EFFECT OF PHYTIC ACID FROM RICE AND CORN ON MORPHOLOGY , CELL PROLIFERATION , APOPTOSIS AND CYCLOOXYGENASE-2 EXPRESSION IN SWINE JEJUNAL EXPLANTS

Phytic acid (IP6) is a potent antioxidant present in several natural foods. Beneficial effects on colon cancer and inflammation have been associated to IP6 in several studies, however, scarce data about the effect on small intestine are available. The aim of the present study was to evaluate the effect of different doses of IP6 from rice and corn on intestinal morphology, cellular proliferation, apoptosis and cyclooxygenase-2 (Cox-2) expression using swine jejunal explants as experimental model. This report demonstrated that explants treated with 0.5 mM, 2.5 mM and 5 mM of IP6 from rice and 2.5 mM and 5 mM from corn showed higher villi height compared to control. Explants treated with 2.5 mM and 5 mM IP6 from rice exhibited a significant reduction on intestinal histological changes (villi atrophy and fusion, edema, lymphatic vessel dilation, loss of apical enterocytes, cell vacuolation, necrotic debris, morphology of enterocytes and microvilli and number of villi). The cellular proliferation decreased in the explants treated with the dosages of 2.5 mM and 5 mM from rice and a significant decrease in cell apoptosis was observed in the treatments with 2.5 mM IP6 from rice and 5 mM IP6 from corn compared to the control. The explants treated with 2.5 mM and 5 mM IP6 from rice and corn showed a significant reduction of the Cox-2 expression. Higher dosages of IP6 from rice and corn used in this experiment increased the viability and preservation of intestinal tissue as evidenced by morphological and immunohistochemical assays.

During storage, fermentation, germination, and digestion of cereals, IP6 can be dephosphorylated in lower phosphoric esters of myo-inositol (IP5, IP4, IP3, IP2 and IP) by action of endogenous phytase or by acid hydrolysis and enzymatic treatment with phytase (Burbano et al. 1995).The IP4 and IP3 play an important role in cell signalling and calcium mobilization (Shamsuddin;Elsayed;Ullah;1999).The IP6 and IP5 are present within the cells in substantial amounts (10 µM to 1 mM).These levels are much higher than any other inositol phosphates (Szwergold;Grahma;Brown 1987), inducing a high capacity to bind minerals (Sandberg;Carlsson;Svanberg 1989).
The hypothesis for IP6 antioxidant and anticarcinogenic action is inhibition of reactive oxygen species (ROS) generation as a result of a strong chelation with iron (Graf;Eaton, 1985).The mechanisms of degradation and hydrolysis of IP6 in the gastrointestinal system have been elucidated (Schlemmer et al. 2001), however, no data about the effect of IP6 on small intestine morphology are available.The interactions and pathways modulated by IP6 from different origins and a possible beneficial result in the gastrointestinal tract remain unknown.
We used the swine as experimental model based on physiological and morphological similarities with the human gastrointestinal tract, as well as in the absorption of IP6 (Schlemmer et al. 2001).The explant culture technique used in this experiment permits to evaluate tissue morphology, maintaining the complex patterns of differentiation seen in vivo (Randall;Turton;Foster;2011).In this study, we investigated the effects of IP6 from rice and corn in different doses on intestinal morphology, cellular proliferation, apoptosis and Cox-2 expression using swine jejunal explants.

Animals
Six 24-days-old crossbred (Landrace x Large White X Duroc) piglets (7.2 kg ± 0.63) were used in the present study.All animal experimentation procedures were carried out in accordance with the Ethics Committee on the use of animals (CEUA/UEL/Brazil-process n° 8022.2012.40).

Phytic acid
The phytic acid dodecasodium salt from rice (MW: 1080) and corn (MW: 1044) were purchased from Sigma -Aldrich (St.Louis, MO).The salts were dissolved in distillate water, the pH adjusted to 7.2 and the solution stored at -20°C.The range of IP6 concentration used in this study (0.5 mM, 1.0 mM, 2.5 mM and 5.0 mM) was chosen to encompass levels previously used with IPEC-1 (Intestinal Porcine Epithelial Cells) cell line (Pacheco et al. 2012).

Jejunal explants technique
The piglets were euthanized by administration of acepromazine 1% (0.1 mL/10Kg) intramuscular, sodium pentobarbital (40 mg/Kg) intravenous and subsequently KCl 15% solution intravenous.The jejunum was rapidly excised, and fragments of 5 cm length were collected and washed with buffer solution.The explants were collected using a punch of 8 mm diameter.From each animal six explants were collected for each treatment.The total number of explants from each pig was 54.The explants collected were laid in six well plates (three explants/well) with 3mL of the following treatments: control -only culture media (DMEM-Dulbecco's modified Eagle's medium, Gibco-BRL Life Technologies, Carlsbad, CA) plus penicillin/streptomycin (1.25 µL/mL, Gibco-BRL Life Technologies, Carlsbad, CA), gentamicin (10 µL/mL, Novafarma, Goiás, Brazil), fetal bovine serum (100 µL/ mL, Invitrogen, São Paulo, Brazil) and L-glutamine (0.4 µL/mL, Sigma-Aldrich, St. Louis, MO); culture media with IP6 from corn (0.5 mM, 1.0 mM, 2.5mM, 5.0 mM) and culture media with IP6 from rice (0.5 mM, 1.0 mM, 2.5mM, 5.0 mM).The explants were incubated during four hours at 37° C under orbital shaking and 5% of CO 2 .All the experimental procedures were performed in duplicated.

Histological assessment
After the incubation period, the explants were fixed in 10% neutral buffered formalin solution and routinely processed for histopathological and immunohistochemical evaluation.An intestinal histological score previously described (Bracarense et al. 2012) was adapted to compare histological changes between the treatments.The maximum score (22 points) indicates the overall integrity of the intestine.Each explant was evaluated to observe the presence and intensity of the criteria disposed on table 1.
The total score was calculated by taking into account the sum of each histological criteria.The villi height was measured randomly on 10 villi using the Motic Image Plus 2.0 software (Motic Instruments, Richmond, Canada).Sections of jejunum were submitted to Alcian blue staining to evaluate goblet cell density.Positively stained goblet cells were counted randomly in 10 villi per explant at 400x magnification.

Immunohistochemical assessment
Evaluation of cell proliferation, apoptosis and Cox-2 expression were performed using antibodies against Ki-67 (clone 7B11, 1:50 dilution, Zymed, Carlsbad, CA) and cleaved caspase-3 (CCasp3) (clone Asp 175, 1:200 dilution, Cell Signalling Technology, Beverly, MA) and Cox-2 (clone CX-294, 1:100 dilution, Dako, Glostrup, Denmark), respectively.The protocols, positive and negative controls were used according to manufacturer's instructions.The immunoexpression of Ccasp3 and Ki-67 was estimated by counting strongly positive immunostaining of cytoplasm and nucleus cells, respectively, in five random fields in crypt region /explant at 400X magnification.The expression of Cox-2 was estimated by evaluation of positive immunostaining of cytoplasm cells in five fields in crypt region/explant at 200x magnification.Fields were considered positive when it showed 75% or more positive immunostaining cells.

Statistical analysis
The experimental design used in the present study was entirely randomized with six replicates (each explant represent one replicate).The means of lesional score, intestinal morphometry, number of goblet cells and positive immunostaining for Ki-67 and Ccasp3 and the sum of the positive and negative immunostaining fields for Cox-2 were used for statistical analysis.Data were statistically analyzed by the free software R 2.11.1 using normality (Shapiro-Wilk's test) and homogeneity (Bartlett) tests.When these two assumptions were met, the analysis of variance (ANOVA) was applied, followed by Tukey's test and presented with their standard errors to Ki-67, CCasp-3.The data of Cox-2 expression were exhibited in percentage of positive and negative immunostaining.The P values of ≤ 0.05 were considered significant.

RESULTS AND DISCUSSION
After 4 hours of incubation, untreated explants presented mild to moderate multifocal atrophy and villi fusion.On the other hand, explants exposed to IP6 showed normal villi height and morphology (Figure 1A, 1B).The control explants showed a mean histological score of 16.74.A significant increase in the histological score was observed in explants treated with 2.5 mM (score= 19.00; P = 0.006) and 5.0 mM (score= 19.35; P = 0.001) of rice IP6, whereas the doses of 0.5 mM (score= 17.56) and 1.0 mM (score= 17.56) induced no difference compared to control explants.No significant difference was observed in explants treated with corn IP6 in all experimental doses when compared with untreated explants (0.5 mM score= 17.78; 1.0 mM score= 17.48; 2.5 mM score= 18.06 and 5.0 mM score= 17.51).The P value was > 0.05 (Figure 1C).
In the present study, we have investigated the effect of different doses of phytic acid from rice and corn on the intestine of piglets in order to determine if they have an effect on jejunal morphology, cell turnover and pro-inflammatory stimuli.The IP6 exerts its effects in a dose-dependent manner (Graf;Eaton, 1990) which can explain the response to greater doses of phytic acid.
In an in vitro study, IPEC-1 cells exposed to 0.5 and 1 mM of phytic acid protected cells from deoxynivalenol toxicity, whereas doses of 5 mM induced cell toxicity (Pacheco et al. 2012).We have observed that explants exposed to 2.5 mM and 5 mM of phytic acid improved jejunal morphology and the IP6 from rice was more efficient to preserve the intestinal morphology compared to IP6 from corn.We hypothesize that the different number of sodium and water molecules present in the IP6 from rice (C6H6O24P6.12Na.8H2O) and from corn (C6H6O24P6.11Na.7H2O) could interfere with the absorption of IP6 by intestinal cells reflecting the difference in morphological preservation observed in this experiment.
This improvement could be seen by a decrease in villi fusion and atrophy, as well as normal enterocytes morphology.In this study, villi were significantly higher in explants exposed to concentrations of 2.5 and 5 mM of both phytic acids.An increased villous surface implicates an augmentation in intestinal absorptive capacity (Wijtten; Van Der; Verstegen 2011).Goblet cells secrete a mucus layer that promotes the first line of defence on intestine (Kim;Ho 2010).We observed no difference in goblet cell density in explants submitted to different doses of phytic acid.Probably, the short period of incubation (four hours) was not sufficient for goblet cells migration from crypts to villi.Ciênc.Agrotec., Lavras, v.38, n. 3, p.278-285, maio./jun., 2014 Time of incubation is a limitation in the explant model.In previous studies we have observed an increase in histological changes (30%) in explants incubated for 4 hours compared to non-incubated explants (unpublished data).The explants are submitted to a relative status of hypoxia.Whenever oxygen level decrease in the surrounding environment, the mitochondria increase the levels of cytosolic reactive oxygen species (ROS) to activate hypoxia-inducible factor (HIF) with the objective to facilitate cellular adaptation to hypoxia (Bell et al. 2007).However, if the cell was submitted to persistent hypoxia as in the present study, these ROS may induce a shift in iron redox state from Fe 2+ to Fe 3+ (Fenton reaction), limiting an essential cofactor of the PHDs (prolyl hydroxylase enzymes), resulting in an inhibition of hydroxylation of HIF protein (Gerald et al. 2004).Furthermore, ROS can activate the MAPKs (mitogen-activated protein kinases) that have been associated to cell proliferation, apoptosis and regulation of Cox-2 gene (Wang et al. 1998).Cox-2 is an enzyme involved in arachidonic acid metabolism and is not expressed in most of the normal tissues, but is strongly induced by mitogenic and proinflammatory stimuli.High levels of Cox-2 have been reported in inflammatory bowel disease and colon cancer (Cianchi et al. 2006). ).In this study, we observed that the IP6, mainly from rice, decreased the cell proliferation, apoptosis and Cox-2 expression due their antioxidant potential by inhibition of ROS production and capacity of modulate the signaling intracellular to preserve the tissue viability.Ciênc. Agrotec., Lavras, v.38, n. 3, p.278-285, maio./jun., 2014 IP6 can interfere with the Fenton reaction, stabilizing the iron in the Fe 3+ (inert form) (Graf;Eaton, 1985), enabling a signalling for cellular adaptation to hypoxia, inhibiting the activation of MAPKs and preventing cellular injury, proliferation, apoptosis and Cox-2 expression by the inhibition of ROS production as observed in this experiment.The preservation of cellular morphology, increased villi height and decreased apoptosis and cell proliferation, promoted increased intestinal homeostasis and integrity, and in consequence better absorption of nutrients and defence against aggressors.

CONCLUSIONS
The IP6 from rice increased the viability and preservation of intestinal tissue by improvement on intestinal morphology, decrease of cellular proliferation, apoptosis and Cox-2 expression and the IP6 from corn increased the villi height and decreased the apoptosis and the Cox-2 expression.Nevertheless, more studies are necessary to elucidate biochemical pathways and molecular mechanisms of IP6 on the gastrointestinal tract and possible protective effects against infectious agents and xenobiotics.

ACKNOWLEDGMENTS
This study was supported by Grant n° 305274/2011-2 from CNPq, Brazil.E.O.S. and J.R.G. were supported by fellowships from CAPES.A.P.F.R.L.B. was supported by fellowship from CNPq.The authors wish to thank the Professor Dr. Caio Abércio da Silva for the supply and care of the animals and the Professor Dr. Elza Iouko Ida for helpful discussion about the antioxidant effect of phytic acid.

Figure 1 -
Figure 1 -Effect of IP6 from rice and corn on jejunum explants histology.A-Control with moderate lesions, villi atrophy (black arrow) and villi fusion (red arrows), , H&E, bar 50 µm.B-Jejunal explant incubated with 5 mM IP6 from rice showed decrease of lesions compared to control, H&E 50 µm.C-Histological score after examination according to the occurrence of lesions in explants submitted to IP6 from rice and corn .(AU-Arbitrary units).D-Villi height of explants submitted to IP6 from rice and corn.Values are mean height (µm).Values are means with standard errors of the mean represented by vertical bars (n 6 animals).Mean values with unlike letters were significantly different (P<0.05).

Figure 2 -
Figure 2 -Effect of IP6 from rice and corn on jejunum explants cellular proliferation, apoptosis and Cox-2 expression.Immunohistochemistry using the immunoperoxidase method and hematoxylin counterstain.A-Positive cell immunostaining for Ki-67 in explants submitted to 2.5mM and 5 mM from rice and corn.Values are mean positive cells per field.B-Immunostaining for CCasp3 in explants submitted to 2.5mM and 5 mM from rice and corn.Values are mean positive cells per field.Values are means with standard errors of the mean represented by vertical bars (n 6 animals).C-Immunostaining for Cox-2 in explants submitted to IP6 from rice.D. Immunostaining for Cox-2 in explants submitted to IP6 from rice.Values are expressed as percentage of positive and negative immunostaining fields represented by vertical bars (n 6 animals).Unlike letters were significantly different (P<0.05).