Journal of Medicine and Health (JMH) Vol. 7 No. 2 August 2025 e-ISSN: 2442-5257 https://doi.org/10.28932/jmh.v7i2.9540 Research Article Comparative Histopathological Effects of Several Non-Selective NSAIDs on Gastric Mucosa in Wistar Rats Komparatif Efek Histopatologis Beberapa NSAID Non-selektif terhadap Mukosa Lambung pada Tikus Wistar Syarinta Adenina1*, Patwa Amani2, Kamalia Layal3 1Bagian Farmakologi, Fakultas Kedokteran, Universitas Sriwijaya, Palembang, Indonesia Jl. Dokter Muhammad Ali, Sekip Jaya, Kec. Kemuning, Kota Palembang, Sumatera Selatan 30114, Indonesia 2Bagian Fisiologi, Fakultas Kedokteran, Universitas Trisakti, Jakarta, Indonesia Jl. Letjen S. Parman No.1, RT.6/RW.16, Grogol, Kec. Grogol petamburan, Kota Jakarta Barat, Daerah Khusus Ibukota Jakarta 11440, Indonesia 3Fakultas Kedokteran dan Ilmu Kesehatan, Universitas Bangka Belitung, Bangka Belitung, Indonesia Gang IV No.1, Balun Ijuk, Kec. Merawang, Kabupaten Bangka, Kepulauan Bangka Belitung 33172, Indonesia *Penulis korespondensi Email: masayusyarintaadenina@fk.unsri.ac.id Received: July 23, 2024 Accepted: April 23, 2025 Abstract Non-selective NSAIDs are widely used for their anti-inflammatory effects. However, their use is limited by significant gastrointestinal side effects. Little is known about which NSAIDs cause more damage to the gastrointestinal. This study aims to compare the mucosal damage induced by various non-selective NSAIDs. Adult male Wistar rats were divided into six groups and administered different NSAIDs: mefenamic acid, diclofenac sodium, piroxicam, ibuprofen, aspirin, and a control group with distilled water. After five days of treatment, the rats were sacrificed, and their gastric tissues were examined both macroscopically and microscopically for signs of damage, including hemorrhagic injury, mucosal edema, epithelial cell loss, and inflammatory cell infiltration. All NSAID-treated groups exhibited significant gastric damage compared to the control group. Diclofenac sodium and piroxicam groups showed the most pronounced hemorrhagic injury, while piroxicam induced the most severe mucosal edema. Ibuprofen caused the highest level of inflammatory cell infiltration. Aspirin resulted in the least gastric histopathological damage score among the tested drugs. Among the tested NSAIDs, aspirin exhibited the least histopathological damage, suggesting a lower risk of gastric injury in this model Keywords: NSAIDs; non-selective; gastric mucosal damage;histopathological examination How to Cite: Adenina S, Amani P, Layal K. Comparative histopathological effects of several non-selective NSAIDs on gastric mucosa in wistar rats. Journal of Medicine and Health. 2025; 7(2): 116-28. DOI: https://doi.org/10.28932/jmh.v7i2.9540 J Med Health.2025;7(2):116-28 116 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Research Article © 2025 The Authors. This work is licensed under a Creative Commons AttributionNonCommercial 4.0 International License. Abstrak NSAID non-selektif masih sering digunakan karena memiliki efek anti-inflamasi yang kuat. Akan tetapi penggunaannya terbatas oleh efek samping gastrointestinal yang signifikan. Informasi mengenai jenis NSAID yang menyebabkan kerusakan saluran gastrointestinal yang lebih parah masih terbatas. Penelitian ini bertujuan untuk membandingkan kerusakan mukosa yang diinduksi oleh berbagai jenis NSAID non-selektif. Tikus Wistar jantan dewasa dibagi menjadi enam kelompok dan diberi perlakuan dengan berbagai jenis NSAID, yaitu asam mefenamat, natrium diklofenak, piroksikam, ibuprofen, aspirin, serta satu kelompok kontrol yang hanya diberikan air suling. Setelah lima hari perlakuan, tikus-tikus tersebut dikorbankan, dan jaringan lambungnya diperiksa secara makroskopik maupun mikroskopik untuk mendeteksi tanda-tanda kerusakan, seperti cedera hemoragik, edema mukosa, kehilangan sel epitel, dan infiltrasi sel inflamasi. Semua kelompok yang menerima perlakuan NSAID menunjukkan kerusakan lambung yang signifikan dibandingkan dengan kelompok kontrol. Kelompok yang menerima natrium diklofenak dan piroksikam menunjukkan cedera hemoragik yang paling parah, sedangkan piroksikam menginduksi edema mukosa yang paling berat. Ibuprofen menyebabkan tingkat infiltrasi sel inflamasi tertinggi, sementara aspirin menghasilkan skor kerusakan histopatologis lambung terendah di antara obat-obatan yang diuji. Disimpulkan, di antara NSAID yang diuji, aspirin menunjukkan kerusakan histopatologis paling ringan, yang mengindikasikan risiko cedera lambung yang lebih rendah dalam model ini. Kata kunci: OAINS; non-selektif; kerusakan mukosa gaster; pemeriksaan histopatologis Introduction More than 30 million people routinely consume NSAIDs as anti-inflammatories, painkillers, antiplatelet drugs, and antithrombotics. NSAIDs have a wide range of side effects such as gastrointestinal ulcers, cardiovascular, kidney toxicity, congestive heart failure, and increased blood pressure. Among these side effects, the most common effect is on the gastrointestinal tract.1–3 Gastrointestinal tract abnormalities range from petechia to ulceration, with complications of hemorrhage and perforation.4 Not only prolonged use, some studies have mentioned that short-term use of NSAIDs is at risk of GI complications, such as gastritis.4,5 Based on their chemical structure or mechanism of action, NSAIDs consist of acetylicylic acid (aspirin), propionic acid derivatives (ibuprofen), acetic acid derivatives (indomethacin and diclofenac), enolic acid derivatives (piroxicam), anthranilic acid derivatives (mefenamic acid), and COX-2 inhibitors (celecoxib).6 Salicylates, acetic acid, enolic acid, and anthranilic acid derivatives are nonselective COX inhibitors.7 COXs are key enzymes that play a role in the prostaglandin synthesis from arachidonic acid. Nonselective NSAIDs can inhibit COX-1 and COX-2. COX-1 plays a role in maintaining the integrity of the gastrointestinal tract. These enzymes play a role in the production and secretion of mucus and bicarbonate, increasing mucosal blood flow and promoting the proliferation of J Med Health.2025;7(2):116-28 117 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Research Article epithelial cells. So that if the COX-1 enzyme is inhibited, unwanted side effects will occur, especially in the gastrointestinal tract.1,2,7,8 COX-2 selective inhibitors have been subsequently developed to avoid this risk, but some COX-2 selective inhibitors have been withdrawn due to increased cardiovascular risk.7 Nonselective NSAIDs remain widely used for their anti-inflammatory effects. Among the existing nonselective NSAIDs, studies have reported varying degrees of selectivity in inhibiting COX isoforms.9 The ratios COX-1/COX-2 ibuprofen, meloxicam, and diclofenac are 0,56, >1, and >1, respectively. Ibuprofen has a ratio of <1, which indicates that this drug is more selective against COX-1, while meloxicam and diclofenac have a ratio of >1, so it is more sensitive to COX-2.8,10 Other studies have presented different conclusions, with some describing ibuprofen as essentially nonselective. There are also conflicting reports on diclofenac's selectivity, with some studies categorizing it as nonselective and others as COX-2 selective. Piroxicam has been found to be COX-1 selective in murine models but nonselective in human blood.8,10 From these previous studies, we can conclude that non-selective NSAIDs have different degrees of effects on the gastrointestinal tract. Although many studies have researched about NSAID gastrointestinal toxic effects on the human gastrointestinal mucosa, however, which nonselective NSAIDs cause damage in gaster still unknown.11 The aim of this study was to compare the mucosal damage in the gaster induced by various non-selective NSAIDs. Methods Animals Adult male Rattus norvegicus, Wistar strain rats (200-300 g, 2-3 months old) were purchased from Abduh Tikus Palembang and used in the present investigation. To prevent bias, adult male rather than female rats were chosen because of physiological variability of female.12,13 Animals were acclimatized to standard laboratory conditions at a constant temperature (25 ± 0.5°C) and light/dark (12/12 h) cycles for 14 days. Rats were kept in polycarbonate cages of 40 cm × 25 cm × 20 cm, with a density of 2 animals per cage to ensure animal welfare and minimize stress due to overcrowding. The cage is placed in a room with controlled environmental conditions including humidity (40–60%), and access to water and feed (Ad libitum). The standard diet composition includes water content (max 14%), protein (17-19%), fat (max 7%), crude fiber (max 7%), ash (max 8%), calcium (0.9-1.2%), total phosphorus (with enzymes, 0.6-1%), total alphatoxin (max 50 µg/kg), amino acid lysine (min 0.9%), methionine (min 0.3%), methionine + cystine (min 0.6%). The feed ingredients include corn, rice bran, soybean meal, copra, meat and J Med Health.2025;7(2):116-28 118 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Research Article bone meal, CGM, rice fragments, DDGs, and palm oil. The protocols for animal research were systematically evaluated by the Ethics Committee Medical Faculty of Sriwijaya University (No 177-2022) Drugs, Chemicals and Reagent Kits All used chemicals, solvents and reagents were purchased and obtained from authorized sources and of analytical grade. Ibuprofen (Ibuprofen 400 mg tablets; Phapros Tbk, Indonesia), Piroxicam (Piroxicam 20 mg tablet; Kimia Farma, Indonesia), Mefenamic acid (Mefenamic Acid 500 mg coated tablet; Dexa Medica, Indonesia), Diclofenac sodium (Diclofenac sodium 50 mg, coated tablet; Kimia Farma, Indonesia), and Aspirine (Acetylsalicylic acid 80 mg, tablet; Medifarma Laboratories, Indonesia) were used throughout the study. The doses of each drugs were determined by using Laurence & Bacharach conversion dosage methods.14 The therapeutic dose for rat weighing 200 g = 18/1000 × adult human therapeutic daily dose (Ibuprofen 400 mg, Piroxicam 20 mg, Mefenamic acid 500 mg, Diclofenac sodium 50 mg and aspirin 80 mg) = 7,2 mg, 0,36 mg, 9 mg, 0,9 mg and 1,476 mg.15 The exact LD₅₀ for gastric-specific lethality remains undefined for each drugs, however the dosages used in this study were based on the standard therapeutic doses for humans, adjusted using the Laurence and Bacharach conversion method. Moreover, previous studies have demonstrated that doses exceeding those administered in our study have been associated with gastritis in rats. Studies have shown that ibuprofen’s gastric toxicity in Wistar rats is dosedependent, with mucosal damage observed even at moderate doses (e.g., 44 mg/kgBW or 8,8 mg/200 gr).16 Lisna and Stepchenko have proved that ibuprofen 400mg can induced morphological changes such as petechial hemorrhages, foci of necrosis and the formation of erosions.17 Others also stated that Iburpofen (50 mg/ kg or 10 mg/200 gr) can cause gastric ulceration by inhibition of serum PGE2 and TXA2 levels.18 The use of piroxicam (30 mg/kgBW or 6 mg/200mg) resulted in large number of lesions on the gastric mucosa.19 Meanwhile Tilarso et all has proved that piroxicam 1.8 mg/kgBW or 0,36 mg/200 mg can caused ulcer and bleeding in gaster.20 Mefenamic acid is a non-steroidal antiinflammatory drug (NSAID) that is commonly used for pain relief and to reduce inflammation. The oral LD₅₀ of mefenamic acid in rats is approximately 740 mg/kg or 148mg/200gr).21 The therapeutic doses of mefenamic acid in rats typically range from 25 mg/kg to 200 mg/kg, depending on the specific experimental context and objectives.22,23 9 mg mefenamic acid/200 g BW also have been used by Ariasti and Muhsin to study the analgesic effect of the drugs.24 J Med Health.2025;7(2):116-28 119 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Research Article Elnashar et al. stated that Sprague-dawley rats which received diclofenac (3,5 mg/kgBW p.o) had small ulcers in the stomach.25 Crowe and Kinsey have stated that diclofenac can induce gastric hemorrhages at doses of ≥ 33.33 mg/kg in male mice and 100 mg/kg in female mice.26 Aspirin oral induction dose of 400 mg/kg BW or 80 mg/200 gr have been used to induced ulcers.27,28 Meanwhile, Marcellinus et all shows aspirin 200 mg/kg BW or 40 mg/200 mg will caused ulceration of the gastrointestinal tract (GIT) lining.29 All substances used in this study were dissolved in distilled water. Experimental Design After acclimatization, animals were divided randomly into 6 groups, each group contain of 2 rats. The treatments were administered orally once daily using oral feeding tube. Before oral administration of drugs, animals were fasted for 16 h. Group I: normal control group receiving distilled water, group II: receiving mefenamic acid (63 mg/kg), group III: receiving diclofenac acid (6.3 mg/kg), group IV: receiving piroxicam (2.52 mg/kg), group V: receiving ibuprofen (5.4 mg/kg) and group VI receiving aspirin (63 mg/kg). After 5 days treatments, animals were anesthetized by ether inhalation after 1 h fasting. Experimental studies show that NSAIDs can induce measurable gastric damage within a short timeframe (from hours to days of administration).30–32 Previous studies have proved that 5 days of NSAID resulted in significant histopathological changes in Wistar rats, indicating that this duration is sufficient to observe NSAID-induced gastric lesions.33 While acute damage can occur within hours or days, shorter durations may not allow sufficient time for cumulative effects to manifest fully. Prolonged exposure could also lead to complications by causing compensatory mechanisms that could affect the study outcomes.34 Animals were carefully dissected, and the stomachs were removed by pyloric ligation for gastric juice collection. After that, the animals were sacrificed by cervical dislocation. The gaster then prepared to morphological and microscopic examination. Determination of gastric acidity Gastric content was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. The pH of the supernatant was examined by pH universal strips.35,36 Morphological and Microscopic Histopathological Examination Rat’s gasters were cut along the smaller curvature, washed with saline (0,9% NaCl) expand, and fixed on the dissection plate with their edges held with the aid of office pins. Gaster J Med Health.2025;7(2):116-28 120 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Research Article then photographed using an appropriate digital camera. The ulcer area will look redder in color than the normal gastric area.37 After visualization, gaster tissues were prepared for microscopic examination. The gaster tissue was fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 4 µM thick sections by slide microtome. To detect microscopic gastric injury, the sections were stained with hematoxylin and eosin using standard procedures, followed by examination under light microscopy. Histopathological changes were assessed by an experienced and qualified pathologist blinded to treatments. Gastric structure was determined on a 0-4 scale, and each tissue section was examined for hemorrhagic damage (0–4), mucosal edema (0–4), presence of inflammatory cells infiltration (0–3), and epithelial cell loss.38 Results The measured gastric pH values in each group are presented in Table 1. The pH of gastric juice in the stomachs of rats in the NSAID groups was slightly decreased compared to the normal group. The macroscopic examination of the gastric mucosa from the control group revealed healthy pink-colored gastric mucosa without any lesions, showing no visible signs of gastritis or ulcers. In contrast, redness was observed in the gaster tissue of all NSAID groups (mefenamic acid, diclofenac sodium, piroxicam, ibuprofen, and aspirin). Additionally, multiple erosions were found in the mefenamic acid group (Figure 1). In the control group, microscopic examination showed a normal histological structure lined by stratified squamous keratinized epithelium with underlying submucosa and muscularis, with no signs of hemorrhages, congestion, or mucosal epithelium disturbances (Fig. 2a). Table 1 Gastric Content Acidity Group I II III IV V VI J Med Health.2025;7(2):116-28 Groups Aquadest Mefenamic Acid Diclofenac Natrium Piroxicam Ibuprofen Aspirin pH 6 4-5 4-5 4-5 5 5 121 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Research Article Administration of mefenamic acid, diclofenac sodium, piroxicam, ibuprofen, and aspilet all resulted in substantial variations in the gaster’s histological appearance, including epithelial cell loss, hemorrhagic injury, edema, and inflammatory cell infiltration. The differences between the groups were notable, with some changes being more prominent than others. Hemorrhagic injury was more dominant in the diclofenac sodium and piroxicam groups. Edema was more pronounced in the piroxicam group. Ibuprofen administration caused more inflammatory cell infiltration than other NSAIDs. Overall, based on the total score, aspilet caused the least damage compared to other NSAIDs. Group I Group II Group III Group IV Group V Group VI Figure 1 Gaster Macroscopic I: Aquadest; II: Mefenamic Acid; III: Diclofenac Acid; IV: Piroxicam; V: Ibuprofen; VI: Aspilet J Med Health.2025;7(2):116-28 122 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Research Article (a) Group I, 40x and 400x (b) Group II, 40x and 400x (c) Group III, 40x and 400x (d) Group IV, 40x and 400x (e) Group V, 40x and 400x (f) Group VI, 40x and 400x Figure 2 Histological Structure of Gaster with H&E Stain, 40x and 400x Blue arrow shows epithelial erosion, green arrow shows inflammatory cells infilitration (lymphocyte), and yellow arrow shows bleeding. I: Aquadest; II: Mefenamic Acid; III: Diclofenac Acid; IV: Piroxicam; V: Ibuprofen; VI: Aspilet.. (E): Stratified squamous keratinized epithelium; (MU): Underlying submucosa (MU); (MM): Muscularis; (G): Gastric glands. J Med Health.2025;7(2):116-28 123 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Research Article (a) (b) (c) (d) Figure 3 Histopathological Examination Tabulation I: Aquadest; II: Mefenamic Acid; III: Diclofenac Acid; IV: Piroxicam; V: Ibuprofen; VI: Aspilet. Discussion NSAID are anti-inflammatory drugs that have been used widely in the world. Many studies have reported GI complications caused by these drugs. The most common diseases by NSAID usage are erosive-hemorrhagic gastritis (20-40%) and peptic ulcer (10-30%).3 In this study, we observed that gastric juice pH are slightly lower compare with the control group. Normal gastric juice pH rats from other studies are around 6,5±0,08.39 Gastric pH secretion occurs due to the activity of the parietal cells H+/K+ ATPase which is controlled by acetylcholine, gastrine, histamine and intracellular signals (cAMP and calcium signalling pathways).40 The decrease in pH occurs due to an increase in the gaster epithelial layer's permeability, resulting in the acid's reverse diffusion into the lumen. The prostaglandin inhibitory effect by NSAIDs also leads to increased gastric acid secretion.41 However, other studies have stated that ulcers caused by NSAIDs is independent of gastric acid.42 21 NSAIDs caused by ulcers are characterized by hyperplasia and mucosal edema, but only a small infiltration of inflammatory cells.43 The gaster has a mucosal lining as the first line of defense against external stimuli.44 Several studies have indicated that NSAIDs can induce gastrointestinal damage in the mucosa, J Med Health.2025;7(2):116-28 124 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Research Article such as ulcers, erosion especially in epithelial cells, cell infiltration, congestion of blood vessels, and leukocyte aggregation.45,46 Macroscopic and microscopic examinations revealed no significant differences among the NSAID groups, although some groups exhibited more pronounced damage in specific aspects, possibly due to the duration and dose of NSAID administration.5 The condition of gastric mucosal in this study was similar to Santos et all, Tandoh et all, Eliot et all, Wang et all, and Firdaus et all. So, it can be concluded that mefenamic acid, diclofenac natrium, piroxicam, ibuprofen and aspirin could damage gaster.37,41,47–50 These studies proved all NSAID can induce epithelium cell loss. Gastric mucosal lession happen through local and systemic. Some NSAID can directly damage epithelial cells. Various mechanisms have been proposed for this cytotoxic action. NSAIDs with a carboxylic acid residue can enter epithelial cells in the gaster. When these cells are intracellular, the drug will be converted into an ionic form and cannot escape. This is known as ion trapping. The more drugs accumulate in the epithelial cells, the swelling of the epithelial cells will occur and cause lysis.51 Meanwhile the systemic actions occur through its ability to suppress gastric prostaglandin synthesis.52 Inhibition in COX-1 will cause a decrease in prostaglandins. These prostaglandins play an important role in maintaining mucosal integrity, blood flow and stimulating mucus and bicarbonate secretions.41 A decrease in mucus and bicarbonate secretion will lead to a decrease in the effectiveness of the pH gradient of juxtamucosal that protects the epithelium.51 NSAIDs can also trigger the production of free radicals that can interact with adenyl cyclase and alter the concentration of cAMP. The use of NSAIDs also causes microvascular damage, decreased blood flow, and increased expression of adhesion molecules and neutrophil molecules in the vascular epithelium.37,47–49 NSAID drugs effect was dose-dependent. Drugs used in this study have lower dose compared to the previous study.41 In this study, hemorrhage was most frequently observed in the piroxicam and diclofenac sodium groups. Diclofenac is known to have a selective effect in inhibiting COX-2 compared to COX-1. Prostaglandins derived from COX-2 are protective against damage induced by hepatotoxins. Rats given diclofenac showed gastrointestinal damage evident in histopathological changes. Similar to the studies conducted by Mostafa et al, the diclofenac group exhibited abnormal histological structures manifested by epithelial erosion, mucosal edema, bleeding, along with inflammatory cellular infiltration in the submucosa.53 In this study the piroxicam group was the most severe mucosal edema. Gastritis mucosal oedema can be caused by vascular changes due to inflammation.54 The administration of aspirin in this study caused the least amount of bleeding. Low-dose aspirin in rats is known not to cause bleeding, but it can result in mucosal damage and epithelial cell exfoliation43,51,55 J Med Health.2025;7(2):116-28 125 Journal of Medicine and Health Vol. 7 No. 2 August 2025 Comparative Histopathological Effects of… e-ISSN: 2442-5257 Research Article This study has limitations that should be acknowledged, such as a small sample size will reduce the statistical power of the findings and the method used to assess gastric pH. Future studies should be conducted to investigate the effect of NSAIDs by using larger sample sizes and standardized pH meter measurements. Conclusion The study demonstrates that all tested NSAIDs induced histopathological damage to the gastric mucosa, although the severity and specific patterns of injury varied among them. Piroxicam induced the most pronounced mucosal edema, while ibuprofen led to the greatest inflammatory cell infiltration. 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