Effect of renin-angiotensin-aldosterone system blockade on intestinal injury induced by indomethacin in rats

Asmaa Abdulaziz A. Rabee; Yahya Saber E. Mansour; Huda A Mariz; Salwa Muftah Eljamay

doi:10.4103/LJMS.LJMS_95_20

ORIGINAL ARTICLE

Year : 2021 | Volume : 5 | Issue : 1 | Page : 6-10

Effect of renin-angiotensin-aldosterone system blockade on intestinal injury induced by indomethacin in rats

Asmaa Abdulaziz A. Rabee¹, Yahya Saber E. Mansour², Huda A Mariz³, Salwa Muftah Eljamay⁴
¹ Department of Pharmacology, Faculty of Medicine, Omar Elmokhtar University, Derna, Libya
² Department of Pharmacy and Toxicology, Faculty of Pharmacy, Omar Elmokhtar University, Elbieda, Libya
³ Department of Pharmaceutical Technology, College of Medical Technology, Derna, Libya
⁴ Department of Public Health Technology, College of Medical Technology, Derna, Libya

Date of Submission	18-Nov-2020
Date of Acceptance	14-Mar-2021
Date of Web Publication	10-Apr-2021

Correspondence Address:
Dr. Salwa Muftah Eljamay
Department of Public Health Technology, College of Medical Technology, Derna
Libya

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/LJMS.LJMS_95_20

Abstract

Aim: The aim of this experimental study was to evaluate the possible cytoprotective effects of renin-angiotensin-aldosterone system (RAAS) blockade, captopril (10 mg/kg), telmisartan (10 mg/kg), and a proton-pump inhibitor (lansoprazole) on intestinal injury induced by indomethacin in rats. Materials and Methods: This effect was evaluated through the assessment of intestinal inflammatory biomarkers and oxidative stress parameters. Forty adult male albino rats weighing 170–200 g were used and divided equally into five groups. After the treatment, the following were assessed: Myeloperoxidase (MPO) enzyme activity, pro-inflammatory (interleukin-1beta [IL-1 β]) content and anti-inflammatory (IL-10) content, malondialdehyde (MDA) content, reduced glutathione (GSH) content, and superoxide dismutase (SOD) enzyme activity, and histopathological examination of the intestinal tissues was conducted. Results: Indomethacin group induced a significant increase in MDA content, also in inflammatory biomarker (MPO enzyme activity, IL-1 β content, and IL-10 content) compared to normal control, while indomethacin group induced a significant decrease in GSH content, SOD enzyme activity compared to normal control group. Captopril, telmisartan, lansoprazole administration before indomethacin-induced significant increase in GSH content, SOD enzyme activity, while induced significant decrease in MDA content as well as in inflammatory biomarker compared to indomethacin group, which indicate the antioxidant, anti-inflammatory effects, and the gastroprotective properties of RAAS blockers, which also shown in the histopathological examination of intestinal tissues. Conclusion: This study showed the possible antioxidant and anti-inflammatory effects as well as the gastroprotective properties of RAAS blockers. Telmisartan has more antioxidant and anti-inflammatory effect as well as cytoprotective action than captopril.

Keywords: Captopril, indomethacin, lansoprazole, renin-angiotensin-aldosterone system blockade, telmisartan

How to cite this article:
A. Rabee AA, E. Mansour YS, Mariz HA, Eljamay SM. Effect of renin-angiotensin-aldosterone system blockade on intestinal injury induced by indomethacin in rats. Libyan J Med Sci 2021;5:6-10

How to cite this URL:
A. Rabee AA, E. Mansour YS, Mariz HA, Eljamay SM. Effect of renin-angiotensin-aldosterone system blockade on intestinal injury induced by indomethacin in rats. Libyan J Med Sci [serial online] 2021 [cited 2023 Mar 30];5:6-10. Available from: https://www.ljmsonline.com/text.asp?2021/5/1/6/313532

Introduction

Peptic ulcer disease is a complex and multi-causal disease that occurs when biological balance between defense and aggressive factors in the gastrointestinal tract is disturbed.^[1] Among aggressive factors are endogenous factors such as gastric acid and pepsin secretion,^[2] active free radicals and oxidants, leukotrienes,^[3] and endothelins^[2] as well as exogenous factors such as ethanol^[4] or nonsteroidal anti-inflammatory drugs (NSAIDs).^[5] Indomethacin induces its gastrointestinal toxicity by interfere with mucosal cell regeneration through inhibition of prostaglandin-E₂ (PGE₂) synthesis, production of free radicals, reduction of gastric nitric oxide level, and invasion of activated neutrophils as well as induction of gastric cells apoptosis.^[6] Angiotensin II, a key peptide hormone in the renin-angiotensin-aldosterone system (RAAS), induces oxidative stress and inflammation.^[7] Telmisartan that can concurrently block the angiotensin II T1-receptor and activate peroxisome proliferator-activated receptor gamma (PPAR-gamma).^[8] PPAR-gamma, a ligand-dependent nuclear receptor, has been reported to exert a variety of pleiotropic effects, including anti-oxidative and anti-inflammatory effects, also has the potential to be implicated in the protection against gastric ulcer.^[9] Proton-pump inhibitors, a strong anti-secretary agent, have anti-inflammatory action beyond acid suppression agents that act on gastric (H+/K+) ATPase of parietal cells,^[10] have been extensively used for therapeutic control of acid-related disorders, including gastroesophageal reflux disease and peptic-ulcer diseases caused by stress, NSAIDs, and Helicobacter pylori infection.^[11]

Angiotensin II is involved in the several key steps of the inflammatory cascade that ultimately provokes intestinal injury and ulceration including polymorphonuclear leukocyte infiltration, probably, through the upregulation of adhesion molecules. The aim of the present experimental study is to investigate and compare the reffect of pretreatment with captopril and telmisartan on indomethacin-induced small intestinal injuries in rats.

Materials and Methods

Animals and experimental design

Forty adult male albino rats weighing 170–200 g were used and divided equally into five groups; each contained eight rats, in clean capacious macrolane cages under standard laboratory conditions, as the following: Group I: Control group. Group II: indomethacin was administered in a dose of 10 mg/kg i. p. for 3 days.^[12] Group III: Captopril was administered in a dose of 10 mg/kg p. o. for 14 days^[13] and indomethacin was administered in a dose of 10 mg/kg i. p. in last 3 days. Group IV: temisartan was administered in a dose of 10 mg/kg p. o. for 14 days^[14] and indomethacin was administered in a dose of 10 mg/kg i. p. in last 3 days. Group V: Lansoprazole was administered (as a reference drug) in a dose of 50 mg/kg, p. o. for 14 days^[15] and indomethacin was administered in a dose of 10 mg/kg i. p. in last 3 days. Experiments were conducted to study the effect of the drugs under investigation on indomethacin-induced small intestinal injury through measuring the following parameters:

Oxidative stress parameters

Malondialdehyde (MDA) content, reduced glutathione (GSH) content, and superoxide dismutase (SOD) enzyme activity.

Inflammatory biomarkers

Myeloperoxidase (MPO) enzyme activity, pro-inflammatory interleukin-1beta (IL-1 β) content, and anti-inflammatory (IL-10) content, in addition to histopathological examinations.

Drugs and chemicals

Indomethacin vials, 50 mg, EL Nile Co. for pharmaceutical and chemical industries (Egypt)
Captopril tablet, 25 mg, Epico (Egypt)
Telmisartan tablets, 80 mg (Hikma Pharmaceuticals USA Inc.)
Lansoprazole capsules 30 mg, Sedico (Egypt)
Tween 80 (Sigma-Aldrich): It was used for the emulsification of captopril, telmisartan, and lansoprazole. All other chemicals used were of good quality and analytical grade.

Ready-made kits

MPO activity, Pro-inflammatory IL-1 β content and anti-inflammatory (IL-10) content measured using a rat-specific enzyme-linked immunosorbent assay kits.

Procedures

All animals were scarified by cervical dislocation and their small intestine were quickly excised and washed with saline and used for the preparation of tissue homogenate 20% (in 0.9% NaCl using homogenizer (X620 CAT, made in Germany) and stored at − 80°C until analysis. The small intestine of two animals from each group was kept in 10% formalin and used for histopathology examination.

Ethical consideration

The animals were handled according to the guidelines of local ethical committee which comply with the international laws for use and care of laboratory animals. Animal Ethics Committee Approval No. Ethics/research/OMU/2020-87.

Methods

Determination of oxidative stress parameters

The antioxidant parameters were determined in small intestine homogenate (20%) after centrifugation of tissues at 3.000 rpm for 10 min, and the supernatant was used.

Determination of malondialdehyde content^[16]

The colorimetric determination of Thiobarbituric acid reactive substances (TBARS) content is based on the reaction of one molecule of MDA with two molecules of thiobarbituric acid in acidic medium at the temperature of 95°C for 45 min. The resultant pink pigment product is extracted by n-butanol, and then its absorbance is determined at 535 nm and 520 nm. The difference in absorbance between the two determinations was used to calculate the TBARS value.

Determination of reduced glutathione content^[17]

Determination of nonprotein sulfhydryl compounds (indicative to GSH content) was done according to the method of Ellman. The method depends on the reduction of Ellman's reagent (5, 5'-dithio-bis (2-nitrobenzoic acid) by SH group in GSH to form an intense yellow product (5–thio-2 nitrobenzoic acid) which can be measured colorimetrically at 412 nm.

Determination of superoxide dismutase activity^[18]

SOD activity in the homogenate was determined by the difference between auto-oxidation of pyrogallol alone and in the presence of homogenate that contain SOD.

Determination of inflammatory biomarkers

Determination of MPO enzyme activity (ELISA kit CAT. NO. SEA601Ra)
Determination of IL-1 β content (ELISA kit CAT. NO. EL10028)
Determination of IL-10 content (ELISA kit CAT. NO. ELR-IL10-001C).

Statistical analysis

Statistical analysis was done using the computer program (prism 5). The quantitative data were presented in the form of mean ± standard error of the mean. Statistical analysis of the difference between the groups was performed using the one–way analysis of variance followed by Tukey-Kramer test for multiple comparisons.

Results

Indomethacin group, induced a significant increase in MDA content compared to normal control group, while pretreated of captopril-induced significant decrease in MDA content compared to indomethacin group as well as with telmisartan, and with lansoprazole indomethacin group induced a significant decrease in GSH content, SOD enzyme activity compared to normal control group.

Captopril, telmisartane, lansoprazole administration before indomethacin induced significant increase in GSH content, SOD enzyme activity compared to indomethacin group. Indomethacin group induced a significant increase in inflammatory biomarker (MPO enzyme activity, IL-1 β content and IL-10 content compared to normal control while decreased with other pretreated groups [Table 1] and [Figure 1].

Table 1: Results of biochemical tests among the four groups and compare with control group

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Figure 1: Comparison of results of biochemical tests among groups

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Histopathological examination of rat intestinal tissues in the four groups (i.e., indomethacin group [Figure 2], captopril and indomethacin group, valsartan and indomethacin group, and lansoprazol and indomethacin group), as compared with control group showed inflammatory cells infiltration in the lamina propria of the villi with diffuse goblet cells formation in the lining mucosal epithelium and severe congestion in the blood vessels in submucosa.

Figure 2: Transverse section of rat small intestine in indomethacin group showing inflammatory cells infiltration in the lamina propria of the villi with diffuse goblet cells formation in the lining mucosal epithelium

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Discussion

NSAIDs such as indomethacin are extensively recommended in the management of pain, fever, and inflammation while its gastrointestinal tract adverse effects disturb its use.^[19] Indomethacin induces its gastrointestinal toxicity through several mechanisms such as an increase in gastric acid secretion; interfere with mucosal cell regeneration through inhibition of PGE₂ synthesis, production of free radicals, reduction of gastric nitric oxide level, and invasion of activated neutrophils as well as induction of gastric cells apoptosis.^[6] Many previous studies have been done by damaging effect of indomethacin on gastrointestinal mucosa.^[20],[21] The present study revealed that indomethacin-induced duodenal ulcer through increase gastric juice volume, free and total acidity as well as a decrease in gastric protective prostaglandin. Pretreatments with captopril or talmisartane have ability to decrease gastric acid secretion induced by indomethacin. Gastric secretion is a controlled by captopril or telmisartan is characterized by the property of stimulating PPARgamma. Telmisartan reduced the ulcer index in both ulcer models. These results are supported by the findings of Nakagiri et al.^[22]

Indomethacin group, induced a significant increase in MDA content compared to normal control group, which indicates that tissues exposed to oxidative stress include large amounts of toxic oxygen radicals, which induce lipid peroxidation leading to MDA formation.^[23],[24] The lowest MDA values were detected in the groups treated by captopril, telmisartan, and lansoprazole compared with indomethacin group, there was an inhibition in SOD as well as GSH content in indomethacin treated rats compared to the corresponding control. The decline in SOD and GSH level in the intestinal mucosa of rats treated with indomethacin hence may render the mucosa more susceptible to injury by indomethacin. Thus, the inhibition of antioxidants leads to the accumulation of ROS.^[25],[26] On the other hand, pretreated of rats with captopril, telmisartan, lansoprazole before indomethacin induced significant increase in GSH content, SOD enzyme activity compared to indomethacin group which indicates the cytoprotective activity of these drugs.

Indomethacin group induced a significant increase in inflammatory biomarker (MPO enzyme activity, IL-1 β content, and IL-10 content compared to normal control. Indomethacin prevents synthesis of PGE₂, which is the main factor that involves in gastric mucosal defense mechanism through many of its actions. First, it regulates gastric blood flow to maintain the level of mucus and bicarbonate secretion as these two substances are secreted in flow-dependent fashion.^[27] Furthermore, endogenous PGE₂ also controls the epithelial cell proliferation and restitution, mucosal immunocyte function, and basal acid secretion,^[28] also increased oxygen-free radicals increases the protease secretion, and capillary blood flow obstruction which will result in gastric injury.^[29],[30] Besides interfering of PGE₂ pathway, NSAIDs also raise the nitric oxide synthase activity which causes increase in pro-inflammatory cytokine and damage of intestinal mucosa^[19] while decreased with captopril, and telmisartan groups which indicate anti-inflammatory and cytoprotective effect of RAAS blockade. Angiotensin II, through the binding with angiotensin receptor, is also capable of promoting inflammation indirectly through the stimulation of several transcription factors, also Angiotensin II stimulates the production of proinflammatory cytokines and chemokines, such as IL-6, IL-8, which is an important mediator for the direct migration of monocytes.^[31]

The results of telmisartan is nearly to the result of standard gastroprotective (lansoprazole), which indicates that telmisartan has better antioxidant and cytoprotective properties than theirs of captopril. In histopathological examination, we found lymphoid hyperplasia in the follicles of the submucosal layer associated with inflammatory cells infiltration in the mucosa and diffuse goblet cells formation in the lining mucosal epithelium of the villi in indomethacin group, but in pretreating groups of telmisartan, captopril, and lansoprazole, we found diffuse goblet cells formation in the lining mucosal epithelium of the villi which indicate anti-inflammatory and cytoprotective action of RAAS blockade.

Conclusion

This study showed the possible antioxidant and anti-inflammatory effects as well as the gastroprotective properties of RAAS blockers. Telmisartan has more antioxidant and anti-inflammatory effect as well as cytoprotective action than captopril.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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