Use of Callistemon citrinus as a gastroprotective and anti-inflammatory agent on indomethacin-induced gastric ulcers in obese rats

Abstract

Background: Obesity leads to an elevated risk of developing gastrointestinal disease such as gastric ulcers. Callistemon citrinus leaf extract has shown antioxidant, antimicrobial, hepatoprotective, and chemoprotective effects against colon cancer. The aim of this study is to evaluate the gastroprotective effect of C. citrinus leaf extract on indomethacin-induced gastric ulcers in obese rats.

Methods: Gastric ulcers were induced in female obese Wistar rats using a single oral dose of indomethacin (IND). In the first stage, the rats were fed with a high fat sugar diet (HFSD) for 15 weeks to induce obesity and, at the same time, the diet of the other group of animals included daily administration of ethanolic C. citrinus leaf extract (250 mg/kg) in addition to HFSD. In the second stage, gastric ulcers were induced with IND (30 mg/kg). The gastroprotective activity of C. citrinus, the inflammatory enzyme activities, and cytokines in the stomach were determined.

Results: C. citrinus produced a reduction of gastric lesions caused by IND. Myeloperoxidase (MPO), cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) activities also decreased. Although inflammatory biomarkers such as TNFα, IL-6, AOPP, and leptin were significantly decreased by C. citrinus, adiponectin levels increased. Moreover, C. citrinus decreased weight gain and morphological and biochemical parameters.

Conclusion: The use of indomethacin in rats fed with a high fat-sugar diet increased gastric ulcers. Gastroprotective effect of C. citrinus in obese rats is attributed to the reduction of pro-inflammatory cytokines and the inflammatory enzymes.

Keywords: Biomarkers; Callistemon citrinus; Gastric ulcers; Indomethacin; Inflammatory; Obesity.

Figure 1. Final weight gain of the groups fed for 15 weeks with a diet high in fat and sugar (HFSD) and extract of C. citrinus (250 mg/kg) (C.c), and control group (C).

Data shown as mean ± S.E.M. (ANOVA one-way followed by Tukey test, values statistically different (^A,B) among groups (*p < 0.05), n = 6).

Figure 2. Gastroprotective effect of Callistemon citrinus.

Rat stomach showing protective effect of Callistemon citrinus (C.c) on the indomethacin (IND) induced gastric ulcer in Wistar rat. Control (A); HFSD + IND (B); IND (C); HFSD + C.c 250 mg/kg + IND (D); C.c 250 mg/kg + IND (E); Omeprazole (OME) + IND (F). Indomethacin was administrated at 30 mg/kg for a single dose in B to F. CA, Cardial region; FU, Fundus region and PY, Pyloric region. (A) showed intact gastric mucosa. (B and C) presented several dark red submucosal regions (red arrows). In (D–F) showed a normal gastric mucosa with few lesions.

Figure 3. Effect of Callistemon citrinus on gastric lesions generated by indomethacin (IND) in rats fed with a high-fat diet (HFSD). Microphotographs show representative cuts of the fundic region.

(A) The graph shows the values of the histopathological score that considers jointly the epithelial cell loss of the gastric mucosa (a), hemorrhage (b), the inflammatory cell infiltration (c), the lamina propria mucosae erosions (d), and the edema and disruption in the submucosa (e). Gastric mucosa (MU), Gastric epithelium (GE), Muscularis mucosae (MM), Submucosal (SM), external muscular (ME) and lamina propria (LP). Scale bar = 200 µm. (B, left) The control rats fed with normal diet, one group fed with HFSD and other group fed with HFSD + C. citrinus, chronically treated during 15 weeks; after this period, one single dose of indomethacin was given for the last two groups. (B, right) One group treated with a single dose of indomethacin, one group treated with a single dose of indomethacin + C. citrinus and the last group treated with a single dose of indomethacin + omeprazole. Data shown as mean ± S.E.M. (ANOVA one-way followed by Tukey test, values statistically different (^A,B,C) among groups (*p < 0.05), n = 6). Score assignment was done according to previously described criteria (Siriviriyakul et al., 2020).

Figure 4. Myeloperoxidase activiy.

Enzyme activity of myeloperoxidase in experimental groups. (A) The control rats fed with normal diet, one group fed with HFSD and other group fed with HFSD + C. citrinus, chronically treated during 15 weeks; after this period, one single dose of indomethacin was given for the last two groups. (B) One group treated with a single dose of indomethacin, one group treated with a single dose of indomethacin + C. citrinus and the last group treated with a single dose of indomethacin + omeprazole. Data shown as mean ± S.E.M. (ANOVA one-way followed by Tukey test, values statistically different (^A,B) among groups (*p < 0.05), n = 6).

Figure 5. Cyclooxygenase-1 activity.

Effect of indomethacin and C. citrinus extract on the activity of COX-1. (A) The control rats fed with normal diet, one group fed with HFSD and other group fed with HFSD + C. citrinus, chronically treated during 15 weeks; after this period, one single dose of indomethacin was given for the last two groups. (B) One group treated with a single dose of indomethacin, one group treated with a single dose of indomethacin + C. citrinus and the last group treated with a single dose of indomethacin + omeprazole. Data shown as mean ± S.E.M. (ANOVA one-way followed by Tukey test, values statistically different (^A,B) among groups (*p < 0.05), n = 6).

Figure 6. Cyclooxygenase-2 activity.

Effect of indomethacin and C. citrinus extract on the activity of COX-2. (A) The control rats fed with normal diet, one group fed with HFSD and other group fed with HFSD + C. citrinus, chronically treated during 15 weeks; after this period, one single dose of indomethacin was given for the last two groups. (B) One group treated with a single dose of indomethacin, one group treated with a single dose of indomethacin + C. citrinus and the last group treated with a single dose of indomethacin + omeprazole. Data shown as mean ± S.E.M. (ANOVA one-way followed by Tukey test, values statistically different (^A,B,C) among groups (*p < 0.05), n = 6).

Figure 7. 5-Lipooxygenase activity.

Effect of indomethacin and C. citrinus extract on the 5-LOX activity. (A) The control rats fed with normal diet, one group fed with HFSD and other group fed with HFSD + C. citrinus, chronically treated during 15 weeks; after this period, one single dose of indomethacin was given for the last two groups. (B) One group treated with a single dose of indomethacin, one group treated with a single dose of indomethacin + C. citrinus and the last group treated with a single dose of indomethacin + omeprazole. Data shown as mean ± S.E.M. (ANOVA one-way followed by Tukey test, values statistically different (^A,B) among groups (*p < 0.05), n = 6).