Curcumin reduces enteric isoprostane 8-iso-PGF2α and prostaglandin GF2α in specific pathogen-free Leghorn chickens challenged with Eimeria maxima

The purpose of this pilot study was to evaluate and determine the concentration of prostaglandin GF2α (PGF2α) and isoprostane 8‐iso‐PGF2α in plasma and intestine of specific pathogen-free (SPF) Leghorn chickens challenged with Eimeria maxima, with or without dietary supplementation of curcumin using solid‐phase microextraction and ultra‐performance liquid chromatography/tandem mass spectrometry. Eighty 1-day-old male SPF chickens were randomly allocated to one of four groups with four replicates (n = 5 chickens/replicate). Groups consisted of: (1) Control (no challenge), (2) Curcumin (no challenge), (3) Eimeria maxima (challenge), and (4) Eimeria maxima (challenge) + curcumin. At day 28 of age, all chickens in the challenge groups were orally gavaged with 40,000 sporulated E. maxima oocysts. No significant differences (P > 0.05) were observed in the groups regardless of the treatment or challenge with E. maxima. Enteric levels of both isoprostane 8‐iso‐PGF2α and PGF2α at 7 days and 9 days post-challenge were significantly increased (P < 0.01) compared to the non-challenge control chickens. Interestingly, the enteric levels of both isoprostane 8‐iso‐PGF2α and PGF2α at 7 days post-challenge were significantly reduced in chickens fed curcumin, compared to control chickens challenge with E. maxima. At 9 days post-challenge, only levels of isoprostane 8‐iso‐PGF2α in the enteric samples were significantly reduced in chickens challenged with E. maxima supplemented with curcumin, compared with E. maxima challenge chickens. No differences of isoprostane 8‐iso‐PGF2α or PGF2α were observed in plasma at both days of evaluation. Similarly, no significant differences were observed between the challenge control or chickens challenge with E. maxima and supplemented with curcumin at both times of evaluation. The results of this pilot study suggests that the antioxidant anti-inflammatory properties of curcumin reduced the oxidative damage and subsequent intestinal mucosal over-production of lipid oxidation products. Further studies to confirm and extend these results in broiler chickens are required.


Results
The evaluation of body weight and body weight gain (in grams) of specific pathogen-free Leghorn chickens without or with Eimeria maxima challenge (7 days post-challenge) are summarized in Table 1. In the present study, challenge with 40,000 sporulated oocysts of E. maxima did not affect the body weight or body weight gain of SPF Leghorn chickens. No significant differences (P > 0.05) were observed in the groups regardless of the treatment or challenge with E. maxima (Table 1). Table 2 presents the results of the evaluation of isoprostane 8-iso-PGF2α and PGF2α from jejunum and plasma in SPF chickens challenged with E. maxima at 7-and 9-days post-challenge. Enteric levels of both isoprostane 8-iso-PGF2α and PGF2α at 7 days and 9 days post-challenge were significantly increased (P < 0.01) compared to the non-challenge control chickens (Table 2; Fig. 1). Interestingly, the enteric levels of both isoprostane 8-iso-PGF2α and PGF2α at 7 days post-challenge were significantly reduced in chickens fed with curcumin compared to control chickens challenge with E. maxima. At 9 days post-challenge, only levels of isoprostane 8-iso-PGF2α in the enteric samples were significantly reduced in chickens challenged with E. maxima supplemented with curcumin, as compared with E. maxima challenge chickens. No differences of isoprostane 8-iso-PGF2α or PGF2α were observed in the plasma at both days of evaluation (Table 2; Fig. 1). Table 1. Evaluation of body weight and body weight gain (in grams) of specific pathogen-free Leghorn chickens with or without Eimeria maxima challenge (7 days post-challenge). Data expressed as mean ± standard error. P > 0.05. www.nature.com/scientificreports/ The results of the evaluation of E. maxima oocyst per gram in the feces of specific pathogen-free Leghorn chickens at 7-and 9-days post-challenge are summarized on Table 3. No significant differences were observed between the challenge control or chickens challenged with E. maxima and supplemented with curcumin at both times of evaluation (Table 3). Table 2. Evaluation of isoprostane 8-iso-PGF2α and prostaglandin GF2α from enteric (jejunum) and plasma of specific pathogen-free Leghorn chickens at 7-and 9-days post-challenge. Data expressed as mean ± standard error. a,b Different superscripts within columns and days indicate a significant difference at P < 0.01.

Prostaglandin GF2α
Isoprostane 8-iso-PGF2α   Table 3. Eimeria maxima oocyst per gram in the feces of specific pathogen-free Leghorn chickens at 7-and 9-days post-challenge. Each value represents the mean (median). a,b Values within groups columns with different superscripts differ significantly at P < 0.05.

Group 7 days post-challenge 9 days post-challenge
Non-challenge control

Discussion
Coccidiosis remains one of the most critical diseases in the poultry industry. Due to international regulations and consumer pressures, there is a need to develop alternatives for antibiotic growth promoters in animal and poultry feed. Phytogenics seem to be candidates of interest as alternatives to antibiotic growth promoters because they have been shown to control of Eimeria infections due to the association of coccidial infection with lipid peroxidation of the intestinal mucosa 31 . Other studies have confirmed the benefits of phytogenics in reducing gastrointestinal infections and increasing performance [32][33][34] . Moreover, several studies have confirmed the reduction E. maxima infection severity in broiler chickens due to curcumin's antioxidant properties [17][18][19] .
In addition to the critical job of absorbing water and nutrients, enterocytes also play an essential role in the mucosal immune response, maintaining tolerance to beneficial microbiota, and identifying luminal pathogens. The invasion of Eimeria spp. in intestinal epithelial cells is a complex process that includes several events, beginning with the excystation of sporozoites after oral ingestion of the oocysts 35,36 . As intracellular parasites, attachment and invasion of the sporozoites to the host cell is recognized by Toll-like receptors 4 and 15, involved in pathogen recognition and activation of the mucosal inflammasome IL-1/IL-18 axis, which is responsible for recruiting and activating heterophils, natural killer cells, mast cells, macrophages, and increased production of transcription factor NF-κB [37][38][39][40] . Nevertheless, sporozoites have evolved a unique molecular system fueling motility and invasion of epithelial cells through gliding motility, allowing them to rapidly invade host cells and form an intracellular parasitophorous vacuole that protects them from the intracellular hostile environment [41][42][43][44] . Within this vacuole, these Apicomplexa parasites gain precious time to continue with their multifaceted life cycle. Each phase of the sexual, asexual, intracellular, or extracellular stages of this prehistoric and remarkable parasite are associated with severe local inflammation, autophagy, apoptosis, cellular death, hemorrhages, and necrosis in the intestinal mucosa [42][43][44][45][46][47] . Hence, coccidia infections are characterized by excessive tissue damage caused by the parasite infection and chronic inflammation of the host immune response elicited against the invaders. In chickens, macrophages are the primary sources of nitric oxide, superoxide, and hydrogen peroxide as essential mediators of both innate and acquired immunity, thus increasing during coccidia infections [48][49][50][51][52] . In the present study, chickens challenged with E. maxima presented with a significant increase (P < 0.01) in enteric PGF2α at 7-and 9-days post-challenge when compared with non-challenged chickens. However, the serum levels of PGF2α remained similar in both groups. Interestingly, chickens challenged with E. maxima and supplemented with curcumin showed a significant reduction of PGF2α levels at 7 days post challenge when compared with E. maxima control chickens. PGs are produced from arachidonic acid release from phospholipids in the cellular membrane by cyclooxygenases (COXs). They are fundamental in generating inflammatory responses against pathogens 53,54 . While they have a rapid response during the acute phases of the inflammatory response, there is crosstalk with cytokines to synergistically activate NF-κB factor and induce gene expression of pro-inflammatory cytokines and more COXs, mediating positive feedback loops and consequently, chronic inflammation 55,56 .
Since the cellular components that suffer immediate damage are the lipids and proteins of the cell membrane and mitochondrial membrane by lipid peroxidation, the whole-cell physiology is then compromised. One of the cellular mechanisms to revert oxidative stress is the production of several heat shock proteins that repair damage proteins and regulate apoptosis [57][58][59] . A noteworthy result observed in this study was the significant increase in isoprostane 8-iso-PGF2α in the jejunum of chickens challenged with E. maxima at 7-and 9-days post-challenge compared to the non-challenge control chickens. Furthermore, chickens in the group supplemented with curcumin showed a significant reduction in isoprostane 8-iso-PGF2α in the jejunum of chickens challenged with E. maxima at both days of evaluation post-challenge compared to the E. maxima challenge control chickens. Excessive generation of reactive oxygen species has been implicated in a variety of pathological events. However, lipid peroxidation is the primary marker of oxidative stress in many pathological conditions, so isoprostanes are reliable evaluation biomarkers evaluate 60,61 . In contrast, F2-isoprostanes (8-Iso-PGF2α) have harmful and potent bioactivities, including vasoconstriction, platelet aggregation, and cardiac hypertrophy [62][63][64][65] . As far as we know, this is the first report of detection of 8-Iso-PGF2α following a challenge of E. maxima in the jejunum, as well as demonstrating the protective antioxidant properties of curcumin reducing the enteric levels of 8-Iso-PGF2α, despite plasma levels of 8-Iso-PGF2α remaining similar in all groups, regardless of the challenge with E. maxima. It is known that in humans, the plasma half-life of 8-Iso-PGF2α is one minute at the distribution stage and the removal stage half-life is four minutes 66 . Hence, the half-life in chicken plasma may also be short, which may be why we were not able to detect it. However, pharmacokinetic and metabolic studies evaluating earlier points as well as daily oocyst count are required to confirm and extend these results.
In summary, in the present study, SPF Leghorn chickens challenged with E. maxima showed an inflammatory response associated with a significant increase at 7 days and 9 days post challenge in enteric PGF2α. These changes were related to a significant increase of enteric 8-Iso-PGF2α and oocyst excretion at both days of evaluation, suggesting that the active disease phase was accompanied by inflammation and oxidative stress within the intestinal layer. Nevertheless, dietary supplementation of curcumin reduced the levels of PGF2α and 8-Iso-PGF2α at 7 days post challenge, and 8-Iso-PGF2α at 9 days post challenge compared with E. maxima challenged control chickens. Since polyunsaturated fatty acids and cholesterol are the principal targets of oxidative stress, lipid peroxidation end products, such as 8-Iso-PGF2α, are also a part of the pathogenesis of inflammation-related changes caused by E. maxima, confirming the role of 8-Iso-PGF2α as a sensitive biomarker of oxidative stress in chickens. The results of this pilot study suggest that the antioxidant and anti-inflammatory properties of curcumin are able to reduce oxidative damage and subsequently intestinal mucosal over-production of lipid oxidation products. Further studies to confirm and extend these results in broiler chickens are required. www.nature.com/scientificreports/ 28 of age, all chickens in the challenge groups were orally gavaged with 40,000 sporulated E. maxima oocysts in a volume of 1 mL of sterile phosphate-buffered saline solution (PBS). The dose used in the present study did not cause clinical coccidiosis in SPF Leghorn chickens. The dose was selected based on a previous trial conducted to determine a challenge dose causing sub-clinical coccidiosis as described previously 13 . Negative control chickens were sham inoculated with 1 mL of PBS. Seven days after challenge, all chickens were bled, and half of them were euthanized to collect the second half of the jejunum to determine plasma and enteric concentrations of isoprostane 8-iso-PGF2α and PGF2α. At 9 days post-challenge, remaining chickens from all groups were bled and jejunum was collected to perform the evaluations. Oocysts per gram (OPG) of feces were evaluated at 7-and 9-days post-challenge.
The standards for 8-iso-PGF2α and 8-iso-PGF2α-d4. The standards for 8-iso-PGF2α and 8-iso-PGF2α-d4 (internal standard) were purchased from Cayman Chemicals (Ann Arbor, MI), while the standard for PGF2α was obtained from Sigma-Aldrich (St Louis, MO). Acetonitrile and methanol (HPLC grade) were purchased from JT Baker. Milli-Q water (Millipore system) was used throughout the experiments. Formic acid (FA: 95%, reactive grade) and isopropanol (LC/MS grade) were purchased from Sigma-Aldrich (St Louis, MO). Ammonium hydroxide (NH 4 OH, reactive grade, 29.60%) and potassium hydroxide (KOH) were purchased from JT Baker. For solid-phase microextraction (micro-SPE), 96-well Oasis® MAX μElution cartridges containing a water-wettable reversed-phase strong ammonium exchange mixed-mode polymer, which is selective for acids and stable in organic eluents, were used. A Positive Pressure-96 processor purchased from Waters was also used. Figure 1 shows the chromatograms of standards.

Procedure for the extraction of 8-iso-PGF2α and PGF2α in chicken plasma. Extraction of 8-iso-
PGF2α and PGF2α were determined as previously described 71 . An aliquot of 500 µL chicken plasma was transferred to 2 mL vials, followed by Ethics. This study was carried out in accordance with the guidelines for the management of chickens as recommended by the Internal Committee for Care and Use of Experimental Animals (CICUAE, from its abbreviation in Spanish) of the National Autonomous University of Mexico (UNAM), Ethical approval code CICUAE: C20_06, and the study is in compliance with the ARRIVE guidelines where animals are involved.
Quantification of oocysts. The quantification of OPG from feces was performed at 7-and 9-days postchallenge by using the McMaster technique as previously described 67 .
Data and statistical analysis. PGF2α and 8-iso-PGF2α data are presented as means with standard deviation (S.D.). The number of samples per variable group was 20, implying a normal distribution (Shapiro-Wilk test), and the homoscedasticity was verified (Levene's test). Accordingly, the parametric test of analysis of variance (ANOVA) was performed, and the differences between the means were evaluated using Tukey's honestly significant difference (HSD) test, and the P value was established with an alpha level of P < 0.01. OPG data are presented as means with median. The number of samples per variable group was 20; however, the hypotheses of normal distribution (Shapiro-Wilk test) and homoscedasticity (Levene's test) were not confirmed. Consequently, non-parametric tests of non-parametric tests of the two-tailed Kruskal-Wallis was applied and subsequently the Mann-Whitney's U test to compare between pairs of groups was applied with an alpha level P < 0.05 72 .