Received: 14/11/2023 Accepted: 05/01/2024 Published: 11/03/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34351
This study investigated the histopathological and
immunohistochemical effect on the intestine and liver tissues with
addition of the soybean meal (SBM), wheat Gluten meal (WGM) and
Corn gluten meal (CGM) to rat diet. A total of 24 average twenty–
day–old male rats (Wistar albino) were used in the study. The rats
were randomly divided into 3 groups with 8 animals in each group
(Control, Wheat and Corn groups). The diet provided to all three groups
contained proteins, which were SBM, WGM and CGM in the Control,
Wheat and Corn groups, respectively. In the study, the group fed with
SBM was used as the Control group. Rats were fed a diet containing
22% crude protein and 2,598 kcal·kg
metabolic energy throughout
the experimental period. The feeding trial was continued for a period
of 50 days. Degenerative changes of varying severity in intestinal
epithelial cells and atrophy in villi were observed. Similarly, the
degenerative changes, especially vacuolar or hydropic degeneration
were determined in hepatocytes. It was determined that the CD4 level
compared to the Control group (P<0.01) on intestine tissue. Also, it was
of the Wheat and Corn groups in liver tissue. (P<0.05). As a result, it
was observed that the histopathological and immunohistochemical
parameters of the intestine and liver tissues of the rats fed with diets
containing highly WGM and CGM were limitedly affected.
Key words: Autoimmune; gluten; histopathology; intestine; liver
Este estudio investigó el efecto histopatológico e inmunohistoquímico
en los tejidos del intestino y el hígado con la adición de harina de soja
(SBM), harina de gluten de trigo (WGM) y harina de gluten de maíz (CGM)
a la dieta de ratas. En el estudio se utilizaron un total de 24 ratas
macho (Wistar albino) de veinte días de edad promedio. Las ratas se
dividieron aleatoriamente en 3 grupos con 8 animales en cada grupo
(grupos Control, Trigo y Maíz). La dieta proporcionada a los tres grupos
contenía proteínas, que eran SBM, WGM y CGM en los grupos Control,
Trigo y Maíz, respectivamente. En el estudio, el grupo alimentado con
SBM se utilizó como grupo Control. Las ratas fueron alimentadas
de energía metabólica durante todo el período experimental. La
prueba de alimentación continuó durante un período de 50 días. Se
observaron cambios degenerativos de diversa gravedad en las células
en los hepatocitos se determinaron los cambios degenerativos,
especialmente la degeneración vacuolar o hidrópica. Se determinó
en los grupos de trigo y maíz en comparación con el grupo de control
(P<0,01) en el tejido intestinal. Además, se determinó que el nivel de
hepático de los grupos Trigo y Maíz. (P<0,05). Como resultado, se
observó que los parámetros histopatológico e inmunohistoquímico
de los tejidos del intestino y del hígado de las ratas alimentadas con
dietas que contenían un alto contenido de WGM y CGM se vieron
afectados de forma limitada.
Palabras clave: Autoinmune; gluten; histopatología; intestino;
The effect of adding wheat and corn gluten to the diet of rats on the
autoimmune and histopathological parameters in the intestine and liver
El efecto de la adición de gluten de trigo y maíz a la dieta de ratas sobre los
parámetros autoinmunes e histopatológicos en el intestino y el hígado
Recep Gümüş
* , Kübra Asena Terim Kapakin
, Esra Manavoğlu Kirman
, İsmail Bolat
, Aybuke İmik
, Nazlı Ercan
Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Animal Nutrition and Nutritional Diseases. Sivas, Türkiye.
Ataturk University, Faculty of Veterinary Medicine, Department of Veterinary Pathology. Erzurum, Türkiye.
Selçuk University, Faculty of Health Sciences, Department of Nutrition and Dietetics. Konya, Türkiye.
Sivas Cumhuriyet University, Faculty of Veterinary Medicine, Department of Biochemistry, Sivas, Türkiye.
*Corresponding author:
Dietary Wheat & Corn Gluten: Autoimmunity & Intestinal/Liver Pathology in Rats / Gümüş et al. ___________________________________
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Cereals are one of the main foods for both humans and animals.
Wheat (Triticum aestivum L.) and corn (Zea mays L.), which are the
main cereal products, are among the most important food products
in the World that are grown and consumed Worldwide [1]. Protein in
wheat consists of 10–15% of albumin/globulin and 85–90% of glüten
[2]. Gluten is a complex mixture of hundreds of different proteins,
primarily gliadin and glutenin. Gliadin and glutenin proteins are termed
prolamins, which represent seed proteins that are insoluble in water
but can be extracted in aqueous ethanol and are characterized by
high levels of glutamine (38%) and proline residues (20%) [2, 3]. Corn
is the most produced product among cereals after wheat, and corn
gluten meal obtained from corn is accepted as a high quality protein
source with desired functionality for food application [4]. Corn gluten
contains approximately 62–74% zein as a protein fraction [5].
Wheat gluten is the main factor that causes some diseases and
allergies in living beings with carrying the HLA–DQ2/8 genes [6].
Due to the harmful immune response to the gluten proteins found in
wheat, cases of gluten intolerance have been reported which people
cannot tolerate wheat consumption [7]. The most common disorders
associated with gluten intake are celiac disease, gluten intolerance,
non–celiac gluten sensitivity (NCGS), wheat allergy, and dermatitis
herpetiformis [3]. It has been reported that gluten causes intestine
and liver damage with an increase in the transglutaminase enzyme and
gliadin level due to liver dysfunctions plays a role in the pathogenesis
in celiac patients with an autoimmune disease [8, 9].
This study was aimed to determine the effects of glutens (WGM and
CGM), incorporated as protein sources into rats (Wistar albino) diet,
on histopathological and immunohistochemical effects on intestine
and liver tissues.
Animals, experimental design and diets
The experimental protocol of the study was approved by the Sivas
Cumhuriyet University Animal Experiments Local Ethics Committee's
decision dated 2021 and numbered 462.
A total of 24 average twenty–day–old male rat (Wistar albino)
were used in the experiment. The rats were randomly divided into 3
groups with 8 animals in each group. The feed diets provided to all
three groups contained of proteins, which were SBM (24.85%), WGM
(24.85%) and CGM (16.80%) in the Control, Wheat and Corn groups,
respectively (TABLE I). The feeding trial was continued for a period
of 50 days. Feed and water were provided ad libitum. The animals
were housed at the comfort temperature (22°C) and were fed on a
diet containing 22% of crude protein and 2,598 kcal·kg
of metabolic
energy throughout the experiment period.
Histopathological examination
At the end of the study eight animals from each group were
sacrificed under anesthesia. The tissue samples were fixed in
10% buffered formalin and routinely processed for histological
cut 4 μm in thickness (Leica RM2125 RTS microtome) and stained
by the Haematoxylin–Eosin for observation under a light microscope
(Olympus Bx51 with a DP72, Tokyo, Japan) [10].
Contents and nutrient composition of diets used in the study (%)
Composition (%)
Control Wheat Corn
Wheat bran 3.24 1.8 4.55
Oat, 11% CP
62.11 68 64.00
Sunower meal, 28% CP
6 13 13
Corn gluten meal, 62% CP
Wheat gluten meal, 75% CP
Soybean meal, 51% CP
Animal fat 2.8 2.2 0.65
Vitamin–mineral premix* 1 1 1
Nutrient composition (calculated)
Metabolisable energy, (kcal·kg
) 2598 2598 2598
Crude protein (%) 22 22 22
*The vitamin–mineral premix provides the following (per kg): vitamin A: 6,000,000 IU,
vitamin D3: 800,000 IU, vitamin E: 8,000 mg, vitamin K3: 2,000 mg, vitamin B1: 1,200
mg, vitamin B2: 3,000 mg, vitamin B6: 2,000 mg, vitamin B12: 8 mg, niacin: 10,000 mg,
folic acid: 400 mg,; d–biotin: 20 mg, choline chloride: 160,000 mg, manganese: 32,000
mg, iron: 16,000 mg, zinc: 24,000 mg, copper: 2,000 mg, iodine: 800 mg, cobalt 200
mg, selenium: 60 mg, Cal–D–Pan: 4,000 mg, antioxidant: 4,000 mg.
CP: Crude protein.
Image analysis
Tissue sections were evaluated by high–power light microscopic
examination using an Olympus Bx51 with a DP72 (Tokyo, Japan) camera
system. Each specimens were examined in 10 randomly selected areas
with an 40× objective. The scores were derived semi–quantitatively
using light microscopy on the preparations from each rat and were
reported as follows: Grade 0 = - (negative); Grade 1 = +1 (mild); Grade 2
= +2 (moderate); Grade 3 = +3 (severe); Grade 4 = +4 (most severe) [11].
Immunohistochemical examinations
Four μm sections from all of the tissue samples were cut and
processed for immunohistochemical examination by a standard
avidin–biotin–peroxidase method that the producer described.
Rabit policlonal antibodies that react with rat transglutaminase 2
(TG2) (Catalog No:NB600–547), gliadin (Catalog No: BS–13374–R),
IgA (Catalog No: BS–0648–R10491–R), IgG (Catalog No: BS– 0392–R),
CD4 (Catalog No: BS–0647R, ThermoFisher), and CD8 antibodies
(Catalog No: BS–0648–R) were used for 60 min. A secondary antibody
was used according to the manufacturers protocol (expose mouse
PBS, the sections were incubated with 3,3–diaminobenzidine (Dako
Cytomation, Glostrup, Denmark) and counterstained with Mayers
hematoxylin (Dako Cytomation) [12, 13].
Statistical analysis
For all analyses, SPSS
and P14
expressed as mean ± standard error of the mean (SEM).