Study of Some Parameters that Play Role in Pathogenesis of Celiac Disease.
Rehab Abdul-Razzaq Abdul-Hassan Issam S. Ismail,(MPH) Alaa Irhayyim Ali
*Department of Biology, college of Science, Wasit University, Iraq
* college of medicine, Wasit University, Iraq Abstract:
Background:Coeliac disease defines as autoimmune disease induced by the effect of wheat proteins which represented by gluten and related proteins from other grains (barley or rye).when exposure to gluten in susceptible individuals, induce the activation of Th1- lymphocytes and up-regulation of interferon-gamma in genetically predisposed individuals, which lead to inflammation in the intestinal mucosa of celiac disease patients. However, a certain level of ROS under physiological conditions in addition to abundant of antioxidant capacity, the cell can tolerate tis oxidative conditions , which is critical for intestinal homeostasis, but when the excessive ROS generation or decreased reduction reactions this lead to increased oxidant load , as well as reduce the antioxidant activity lead to enhance membrane permeability, in addition to various effect such as inflammatory response and result in lipid and protein modifications, as well as,DNA damage, carcinogenesis, and apoptosis,. Aim of study: investigation some of markers to confirm the celiac disease stimulated factors.Patients and Methods: Study included 20 case CD patients with 12 healthy controls, Serum patients and controls examination in Al-Zahraa hospital, Dep.
Immunology.Results: Serum IL-12 levels in patients (-7.20) and controls (-11.23) with highly significant differences (0.034)which mean that IL-12 activated due to ROS produce, MMP-3 in patients (6.27) and controls( 11.72)with sig.0.001,compared with the levels of TIMP-3 which few increased to inhibit the MMP-3 stimulated (11.20) and in controls was (25.33)with sig.o.ooo1. due to the activate of IL-12 in patients serum ,this lead to stimulate production of IFN-gamma which was ( 19.30) in patients , but (11.83) in controls with sig.0.029. Total oxidative status which used to estimate the overall oxidation state of the body was (4.53) in patients, (8.20) in control with sig.0.009, compared with the activity of antioxidant parameter SOD which levels in patients was (12.63) and (21.33) with sig. 0.008.
Keywords:CD,IFN-gamma,IL-12, MMP_3,TIMP3, SOD,TOS, NOS.
Introduction:
In fact, in HLA-DQ2- or -DQ8 positive individuals, the effect the ingestion of gluten leads to oral tolerance losing , and triggers celiac disease (Catassi et al.,2010 ). Depending on some studies idea they indicate that the perfect mechanism that cause induce inflammation in addition to small intestinal mucosal damage development of in coeliac disease is not fully known. On the same side, some studies confirm that from genetically susceptible individuals just a small ratio of them will develop active disease, this based on both environmental and immunologic factors activity role in inducing complex coeliac disease (Kupfer, 2012 (.
Additionally, In lamina propria, once the immunogenic gliadin peptides reached( p57-68) they can stimulate an adaptive immune response in the patients with CD (Arentz-Hansen et al.,2000; Shan et al., 2002;Qiao et al., 2004).On the same side, these peptides represent excellent substrates for TG2(Bruce,1985; Piper,2002), which act by deamidation process to converts of distinct glutamine residues to negatively charged glutamate(Molberg et al., 1998;van de Wal et al.,1998) .Furthermore, for gliadin peptides presentation,CD4 t-cells activation occur by APCs induce (Meresse,2012; Qiao,2012).Moreover, immune cell that affected by gluten contributes to the pathogenesis of coeliac disease mainly by of pro- inflammatory cytokines as IFN-γ secretion (Nilsen et al.,1998; Perera et al.,2007). These cytokines play an important role in the triggered of mucosal disrupter alongside other inflammatory mediators like tumor necrosis factor (TNF)-α (Deem,1991; Bajaj- Elliott,1998).
On the other hand, the barrier that detach between luminal contents and the underlying interstitium represented the main function of intestinal mucosa , as well as the integrity of the intestinal barrier, play a fundamental role in provide normal intestinal function and protected the tissue from damage. On the same side, all processes (such as oxidative stress, inflammation, and promote epithelial cell apoptosis)lead to happens damage of intestinal mucosal , absence the functions of epithelial barrier , in addition to increased permeability of intestinal mucosa. (Farhadi,2003 ).Furthermore, in the pathomechanism of celiac disease which leading to villous atrophy, also matrix metalloproteinases act to degradation of the extracellular matrix. In general, matrix metalloproteinases is one of zinc-dependent proteins, these enzymes normally generated at reduced concentrations . functionally, the primary role of MMPs is repairing tissue injury and remodeling (Zitka et al.,2010).Moreover, several studies and experiments, confirmed that the celiac disease patients have increased levels in
the expression of matrix metalloproteinase-1, matrix metalloproteinase-3 and a tissue inhibitor of metalloproteinases, which observed the main role of these proteases and protease inhibitors in the pathomechanism of coeliac disease (Daum et al.,1999).On the other hand, nitric oxide (NO) which is involved in the inflammatory process and endothelium dysfunction may be implicated in the pathogenesis of CD or its complications. As well as, in untreated of celiac disease, the concentration of NO in the small intestine was Increased(Calasso et al.,2012 ).
Patients and Methods:
Study included 20 case CD patients with 12 healthy controls, Serum patients and controls examination in Al-Zahraa hospital, Dep. Immunology .
Methods:
TOS :
Assay Protocol
1_Firstly,must be equilibrated reagent and samples before test.in addition to shake the unknown sample for homogenation well.
2_By using pipette take 35µL standards (20µM -0µM) and samples and add to related wells.
3_ 225µL Reagent 1 reagent by pipette then add to the all wells.
4_After that must be mix well and read the absorbance with microplate reader/ using ELISA reader at 560nm (OD1).
5_then add 11µL Rreagent 2 reagent to the all micro plate wells.
6_After that mix wells than incubation at room temperature for 10 min .
7_In the end must read the absorbance with microplate reader/ELISA readerat 560nm (OD2).
SOD:
Reagent preparation
1. Preparation of 1X R1: Dissolve 13 mI R1 with 13mL double distilled water.
2. Chromogen preparation: Dissolve R3 total powder into a total R4 reagent.
Assay Protocol:
In this assay, each unknown sample used as its blank too.
1_All reagents and samples must be equilibrated to RT before the test. Shake the unknown sample for homogenation well.
Measured sample Blank sample
Sample 10µL 10 µL
Diluted R1(1X) 250 µL 250 µL
R2 10 µL 10 µL
Add- water 10 µL 10 µL
Chromogen 20 µL -
Add- water - 20 µL
2_ Mix well and read the wells absorbance at time 0 and 2 min with microplate reader/
ELISA reader at 420nm.
3_ Calculate SOD activity in unknown samples based on below formula:
SOD activity (U/ml)= (Vp-Vc)/(Vp)x 60 Vp=OD sample 2 min – OD blank 2 min Vc= OD sample 0 min – OD blank 0 min.
NOS:
Assay Protocol1.
1.All reagents and samples must be equilibrated to RT. Shake the unknown sample for homogenization well.
2. Add 300μL unknown samples to the related test tubes.
3. Add 10μL R1 reagent and mix well.
4. Centrifuge the test tubes for 10min at 3000-4000rpm.
5. Transfer 100μL of the supernatants of the tubes and 100μL standards into related microwells.
6. Add 100μL ready R2, 50μL ready R3 and 50μL ready R4 into the all wells.
7. Mix well and incubate 30min at 37ºC.
8. Read the absorbance at 540 or 550nm.
9. Calculate NO level in unknown samples based on standard curve.
TIMP-3 , MMP-3,IL-12, and IFN-gamma procedure:
Assay procedure : Assay procedure:
1_Firstly, Standard working solution must be add to the first two columns:and each concentration of the standard solution is added in duplicate, to one well each, side by side (100 uL for each well).then the samples adding to the other wells (100 uL for each well).also must be cover the plate by the sealer which provided in the kit.After that Incubate the plate at 37℃ for 90 min . Note: avoid touching the inside wall and causing foaming as much as possible when adding solutions should to the bottom of the micro ELISA plate well.
2_After incubation must be remove the liquid out of each well, without wash , and by using pipette directly add 100 μL of Biotinylated Detection Ab working solution to each well, after that Cover wells again with the Plate sealer, and also mix gently , then incubator at 37°C for 1 hour .
3_After that the solution aspirate or decant from each well,then using pipette to add 350 uL of wash buffer to each well. Soak for 1~2 min , then the solution aspirate or decant from each well and pat it dry against clean absorbent paper.this wash steps must reapeat about 2-3 times .
4_After washing steps , using pipette to add 100 μL of HRP Conjugate working solution to each well.also the plate must be cover by sealer, and incubator at 37°C for 30 min.
5_Likewise,the solution must aspirate or decant from each well, and wash the plate 5 times.
6_Using pipette to add 90 μL of Substrate Reagent to each well.then the plate must be cover by sealer.in the end of this phase the plate incubator at 37°C for about 15 min .
keep in your mind to protect the plate from light.
7_After the end incubator, using pipette to add 50 μL of Stop Solution to each well. Note:
Adding the stop solution should be done in the same order as the substrate solution.
8_ In the end determine the optical density (OD value) of each well at once with a micro- plate reader set to 450 nm.
Statistical Analysis
Data were organized and analyzed by using SPSS V 0.25. Descriptive statistics)frequency distribution and percentages with tables and figures, and mean ±standard deviation) and inferential statistics (Independent t-test, Mann-Whitney U test, and Pearson/ Spearman Bivariate correlations) were used. P-value of <0.05 considered statistically significant.
Results and discussion:
Table 1: Mean/ mean rank differences of NOS, MMP-3, TIMP-3, SOD, TOS, IFN-G and IL-12 in patients and controls
Factors ▼ Sample (n=32) Independent T-test and Mann-Whitney U test Patients (n=20) controls (n=12) t or U df Sig.
IL-12 -7.20 (4.72)* -11.23 (5.37) -2.218 30 0.034 MMP-3 6.27 (3.77) 11.72 (3.97) -3.880 30 0.001 TOS 4.53 (2.94) 8.20 (4.48) -2.799- 30 0.009
SOD 12.63** 21.33 50.0*** - 0.008
NOS 20.35 10.08 43.0 - 0.003
TIMP-3 11.20 25.33 14.0 - 0.0001
IFN.G 19.30 11.83 64.0 - 0.029
* mean (SD). ** mean rank. *** Mann Whitney U Test
Serum IL-12 levels in patients (-7.20) and controls (-11.23) with highly significant differences (0.034)which mean that IL-12 activated due to ROS produce, MMP-3 in patients (6.27) and controls( 11.72)with highly sig.0.001,compared with the levels of TIMP-3 which few increased to inhibit the MMP-3 stimulated (11.20) and in controls was (25.33)with highly sig.o.ooo1. due to the activate of IL-12 in patients serum ,this lead to stimulate production of IFN-gamma which was ( 19.30) in patients , but (11.83) in controls with high sig.0.029. Total oxidative status which used to estimate the overall oxidation state of the body was (4.53) in patients, (8.20) in control with highly sig.0.009, compared with the activity of
antioxidant parameter SOD which levels in patients was (12.63) and (21.33) with highly sig.
0.008.
Table 2: Bivariate Correlation of all factors in patients group Correlation
Coefficient (r) Age TOS SOD IFN-G IL-12 TIMP-3 MMP-3
TOS
r 0.360
Sig. .119
N 20
SOD
r 0.566** .215
Sig. .009 .364
N 20 20
IFN-G
r -.045- -.343- .236
Sig. .852 .139 .316
N 20 20 20
IL-12
r -.143 -.262- -.228- .212
Sig. .549 .265 .333 .369
N 20 20 20 20
TIMP-3
r .207 .182 .348 -.289- -.475-*
Sig. .380 .443 .133 .217 .034
N 20 20 20 20 20
MMP-3
r .190 -.299- .076 .472* .180 -.293-
Sig. .422 .200 .750 .036 .446 .210
N 20 20 20 20 20 20
NOS
r .261 .251 .265 .104 -.044- .490* -.171-
Sig. .266 .286 .259 .663 .855 .028 .470
N 20 20 20 20 20 20 20
* Correlation is significant at the 0.05 level (2-tailed).
** Correlation is significant at the 0.01 level (2-tailed).
In (table-2) show the correlation between the parameters , correlation between Age and TOS (positive 0.360)with non-sig.as well as a highly significant correlation between age and SOD (sig 0.009, r=0.56). Also inversible correlate between age and IFN gamma, IL-12 respectively (-0.45),( -.14), age with TIMP_3,MMP_3,NOS was positive correlation (r=0.380, 0.422, 0.266 respectively).the correlation results between TOS and (IFN gamma, IL_12,MMP_3)(r=-.34,-.26,-.29 respectively) were inversible with non-sig, but TOS with
(SOD ,NOS ) positive correlate( .215, .251 respectively).SOD positive correlated with IFN gamma, TIMP-3, NOS ,MMP-3(.236,.348, .265, .07 respectively) but inversible correlate between SOD and IL-12 (-.228). additionally, IFN gamma observed +ve correlate with IL- 12(.212 with non sig.) ,and –ve correlate with TIMP (-.289, and non sig) but highly correlate with MMP (.472, and sig .036), IFN gamma show +ve correlate with TOS but non sig (sig=.663) TIMP results show –ve correlate with MMP(-.293) and non sig( .210), but highly correlate with NOS (.490) with sig.( .028). contrastigly ,MMP show –ve correlate (-.171 with NOS) with non sig.
Beginning, due to the studies which confirm that increased cancer disease in patients with celiac disease , as well as, some of the evidence which states that oxidative damaged DNA play important role in the development of cancer . additionally to above, some research studies about this side diagnosis the role of oxidative stress/oxidative DNA damage in the patients with celiac disease. On the same side, the studies result observed that although in partially the diet in celiac disease patients responsible for oxidative stress/oxidative damaged DNA,but there is a factor independent of diet (Anna Szaflarska-Popławska, et al.,2010 ).oxidative stress and the production of free radicals at high concentrations both play an important role in the triggers of celiac disease. Moreover, the oxidative stress induced by either external or internal factors which lead to the increase of ROS, RNS, also extends to reduce the efficiency of the antioxidant system(Halliwell,1989 ).Cells and the production of cytokines act as a network of interactions resulting inflammatory process in the intestinal . As well as, one of the proinflammatory cytokines as IFN-γ is the most active stimulates to the expression of stransglutaminasa2 (TG2), the function of IFN-γ observed as synergistically with TNF-α, furthermore, induce the upregulation of HLA expression, in addition to trigger of fibroblasts to secretion tissue-damaging Metalloproteinases (MMPs), enhance Intraepithelial Lymphocytes (IELs) cytotoxicity against enterocytes as well as acts to rais apoptosis and villous flattening. However,IL-12 plays a necessary role in the development of type 1 response characterized by the production of IFN-γ,but does not represent pro- inflammatory cytokine. additionally, to above, the main role of IL-12 represent by the triggers IFN-γ expression by responding to T and NK cells. On the other hand, the function of IL-12 in CD occurs by contributed of this cytokine in the oral tolerance prevention that normally linked with mucosally administered antigens. Furthermore, the production of IFN- gamma results from IL-12 that synergizes with IL-18. Additionally, the suppressive effect on
the triggers of antigen-specific tolerance occur by IL-12 and IL-18 activity, which lead to creating a more vigorous response on the challenge (Jenkins,1991 ; Park ,2001;Tania,2016).When talking about the causes that increase the development of CD, some prior studies confirm that Th1 cell-mediated intestinal pathologies like CD may increase due to the interplay between IECs and dendritic cells, then lead to disrupted the homeostasis of the gastrointestinal tract.On the same side, some studies indicate that the decreased mRNA expression of a cytokine called Thymic stromal lymphopoietin in intestinal epithelial cells (IECs) can impact the pathogenesis of inflammatory bowel disease(Rimoldi ,2005).The Extracellular matrix consists of a large and various collection of biochemically, as well as, structurally several compositions which include proteins, proteoglycans, and glycoproteins (Daley,2008 ). MMPs represent as precursors that are enzymatically inactive converted to mature proteinases when activated. Tissue inhibitors of metalloproteinases (TIMPs) are effective MMP inhibitors , as well as, TIMPs function occurs by bind to MMPs 1:1approximately , complexes are produced due to this reaction, which can be recognized by a scavenger receptor then phagocyte by macrophages (Page-McCaw,2007; Brew,2010).
However, normal cellular functions characterized by normal proteolysis on the cell surface in addition to the fundamental role of extracellular matrix during development, and in the adult, but it may result in unwanted effects that extend to stimulate some diseases like cancer (Blobel,2000). Recently, the evidence confirms the main roles of zinc-dependent metalloproteinases (metzincins) occurs by cleave the protein components of the extracellular matrix then play the main role in tissue remodeling(Ivan,2003).Studies result in 1999 showed that the numbers of cells positive for MMP-3 (p<0.01)mRNA increase in the subepithelial region of intestinal mucosa of CD patients than in controls. as well as after treated with GFD, MMP-3 mRNA expression returned to normal. On the same side, near concluded that the decreased ratio of collagen I and TIMP-1 mRNA expressing cells to MMP-1 and MMP-3 mRNA expressing cells in untreated CD confirm that the ECM degradation may be occurring.ECM degradation occurs by stimulated sub-epithelial fibroblasts and macrophages(Dauma, et al., 1999 ).Other study agreement the fact that talking about the pathogenic role of MMPs . In 2006 Bashir M Mohamed and his group study the expression levels of MMp-3 and TIMP in CD patients, in the results, they confirm that the cells expressing of these enzymes with their inhibitor were significantly greater in all patients than in the healthy controls (P < 0.0001). Furthermore, these results were very clear in patients with severe damage and less in patients with minimal lesions. When the expression of these
enzymes increased in the intestinal mucosa of patients with CD in addition to increased of enzymes inhibitors , this condition lead to confirm the major role of these enzymes in the tissue remodeling that considered as the common feature of these disorders due to the extracellular matrix degradation. (Bashir, et al.,2006 ).Likewise, show the same suggest by studies results done by von Lampe B and his group, when they examine the MMP and TIMP in patients with celiac disease, the result observed that MMP-1 and MMP-3 mRNA expression correlated well with the histological degree of acute inflammation (von Lampea, et al.,2000).Interferon (IFN)-γ and interleukin (IL)-17 that secreted from T cells play a role in the activate matrix metalloproteinases (MMP), which lead to damage enterocytes in addition to villous atrophy(Consuelo Ortega, et al.,2013).regarding to oxidative stress role in CD, in 2020 some researchers examination Children with CD followed a strict GFD for 2 years compared with healthy control, in the result they observed no differences were found in oxidative stress between CD patients and control, in addition, IFN-gamma levels were higher in CD patient than the control group. by the way, maybe conclude, treated by GFD in CD patient is enough to decrease the oxidative stress contrastingly with the untreated patients which have the main role of oxidative stress in the initiation of inflammatory response(Javier Diaz-Castro, et al.,2020 ).
S. Björck in 2015 (Björck, 2015).and his grouped observe identical results with the present study, they confirm that levels of IFN-γ and IL-12 both are increased in children suffer from CD compared with healthy control, furthermore, other prior studies have same evidence when suggesting that high levels of IFN gamma in that intestinal mucosa of CD patients, as well as in children serum(Lahat, et al.,1999; Lahdenpera, et al.,2011).Moreover, our observed irregular IFN gamma response detecting in young children, in addition to elevated IL-12 levels which considered as IFN-gamma inducer(GiorgioTrinchieri, et al.,1992 ).
On the other hand, other studies continue to investigate cytokine levels until after treatment and as a result, they confirm reduced the IFNgamma and IL-12 after treatment with a gluten-free diet, likewise, various studies referred to alterations in cytokine levels occurred after starting a gluten-free diet compared to the time of investigating (Hansson 1999; Romaldini,2002; Lettesjo,2005; John SanilManavalan, et al.,2010 ).but, but, other studies indicate that some of the serum cytokines continue to be increased compared to controls until after 1 year of treatment (Romaldini,2002 ).In 2005, some researchers work-
study to determined NOs levels in the celiac disease pediatric patients, then compared with the patients on GFD after one year, the results show elevated of serum NOs levels in children who had not complied with the gluten-free diet, this affirms the important role of serum nitric oxide as an indicator of diet compliance (Ertekin, et al.,2005 ). On the other hand, Oxidative imbalance which triggers by gliadin peptides in enterocytes play important role in the stimulation of the transcription both pro-inflammatory cytokines and enzymes, such as nitric oxide synthase (NOS) which lead to increased production of nitric oxide (NO) metabolites supporting to oxidative stress (Ferretti, et al.,2012 ). when talking about the prior studies that worked on nitric oxide effect in CD, they suggested that regardless of GFD, the nitrosative stress is permanent as well as they suggest that maybe a responsible role for persistent histopathologic alteration and that GFD is only as factor improve the oxidative imbalance . as well as , serum NO levels act as the hallmark of treatment efficacy(Piątek- Guziewicz,et al.,2017).Additionally,prior studies results observed high serum level of NO in active celiac disease patients,On the same side, in celiac disease, the high levels of NO extend to the weakness of tight junction in the small intestinal because downregulating the expression of zonula occludens 1(Salvador Pérez, et al.,2017).
In fact, few studies focus on the role of some markers like total oxidative capacity TAC, TOC, ox-LDL, and OSI to investigate oxidative/antioxidant imbalance in children with celiac disease. However, the GFD represents the main treatment against CD and leads to decrease various parameters that play an important role in Deterioration of small intestinal functions , but about 50% of children with Celiac disease had comparatively low TAC levels and high TOC levels, that confirm the role of oxidative stress (Grażyna, et al.,2018 ).Other studies in 2011, shown that children with symptomatic, untreated CD have a high concentration of NO in serum and urine. Same result repeated in 2012 by Gianna Ferretti (Högberg, et al.,2011;
Gianna Ferretti, et al.,2012).
in 2014, a study done by I D Uspenskaya to examine serum nitric oxide (NO) metabolites and detection the correlate with a gluten-free diet (GFD) compliance, the results show the CD patients on a strict GFD had no significant differences in NO, while in patients violating GFD these parameters were significantly higher(Sarah Moretti, et al.,2014 ).
Beginning, the reduced antioxidant defenses may be a primary phenomenon that severely affects the intestinal mucosa and therefore increase susceptibility to oxidative tissue damage(Ioannise Koutroubakis, et al.,2004 ).
Some of studies result observed that the activity of SOD is markedly increased in pediatric patients with CD(Ferretti,et al.,2012 ). As well as , The increased activity of SOD in celiac disease patients probably indicate to the physiological response which occur due to increased pro-oxidant pressure in celiac disease patients. Namely, some prior reports have observed that, in CD patients, gluten activates pro-oxidant processes which leads to an overproduction of free radicals (Boda, 1999 ).
in 2017 some of the researchers confirm that an increase in SOD activity plays an important role against ROS but is not enough to maintain the normal level of free radicals, because of the imbalance between antioxidant and pro-oxidant, which lead to enhanced lipid peroxidation. These processes persist even in some patients on a long-term GFD(Vesna Stojiljković,et al.,(2017 ).
in 2009 some studies design to explain the role of stress on the different forms of celiac disease pathology, the results showed an increase in the activities of superoxide dismutase in patients with active and silent celiac disease(Vesna Stojiljković ,et al.,2009 ).
in fact, oxidative stress, recognized mainly by the increased levels free radicals, this occurs due to environmental factors or internal conditions. As well as, some studies confirm that the resultant cellular damage, have an essential role in the etiology of gluten-sensitive enteropathy disease (Chiurchiu,2011 ). Oxidized metabolites, and free radicals that produce in celiac disease happen due to the oxidation in proteins structures in addition to lipids that bound to specific gliadin peptides, then lead to oxidative imbalance. (Heyman,2009; Klaus- Peter Zimmer, et al.,2010 ).
In 2017 the study results observed a reduction in TAS level and increased levels of OSI and TOS level in CD patients when compared with control group. Additionally, the researchers divided patients with the celiac disease into two groups, the first group contain the patients undergo to gluten-free routine ,and the second group contains the patients that have resistant to GFD, the results they observed that OSI rate was higher in group one which consist of the patients agreeable with GFD routine. However, the NOS increased levels reported in the literature, in the case of oxidative stress, but reduced in celiac disease.On the same side, they suggested that the NOS level gradually return to normal directly when the patient start to respond with the gluten diet (Mustafa Kaplan,et al.,2017 ).
In 2015, researchers study to examine some of the oxidative markers as well as evaluate the effects of the gluten free diet on the case of oxidative stress in CD patients. in fact, there is a decrease in the number of studies examining the effect of oxidative stress before and after adopting a gluten diet . for this reason, Ersin Sayar and his group compared the levels of oxidative parameters before and after the gluten free diet, and observed that the TOS levels were significantly lower after the GFD. Similarily to the present study, the levels of TAC were showed to be significantly higher when compared to the values before the diet, this indicates that the serum sample collected from patients with celiac disease on GFD, these findings confirm that the oxidative stress effect reduces after the GFDin celiac patients(Ersin Sayar,et al.,2015 ).
Conclusion:
From our studies and depending on the results of previous study, concluded that the exposure to gluten in susceptible individual cause various type of deterioration in the small intestine represented by increased the production of ROS that effected and play main and important role in the imbalance between pro-oxidative and antioxidant. Likewise, the common and successful factors which dependent in CD patients was a gluten-free diet as well as supplementation of nutrient and vitamin to return the small intestine function to normal or semi- normally.
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