Puerarin enhances intestinal function in piglets infected with porcine epidemic diarrhea virus

Puerarin has been reported to be an excellent antioxidant, anti-inflammatory and antimicrobial agent, but the potential effect of puerarin on porcine epidemic diarrhea virus (PEDV) is unclear. This study aimed to determine whether puerarin could alleviate intestinal injury in piglets infected with PEDV. A PEDV (Yunnan province strain) infection model was applied to 7-day-old piglets at 104.5 TCID50 (50% tissue culture infectious dose). Piglets were orally administered with puerarin at the dosage of 0.5 mg/kg body weight from day 5 to day 9. On day 9 of the trial, piglets were inoculated orally with PEDV. Three days later, jugular vein blood and intestinal samples were collected. Results showed puerarin reduced morbidity of piglets infected with PEDV. In addition, puerarin reduced the activities of aspartate aminotransferase and alkaline phosphatase, the ratio of serum aspartate aminotransferase to serum alanine aminotransferase, the number of white blood cells and neutrophils, and the plasma concentrations of interleukin-6, interleukin-8 and tumor necrosis factor-α, as well as protein abundances of heat shock protein-70 in PEDV-infected piglets. Moreover, puerarin increased D-xylose concentration but decreased intestinal fatty acid-binding protein concentration and diamine oxidase activity in the plasma of piglets infected with PEDV. Puerarin increased the activities of total superoxide dismutase, glutathione peroxidase and catalase, while decreasing the activities of myeloperoxidase and concentration of hydrogen peroxide in both the intestine and plasma of PEDV-infected piglets. Puerarin decreased mRNA levels of glutathione S-transferase omega 2 but increased the levels of nuclear factor erythroid 2-related factor 2. Furthermore, puerarin increased the abundance of total eubacteria (16S rRNA), Enterococcus genus, Lactobacillus genus and Enterobacteriaceae family in the intestine, but reduced the abundance of Clostridium coccoides in the caecum. These data indicate puerarin improved intestinal function in piglets infected by PEDV and may be a promising supplement for the prevention of PEDV infection.

Plasma biochemical parameters and blood cell counts. Data on plasma biochemical parameters and blood cell counts are summarized in Table 1. Compared with non-infected pigs, PEDV-infected pigs had higher activities of aspartate aminotransferase (AST) and alkaline phosphatase (ALP), total cholesterol (CHOL) level, and AST/ALT ratio, but lower alanine aminotransferase (ALT) activity and levels of total bilirubin (TBIL), triglyceride (TG), and urea in the plasma (P < 0.05). However, PR-administered piglets exhibited lower activities of AST and ALP and lower concentrations of CHOL in plasma, but higher activity of ALT and higher concentrations of TBIL, TG and urea in plasma, compared with PEDV-infected pigs without receiving PR (P < 0.05). PEDV increased the numbers of white blood cells (WBC), neutrophils (NEU), lymphocytes (LYM) and red blood cells (RBC), while decreasing the numbers of monocytes (MONO), monocyte ratio (MONOR), mean platelet volume (MPV), and platelet distribution width ratio (PDWR) in the blood (P < 0.05). In addition, PR administration reduced the numbers of WBC, NEU and RBC, but elevated the numbers of MONO, MONOR, MPV and PDWR (P < 0.05) in comparison with PEDV-infected pigs.

DAO activity, D-xylose and I-FABP concentrations in plasma.
Data on blood DAO activity, D-xylose and I-FABP concentrations are summarized in Fig. 2. PEDV infection decreased D-xylose concentration in plasma, but increased I-FABP concentration and DAO activity in plasma, when compared with the control (P < 0.05). On the contrary, PR administration to PEDV-infected piglets increased D-xylose concentration in plasma, but decreased I-FABP concentration (P < 0.05).
Anti-oxidative enzymes and oxidation-relevant products in plasma and intestinal mucosae. Data on activities of T-SOD, GSH-PX, CAT and MPO, as well as the concentrations of MDA and H 2 O 2 are summarized in Fig. 3. Compared with the control, PEDV infection reduced the activities of CAT in the plasma, duodenum and colon, T-SOD in the colon, and GSH-Px in the duodenum, jejunum and colon, but increased the activities of MPO in the plasma and colon, and the concentrations of MDA in the ileum and of H 2 O 2 in the plasma and ileum (P < 0.05). However, compared with the PEDV group, the PEDV + PR group had higher activities of T-SOD in the duodenum and colon, GSH-Px in the duodenum, jejunum, ileum and colon, and CAT in the plasma and colon, but lower levels of MPO in the plasma, duodenum and colon, as well as lower levels of H 2 O 2 in the plasma and ileum (P < 0.05). Additionally, as shown in Fig. 3 www.nature.com/scientificreports/ PEDV infection increased the mRNA levels of GSTO2, but reduced the mRNA levels of nuclear factor carotenoid 2 related factor 2 (Nrf2). In contrast, PR administration decreased the mRNA levels of GSTO2, but increased the mRNA levels of Nrf2.

Concentration of cytokines in plasma. PEDV infection increased the concentrations of IL-6, IL-8, and
TNF-α in plasma, as compared with the control (P < 0.05). However, PR intervention reduced the concentrations of IL-6, IL-8, and TNF-α in the plasma of the PEDV-infected piglets (P < 0.05). The results are shown in Fig. 4.
HSP70 and villin protein abundances. PEDV infection significantly increased the abundance of the HSP70 protein, but decreased the abundance of the villin protein, compared to the control (P < 0.05). However, PR administration decreased the abundance of the HSP70 protein (P < 0.05) in comparison with the PEDV group (Fig. 5).

Abundance of selected intestinal bacteria.
Compared with the control, PEDV infection reduced the number of Enterococcus genus, but increased the number of Lactobacillus genus in the jejunum, ileum, caecum and colon. Moreover, PEDV infection reduced the abundances of total eubacteria in the jejunum and ileum, Clostridium coccoides and Enterobacteriaceae family in the jejunum, while increasing the abundances of total bacteria in the caecum and colon, Clostridium coccoides in caecum and Enterobacteriaceae family in the caecum and colon (P < 0.05). Compared with the PEDV group, PR administration increased the abundances of Enterococcus genus in the jejunum and caecum, Lactobacillus genus in the jejunum, ileum and caecum, total bacteria www.nature.com/scientificreports/ in the jejunum and ileum, Enterobacteriaceae family in the caecum, but reduced the abundance of Lactobacillus genus and total eubacteria in the colon, and Clostridium coccoides in the caecum (P < 0.05) (Fig. 6).

Discussion
PEDV infection is a significant challenge to swine production worldwide [9][10][11] . Its clinical symptoms include diarrhea and vomiting, as well as reductions in body weight. Results of our study indicated that PR intervention reduced morbidity in infected piglets, as compared with the PEDV group without PR administration. As an antioxidant phytochemical, PR could be used as herbal medicine to inhibit intestinal inflammation and improve intestinal function. This can be translated into sustaining global pig production.   www.nature.com/scientificreports/ The immune system status of animals could be evaluated by the numbers of white blood cells, including neutrophils, lymphocytes, and monocytes 19,20 . These cells play a key role in the process of inflammation and tissue damage. Recruitment or extravasation of leukocytes is their migration toward an area of inflammation, injury, or infection 21 . There are pieces of evidence that blood lymphocytes are significantly elevated after PEDV infection or vaccination 22,23 . Infiltration of lymphocytes, eosinophils, neutrophils and other mononuclear cells were found in the lamina propria of the small intestine after PEDV infection 24 . In good agreement with these studies, our results demonstrated that the numbers of blood WBC, NEU and LYM significantly increased in piglets after PEDV infection. Monocytes are a subset of circulating white blood cells that can further differentiate into tissue macrophages and dendritic cells (DCs) 25 . Recruitment of monocytes is essential for mediating the host antimicrobial defense and is also implicated in many inflammatory diseases 26 . PEDV infection of neonatal  www.nature.com/scientificreports/ piglets triggered a strong and rapid induction in type I interferon 27 . DC is known to secret cytokines such as type I interferon, IL-12, IL-10, and chemokine to regulate the subsequent immune responses 28 . In the present study, the decrease of blood monocytes in piglets after acute PEDV infection may be that monocytes are rapidly recruited to the intestinal injury site and differentiated into dendritic cells to participate in the immune response. More studies are needed to test this hypothesis. One study has shown the upregulation of genes for IL-1β, IL-6, IL-8, and TNF-α in IPEC-J2 cells infected with PEDV 29 . Consistently, we previously reported that the mRNA levels of related inflammatory factors in Vero cells and the levels of inflammatory factors in the ileal tissue are elevated in PEDV-challenged piglets 8 . However, blood circulation is vital in the development of systemic inflammation.
In the current study, the concentrations of pro-inflammatory cytokines, such as IL-6, IL-8, and TNF-α, were increased in the plasma of PEDV-infected pigs. Taken it together, these findings indicated that PEDV infection induced systemic inflammation in piglets. Notably, we found that PR administration significantly decreased the numbers of immune cells and the concentrations of blood pro-inflammatory cytokines in PEDV-infected piglets, thereby potentially mitigating inflammation and tissue injury in the animals. Our results are consistent with those that PR exerted protective effects against neutrophil infiltration in rats 30 , decreased neutrophil adhesion in cultured endothelial cells 31 , and prevented LPS-induced acute injury in mice 32 and rats 33 . Therefore, PR may attenuate systemic inflammation by suppressing the production of pro-inflammatory mediators. www.nature.com/scientificreports/ Another indicator of tissue damage is the levels of enzymes in plasma. For example, abnormal hepatic biochemical parameters in patients with inflammatory bowel disease (IBD) are associated with increased activities of AST, ALT and ALP in plasma 34 . In the present study, the decreases in plasma AST and ALP levels after PR intervention provided further evidence for the anti-inflammatory effect of PR. Additionally, AST, ALT, AST/ALT ratio, and ALP in plasma are good indicators of liver function [35][36][37] . Furthermore, one study found that PEDV-induced up-regulated proteins were involved in 22 diseases and disorders, such as hepatic diseases, gastrointestinal diseases, and inflammatory responses 9 . It appears that PR could attenuate the PEDV-induced liver dysfunction because PR administration attenuated the rise in plasma AST, AST/ALT ratio and ALP in PEDV-infected piglets. The underlying mechanisms are largely unknown and warrant to be elucidated. PEDV damage the pig intestinal epithelium, leading to reduced integrity and function of the mucosal barrier 38 . Intestinal mucosal barrier function can be commonly assessed by several indicators, including D-xylose concentration and DAO activity in plasma 39,40 . DAO is a highly active intracellular enzyme produced by the intestinal epithelial cells and only presents in the intestinal mucosa and ciliated cells 41 . In the case of malabsorption, the entry of D-xylose from the intestinal cavity to the portal vein is destructed, thereby reducing the concentration of D-xylose in the blood. Therefore, plasma D-xylose level and DAO activity can be used as indicators of intestinal absorption function and integrity 42 . Additionally, among the FABP family proteins, I-FABP is particularly abundant in epithelial cells of the small intestinal mucosa and it is rapidly released into the circulation when the small intestinal mucosa is impaired. Therefore, I-FABP could be used as a tissue-specific injury marker 43,44 . In our previous study, histologic lesions were present in the jejunum and ileum of PEDV-infected piglets, including severe villous atrophy 8 . In the present study, PEDV decreased plasma D-xylose and I-FABP concentrations, while increasing DAO activity in the plasma, indicating that PEDV induced intestinal dysfunction. Importantly, PR administration enhanced intestinal function, as indicated by an increase in the plasma concentrations of D-xylose and I-FABP and a decrease in plasma DAO activity. Previous studies also reported that PR could improve intestinal mucosal barrier function in mice 45,46 . Furthermore, we found that PR administration attenuated the PEDV-induced decrease in the expression of villin, a marker of villus cell differentiation in ileal mucosae 47,48 . Based on molecular level, detection indicators, the current results further support our previous conclusion. Taken together, these data indicated that PR could alleviate intestinal injury and improve intestinal function in PEDV-infected piglets.
Hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) levels in the intestinal mucosa and plasma are important indicators of oxidative stress 49 . Neutrophil myeloperoxidase (MPO) is a rich granule enzyme that catalyzes the production of potent ROS, the latter serves as a biomarker for oxidative damage and is raised in patients with inflammatory bowel disease (IBD) 50 . Glutathione S-transferase is also an important biomarker for inflammation and oxidative stress 51 . GSTO2 plays a protective role in counteracting oxidative stress 52 . In the present study, the levels of MDA, H 2 O 2, MPO and GSTO2 were elevated but CAT, T-SOD and GSH-Px activities in the plasma and intestine were decreased in PEDV-infected pigs. These results were consistent with our previous report that supplementation with N-acetylcysteine (an antioxidant) alleviated intestinal injury in piglets infected by PEDV 53 . In addition, the expression of HSP70, one of the most important cellular defense mechanisms, is induced under stressful conditions such as infection 54 . Interestingly, HSP70, GSH-Px and total antioxidant capacity are significantly up-regulated in pigs and rats after PEDV infection [55][56][57] . A study found that Nrf2 may increase the expression of antioxidant related genes by acting on the antioxidant response element (ARE) in the gene promoter, therefore control abnormal oxidative stress 58 . In the present study, PR increased the mRNA levels of Nrf2 under PEDV infection. The result was similar to previous studies demonstrating that Nrf2 is a key regulator on puerarin preventing oxidative stress damage in rodents [59][60][61] . Collectively, our results indicated that PR could effectively improve the redox status and alleviate oxidative damage in piglets infected with PEDV.
The intestinal microbiota is important for gastrointestinal function and health. Dynamic changes in the gut microbiota of piglets may contribute to decreases in morbidity and mortality in the older PEDV-infected piglets 62 . In the current study, although Enterobacteriaceae family and Clostridium coccoides were reduced in the jejunum, they were increased in the caecum and colon after PEDV infection. Enterobacteriaceae family is commonly present in the gastrointestinal tract and represents a group of potentially pathogenic microflora 63 . One study observed a statistically significant predominance of Clostridium_Sensu_Stricto_1 in 2-week-old piglets after PEDV infection 64 . Enterococcus genus, which has been studied as a probiotic 65 , was reduced in the jejunum, ileum and cecum after PEDV infection. Lactobacillus genus is commonly investigated as a probiotic agent 66 . L. johnsonii, one of the lactobacilli, has been shown to enhance the resistance of cells to E. coli challenge and can be used as a probiotic to handle the problem of piglet diarrhea 67,68 . However, another study found Lactobacillus was elevated in children diagnosed with irritable bowel syndrome 69 . It is still unclear whether the increase of Lactobacillus in the intestine could protect animals against PEDV. Another study found that total bacteria in the small intestine was reduced after transmissible gastroenteritis virus (TGEV) infection in swine 70 , which was consistent with our results. However, the increase of total eubacteria in the large intestine may be attributed to the increase of Lactobacillus genus, Enterobacteriaceae family, and Clostridium coccoides. Consistent with this notion, PR can be used as a preventive treatment for Clostridium difficile-associated diarrhea in a mouse model 71 . A lower abundance of Clostridium coccoides was detected in the caecum, while a higher abundance of the Enterococcus genus in the jejunum and caecum after PR intervention. Intriguingly, there is a significant increase in the number of Lactobacillus genus in the PEDV + PR group as compared with the PEDV group and, even to a greater extent with the control group. Collectively, PR intervention could maintain the balance of intestinal microflora and increase the number of beneficial bacteria. Considering the reduced morbidity, the improvements of intestinal mucosal barrier function indicators and anti-oxidative function, we believe that the changes in the abundance of selected microorganisms in the gut in response to PR intervention are beneficial for intestinal health in PEDV-infected piglets. Because the digestion, absorption and metabolism of nutrients are crucial for animal growth and survival 72 , regulating the intestinal bacteria may be an alternative measure to prevent or treat PED. Further researches are necessary to identify specific changes in the intestinal flora at the www.nature.com/scientificreports/ species level, especially the Lactobacillus genus. This is the first time to report the effect of PR on anti-oxidation and some specific bacteria in the intestine of PEDV-infected piglets.

Conclusion
Oral administration with PR reduced morbidity in piglets infected with PEDV. The beneficial effects of PR on intestinal function were associated with the following: (1)

Animals and treatments.
Twenty-four crossbred (Duroc × Landrace × Large White) 7-day-old healthy piglets, half male and half female with similar body weight (3.17 ± 0.51 kg), were randomly divided into one of three groups (Control, PEDV, and PEDV + PR) with 8 replicates per group. All pigs were purchased from a PEDV-free farm. These piglets were not vaccinated with a PEDV vaccine. The experiment lasted for 12 days. During day 5 to day 9 of the trial, piglets in the PEDV + PR group were orally administered with PR at the dosage of 0.5 mg/kg body weight (dissolved in a liquid milk replacer), whereas the other two groups were given the same volume of liquid milk replacer. The dose of PR was chosen according to our preliminary study that PR (0.5 mg/kg BW) could exert antiviral and anti-inflammatory effects in piglets infected with PEDV 8 . On day 9 of the trial, piglets in the PEDV and PEDV + PR groups were orally inoculated with 3.3 mL of PEDV solution at 10 4.5 TCID 50 (50% tissue culture infectious dose) per pig, while those in the control group were orally inoculated with an equal volume of sterile saline. D-xylose (0.1 g / kg body weight) was orally administered to all pigs on day 12 of the trial. One hour later, blood samples were obtained from the jugular vein, and thereafter all piglets were weighed and sacrificed by injection of sodium pentobarbital (50 mg/kg Body weight) to collect intestinal tissues and chyme as described previously 53,73 . Briefly, the intestine was dissected free of the mesentery tissue, and segments were obtained from the distal duodenum, mid-jejunum, mid-ileum, mid-caecum, and mid-colon, respectively. Intestinal contents were collected carefully and then intestine was flushed with ice-cold PBS. The mucosa was collected through scraping using a sterile glass microscope slide. All the sampling procedures were conducted at a chilled glass plate. All samples were stored at -80 ℃ until further analysis. The experimental basic feed (liquid milk substitute) formulated to meet all the nutrients required by suckling pigs was purchased from Wuhan Anyou Feed Co., Ltd. (Wuhan, China). Piglets were housed in clean pens with the strict prevention of cross-infection. The control group was quarantined from the infection groups and in different rooms. Piglets were observed daily and weighed to analyze the health status (body weight, morbidity). For the assessment of morbidity, clinical signs were recorded in all piglets, including diarrhea (defined as any loose or soft stool observed during defecation or seen on the piglets), dehydration (defined as sunken eyes and the appearance of wrinkled skin), and vomiting 74 . The specific evaluator was blind to the experimental design and objectively registered the signs. The animal use protocol for the present study was approved by the Animal Care and Use Committee of Wuhan Polytechnic University (Index number: 011043145-029-2013-000,009).

Plasma biochemical parameters and blood cell counts.
The method was presented in the previous study 53 . In short, one hour after oral administration of D-xylose on day 12 of the experiment, blood samples were collected from the anterior vena cava into heparinized vacuum tubes (Becton-Dickinson Vacutainer System, Franklin Lake, NJ, USA) and then centrifuged at 3000 rpm for 10 min at 4 ℃ to obtain plasma. Plasma biochemical parameters were measured according to the manufacturer's instructions by a Hitachi 7060 Automatic Biochemical Analyzer (Hitachi, Japan) 75 . Blood cell counts were performed on the Siemens ADVIA 2120i Hematology Analyzer (Siemens Healthcare Diagnostics, Deerfield, Illinois, USA).

Determination of D-xylose and diamine oxidase (DAO) in plasma.
The kit for detecting D-xylose in plasma was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), and operated following the procedure described previously 76 . 50 μL of the plasma was added to 5 mL of the phloroglucinol color reagent solution (Sigma Chemical Inc., St. Louis, MO, USA), then heated at 100 ℃ for 4 min in a water bath. After the samples were cooled to room temperature, the absorbance of the mixed solution at 554 nm was measured using a SpectraMax i3x Multi-Mode Detection Platform (Molecular Devices, LLC, Sunnyvale, CA, USA). The standard solution of 0 mmol/L D-xylose was considered as the blank. In addition, DAO activities in plasma were determined by using spectrophotometry according to the manufacturer's instructions 77 . The assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Western blot analysis. Western blot was performed according to the previous method 78 . In brief, proteins were extracted from intestinal mucosae and the concentrations were determined by using the bicinchoninic acid assay (Thermo Scientific, USA). Equivalent quantities of proteins from the independent biological replicates were denatured in 5 × sample loading buffer by heating at 100 ℃ for 5 min and separated by 10% SDS-PAGE. Separated proteins were then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane and blocked with 5% w/v skim milk in Tris-buffered saline containing Tween 20 (TBST) for 1.5 h at room temperature. Membranes were incubated with primary antibodies at 4 ℃ overnight: heat shock protein 70 (HSP70, Enzo, 1: 1000), villin (Santa, 1: 1000). After washed three times with TBST, membranes were incubated with the anti-rabbit (mouse) immunoglobulin G horseradish peroxidase conjugated secondary antibody (Beijing Zhong-Shan Golden Bridge Biological Technology Co. Ltd, Beijing, China; 1:5000 dilution). After being washed with TBST, blots were detected by enhanced chemiluminescence Western blotting kit (ECL-plus, Amersham Biosciences, Sweden). β-actin (Invitrogen, 1:4000) was determined as an internal reference.  79 . Ribosomal protein L4 (RPL4) was used as the reference gene in the ileum. Genomic DNA was extracted from chyme by using QIAamp Fast DNA Stool Mini Kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions and the concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The ddPCR was conducted in a QX200 Droplet Digital PCR system (Bio-Rad) as described previously 80 . In short, the final volume of each assay mixture was 20 μL, which contained 10 μL of 2x ddPCR supermix,100 nM primers and 4 μL of extracted DNA. Then the QX200 Droplet Generator (Bio-Rad) was used to generate the droplets according to the manufacturer's instructions. PCR amplification was performed using the following conditions: 1 cycle of 95 ˚C for 10 min, 40 cycles of 94 ˚C for 30 s, 55 ˚C for 1 min, and 98 ˚C for 10 min. The droplets were quantified by QX200 Droplet Reader (Bio-Rad). Since no standard curve was required for the ddPCR 81 , therefore, the fluorescent signal events above the threshold line were analyzed by QuantaSoft Software (Bio-Rad). According to preliminary experiments, the threshold of 10,000 was selected to separate positive and negative droplets. The universal primers were used to analyze total eubacteria. Specific 16S rRNA genes were targeted for these selected bacteria (Enterococcus genus, Enterobacteriaceae family, Clostridium coccoides and Lactobacillus genus). The relative abundance levels of genes in the Table 2. The sequences of primers used in the present study. RPL4 ribosomal protein L4. GSTO2 glutathione S-transferase omega 2. Nrf2 nuclear factor erythroid 2-related factor 2. RPL4  F:5′-GGA AAC CGT CGC GAGA-3'   8   R:5′-GCC CCA GAG ACA GTT-3'   GSTO2  F:5′-GCC TTG AGA TGT GGG AGA GAA-3'   82   R:5′-AAG ATG GTG TTC TGA TAG CCA AGA -3'   Nrf2  F:5′-ATC ACC TCT TCT GCA CCG AA-3'  Present study  R:5′-GCT TTC TCC CGC TCT TTC TG-3 www.nature.com/scientificreports/ treatment groups were normalized to the control group to determine the differences among the groups, as we described previously 73 . Primers used in the present study are listed in Table 2.

Gene name Sequence References
Statistical analysis. Data were reported as means with SD and were analyzed by one-way ANOVA in the SPSS 17.0 statistical software (SPSS Inc. Chicago, USA). The data of the morbidity was analyzed by χ2 analysis and expressed as a percentage. Following the 2 −ΔΔCt method, the mean value of the ileal gene expression of piglets in the control group was set to 1.00. The mean value of the relative abundance of bacteria in the control group was also set to 1.00. Multiple comparisons of means by the Duncan test was performed when the difference is significant. P-values ≤ 0.05 were taken to indicate statistical significance.
Ethics statement. All animal works were conducted according to the guidelines for the care and use of experimental animals approved by the Animal Care and Use Committee of Wuhan Polytechnic University (Index No.: 011043145-029-2013-000009) and the ARRIVE guidelines.

Data availability
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