribution-NonCommercial 3.0 Unported Licence. To demonstrate the effect of diet supplemented with ICA on DAO, D-lactate, and zonulin. The levels of plasma and colonic growth performance, we examined BW, ADG, and diarrhea rate endotoxin, DAO, D-lactate, and zonulin were markedly reduced (Table 1). On days 1 and 14 of the experimental period, there (P < 0.05) in the piglets from the ICA groups (Fig. 3). was no signicant change in the BW between the two groups (P > 0.05). However, on day 28, the BW of piglets in the Effect of diet supplemented with ICA on intestinal tight ICA group was signicantly higher than that in the CON group junction protein in weaned piglets (P < 0.05). In addition, there was no difference in the ADG and diarrhea rates between the two groups during the rst two To investigate the effect of diet supplemented with ICA on

This article is licensed under a weeks of the experimental period (P > 0.05). During the last two intestinal tight junction protein, we determined the gene and weeks of the experimental period, the ADG of the ICA group was protein expression of ZO-1, Occludin, and Claudin-1 in the signicantly higher than that of the CON group (P < 0.05) and colonic mucosa. The gene and protein abundance of ZO-1, Occludin, and Claudin-1 was dramatically increased (P < 0.05)

Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. in the piglets from ICA groups (Fig. 4). Table 1 Effect of diet supplemented ICA on growth performance and diarrhea incidence in weaned pigletsa Effect of diet supplemented with ICA on NF-kB signal pathway Groups in weaned piglets Items CON ICA P-value To investigate the potential mechanism by which diet supplemented with ICA increases the intestinal barrier function, we BW, kg k examined the NF- B signaling pathway in the colonic mucosa. 1 d 7.0 0.1 7.0 0.1 0.98 k 14 d 13.1 0.2 13.6 0.1 0.28 The gene and protein abundance of TLR4, MyD88, and NF- B 28 d 17.8 0.2 19.5 0.2 0.02 was dramatically increased (P < 0.05) in the piglets from the ICA groups (Fig. 5). 1–14 d ADG, g 320 18 333 22 0.21 Diarrhea rate, % 5.0 0.1 4.5 0.1 0.33 Effect of diet supplemented with ICA on microbial 14–28 d metabolites in weaned piglets ADG, g 401 24 453 28 0.03 To analyze the effect of diet supplemented with ICA on intes- Diarrhea rate, % 6.8 0.1 4.4 0.1 0.01 tinal microbiota metabolism, we focused on the colonic and 1–28 d plasma SCFAs of piglets from either CON or ICA groups (ESI ADG, g 360 14 393 18 0.04 Fig. 1†). The results showed that the levels of colonic (ESI Diarrhea rate, % 5.8 0.1 4.3 0.1 0.04 Fig. 1A†) and plasma (ESI Fig. 1B†) acetate, propionate, buty- a Data were shown as mean SEM (n ¼ 12). rate, and total SCFAs in the piglets from ICA group were higher (P < 0.05) than those from the CON group.

37948 | RSC Adv.,2019,9, 37947–37956 This journal is © The Royal Society of Chemistry 2019 View Article Online Paper RSC Advances

Fig. 1 Eﬀect of diet supplemented with ICA on plasma and colonic cytokines in weaned piglets. (A) Plasma IL-1b concentration. (B) Plasma IL-6 concentration. (C) Plasma IL-8 concentration. (D) Plasma TNF-a concentration. (E) Colonic IL-1b concentration. (F) Colonic IL-6 concentration. (G) Colonic IL-8 concentration. (H) Colonic TNF-a concentration. Data are shown as mean SEM (n ¼ 6). *, P < 0.05.

Eﬀect of diet supplemented with ICA on intestinal microbial displayed in ESI Fig. 3.† Firmicutes and Bacteroidetes were the community in weaned piglets most dominant phyla in both the CON and ICA piglets, followed by Proteobacteria, Spirochaetae, and Actinobacteria. Other The microbiota composition and diversity of the colonic digesta phyla were present at very low relative abundances (ESI

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. samples in the piglets were assessed by deep sequencing of the Fig. 3A†). At the genus level, we also revealed the predominant V3–V4 region of the 16S rRNA genes. A total of 533 524 high- genera that are presented in ESI Fig. 3B.† Signicant differences quality 16S rRNA gene sequences were generated from twelve in the relative abundance of the phylum and genus between the colonic digesta samples. The average numbers of high-quality colonic digesta samples in the CON and ICA groups were sequences generated per sample were 44 460 from the micro- further investigated (Fig. 6). We performed differential analysis bial populations. The colonic digesta samples of piglets in the of the top 10 bacteria between the two groups at the phylum and CON and ICA groups showed 819 and 897 OTUs, respectively. genus level. The relative abundances of the phyla Bacteroidetes, Among them, 781 shared common OTUs and total 154 indi- Spirochaetae, Verrucomicrobia, and Saccharibacteria were vidual OTUs were isolated in the two groups (ESI Fig. 2E†). The  This article is licensed under a signi cantly elevated and the phyla Firmicutes and Proteobac- differences in the microbial a-diversity in the colonic digesta teria were signicantly reduced in piglets from the ICA group samples between two groups are shown in ESI Fig. 2A–D.† The compared with those from the CON group (Fig. 6A). At the genus Sobs, Shannon, Ace and Chao index increased signicantly (P < level, Ruminococcaceae_NK4A214_group and Pre- 0.05) in the ICA group. Besides, PCoA based on the Bray–Curtis Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. votellaceae_NK3B31_group exhibited increased relative abun- distances showed a shi in the microbial beta diversity of the dances in piglets from the ICA group. In contrast, there was colonic digesta samples between two groups (ESI Fig. 2F†). a remarkable decrease in the relative abundances of the genera The top 15 phyla and the top 30 genera in relative abundance of the colonic digesta microbiota that are present in piglets are

Fig. 2 Eﬀect of diet supplemented with ICA on plasma and colonic oxidative status in weaned piglets. (A) Plasma MDA content. (B) Plasma SOD activity. (C) Plasma GSH-px activity. (D) Plasma CAT activity. (E) Colonic MDA content. (F) Colonic SOD activity. (G) Colonic GSH-px activity. (H) Colonic CAT activity. Data are shown as mean SEM (n ¼ 6). *, P < 0.05.

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Fig. 3 Eﬀect of diet supplement with ICA on intestinal permeability in weaned piglets. (A) Plasma endotoxin content. (B) Plasma DAO content. (C) Plasma D-lactate content. (D) Plasma zonulin content. (E) Colonic endotoxin content. (F) Colonic DAO content. (G) Colonic D-lactate content. (H) Colonic zonulin content. Data are shown as mean SEM (n ¼ 6). *, P < 0.05.

Enterococcus, Escherichia-Shigella, and Globicatella in pigs Functional proling of the microbial communities from the ICA group (Fig. 6B). To investigate the functional proles of the colonic digesta We further performed LEfSe analysis to identify the bacteria bacterial community, we used PICRUSt to predict the gene that were signicantly diﬀerent between the CON and ICA

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. family abundances of bacterial communities. The results groups in this study (ESI Fig. 4†). All diﬀerential bacteria were demonstrated that the relative abundances of the genes demonstrated from the phylum to genus level in the cladogram involved in replication and repair, amino acid metabolism, of LEfSe between the CON and ICA groups (ESI Fig. 4A†). The energy metabolism, metabolism of cofactors and vitamins, circle from inside to outside represents distinct bacteria from genetic information processing, enzyme families, cell growth the phylum to the genus level, respectively. The yellow dots and death, endocrine system, environmental adaptation, inserted in the circle suggest no signicant diﬀerence in nervous system, immune system, digestive system, and excre- bacteria among the two groups. Our LEfSe analysis revealed that tory system were signicantly elevated and involved in infec- the phyla Firmicutes and Proteobacteria were signicantly tious diseases, and immune system diseases were signicantly

This article is licensed under a enriched in colonic digesta samples of piglets from the CON reduced in the ICA group compared with those from the CON group and phylum Bacteroidetes was signicantly enriched in group (P < 0.05, Fig. 7). colonic digesta samples of piglets from the ICA group. At the genus level, genera, Enterococcus, Escherichia_Shigella, and Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. Globicatella were signicantly enriched in colonic digesta Discussion samples of piglets from the CON group and genus Pre- The integrity of the intestinal barrier is important to ensure the votellaceae_NK3B31_group was signicantly enriched in the absorption of nutrients and the prevention of harmful colonic digesta samples of piglets from the ICA group (ESI substances.25 Due to their high safety and low cost, natural Fig. 4B†). products are increasingly used to improve bodily functions. ICA has been shown to have excellent preventive eﬀects against many disease processes by exerting their potent anti-

Fig. 4 Eﬀect of diet supplemented with ICA on intestinal tight junction protein in weaned piglets. (A) mRNA expression of ZO-1, Occludin, and ZO-1. (B and C) Protein expression of ZO-1, Occludin, and ZO-1. Data are shown as mean SEM (n ¼ 6). *, P < 0.05.

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Fig. 5 Eﬀect of diet supplement with ICA on NF-kB signal pathway in weaned piglets. (A) mRNA expression of TLR4, MyD88, and NF-kB. (B and C) Protein expression of TLR4, MyD88, and NF-kB. Data are shown as mean SEM (n ¼ 6). *, P < 0.05.

inammatory and antioxidant properties.26,27 In this study, we Intestinal integrity is critical for the health and survival of used weaned piglets as a model to explore the eﬀect of ICA on newborns and animals. Damage to intestinal integrity increases intestinal barrier function. Besides, we systematically evaluated intestinal permeability, leading to bacterial translocation and the local inammatory response, oxidative stress, NF-kB subsequent intestinal infection.28 The chemical markers signaling pathway, and the composition and function of the (endotoxin, DAO, D-lactate, and zonulin) that are usually low in intestinal microbiota to explore the underlying mechanisms of the circulation system in healthy individuals will show ICA aﬀecting intestinal barrier function. a signicant increase in the circulation system during the Creative Commons Attribution-NonCommercial 3.0 Unported Licence. This article is licensed under a Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM.

Fig. 6 Effect of diet supplemented with ICA on the microbial difference in the colonic digesta in weaned piglets. (A) Difference analysis at the phylum level. (B) Difference analysis at the genus level. *, P < 0.05; **, P < 0.01.

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Fig. 7 Abundances of KEGG pathways in the functional prediction by PICRUSt. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

destruction of the intestinal barrier.29 Our data showed that the previous study has shown that oxidative stress is a common levels of endotoxin, DAO, D-lactate, and zonulin in plasma and mechanism that destroys intestinal tight junction.35 Our data Creative Commons Attribution-NonCommercial 3.0 Unported Licence. colonic mucosa were signicantly decreased in the ICA group, clearly showed that dietary intake ICA signicantly reduced the which implied decreased intestinal permeability of the ICA products of oxidative stress and increased the antioxidant piglets. Tight junctions (TJs) are the primary component of the capacity of piglets. As a common nuclear transcription factor, NF- intestinal epithelium that maintain selective permeability.30 kB has been shown to have the potential to regulate inamma- The TJs mainly include the Claudin, Occludin, and ZO families, tory responses and oxidative stress.36,37 A previous study demon- which together constitute the main physical barrier against strated that ICA can eﬀectively regulate the activation of NF-kB.38 intestinal pathogenic microbial invasion and spreading.1 In the In this study, we found that the TLR4-MyD88-NF-kB signaling current study, our data showed that dietary intake of ICA pathway was signicantly inhibited in the colonic mucosa of 

This article is licensed under a signi cantly elevated the gene and protein expression of ZO-1, piglets in the ICA group. These results strongly suggested that Occludin, and Claudin-1 in colonic mucosa of piglets. ICA suppresses inammatory responses and oxidative stress by Impaired intestinal barrier function is the direct cause of diar- inhibiting the NF-kB signaling pathway, thereby enhancing rhea in piglets. We observed that the diarrhea rate of piglets in intestinal barrier function.

Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. the ICA group was signicantly lower than that of the control Intestinal microbial diversity is closely related to the health group. These results indicated that the dietary intake of ICA status of humans and animals.39 Our results suggested that the effectively enhances colonic barrier function and reduces diar- addition of ICA to diet signicantly increased the diversity of pigs' rhea in piglets. gut microbiota. As an important component of the intestinal To reveal the underlying mechanisms by which ICA enhances epithelial barrier, intestinal microbes and their metabolites exert intestinal barrier function, we investigated the effects of ICA on benecial effects on the host by various means such as regulation the inammatory responses and oxidative stress. Previous of mucosal immunity, inhibition of intestinal pathogen coloni- studies have shown that intestinal invasion by pathogens is zation, and production of antimicrobial peptides.40–42 Similar to accompanied by high expression of pro-inammatory cytokines the previous report,43 Firmicutes and Bacteriodetes represented such as IL-1b, IL-6, IL-8, and TNF-a.5 TNF-a has a signicant approximately 90% of total gene sequences in the colonic digesta destructive effect on the expression and distribution of TJ samples of piglets. A study of gut microbial transplantation protein. IL-1b, IL-6, and IL-8 not only regulate the reorganization showed that the transfer of fecal microbes from lean pigs of cytoskeletal proteins but also directly lead to the rearrange- (Landrace pigs) to antibiotic-treated mice caused signicant ment of TJs proteins, thereby reducing barrier function.31,32 In the changes in the intestinal bacterial community structure, mainly current study, our data showed that the dietary intake of ICA leading to a decrease in the ratio of Firmicutes to Bacteriodetes.44 signicantly decreased the concentrations of IL-1b, IL-6, IL-8, and Moreover, it has been recently reported that Lean pigs have TNF-a in the plasma and colonic mucosa of piglets. Oxidative a higher abundance of Verrucomicrobia than obese pigs.45 Our stress is one of the most important potential mechanisms by data showed that the ratio of Firmicutes to Bacteriodetes in the which toxic substances affect mammalian cells.33 When animals colonic digesta of pigs in the ICA group was signicantly reduced, and humans are under oxidative stress, the antioxidant system while Verrucomicrobia was signicantly elevated in the ICA will be destroyed, leading to oxidative damage to DNA and group, suggesting that the addition of ICA to the diet may abnormal protein expression, and nally physical damage.34 A increase the lean rate of piglets. Proteobacteria has been shown

37952 | RSC Adv.,2019,9, 37947–37956 This journal is © The Royal Society of Chemistry 2019 View Article Online Paper RSC Advances

to be a mucosa-associated pathogen that increases the risk of that enhance the gut health and provide new ideas for the colon-related diseases in humans.46 In our study, the ICA group rational formulation of nutritional intervention strategies. showed a lower abundance of Proteobacteria compared to the CON group. The relative abundance of the phylum Saccharibac- teria was signicantly elevated in pigs from the ICA group, Materials and methods  similar to previous ndings in the intestinal microbiota of pigs Ethical approval aer feeding with prebiotics.47 In addition, our data suggested that the functional alteration of the intestinal bacterial commu- All animal experiments conformed to the Guide for the Care nity in the ICA piglets was characterized by the signicant and Use of Laboratory Animals published by the US National increase in the metabolism, immunity, digestive function, and Institutes of Health (NIH Publication, Eighth edition, 2011) and marked decrease in the infectious and immune system diseases. was approved by the Laboratory Animal Welfare and Ethics Our study revealed that two genera of intestinal microbiota Committee of Southwest University. The experimental regula- in piglets fed ICA were signicantly elevated (Ruminococca- tions and methods were approved and then performed ceae_NK4A214_group and Prevotellaceae_NK3B31_group), while according to relevant criteria. the three genera were markedly reduced (Enterococcus, Escherichia-Shigella, and Globicatella). Ruminococca- Animals and dietary treatments ceae_NK4A214_group belongs to the Firmicutes phylum, which was considered as a brolytic bacteria that ferments complex Twenty four Duroc (Landrace Yorkshire) crossbred piglets components of plant cell wall into the SCFAs.48 A study has were allotted into 2 dietary groups randomly by considering the shown that the long-term consumption of foods with high ber sex and weight of pigs. The piglets were 28 days old (weaned at  content leads to increased microbial diversity and a predomi- 21 d and fed creep feed a er weaning for one week). The – nance of Prevotella over other bacteria.49 Thus, these results led temperature of the pig house was maintained at 25 28 C and the humidity at 65–75%. This experiment lasted 28 d, feed and

Creative Commons Attribution-NonCommercial 3.0 Unported Licence. us to analyze intestinal microbiota metabolism in piglets from the ICA group and we focused on the colonic digesta and water were provided ad libitum, all piglets were healthy, and plasma SCFAs of piglets. Consistently, we found that acetate, none received antibiotic treatment during the experimental propionate, butyrate, and total SCFAs were increased in the period. The piglets in the two groups were fed either a control colonic digesta and plasma of the piglets. Enterococcus is diet based on corn-soybean meal (CON group), or a control diet 1 a large genus of lactic acid bacteria of the phylum Firmicutes, supplemented with icariin (1 g kg BW; ICA group). Before which is a pathogen that can cause diseases such as urinary feeding the animals, we mixed ICA into the diet in proportion. ﬃ 1 tract infections, bacteremia, and diverticulitis.50 Globicatella is In addition, we ensured that the su cient ICA dose (1 g kg a non-spore-forming and non-motile genus of bacteria from the BW) of each pig was adhered to by recording the feed intake of

This article is licensed under a family of Aerococcaceae, which was found in the digestive tract each pig and feeding them to the appropriate weight of the diet. of pigs and was associated with the expression of cytokines.51 The diets were formulated according to the nutritional Escherichia-Shigella is a potential pathogen that was signi- requirements of the National Research Council (NRC, 1998, the cantly elevated in the feces of diarrhea piglets.52 Regarding the United States) for piglets. Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. results of functional proling of microbial communities, the genes involved in immune system diseases were signicantly Sample collection reduced in the ICA group compared with the control group, suggesting that the animals in the ICA group were more resis- On day 28, blood and colon samples were collected. Blood tant to inammation. The process of infectious diseases is oen samples were collected from pigs by vena jugularis, with accompanied by the presence of oxidative stress. Our data a minimum amount of stress, into heparinized tubes (5 mL). showed that the genes involved in the infectious diseases of the Plasma samples were then obtained by centrifuging the blood ICA group were also signicantly lower than the control group, samples at 3000g and 4 Cfor10min,andwerestoredat suggesting that the animals in the ICA group may have 80 C until analysis. The colonic segments were collected a stronger ability to resist oxidative stress. In our study, the from approximately the middle positions in the intestinal results of functional proling of the microbial communities tracts. The intestinal epithelium was separated from the showed that the genes involved in replication and repair, amino muscular layers by blunt dissection and then stored at 80 C acid metabolism, and energy metabolism were relatively prior to further analysis. Digesta from the proximal colon was abundant but their biological signicance still requires further aseptically collected and kept on ice until storage at 80 C research. until analysis. In conclusion, our data highlighted that ICA was an eﬀective natural product for promoting growth performance and reducing diarrhea rates in piglet. Because of its excellent ability Determination of growth performance to regulate NF-kB signaling pathways and intestinal microbiota, The BW of piglets and feed intake per pen were measured to ICA eﬀectively promoted intestinal barrier function. Therefore, measure the weight gain (ADG) of piglets on day 0, 14, and 28 of our ndings help to better understand the mechanisms of ICA the trial. The diarrhea frequency of the piglets was also esti- mated according to a previous report.53

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0 0 Determination of short-chain fatty acids (SCFAs) GGCAGCAG-3 ) and the reverse primer 806R (5 -GGAC- TACHVGGGTWTCTAAT-30), as described in a previous study.54 The short-chain fatty acid (acetate, propionate, butyrate, and The amplied products were detected using agarose gel elec- total SCFAs) concentrations in colonic digesta and plasma trophoresis (2% agarose), recovered by AxyPrep DNA Gel were analyzed by using the method described in a previous Recovery Kit (Axygen Biosciences, Union City, CA, United report.47 Briey, approximately 1.5 g of colonic digesta was States), and then quantied by Qubit 2.0 Fluorometer (Thermo rst homogenized in 1.5 mL of deionized water. The samples Fisher Scientic, Waltham, MA, United States) to pool into were centrifuged at 12 000g for 10 min. The supernatants of colonic digesta and plasma samples were acidied with 25% equimolar amounts. Amplicon libraries were sequenced on the meta-phosphoricacidata1:5ratio.Thesampleswerethen Illumina MiSeq 2500 platform (Illumina, San Diego, CA, United States) for paired-end reads of 250 bp. centrifuged at 12 000g 10 min. The supernatant (1 mL) was separated and analyzed using a gas chromatograph (GC RNA isolation, cDNA synthesis, and real-time quantitative 2010; Shimadzu, Japan) equipped with a CP-Wax 52 CB PCR columnof30.0mby0.53mminternaldiameter.SCFAswere quantied using external standard curves from 0.5 to 100 Total RNA was extracted from 100 mg of each colonic mucosa mmol mL 1 of the respective authentic organic acids (Fluka, specimen with Trizol reagent. The concentration and quality of Switzerland). the RNA were measured by NanoDrop ND-1000 Spectropho- tometer (Thermo, USA). Then, two micrograms of total RNA Determination of cytokines and oxidative stress indicators were treated with RNase-Free DNase and reverse transcribed according to the manufacturer's instructions. Two microliters One hundred mg frozen colonic mucosa specimen were of diluted cDNA (1 : 20, vol/vol) were used for real-time PCR, minced and homogenized in 1 mL of ice-cold cytoplasm RIPA which was performed in Mx3000P (Stratagene, USA). GAPDH, containing the protease inhibitor cocktail complete EDTA-free which is not aﬀected by the experimental factors, was chosen as (Roche, Penzberg, Germany). The homogenates were centri- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. the reference gene. All the primers that were used in this study fuged at 12 000g for 15 min at 4 C and then the supernatant are listed in Table S1† and were synthesized by Generay was collected. Protein concentration was determined using DD Company (Shanghai, China). The method of 2 Ct was used to a BCA Protein Assay kit (Pierce, Rockford, IL, USA) and then analyze the real-time PCR results and gene mRNA levels were diluted to the same concentration for subsequent analysis. expressed as the fold change relative to the mean value of the The plasma and colonic interleukin (IL)-1b,-6,-8,tumor control group. necrosis factor (TNF)-a, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and Protein expression analysis by western blotting catalase (CAT) were determined by using porcine enzyme-

This article is licensed under a linked immunosorbent assay kits (Shanghai Enzyme-linked Colonic tissue was minced and homogenized in 1 mL of ice-cold ﬀ Biotechnology Co. Ltd, Shanghai, China), according to the homogenization bu er RIPA containing the completely EDTA- manufacturer's instructions. free protease inhibitor cocktail (Roche, Penz-berg, Germany). The homogenates were centrifuged at 12 000 rpm for 20 min at

Open Access Article. Published on 20 November 2019. Downloaded 9/28/2021 8:28:15 PM. 4 C, and the supernatant fraction was collected. Protein Determination of intestinal permeability indicators concentration was determined using a BCA Protein Assay kit One hundred mg frozen colonic mucosa specimen were minced (Pierce, Rockford, IL, USA). Protein extract from each sample and homogenized in 1 mL of ice-cold cytoplasm RIPA contain- amounting to 80 mg was then loaded onto 7.5% and 15% SDS- ing the completely EDTA-free protease inhibitor cocktail PAGE gels, and the separated proteins were transferred onto (Roche, Penzberg, Germany). The homogenates were centri- nitrocellulose membranes (Bio Trace, Pall Co, USA). Post- fuged at 12 000 g for 15 min at 4 C and then the supernatant transfer, the membranes were blocked for 2 h at room temper- was collected. Protein concentration was determined using ature in a blocking buffer and then incubated with the following a BCA Protein Assay kit (Pierce, Rockford, IL, USA) and then primary antibodies: Claudin-1, Occludin, ZO-1, and GAPDH in diluted to the same concentration for subsequent analysis. The a dilution buffer overnight at 4 C. Aer several washes in Tris- plasma and colonic endotoxin, diamine peroxidase (DAO), D- Buffered-Saline with Tween (TBST), the membranes were incu- lactate, and zonulin were determined by using porcine enzyme- bated with horseradish peroxidase (HRP)-conjugated secondary linked immunosorbent assay kits (Shanghai Enzyme-linked antibodies in a dilution buffer for 2 h at room temperature. Biotechnology Co. Ltd, Shanghai, China), according to the Finally, the blot was washed and detected by enhanced chem- manufacturer's instructions. iluminescence (ECL) using the LumiGlo substrate (Super Signal West Pico Trial Kit, Pierce, USA), the signals were recorded with DNA extraction, 16S rRNA gene amplication, and sequencing the help of an imaging system (Bio-Rad, USA), and analyzed using  Total DNA was extracted from 200 mg of each colonic digesta Quantity One so ware (Bio-Rad, USA). specimen by using the QIAamp R Fast DNA Stool Mini Kit (Qiagen Ltd., Germany) in accordance with the manufacturer's Analysis of sequencing data instructions. The V3–V4 region of the 16S rRNA gene was The raw paired-end reads were assembled into longer amplied with universal primers 341F (50-ACTCCTACGGGA sequences and quality ltered by PANDAseq (version 2.9) to

37954 | RSC Adv.,2019,9, 37947–37956 This journal is © The Royal Society of Chemistry 2019 View Article Online Paper RSC Advances

remove the low-quality reads with a length of <220 nucleotides 2 F. Sanchez de Medina, I. Romero-Calvo, C. Mascaraque and (nt) or >500 nt, an average quality score of <20, and sequences O. Martinez-Augustin, Inammatory Bowel Dis., 2014, 20, containing >3 nitrogenous bases.55 The high-quality sequences 2394–2404. were clustered into OTUs with a 97% similarity using UPARSE 3 L. W. Peterson and D. Artis, Nat. Rev. Immunol., 2014, 14, (version 7.0)56 in QIIME (version 1.8)57 and the chimeric 141–153. sequences were removed using UCHIME.58 Taxonomy was 4 F. E. O. Holmberg, J. Pedersen, P. Jorgensen, assigned to the OTUs using the RDP classier59 against the C. Soendergaard, K. B. Jensen and O. H. Nielsen, J. Tissue SILVA 16S-rRNA gene database,60 with a condence threshold of Eng. Regener. Med., 2018, 12, 923–935. 70%. The data were analyzed on the free online platform of 5 H. Zhu, H. Wang, S. Wang, Z. Tu, L. Zhang, X. Wang, Y. Hou, Majorbio I-Sanger Cloud Platform (www.i-sanger.com). C. Wang, J. Chen and Y. Liu, Mol. Nutr. Food Res., 2018, 62, e1700814. Statistical analysis 6 G. Bevivino and G. Monteleone, Expert Rev. Gastroenterol. – Data are presented as mean SEM. Statistical signicance was Hepatol., 2018, 12, 907 915. assessed by the independent sample t-test using SPSS (SPSS v. 7 P. Sheth, S. Basuroy, C. Li, A. P. Naren and R. K. Rao, J. Biol. – 20.0, SPSS Inc., Chicago, IL, USA) soware packages. The data Chem., 2003, 278, 49239 49245. were considered statistically signicant when P < 0.05. The 8 J. R. Marchesi, D. H. Adams, F. Fava, G. D. Hermes,  number of replicates used for statistics are noted in the Figures. G. M. Hirsch eld, G. Hold, M. N. Quraishi, J. Kinross, H. Smidt, K. M. Tuohy, L. V. Thomas, E. G. Zoetendal and A. Hart, Gut, 2016, 65, 330–339. Authors' contributions 9 G. Tomasello, M. Mazzola, A. Leone, E. Sinagra, G. Zummo, W. X. designed the research. W. X. and H. M. conducted the F. Farina, P. Damiani, F. Cappello, A. Gerges Geagea, research. Z. Z., M. J., J. W., and Y. X. analyzed the data. The A. Jurjus, T. Bou Assi, M. Messina and F. Carini, Biomedical

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