INTRODUCTION:

Gastric mucosal injury frequently arises from various pathological states in both adult and pediatric populations^1,2. Over the years, several studies have been conducted to extensively elucidate the etiology of gastric mucosal injury³.

Medication usage, especially nonsteroidal anti-inflammatory drugs (NSAIDs), is widely recognized as a significant factor contributing to gastric mucosal injury^4,5. Ibuprofen is an analgesic and antipyretic drug belonging to the NSAID class^6,7 and is a non-selective inhibitor of both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)^8,9. Notably, ibuprofen-induced inhibition of cyclooxygenase activity plays a role in suppressing prostaglandin (PG) synthesis, which alleviates pain, inflammation, and fever⁹. However, ibuprofen intake causes gastrointestinal side effects, that vary depending on the dose and patient population¹⁰. Importantly, these side effects include ulceration of the gastric mucosa, enhanced ulcerogenic response to stress, and hindered gastric ulcer healing^11–13.

Various gastric ulcer and lesion models have been established to evaluate the efficacy of novel therapeutics for protecting gastric mucosa. However, a more detailed comparative analysis of these models is required. Asebayo–Gege et al., induced gastric ulceration using ibuprofen at a dose of 400mg/kg BW. Lanza et al. revealed that the administration of three doses of 200mg of ibuprofen for seven days resulted in five (22.7%) cases of gastric bleeding, eight (36.4%) cases of gastric erosion, and one (4.5%) case of gastric ulcer¹⁴. Additionally, Laine et al., reported that the administration of 800mg of ibuprofen for 24 weeks significantly induced (p = 0.004) the development of stomach ulcers¹⁵. Moreover, the findings of mechanistic studies suggest that factors such as high neutrophil levels and free radicals are involved in the development of stomach ulcers^16,17. Notably, the gastric-damaging effects of NSAIDs are attributed to COX-1 and COX-2 inhibition^2,17^,¹⁹. Although previous studies have compared the effects of various dosages of ibuprofen or its efficacy against other drugs used for inducing stomach ulcers, no research has compered the effects of minimal doses of ibuprofen. Therefore, this study aimed to determine whether the administration of minimal doses of ibuprofen (200 and 300mg/kg BW) can induce gastric mucosal damage.

MATERIALS AND METHODS:

Study design:

This study employed a randomized post-test-only control group design. Notably, the study was approved by the Animal Care and Use Committee (ACUC) of the Veterinary School of Medicine, Universitas Airlangga, Indonesia, on March 15, 2021 (Ethical Clearance: 2.KE.024.03.2021).

Animal model:

White male Wistar rat aged 12 weeks with body weights ranging from 200-250g were used for this study. Animals that were ill or exhibited clinical changes such as weight loss or altered breathing patterns were excluded. Additionally, animals showing signs of physical or mental stress or those that sustained damage to organs or tissues during immunohistochemical sampling were excluded. Briefly, 32 rats were randomly assigned to two main doses of ibuprofen: Group 1 (G1;200mg/kg) and Group 2 (G2;300mg/kg). Each group was further divided into four subgroups, each comprising four male white Wistar rats. Each animal was assigned a color code to distinguish the results. Importantly, all animals were acclimatized for 1 week in biochemistry laboratory cages prior to any interventions.

Ibuprofen:

After a 7-day acclimation period, the rats were administered specific doses of ibuprofen dissolved in 2 ml of sterile water, using a gastric tube (no. Reg GTL9907111310A1 PT First Medipharma Indonesia). Rats in G1 received ibuprofen at 200mg/kg BW, whereas those in G2 received 300mg/kg BW. All the groups were administered a single dose of ibuprofen orally on day 0. Distilled water was administered following ibuprofen for the control group. Gastric tissue examinations were conducted at 1-, 3-, 5-, and 7-days post-ibuprofen administration. This method is similar to the previous study²⁰.

Scoring system of gastric mucosa:

At the end ibuprofen treatment, the stomachs of the rat were cleaned with 10ml of saline and fixed in 1% formalin for 1 h. At 1-, 3-, 5-, and 7-days post-ibuprofen administration, rats in all groups underwent surgery under ether anesthesia. Antral tissue slices were cleaned, fixed in 10% buffered formalin solution, and paraffin- embedded for histopathological analysis at the Anatomical Pathology Laboratory, Faculty of Veterinary Medicine, Universitas Airlangga.

Additionally, paraffin blocks containing the anterior stomach tissue were cut into 4-mm sections, deparaffinized, dehydrated, cleared, and stained with hematoxylin and eosin (H&E). Stomach damage was assessed based on the following five elements: inflammation, epithelial defects, mucosal atrophy, bleeding, and submucosal edema. Stained sections were viewed, photographed, and scored by an independent examiner using a Nikon E100 microscope (Nikon Instruments Inc., Tokyo, Japan) and a light microscope (Olympus CX21) at the Biomolecular Biochemistry Laboratory, Department of Biomolecular Biochemisty, Faculty of Medicine, Universitas Airlangga. Finally, tissue samples were assessed and graded using Roger’s modified (2012) (table 1) histology activity index (HAI) by an observer blinded to the treatments²¹.

Table 1. Roger’s Modified Criteria

Criteria	Score
No abnormalities	0
Abnormalities <25% visual field	1
Abnormalities 25–50% visual field	2
Abnormalities of 50–75% visual field	3
Abnormalities >75% visual field	4

Statistic analysis:

Roger’s modified HAI scores of stomach damage resulting from ibuprofen treatment are presented as mean ± standard deviation (SD). Mean comparisons were performed using Mann–Whitney U test for non-normally distributed data. All statistical analyses were performed using IBM's Statistical Product and Service Solutions (SPSS) software, version 20.0 and statistical significance was set at p<0.05.

RESULT:

Regarding gastric inflammation severity (figure 1), significant variation was observed between days 1 and 3 and between days 3 and 5 in G1, but not between days 5 and 7. However, there was no significant difference (p = 0.937) in stomach inflammation severity among the four groups (1-, 3-, 5-, and 7-days post-ibuprofen administration). In contrast, there was a significant difference in stomach inflammation severity in G2 between days 1 and 3, 3 and 5, and 5 and 7. However, no discernible difference (p = 0.204) in inflammation was observed among the four groups (1-, 3-, 5-, and 7-days post-treatment). Compared with that in the control group, there was a significant increase in stomach inflammation in G1 and G2 at 1-, 3-, 5-, and 7-days post- ibuprofen administration. In contrast, there was no significant difference in the severity of stomach inflammation between the G1 and G2 at the different time points. Moreover, there were significant differences (P < 0.05) in inflammation severity among the control group, G1, and G2 at 1-day post-treatment. However, there were no significant differences among the three groups (control, G1, and G2) at 3-, 5-, and 7-days post- treatment (p = 0.179, 0.146, and 0.430, respectively).

Figure 1. Comparative images of gastric inflammation in rats. (A) control group, (B) G1 (200mg/kg BW), (C) G2 (300mg/kg BW). Arrows point to the inflammation cells, neutrophils.

In the G1, there were significant differences in the severity of epithelial defects between days 1 and 3, and days 3 and 5, but not between days 5 and 7 (figure 2). However, there was no significant difference (p = 0.556) in epithelial defect severity among the four groups (1-, 3-, 5-, and 7-days post treatment). In contrast, there was a significant difference in epithelial defect severity in G2 between days 1 and 3, 3 and 5, and 5 and 7. However, there was no significant difference (p = 0.704) among the four groups (1-, 3-, 5-, and 7-days post-treatment). Although there was no significant difference between the G1 and G2 at 1-, 5-, and 7-days post treatment, epithelial defect severity was significantly higher in G1 than in the control group at 3 days post treatment. In contrast, there was no significant difference between G1 and G2 at 3-days post-treatment. Compared with that in the control group, there was no significant difference in epithelial defect severity in G2 at 1-, 3-, 5-, and 7-days post-treatment. Additionally, although there were no significant differences in epithelial defect severity among the control group, G1, and G2 at 1-, 5-, and 7-days post-treatment (p = 0.075, 0.393, and 0.067, respectively). However, epithelial defect severity was significantly higher (p = 0.004) in the G1 and G2 than in the control group.

Figure 2. Comparative images of gastric epithelium in rats. (A) control group, (B) G1 (200mg/kg BW), (C) G2 (300mg/kg BW). Arrows point to gastric epithelial erosion.

Regarding mucosal atrophy, there were significant differences in severity between days 1 and 3, 3 and 5, and 5 and 7 in G1 (figure 3). Additionally, there was a significant difference (p = 0.0000) in mucosal atrophy severity among days 1, 3, 5, and 7. In the G2, there were significant differences in mucosal atrophy between days 1 and 3, 3 and 5, and 5 and 7. Moreover, there was a significant difference (p = 0.0000) in mucosal atrophy severity among days 1, 3, and 5 in G2. Although there was no significant difference between G1 and the control group at 1-day post-treatment, mucosal atrophy severity was significantly higher in G2 than in G1. Notably, there were no significant differences among the control group, G1, and G2 at 3-, 5-, and 7-days post-treatment. Additionally, there were no significant differences between the control group and G2 at 1-, 3-, 5-, and 7-days post-treatment. Importantly, there were significant differences among the three groups (control group, G1, and G2) at 1-, 3-, and 5-days post-treatment (p = 0.003, 0.003, and 0.003, respectively).

Figure 3. Comparative images of gastric mucous gland atrophy in rats. (A) control group, (B) G1 (200mg/kg BW), (C) G2 (300 mg/kg BW). Arrows point to the proliferation of connective tissue and the loss of glandular structures.

Regarding gastric bleeding (figure 4), there were notable differences in severity in G1 between days 1 and 3, 3 and 5, and 5 and 7, although these differences were not statistically significant. In contrast, there was a significant difference (p = 0.0000) in the severity of gastric bleeding among days 1, 3, 5, and 7. Notably, there were significant differences in gastric bleeding severity in G2 between days 1 and 3, 3 and 5, and 5 and 7. Additionally, there was a significant difference (p = 0.000) in gastric bleeding severity among Days 1, 3, and 5 in G2. Although there was no significant difference between G1 and the control group at 1-day post-treatment, gastric bleeding was significantly higher in G2 than G1. Moreover, there were significant differences in bleeding severity between the control group and G1 and between G1 and G2 at 3-, 5-, and 7-days post- treatment. Compared with that in the control group, gastric bleeding was significantly higher in G2 at 1-, 5-, and 7-days post-treatment. Importantly, there were significant differences in gastric bleeding severity among the control group, G1, and G2 at 1-, 3-, 5-, and 7-days post-treatment (p = 0.003, 0.003, 0.004, and 0.007, respectively).

Figure 4. Comparative images of gastric mucosal hemorrhage in rats. (A) control group, (B) G1 (200 mg/kg BW), (C) G2 (300 mg/kg BW). Arrows point to erythrocyte infiltration in the gastric mucosa.

Furthermore, there were significant differences in the formation of submucosal edema between days 1 and 3 and 3 and 5, but not between days 5 and 7 in G1 (figure 5). In contrast, there was no significant differences (p = 0.909) in the formation of submucosal edema among days 1, 3, 5, and 7. In G2, there were significant differences in submucosal edema formation between days 1 and 3, 3 and 5, and 5 and 7. Additionally, there were significant differences (p = 0.000) among days 1, 3, and 5. Notably, there were significant differences in submucosal edema formation between the control group and G1 and between G1 and G2 at 1-, 3-, and 5-days post-treatment. Additionally, the presence of submucosal edema was significantly higher in G1 than in the control group at 7-days post-treatment. Although there were no significant differences between the control group and G2 at 1- and 3-days post-treatment, submucosal edema formation was significantly higher in G2 than in the control group at 5- and 7-days post-treatment. Importantly, there was a significant difference (p = 0.003) in submucosal edema formation among the control group, G1, and G2 at 1-day post-treatment; however, there were no significant differences among the groups at 3-, 5-, and 7-days post-treatment (p = 0.463, 0.251, and 0.592, respectively; table 3). Figure 6 summarizes the comparisons between control groups and various doses of ibuprofen-induced gastric mucosal damage, encompassing inflammation, epithelial defects, mucous atrophy, hemorrhage, and submucosal edema. Overall, these results indicate that administering ibuprofen at 200mg/kg BW can significantly damage the gastric mucosa and that a higher dose of 300mg/kg BW can exacerbate gastric mucosal damage.

Figure 5. Comparative images of gastric mucosal edema in rats. (A) control group, (B) G1 (200 mg/kg BW), (C) G2 (300 mg/kg BW). Arrows point to fibrin accumulation in the gastric submucosa, indicating the thickening of the submucosal layer.

Table 2. Results of the comparison between the control group and the effects of ibuprofen on the gastric mucosal at doses of 200 and 300mg/kg BW

Dose	Day						p-value
	Day 1 Mean ± SD Median (min–max)	Day 3 Mean ± SD Median (min–max)	Day 5 Mean ± SD Median (min–max)		Day 7 Mean ± SD Median (min–max)
Inflammation
Control	0.60^a	0.60^a	0.60^a		0.60^a
200 mg (G1)	1.05 ± 0.44	1.20 ± 0.16	1.20 ± 0.69		1.30 ± 0.73		0.937^*
	(1.20 (0.40-1.40))^b,1	(1.20 (1.00–1.40))^b, ²	(1.00 (0.60–2.20))^b, ³		(1.30 (0.40–2.20))^b, ³
300 mg (G2)	3.30 ± 0.34	2.05 ± 0.99	2.25 ± 0.86		2.00 ± 1.21		0.204^*
	(3.40 (2.80–3.60))^c, ¹	(1.90 (1.00–3.40))^b, ²	(2.10 (1.40–3.40))^b, ³		(2.20 (0.40–3.20))^b, ⁴
P-value	0.000^#	0.179^#	0.146^#		0.430^#
Epithelial defect
Control	0.60^a	0.60^a	0.60^a		0.60^a
200 mg (G1)	^1.20±^0.32	^1.70±^0.25	^1.70±^0.82		^1.45±^0.64		0.556*
	(1.20 (0.80–1.60))^b, ¹	(1.70 (1.40–2.00))^b, ²	(1.80 (0.80–2.40))^b, ³		(1.50 (0.80–2.00))^b, ³
300 mg (G2)	^2.35±^0.91	^2.65±^0.44	^2.05±^0.92		^2.25±^0.44		0.704*
	(2.70 (1.00–3.00))^b, ¹	(2.60 (2.20–3.20))^c, ²	(1.90 (1.20–3.20))^b, ³		(2.40 (1.60–2.60))^b, ⁴
P-value	0.075^#	0.004^#	0.393^#		0.067^#
Mucosal atrophy
Control	0.60^a	0.60^a	0.60^a		0.60^a
200 mg (G1)	^1.00±^0.51	^0.50±^0.11	^0.60±^0.23		^0.60±^0.81		0.000**
	(1.00 (0.40–1.60))^b, ¹	(0.50 (0.40–0.60))^a, ²	(0.60 (0.40–0.80))^a, ³		(0.30 (0.00–1.80))^a, ⁴
300 mg (G2)	^2.70±^0.34	^1.30±^1.18	^1.15±^1.23		^0.65±^0.10		0.000**
	(2.70 (2.40–3.00))^a, ¹	(0.90 (0.40–3.00))^a, ²	(0.60 (0.40–3.00))^a, ³		(0.60 (0.60–0.80))^a, ⁴
P-value	0.003^##	0.003^##	0.003^##		0.003^##
Hemorrhage
Control	0.60^a	0.60^a		0.60^a		0.60^a
200 mg (G1)	^0.55±^0.30	^1.20±^0.58		^1.20±^0.74		^0.50±^0.25	0.000**
	(0.40 (0.40–1.00))^a, ¹	(1.20 (0.60–1.80))^b, ²		(1.10 (0.40–2.20))^b, ³		(0.50 (0.20–0.80))^b, ³
300 mg (G2)	3.00 ± 0.63	3.20 ± 0.40		3.30 ± 0.38		2.30 ± 0.70	0.000**
	(2.90 (2.40–3.80))^b, ¹	(3.00 (3.00–3.80))^a, ²		(3.20 (3.00–3.80))^c, ³		(2.30 (1.60–3.00))^c, ⁴
P-value	0.003^##	0.003^##		0.004^#		0.007^#
Submucosal edema
Control	0.60^a	0.60^a		0.60^a		0.60^a
200 mg (G1)	^1.05±^0.59	^1.40±^0.51		^1.20±^0.99		^1.10±^0.73	0.909*
	(1.20 (0.20–1.60))^b, ¹	(1.40 (0.80–2.00))^b, ²		(0.90 (0.40–2.60))^ab, ³		(1.20 (0.20–1.80))^b, ³
300 mg (G2)	^2.95±^0.77	^2.00±^1.50		^2.35±^1.21		^1.25±^0.58	0.000**
	(3.30 (1.80–3.40))^a, ¹	(2.00 (0.60–3.40))^ab, ²		(2.70 (0.60–3.40))^b, ³		(1.10 (0.60–2.20))^b, ⁴
P-value	0.003^##	0.463^#		0.251^#		0.592^#

^a ^b ^c in each column (time) showed no difference between groups

¹ ² ³ ⁴ in each row (dose) showed no difference between groups

*ANOVA

**Friedman Test ^#One-way ANOVA ^##Friedman Test

Figure 6. Comparison of ibuprofen's effects on gastric mucosal damage in control groups, Group 1 (200 mg/kg BW), and Group 2 (300 mg/kg BW).

DISCUSSION:

Ibuprofen is an NSAID associated a low incidence of adverse effects and is considered the safest among other classes of NSAIDs. Ibuprofen can ameliorate fever, pain, and inflammation by inhibiting COX-1 and COX-2, which in turn inhibit PG synthesis¹⁸. In this study, we found that all the assessed parameters related to the gastric mucosa showed varying degrees of severity. Ibuprofen administration at a dose of 200 mg/kg BW induced gastric mucosal damage, such as inflammation, epithelial defects, mucosal atrophy, hemorrhage, and submucosal edema. Notably, an increase in the dose to 300 mg/kg exacerbated these symptoms. Similarly, Alhammadi et al., showed that daily intake of NSAIDs can induce gastrointestinal complications, although the difference was not statistically significant when compared to the control group²².

NSAIDs endanger the gastroduodenal mucosa through various mechanisms, including topical irritation of the epithelium, suppression of gastric PG synthesis, impairment of the healing of superficial wounds, deterioration of mucosal barrier characteristics, and decreased blood flow to the gastric mucosa^5,23. Under normal circumstances, PGs, as biomediators, stimulate the gastric mucosa to secrete bicarbonate ions and protective gastric mucus. Notably, this essential pathway ensures a consistent and healthy blood supply to the gastric mucosa and protects the stomach against stomach acids. Gastric ulceration occurs when any mechanism interferes with the production and activity of PGs, which in turn affects the release of stomach acids and mucus²⁴. Additionally, the ability of NSAIDs to inhibit the activities of COX-1 and COX-2 is another side effect. For example, alterations in the activities of Cox-1 and COX-2 result in a decrease in gastrointestinal mucosal blood flow, downregulation of bicarbonate and mucus output, and poor platelet aggregation^3,25. Additionally, reduced angiogenesis in the lamina propria is accompanied by epithelial cell destruction, particularly in the surface mucous cells. Moreover, leukocytes migration to connective tissues exacerbates oxidative cell damage. Furthermore, an increase in reactive oxygen species production in the tissue triggers lipid peroxidation in the cell membrane, initiating cellular damage²⁶.

Compared with that in the control group and G1, ibuprofen treatment at 300 mg/kg BW exacerbated mucosal atrophy, gastric hemorrhage, and submucosal edema. A separate study involving nine patients ranging from 21 months to 5 years in age reported upper gastrointestinal hemorrhage after receiving 2-4 doses of ibuprofen. Among the patients, only one had a superficial stomach ulcer, whereas the others had acute bleeding gastritis, with no case of anemia or coagulopathy²³. In Denmark, studies on ibuprofen and naproxen have shown a clear trend of increased risk of upper gastrointestinal hemorrhage with increasing dosage²⁷. Importantly, severe cases of pediatric gastrointestinal bleeding have also been reported. For example, two pediatric patients presented with melena and significantly decreased hemoglobin levels had a history of ibuprofen use the week before their presentation, although the specific quantities consumed were unknown²⁸. Importantly, one patient required surgical intervention for a perforated ulcer.

Research on the effect of ibuprofen on blood clotting showed that the drug prevents platelets from aggregating and inhibits the production of thromboxane A2 at 1, 3, 5, and 6 h after oral intake of a single dose of 800 mg, with the effect worsening within 24h²⁹. Importantly, increased neutrophil level in the gastric mucosa following NSAID administration regulates the production of reactive oxygen and nitrogen species and proteases. Although neutrophils are white blood cells, they are unable to kill bacteria in the gastric mucosa. Neutrophils produce compounds harmful to normal tissue³⁰. Administration of ibuprofen to the gastric mucosa also leads to erosion of the gastric epitheliu, possibly due to the activities of TNF-α, a potent extracellular modulator that activates pro-apoptotic caspases in vitro¹⁸.

CONCLUSION:

In this study, we showed that the administration of ibuprofen at doses of 200 and 300 mg/kg BW causes inflammation, epithelial defects, mucosal atrophy, hemorrhage, and submucosal edema in the stomach. However, further studies are required to determine the biomolecular specificity of ibuprofen in larger animal samples.

CONFLICT OF INTEREST:

The authors declared no conflict of interest.

ACKNOWLEDGMENTS:

This study was funded by Faculty Research Priority Scheme Universitas Airlangga contract Number: 212/UN3/2021.

REFERENCES:

1. Adinortey MB, Ansah C, Galyuon I, Nyarko A. In Vivo Models Used for Evaluation of Potential Antigastroduodenal Ulcer Agents. Ulcers. 2013; 2013: 1–12. Hindawi Publishing Corporation. http://dx.doi.org/10.1155/2013/796405.

2. Khoder G, Al-Menhali AA, Al-Yassir F, Karam SM. Potential role of probiotics in the management of gastric ulcer (Review). Exp Ther Med. 2016; 12(1): 3–17. DOI: 10.3892/etm.2016.3293.

3. Roberts-Thomson IC. Rise and fall of peptic ulceration: A disease of civilization? J Gastroenterol Hepatol. 2018; 33(7): 1321–6. Journal of Gastroenterology and Hepatology Foundation and John Wiley and Sons Australia, Ltd. doi:10.1111/jgh.14090.

4. Marlicz W, Łoniewski I, Grimes DS, Quigley EM. Nonsteroidal anti-inflammatory drugs, proton pump inhibitors, and gastrointestinal injury: Contrasting interactions in the stomach and small Intestine. Mayo Clin Proc. 2014; 89(12): 1699–709. Mayo Clinic Proc. http://dx.doi.org/10.1016/j.mayocp.2014.07.015.

5. Melcarne L, García-Iglesias P, Calvet X. Management of NSAID-associated peptic ulcer disease. Expert Rev Gastroenterol Hepatol. 2016; 10(6): 723–33. Taylor and Francis. DOI: 10.1586/17474124.2016.1142872.

6. Abraham P, Indirani K, Desigamani K. Nitro-arginine methyl ester, a non-selective inhibitor of nitric oxide synthase reduces ibuprofen-induced gastric mucosal injury in the rat. Dig Dis Sci. 2005; 50(9): 1632–40. Springer Science. DOI: 10.1007/s10620-005-2908-y.

7. Bradbury F. How important is the role of the physician in the correct use of a drug? An observational cohort study in general practice. Int J Clin Pract Suppl. 2004; (144): 27–32. DOI: 10.1111/j.1742-1241.2004.027_e.x.

8. Chavez ML, DeKorte CJ. Valdecoxib: A Review. Clinical Therapeutics. 2003; 25(3): 817–851. DOI: 10.1016/s0149-2918(03)80110-8.

9. Wahbi AA, Hassan E, Hamdy D, Khamis E, Barary M. Spectrophotometric methods for the determination of Ibuprofen in tablets. Pak J Pharm Sci. 2005; 18(4): 1-6. PMID: 16380350.

10. Varrassi G, Pergolizzi J V, Dowling P, Paladini A. Ibuprofen Safety at the Golden Anniversary : Are all NSAIDs the Same ? A Narrative Review. Adv Ther. 2020; 37(1): 61–82. https://doi.org/10.1007/s12325-019-01144-9.

11. Konturek PK, Brzozowski T, Konturek SJ, Dembiński A. Role of epidermal growth factor, prostaglandin, and sulfhydryls in stress-induced gastric lesions. Gastroenterology. 1990; 99(6): 1607–15. DOI: 10.1016/0016-5085(90)90464-c.

12. Ukawa H, Yamakuni H, Kato S, Takeuchi K. Effects of cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal antiinflammatory drugs on mucosal ulcerogenic and healing responses of the stomach. Dig Dis Sci. 1998; 43(9): 2003–11. Plenum Publishing Corporation. doi: 0163-2116/98/0900-2003. DOI: 10.1023/a:1018846912032.

13. Wang JY, Yamasaki S, Takeuchi K, Okabe S. Delayed healing of acetic acid-induced gastric ulcers in rats by indomethacin. Gastroenterology. 1989; 96(2 PART 1): 393–402. Elsevier. DOI: 10.1016/0016-5085(89)91563-1.

14. Lanza FL, Collaku A, Liu DJ. Endoscopic comparison of gastroduodenal injury with over-the-counter doses of new fast- dissolving ibuprofen and paracetamol formulations: A randomized, placebo-controlled, 4-way crossover clinical trial. Clin Exp Gastroenterol. 2018; 11: 169–77. doi: 10.2147/CEG.S153231.

15. Laine L, Kivitz AJ, Bello AE, Grahn AY, Schiff MH, Taha AS. Double-blind randomized trials of single-tablet ibuprofen/high- dose famotidine vs. ibuprofen alone for reduction of gastric and duodenal ulcers. Am J Gastroenterol. 2012; 107(3): 379 –86. Nature publishing group. doi: 10.1038/ajg.2011.443.

16. Hung KKC, Graham CA, Lo RSL, Leung YK, Leung LY, Man SY, et al. Oral paracetamol and/or ibuprofen for treating pain after soft tissue injuries: Single centre double-blind, randomised controlled clinical trial. PLoS One. 2018; 13(2): 1–13. DOI: 10.1371/journal.pone.0192043.

17. Takeuchi K, Tanaka A, Kato S, Amagase K, Satoh H. Roles of COX inhibition in pathogenesis of NSAID-induced small intestinal damage. Clin Chim Acta. 2010; 411(7–8): 459–66. Elsevier. doi:10.1016/j.cca.2009.12.026.

18. Lazzaroni M, Bianchi Porro G. Gastrointestinal side-effects of traditional non-steroidal anti-inflammatory drugs and new formulations. Aliment Pharmacol Ther. 2004; 20(SUPPL.2): 48–58. DOI: 10.1111/j.1365-2036.2004.02037.x.

19. Wallace JL, McKnight W, Reuter BK, Vergnolle N. NSAID-Induced gastric damage in rats: Requirement for inhibition of both cyclooxygenase 1 and 2. Gastroenterology. 2000; 119(3): 706–14. American Gastroenterological Association. doi:10.1053/gast.2000.16510.

20. Darma A, Riawan W, Sumitro KR, Athiyyah AF, Ranuh R, Qorib MF, et al. Effects of Lactobacillus plantarum IS-10506 on gastric protective mucosal factors: an ibuprofen-induced gastric mucosal injury. Bali Med J. 2023; 12(1): 514–8. DOI: 10.15562/bmj.v12i1.3948.

21. Rogers, A. B. Histologic Scoring of Gastritis and Gastric Cancer in Mouse Models. Methods in Molecular Biology. 2012; 921: 189–203. DOI: 10.1007/978-1-62703-005-2_22.

22. Alhamadi N, Asiri AH, Alshahrani FM, Alqahtani AY, Al Qout MM, Alnami RA, et al. Gastrointestinal Complications Associated With Non-steroidal Anti-inflammatory Drug Use Among Adults: A Retrospective, Single-Center Study. Cureus. 2022; 14(6): 1–10. DOI: 10.7759/cureus.26154.

23. Lichtenberger LM, Richards JE, Hills BA. Effect of 16,16-Dimethyl Prostaglandin E2 on the Surface Hydrophobicity of Aspirin- Treated Canine Gastric Mucosa. Gastroenterology . 1985; 88(1): 308–14. doi.org/10.1016/S0016-5085(85)80185-2.

24. Ugan RA, Un H. The protective roles of butein on indomethacin induced gastric ulcer in mice. Eurasian J Med. 2020; 52(3): 265– 70. DOI: 10.5152/eurasianjmed.2020.20022.

25. Halici Z, Polat B, Cadirci E, Topcu A, Karakus E, Kose D, et al. Inhibiting renin angiotensin system in rate limiting step by aliskiren as a new approach for preventing indomethacin induced gastric ulcers. Chem Biol Interact. 2016; 258: 266–75. Elsevier. DOI: 10.1016/j.cbi.2016.09.011.

26. Toktay E, Selli J. Histopathological Overview of Experimental Ulcer Models. Eurasian J Med. 2022; 54: S120–6. DOI 10.5152/eurasianjmed.2022.22312.

27. Mellemkjær L, Blot WJ, Sørensen HT, Thomassen L, McLaughlin JK, Nielsen GL, et al. Upper gastrointestinal bleeding among users of NSAIDs: A population-based cohort study in Denmark. Br J Clin Pharmacol. 2002; 53(2): 173–81. DOI: 10.1046/j.0306-5251.2001.01220.x.

28. Anyanwu LJC, Mohammad AM. Gastrointestinal bleeding following NSAID ingestion in children. Ann Pediatr Surg. 2013; 9(2): 87–9. DOI: 10.1097/01.XPS.0000428237.26252.84.

29. Evans A, Nation R, Sansom L, Bochner F, Somogyi A. Effect of racemic ibuprofen dose on the magnitude and duration of platelet cyclo‐oxygenase inhibition: relationship between inhibition of thromboxane production and the plasma unbound concentration of S(+)‐ ibuprofen. Br J Clin Pharmacol. 1991; 31(2): 131–8. DOI: 10.1111/j.1365-2125.1991.tb05500.x.

30. Yoshikawa T, Naito Y. The role of neutrophils and inflammation in gastric mucosal injury. Free Radic Res. 2000; 33(6): 785–94. DOI: 10.1080/10715760000301301.

Received on 03.08.2024 Revised on 05.12.2024

Accepted on 08.02.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3530-3536.

DOI: 10.52711/0974-360X.2025.00508

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Alpha Fardah Athiyyah^1,2, Andy Darma^1,2*, Khadijah Rizky Sumitro^1,2, Reza Gunadi Ranuh^1,2,

Rabiatul Adawiah^1,2, Wibi Riawan³, Widjiati Widjiati⁴, Subijanto Marto Sudarmo^1,2

MATERIALS AND METHODS:

Study design:

Animal model:

Ibuprofen:

Scoring system of gastric mucosa:

Statistic analysis:

RESULT:

DISCUSSION:

CONCLUSION:

CONFLICT OF INTEREST:

ACKNOWLEDGMENTS:

REFERENCES:

Alpha Fardah Athiyyah1,2, Andy Darma1,2*, Khadijah Rizky Sumitro1,2, Reza Gunadi Ranuh1,2,

Rabiatul Adawiah1,2, Wibi Riawan3, Widjiati Widjiati4, Subijanto Marto Sudarmo1,2

MATERIALS AND METHODS:

Study design:

Animal model:

Ibuprofen:

Scoring system of gastric mucosa:

Statistic analysis:

RESULT:

DISCUSSION:

CONCLUSION:

CONFLICT OF INTEREST:

ACKNOWLEDGMENTS:

REFERENCES:

Alpha Fardah Athiyyah^1,2, Andy Darma^1,2*, Khadijah Rizky Sumitro^1,2, Reza Gunadi Ranuh^1,2,

Rabiatul Adawiah^1,2, Wibi Riawan³, Widjiati Widjiati⁴, Subijanto Marto Sudarmo^1,2