Sumbawa Horse Milk as a Prevention of Inflammatory Bowel Disease

(IBD) in Animal Model based on IL-6 Expression

and Gastric Histopathology

Nurina Titisari

*, Theresa Lidya Pramesti

, Tiara Widyaputri

, Rahadi Swastomo

Physyiology Departement of Veterinary Medicine, University of Brawijaya, PuncakDiengEksklusif, Malang, Indonesia

Student of Veterinary Medicine, University of Brawijaya, PuncakDiengEksklusif, Malang, Indonesia

Clinic Departement of Veterinary Medicine, University of Brawijaya, PuncakDiengEksklusif, Malang, Indonesia

Parasitology Departement of Veterinary Medicine, University of Brawijaya, PuncakDiengEksklusif, Malang, Indonesia

Keywords: Flowcytometri, Gastrointestinal, Inflamation, Rattusnorvegicus.

Abstract: Sumbawa horse milk contains bioactive components as exogenous antioxidants, anti-inflammatory

andantimicrobial which can prevent Inflammatory Bowel Disease. IBD is characterized by ongoing

inflammation of the digestive tract that could changed gastric histology and trigger pro-inflammatory

cytokines. The purpose of this study was to observe the preventive effects of Sumbawa horse milk on gastric

organs inflammation in IBD animal models. Twenty male rats, 8 weeks, 150-200 grams BW, divided into

five treatment groups; K- group (negative control), indometacin induced group; i.e K+ (positive group without

horse milk), P1(horse milk dose 0.5 mL/rat), P2 (horse milk dose1.0 mL/rat), and P3(horse milk dose

1.5mL/rat). Flowcytometry method was conducted to observe IL-6 expression, and then analyzed with one

way ANOVA followed by a tukey test (α 95%). While the gastric histopathological was analyzed

descriptively. The results showed that IL-6 expression were significantly lower (p <0.05) in P2 dan P3

compared with K+group. Whereas in the histological result, the p3 group was able to prevent

histopathological damage of the gastric organs compared to other treatment groups. The conclusion of the

study is Sumbawa horse milk able to prevent increased of IL-6 and gastric mucosal cell erosion, with the best

dose is 1.5 mL/rat.

1 INTRODUCTION

Inflammatory Bowel Disease (IBD) is a disease

characterized by inflammation of the digestive tract

associated with histopathological changes in the

digestive tract mucosa such as the stomach, intestine

and colon in the form of infiltration of inflammatory

cells in the lamina propria mucosa (Washabau, 2008).

Common symptoms of IBD are diarrhea and bleeding

in the digestive tract which is at high risk of causing

damage to the digestive tract (McFarland, 2008). IBD

is divided into two types, i.e ulcerative colitis

(chronic inflammation of the large intestine) and

chron's disease (chronic inflammation of the small

intestine) (Xavier and Podolsky, 2007). According to

Volkman's research (2017), there were 136 cases of

IBD (Inflammatory Bowel Disease) reported at the

Clinic Clinic for Small Animals, Faculty of

Veterinary Medicine, Freie Berlin, Germany

throughout 2017.

The causative factor of IBD is not known for

certain, but it is allegedly influenced by immune

regulation failure, exogenous factors, and the role of

intestinal flora (Thorenson et al. 2007). IBD also

occurs due to the use of non-steroidal anti-

inflammatory drugs (NSAIDs) such as indomethacin

(Podolsky, 2002). Indomethacin is an indole-acetic

acid derivate that is used as a treatment for arthritis.

In addition to its curative effects, indomethacin has

side effects which can inhibit COX-1 cyclooxygenase

which functions against prostaglandin synthesis and

mucous production to protect the small intestinal

mucosa from bacterial and viral infections (Takeuchi,

2003). Another side effect of indomethacin, it can

increase free radicals, which it can damage the

digestive organs marked by microflora reduction

(Strus et al., 2009).

Titisari, N., Pramesti, T., Widyaputri, T. and Swastomo, R.

Sumbawa Horse Milk as a Prevention of Inﬂammatory Bowel Disease (IBD) in Animal Model based on IL-6 Expression and Gastric Histopathology.

DOI: 10.5220/0009587801470151

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 147-151

ISBN: 978-989-758-483-1

147

In this study we examined the ability of Sumbawa

horse milk to prevent IBD in animal model.

According to Saputro (2016), Sumbawa horse milk

has protein componeants namely lysozyme (BM 17

kDa) and lactoferrin (BM 75 kDa) which act as

indigenus proteins that function for antimicrobial

compounds and have bioactive compounds as

antioxidants which play an important role to protect

the body from conditions of oxidative stress due to

inflammatory processes. Elias (2008) states that the

bioactive compounds in Sumbawa horse milk have

the primary structure of the amino acid histidine

which has an imidazole group as a hydrogen giver, a

lipid-peroxyl radical scavenger, and a hydrifobic

potential as an exogenous antioxidant.

2 MATERIAL AND METHOD

2.1 Tools and Materials

Equipment include rats cages, masks, gloves,

micropipets, dissecting sets, glass objects, glass

cover, autoclaves, scales, sonde, filters, magnetic

stirrer, measuring cup, digital scale, incubator,

aluminum foil, blue tip, yellow tip, microtube, sample

pot, cover slip, fixation board, centrifugator,

waterbath.

Materials used in this study include 20 rats

(Rattusnovergicus), Wistar strain, male, 120-150

gram ofbody wieght, Sumbawa horse milk, corn oil,

10% formalin, HE staining (Hematoxyline Eosin),

aquades, xylol, PBS, liquid paraffin, IL-6 antibodies,

chromagen DAB (DiaminoBenzidine) dyes,

Biotinylated Rabbit Anti-Rat IgG Antibody

secondary antibodies.

2.2 Horse Milk Induction

Sumbawa horse milk was from horse farms located in

Dompu Regency, Sumbawa Island, NTB (West Nusa

Tenggara). Sumbawa horse milk was a fresh pure

milk with milking date on 31 December 2018.

Sumbawa horse milk is given as preventive once a

day with a volume of 0.5 mL/rat, 1 mL/rat, and 1.5

mL/rat for 7 days and then induced indomethacin

using gastric sonde on day 8 then continued giving

Sumbawa horse milk on day 9-14 (Reni et al., 2010).

2.3 Indometacin Induction

The IBD animal model in this study used a single

dose of NSAID induction in the form of

indomethacin. The induction dose of IBD with

indomethacin was 15mg / kgBB (Aulanni'am et al.,

2011). The administration of indomethacin in this

study was carried out orally using a gastric sonde.

Previously the indomethacin must be diluted with

corn oil. The dilution of indomethacin as much as 45

mg requires 4 mL of corn oil as the solvent (Bures,

2011).

2.4 Histopathological Preparation

Gastric organs that have been fixed with 10%

formalin then carried out the process of alcohol

dehydration using alcohol concentration levels of

70%, 80%, 90%, absolute alcohol I, absolute alcohol

II, then purified using xylol I and xylol II. The

paraffin process was carried out using paraffin I and

paraffin II. The preparation is put into a mold that

contains half volume paraffin and the sample is

placed vertically and horizontally so that the cross

section is attached to the base of the paraffin. After

starting to rigid, paraffin is added again until the mold

was full and left until the paraffin hardens. The

paraffin blocks are then cut thin 5 micrometers thick

using a microtome. The results of the ribbon-shaped

pieces are spread on warm water with a temperature

of 46 ° C and immediately removed which is useful

for stretching the pieces so they do not multiply or

eliminate the folds caused by cutting. The preparation

is then removed and placed on a glass object and dried

overnight in an incubator at 60 ° C so that staining of

Hematoxylin-Eosin (HE) (Febram, et al., 2010).

2.5 Interleukin 6 (IL 6) Expression

IL-6 expression measurements using the

Flowcytometry method. The initial stage was taken

the rat's gastric, washed and soaked in PBS then the

organs were crushed with the base of a new syringe

in 5 mL Phosphat Buffer Saline (PBS) Solution.

Filtered with wire and put into propylene 15 mL to a

certain volume. Homogenized by centrifuging with a

2500 rpm for 5 minutes at a temperature of 10ºC and

then discarded the supernatant. The pellet that remain

then resuspended with PBS 1 mL. Suspense results

are divided into several 1.5 mL microtubes according

to the needs of the type of coloring combination that

has been filled with PBS ± 0.5 mL, @ 50 μL. The

suspension is centrifuged at 2500 rpm for 5 minutes

at 10ºC then the supernatant is discharged from the

centrifugation until there is a pellet which is then

carried out intracellular staining to determine the

expression of IL-6 by adding a fixative solution of 50

μL, then incubated for 20 minutes at 4 ° C in a dark

room. The 500 μL permeability solution (1X) was

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

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then homogenized with a centrifuge speed of 2500

rpm for 5 minutes at 10ºC then the supernatant was

removed. The pellet which was then added 50 μL

specific antibody solution, after that incubated it

for20 minutes at a 4°C in a dark room. After

incubation is complete, ± 400 μL PBS was added and

transferred to the flowcytometry cuvette for analysis.

2.6 Data Analysis

Flowcytometry test results were analyzed

quantitatively using BD CellquestProTM Software,

Microsoft Exel applications and statistical package

software for the social science (SPSS) for Windows

16 with One Way analysis of variance (ANOVA)

Test. The Tukey test was performed if there were

significant differences (α 95%). As for

histopathological features, descriptive analysis was

performed.

3 RESULT AND DISCUSSION

The test results showed that IL-6 expression of gastric

organ in negative control group (K-) was significantly

different with the positive control group (K+), group

P1 and group P2 but not with group P3 (Table 1). This

is due to the normal state of proinflammatory

cytokines was produced by the body to maintain

hemeostatic conditions in the immune system.

According to Erica et al. (2000), IL-6 is a

proinflammatory cytokine that is produced by

macrophages and is present in almost all tissues in the

body even in small amounts.

The positive control group (K+) showed

significantly different results from the negative

control group (K-), P2 group, and P3 group, but not

significantly different from P1 group. The results in

this study are in accordance with research conducted

by Riyansyah et al (2015), administration of

indomethacin at a dose of 15 mg/kgBW orally in rats

can cause irritation and damaged gastric mucosa,

which can trigger IL-6 expression as a pro cytokine

inflammation. Indomethacin inhibits COX-1

formation which destroys the performance of

mitochondria in the cell so it will interupted the

oxidative postforilation cycle. The disruption of

electron transfer on oxygen molecules can trigger

inflammation (Takeuchi et al., 2003). This can

triggers the formation of excess Reactive Oxygen

Species (ROS) that will stimulate the activation of

NF-kB which is a transcription factor in regulating

the expression of pro-inflammatory cytokine cells

such as IL-6 (Aulanni'am et al., 2012).

Table 1: Table of the average expression of interleukin 6

(IL-6) gastric organs of white rats.

Treatment

Average

IL-6 expression

(% gated) ± SD

K-

19,4925±4,33

K+

36,0475±1,535

31,57±1,489

28,3525±2,464

Note: notations a, b, c show significant differences

between groups.

3.1 Histopathology of the Rat Gastric

Organ (Rattus Novergicus)

Gastric histology with hematoxylin eosin (HE)

staining (Figure 1). Based on on observation on the

gastric preparations of the negativ control group

(healthy mice) (Figure 1.A) showed that the mucosal

layer of tunica mucosa epithelial simplex with gastric

pit formation and gastric gland still arranged and

neatly lined with cylindrical simplex form.

Puspitasari (2008) state mucosal layers contain many

mitochondria and granules to produce pepsinogen

enzymes, while parietal cells are acidophilic because

these cells produce HCL of stomach acid. It also

appears a small number of neutrophill cells, which is

normal because to its role agains pathogenic agents.

Contrast result occur in the positive control group

(Figure 1.B), which showed a severe cell damage in

the tunica lamina propria layer of the gastric mucosa.

The epithelium erosion accompanied by infiltration

of inflammatory cells (neutrophils) when we

compared it with negative controls. Erosion that

occurs in the gastric mucosa suspected as a result of

free radicals from indomethacin induction, that can

stimulate leucocyte toward gastric mucosa cells.

Leucocyte wull produce H

to kill several types of

bacteria and fungi and for cell growth, but neither

attack specific targets. So it will also attack

polyunsaturated fatty acis from cell membranes, cell

organeles, or DNA, which can cause damage to

structure and function cell (Puspitasari, 2008).

The histopathological result showed no

significant difference between group P1 (Figure 1.C)

and K

(Figure 1.B). There were erosion in gastric

mucosal epithelial cells and inflammatory cell

infiltration still visible although its not as much as K

group. In group P2 (Figure 1.D) showed less erosion

of the epithelial cell and lower number of

inflammation cells when we compared with P1 group

Sumbawa Horse Milk as a Prevention of Inﬂammatory Bowel Disease (IBD) in Animal Model based on IL-6 Expression and Gastric

Histopathology

149

(Figure 1.C). In the last horse milk treatment, P3

(Figure 1.E) showed a better result compared to the

other treatment group. Epithelial cells erosion in

lamina propria tunica gastric mucosa was decreasing

and inflammation cells number was much lower when

we compared with P1 and P2 group.

Figure 1: Histopathology of rat gastric mucosa

(Rattusnorvegicus) (gastric preparations; cross sections;

HE staining. 100xenlargement).

Note: (a) Negative control rats (K-); (B) Positive control

rats (K+); (C) Group P1; (D) Group P2; (E) Group P3;

(400x magnification)

(epithelial erosion of the gastric mucosa)

(neutrophil cell infiltration)

(repair of epithelial cells)

4 CONCLUSIONS

Preventive administration of Sumbawa horse milk in

animal model of IBD induced by indomethacin were

able to prevent IL-6 expression and histopathological

changes in gastric organs with the best dose was 1.5

mL/rat. Further research needs to be done to prevent

Inflammatory Bowel Disease (IBD) using Sumbawa

horse milk on the entire digestive tract.

ACKNOWLEDGEMENTS

This research was funded by Veterinary Medicine

Faculty, University of Brawijaya through the

DPPSPP scheme 2019.

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