Chemotherapy modulates intestinal immune gene expression in piglets including surfactant protein-D and DMBT1
ABSTRACT: Cytotoxic therapy leads to marked changes in intestinal structure, function, immunity and microbiota. Information about chemotherapy-induced intestinal gene expression may provide insights into the mechanisms underlying gut toxicity, and help to identify biomarkers and targets for intervention. We investigated jejunal tissue from piglets subjected to two different, clinically-relevant chemotherapy regimens, 1) busulfan plus cyclophosphamide (BUCY) and 2) doxorubicin (DOX). Gene expression analysis identified 1328 and 594 differentially expressed genes in the BUCY and DOX pigs, compared with their respective controls. Similar changes in expression patterns across BUCY and DOX piglets were found for 137 genes (95 genes repressed and 42 induced). Selected genes of potential biological significance were confirmed by real-time polymerase chain reaction. Key innate defense molecules, including surfactant protein-D (SP-D), deleted in malignant brain tumors 1 (DMBT-1) and peptidoglycan recognition protein 2 (PGLYRP2) were among the up-regulated genes for both chemotherapy treatments. Based on the results from the two different treatments, we conclude that chemotherapy induces reduced intestinal adaptive immunity while innate immune functions involved in surveillance, protection and homeostasis of mucosal surfaces are up-regulated. The results may be of value to understand monitor and prevent chemotherapy-induced intestinal mucositis by dietary or medical interventions. Overall design: Twenty-two three day-old crossbred SPF piglets (Large White × Danish Landrace) were used for the experiments. All pigs were delivered to our research facilities after having suckled their mother from birth. The pigs were housed in individual cages and kept in an environmentally controlled room (12/12 light/dark rhythm, ~30 °C). The studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the National Committee on Animal Experimentation (approval number: 2010/561-1760). The manuscript was prepared in accordance to the ARRIVE guidelines for reporting animal research (32). The tissues used for the analyses originated from two separate studies on chemotherapy-induced GI toxicity in piglets. One experiment was based on treatment with a combination of busulfan and cyclophosphamide (BUCY) (26) while another experiment was based on a single dose of the topoisomerase II inhibitor, doxorubicin (DOX) (33). For all details regarding the clinical condition of the piglets and organ and physiological responses to chemotherapy, we refer the reader to these publications (26, 33). Prior to cytotoxic treatment pigs were anesthetized and fitted with orogastric tubes (6F Portex, Kent, UK) and a catheter (Tygon OD 0.070 ID 0.040 Saint-Gobain Performance Plastics, France) inserted in the external jugular vein for blood sampling and administration of drugs and fluids. Surgery was performed under zoletil mix anesthesia (i.m. tiletamine, 0.28 mg/kg; zolazepam, 0.28 mg/kg; xylazine, 0.56 mg/kg; ketamine, 0.56 mg/kg; butorphanol, 0.11 mg/kg). All animals were anesthetized and euthanized by an intracardial injection of sodium pentobarbital (200 mg/kg). Chemotherapeutic drugs were administered intravenously (i.v.) via the jugular vein catheter.
INSTRUMENT(S): Agilent-026440 Sus scrofa (Pig) Oligo Microarray v2 (Probe Name version)
Project description:?-Conglycinin (?-CG), an anti-nutritional factor, is a major allergen in soybeans to induce intestinal dysfunction and diarrhea in neonatal animals, including piglets and human infants. This study with a piglet model determined the effects of N-acetylcysteine (NAC) on intestinal function and autophagy in response to ?-CG challenge. Twenty-four 12-day-old piglets (3.44?±?0.28 kg), which had been weaned at 7 days of age and adapted for 5 days after weaning, were randomly allocated to the control, ?-CG, and ?-CG?+?NAC groups. Piglets in the control group were fed a liquid diet containing 10% casein, whereas those in the ?-CG and ?-CG?+?NAC groups were fed the basal liquid diets containing 9.5% casein and 0.5% ?-CG for 2 days. Thereafter, pigs in the ?-CG?+?NAC group were orally administrated with 50 mg (kg BW)<sup>-1</sup> NAC for 3 days, while pigs in the other two groups were orally administrated with the same volume of sterile saline. NAC numerically reduced diarrhea incidence (-?46.2%) and the concentrations of hydrogen peroxide and malondialdehyde, but increased claudin-1 and intestinal fatty-acid binding protein (iFABP) protein abundances and activities of catalase and glutathione peroxidase in the jejunum of ?-CG-challenged piglets. Although ?-CG challenge decreased the villus height, villus height/crypt depth ratio, and mRNA levels of claudin-1 and occludin, no significant differences were observed in these indices between the control and ?-CG?+?NAC groups, suggesting the positive effects of NAC supplementation on intestinal mucosal barrier function. Moreover, NAC increased the concentrations of citrulline and D-xylose in the plasma, as well as the expression of genes for aquaporin (AQP) 3, AQP4, peptide transporter 1 (PepT1), sodium/glucose co-transporter-1 (SGLT-1), potassium inwardly-rectifying channel, subfamily J, member 13 (KCNJ13), and solute carrier family 1 member 1 (SLC1A1) in the jejunum, demonstrating that NAC augmented intestinal metabolic activity and absorptive function. Remarkably, NAC decreased Atg5 protein abundance and the LC3II/LC3I ratio (an indicator of autophagy) in the jejunum of ?-CG-challenged piglets. Taken together, NAC supplementation improved intestinal function and attenuated intestinal autophagy in ?-CG-challenged piglets.
Project description:BACKGROUND:Chitosan was used as an alternative to promote the growth of weaned piglets. And low-molecular-weight chitosan (LC) is one of chitosan derivatives and maintain beneficial biological properties of chitoson. The present experiment was carried out to examine the effects of LC on the growth performance, intestinal morphology, barrier function, cytokine expression, and antioxidant system of weaned piglets. RESULTS:A total of 40 piglets weaned at 21 d of age, with average body weight 6.37?±?0.08 kg, were randomly assigned (5 pens/diet; 4 pigs/pen) to 2 treatments (a basal diet and the basal diet supplemented with 50 mg/kg LC) and were fed for 28 d. Compared with the control group, average daily feed intake (ADFI), and the expression of intestinal barrier protein ZO-1 was increased (P?<?0.05) when the piglets fed the diet supplemented with LC. No significant differences were found in average daily gain (ADG, P?>?0.05), gain-to-feed ratio (G:F, P?>?0.05), the incidence of diarrhea (P?>?0.05), or the antioxidant capacity (P?>?0.05) between two groups. The expression of IL-1? and TNF-? in jejunal mucosa were significantly decreased (P?<?0.05) in piglets fed the LC-supplemented diet in comparison to the control. CONCLUSION:The results of this study indicate that dietary supplementation with LC at 50 mg/kg was effective for enhancing the growth performance in weaned piglets, improving intestinal barrier function and alleviating intestinal inflammation.
Project description:The previous study in our team found that supplementation of probiotic <i>Bacillus amyloliquefaciens</i> (Ba) instead of antibiotics promote growth performance of piglets. Hence, the present study was carried out to further demonstrate the effect of Ba replacement of antibiotics on digestive and absorption enzyme activity and intestinal microbiota population of piglets. A total of 90 piglets were selected and divided into three groups: G1 group was fed with basal diet supplemented with 150 mg/Kg aureomycin, G2 group was fed with 1 × 10<sup>8</sup> cfu/Kg Ba and half dose of aureomycin, G3 group was used the diet with 2 × 10<sup>8</sup>cfu/Kg Ba replaced aureomycin. Each treatment had three replications of 10 pigs per pen. Results indicated that Ba replacement significantly increased the activities of amylase, disaccharides and Na<sup>+</sup>/K<sup>+</sup>-ATPase. And chymotrypsin activity in different section of intestine was dramatically enhanced in half replacement of aureomycin with Ba. Moreover, Ba replacement maintained the intestinal integrity with the significantly decreased activity of DAO compared with aureomycin group. Besides, supplementation with Ba increased the ?-diversity of intestinal microbiota. Taken together, the current study indicated that diet supplementation with Ba instead of aureomycin increased the growth performance of piglets by improving the digestive and absorb enzyme activities, enhancing the intestinal integrity and regulating the population of intestinal micrbiota.
Project description:In the present study, we investigated the influence of diquat-induced oxidative stress on intestinal barrier, mitochondrial function, and the level of mitophagy in piglets. Twelve male Duroc × Landrace × Yorkshire 35-d-old pigs (weaned at 21 d of age), with an average body of 9.6 kg, were allotted to two treatments of six piglets each including the challenged group and the control group. The challenged pigs were injected with 100 mg/kg bodyweight diquat and control pigs injected with 0.9% (w/v) NaCl solution. The results showed that diquat injection decreased ADFI and ADG. Diquat decreased (P < 0.05) the activities of superoxide dismutase and glutathione peroxidase and increased (P < 0.05) the malondialdehyde concentrations. The lower (P < 0.05) transepithelial electrical resistance and higher (P < 0.05) paracellular permeability of fluorescein isothiocyanatedextran 4 kDa were found in diquat challenged piglets. Meanwhile, diquat decreased (P < 0.05) the protein abundance of claudin-1, occluding, and zonula occludens-1 in jejunum compared with the control group. Diquat-induced mitochondrial dysfunction, as demonstrated by increased (P < 0.05) reactive oxygen species production and decreased (P < 0.05) membrane potential of intestinal mitochondria. Diquat-injected pigs revealed a decrease (P < 0.05) of mRNA abundance of genes related to mitochondrial biogenesis and functions, PPARg coactivator-1?, mammalian-silencing information regulator-1, nuclear respiratory factor-1, mt transcription factor A, mt single-strand DNA-binding protein, mt polymerase r, glucokinase, citrate synthase, ATP synthase, and cytochrome coxidase subunit I and V in the jejunum. Diquat induced an increase (P < 0.05) in expression of mitophagy-related proteins, phosphatase and tensin homologue deleted on chromosome 10-induced putative kinase, and Parkin in the intestinal mitochondria, as well as an enhancement of the ratio of light chain 3-II (LC3-II) to LC3-I content in the jejunal mucosa. These results suggest that oxidative stress disrupted the intestinal barrier, caused mitochondrial dysfunction, and triggered mitophagy.
Project description:This study investigated the different addition levels of iron (Fe) in growing-finishing pigs and the effect of different Fe levels on growth performance, hematological status, intestinal barrier function, and intestinal digestion. A total of 1,200 barrows and gilts ([Large White × Landrace] × Duroc) with average initial body weight (BW; 27.74 ± 0.28 kg) were housed in 40 pens of 30 pigs per pen (gilts and barrows in half), blocked by BW and gender, and fed five experimental diets (eight replicate pens per diet). The five experimental diets were control diet (basal diet with no FeSO4 supplementation), and the basal diet being supplemented with 150, 300, 450, or 600 mg/kg Fe as FeSO4 diets. The trial lasted for 100 d and was divided into the growing phase (27 to 60 kg of BW) for the first 50 d and the finishing phase (61 to 100 kg of BW) for the last 50 d. The basal diet was formulated with an Fe-free trace mineral premix and contained 203.36 mg/kg total dietary Fe in the growing phase and 216.71 mg/kg in the finishing phase based on ingredient contributions. And at the end of the experiment, eight pigs (four barrows and four gilts) were randomly selected from each treatment (selected one pig per pen) for digesta, blood, and intestinal samples collection. The results showed that the average daily feed intake (P = 0.025), average daily gain (P = 0.020), and BW (P = 0.019) increased linearly in the finishing phase of pigs fed with the diets containing Fe. On the other hand, supplementation with different Fe levels in the diet significantly increased serum iron and transferrin saturation concentrations (P < 0.05), goblet cell numbers of duodenal villous (P < 0.001), and MUC4 mRNA expression (P < 0.05). The apparent ileal digestibility (AID) of amino acids (AA) for pigs in the 450 and 600 mg/kg Fe groups was greater (P < 0.05) than for pigs in the control group. In conclusion, dietary supplementation with 450 to 600 mg/kg Fe improved the growth performance of pigs by changing hematological status and by enhancing intestinal goblet cell differentiation and AID of AA.
Project description:The aim of this study was to investigate whether supplementation with chitosan (COS) could reduce diarrhea and to explore how COS alleviates intestinal inflammation in weaned pigs. Thirty pigs (Duroc×Landrace×Yorkshire, initial BW of 5.65±0.27) weaned at age 21 d were challenged with enterotoxigenic Escherichia coli during a preliminary trial period, and then divided into three treatment groups. Pigs in individual pens were fed a corn-soybean meal diet, that contained either 0 (control), 50 mg/kg chlortetracycline, or 300 mg/kg COS for 21 days. The post-weaning diarrhea frequency, calprotectin levels and TLR4 protein expression were decreased (P<0.05) in both the COS and chlortetracycline groups compared with control. Simultaneously, supplemental COS and chlortetracycline had no effect on the mRNA expression of TNF-? in the jejunal mucosa, or on the concentrations of IL-1?, IL-6 and TNF-? in serum. However, COS supplementation improved (P<0.05) the mRNA expression of IL-1? and IL-6 in the jejunal mucosa. The results indicate that supplementation with COS at 300 mg/kg was effective for alleviating intestinal inflammation and enhancing the cell-mediated immune response. As feed additives, chitosan and chlortetracycline may influence different mechanisms for alleviating inflammation in piglets.
Project description:BACKGROUND: Zinc (Zn) supplementation has been shown to reduce the incidence of diarrhea and to protect animals from intestinal diseases, but the mechanisms of this protective effect against virus infection in vivo have not yet been elucidated. Transmissible gastroenteritis virus (TGEV) causes diarrhea in piglets with an age-dependent decrease of severity. RESULTS: We used 60 weaned piglets that were divided into three groups to evaluate the effect of different Zn levels added to a conventional diet (50 mg Zn/kg diet, Znlow, control group). The other groups received the diet supplemented with ZnO at final concentrations of 150 mg Zn/kg diet (Znmed), or 2,500 mg/kg diet (Znhigh). Oral challenge infection with TGEV was performed when the pigs had been fed for 1 week with the respective diet. Half of the piglets of each group were sacrificed at day 1 and 18 after challenge infection. Fecal consistency was improved and body weights increased in the Znhigh group when compared to the other groups, but no direct effect of Zn concentrations in the diet on fecal TGEV shedding and mucosal immune responses was detectable. However, in the Znhigh group, we found a prevention of villus atrophy and decreased caspase-3-mediated apoptosis of jejunal epithelium. Furthermore, pigs receiving high Zn diet showed a down-regulation of interferon (IFN)-?, oligoadenylate synthetase (OAS), Zn transporter SLC39A4 (ZIP4), but up-regulation of metallothionein-1 (MT1), as well as the Zn transporters SLC30A1 (ZnT1) and SLC30A5 (ZnT5). In addition, forskolin-induced chloride secretion and epithelial resistance were controlled at a physiological level in the Znhigh but not the other groups. Finally, in the Znhigh group, we documented an earlier and higher systemic TGEV-specific serum antibody response. CONCLUSIONS: These results suggest that high dietary Zn could provide enhanced protection in the intestinal tract and stimulate the systemic humoral immune response against TGEV infection.
Project description:This study aimed to investigate the effects of dietary bacteriophage supplementation on growth performance, intestinal morphology, barrier function, and intestinal microbiota of weaned piglets fed antibiotic-free diet. A total of 120 weaned piglets were allotted to four dietary treatments with five pens/treatment and six piglets/pen in a 21-d feeding trial. The control diet was supplemented with 25 mg/kg quinocetone and 11.25 mg/kg aureomycin in the basal diet, while the three treatment diets were supplemented with 200, 400, or 600 mg/kg bacteriophage in the basal diet, respectively. There was no difference for growth performance and all measured indices of serum and intestinal tissues between 200 mg/kg bacteriophage group and the control group with antibiotics (<i>P</i> > 0.05). More importantly, compared with the control diet, dietary 400 mg/kg bacteriophage inclusion increased average daily gain and average daily feed intake, and decreased feed/gain ratio and diarrhea incidence of weaned piglets (<i>P</i> < 0.05). Also, piglets fed 400 mg/kg bacteriophage had elevated villi height (VH) in jejunum and ileum, reduced crypt depth (CD) in jejunum and ileum, and elevated VH/CD ratio in duodenum, jejunum and ileum (<i>P</i> < 0.05). Compared to the control group, piglets fed 400 mg/kg bacteriophage had lower interleukin-1? (IL-1?) and tumor necrosis factor-? (TNF-?), and higher interleukin-10 (IL-10) concentration in serum, and higher secretory immunoglobulin A (sIgA), intestinal trefoil factor (ITF), and tumor growth factor-alpha (TGF-?) content in the ileal mucosa (<i>P</i> < 0.05). Besides, dietary addition with 400 mg/kg bacteriophage decreased the D-lactate concentration and diamine oxidase (DAO) activity in serum, and increased the relative mRNA expression of ZO-1, Claudin-1, Occludin, TLR2, TLR4, and TLR9, as well as the relative protein expression of Occludin in the jejunum (<i>P</i> < 0.05). However, the growth performance and all analyzed parameters in serum and intestinal tissues were not further improved when piglets fed 600 vs. 400 mg/kg bacteriophage (<i>P</i> > 0.05). MiSeq sequencing analysis showed that bacteriophage regulated the microbial composition in caecum digesta, as indicated by higher observed_species, Chao1, and ACE richness indices, as well as changes in the relative abundance of Firmicutes, Bacteroidetes, and Tenericutes (<i>P</i> < 0.05). Collectively, 400 mg/kg bacteriophage can be used as an antibiotics alternative for promoting the growth of weaned piglets. The underlying mechanism is associated with a positive effect of bacteriophage on intestinal inflammation, intestinal barrier function and gut microbiota in weaned piglets.
Project description:Vitamin A (VA) is an important nutrient for weaning piglets. It plays a significant role in the normal formation, development, and maintenance of epithelial cells. Previous studies have shown that VA supplements could improve the host's intestinal barrier function. Therefore, we hypothesized that VA supplements can affect intestinal function in weaned piglets by regulating intestinal stem cells. Thirty-two 21-d-old weaned [(Yorkshire × Landrace) × Duroc] piglets with an average weight of 8.34 ± 0.13 kg were randomly divided into 4 treatment groups, with 1) 2 mg/kg (control), 2) 4 mg/kg, 3) 8 mg/kg, and 4) 16 mg/kg doses of VA, respectively. The experiment lasted for 14 d. Weaned piglets were given ad libitum access to food and water during the test. The ADG (linear, P = 0.020) and G:F (linear, P = 0.005) of the piglets were found to increase significantly from days 8 to 14. The Lgr5+ gene expression (P = 0.012) in the jejunum mucosa of the 16 mg/kg VA group was increased. The jejunum villus height (P = 0.027) and villi surface area (P = 0.035) were significantly increased in the 4 mg/kg VA treatment group. The crypt depth increased significantly in the 4 and 8 mg/kg VA treatment groups (quadratic, P = 0.043), and the ratios of villus height to crypt depth significantly increased in the 16 mg/kg VA group (quadratic, P = 0.015). The maltase (P = 0.032), sucrose (P = 0.041), and alkaline phosphatase activity (linear, P = 0.024) were significantly increased when further supplemented with 4 mg/kg VA. Slc2a2 mRNA abundance was significantly increased in the 2 mg/kg VA group (linear, P = 0.024). Moreover, the budding rates, buddings number per organoid, and Chromogranin A and Muc2 expression of piglet intestinal organoids were significantly reduced (P < 0.05) by VA and its metabolites (retinoic acid). Compared with the control group, the expression of Spp1 and Trop2 increased. These results indicated that VA may increase the stemness of intestinal stem cell in vitro. This study suggested that VA could affect growth performance and intestinal function by regulating intestinal stem cells in the jejunum of weaned piglets.
Project description:An elevated level of serum uric acid-hyperuricemia, is strongly associated with the development of gout and chronic kidney disease (CKD) which is often accompanied by a significantly reduced glomerular filtration rate (GFR). In the present study, we investigated the extra-renal elimination of uric acid via the intestine in a healthy pig model and the effect of oral uricase therapy on plasma uric acid concentrations in pigs with induced hyperuricemia and CKD. The experiment was conducted on eleven, ten-week-old pigs (n = 11). The porcine model of CKD was developed by performing 9/10 nephrectomy surgery on eight pigs. A stable model of hyperuricemia was established in only five of the eight nephrectomized pigs by frequent injections of uric acid (UA) into the jugular vein. All pigs (three healthy pigs and five CKD pigs) were operated for implantation of jugular vein catheters and the three healthy pigs also had portal vein catheters inserted. Blood uric acid concentrations were measured spectrophotometrically, using the Uric Acid Assay Kit (BioAssay Systems, Hayward, USA). The piglets with CKD received orally administered uricase (treatment) and served as their own controls (without uricase supplementation). Oral uricase therapy significantly decreased plasma uric acid concentrations in pigs with CKD, whereas hyperuricemia was observed in the pigs whilst not being treated with uricase. Urinary uric acid excretion was similar during both the treatment and control periods during the first 8 h and 24 h after UA infusions in the CKD pigs. To demonstrate the elimination of UA via the intestine, the healthy pigs were infused with UA into the jugular vein. The blood collected from the jugular vein represents circulating UA concentrations and the blood collected from the portal vein represents the concentration of UA leaving the intestine. The final (after 2 h) concentration of UA was significantly lower in blood collected from the portal vein compared to that collected from the jugular vein (3.34 vs. 2.43 mg/dL, respectively, p = 0.024). The latter allows us to suggest that UA is eliminated from the blood via the gut tissue.