Abstract

Introduction:

Aspirin-induced gastritis results from reduced prostaglandin synthesis and increased oxidative stress, compromising gastric mucosal integrity. While omeprazole effectively suppresses gastric acid secretion, it does not directly promote mucosal regeneration. Striatin, a bioactive fraction of Channa striata, has anti-inflammatory and regenerative properties and may enhance mucosal healing.

Purpose:

This study aimed to evaluate whether adjunctive striatin at escalating doses enhances the therapeutic effect of omeprazole in aspirin-induced gastric injury.

Methods:

A controlled experimental study was conducted in 30 male Wistar rats randomly assigned into six groups (n = 5 each): healthy control, negative control, omeprazole monotherapy (20 mg/kg BW), and three combination groups receiving omeprazole plus striatin (500, 1,000, and 1,500 mg/kg BW). Gastric injury was induced using aspirin. Vascular endothelial growth factor (VEGF; day 2) and intestinal trefoil factor (ITF; day 14) were measured as markers of angiogenesis and epithelial restitution, respectively. Histological and macroscopic assessments were performed at the end of treatment.

Results and Discussion:

VEGF levels did not significantly differ across groups. In contrast, ITF significantly increased in the highest-dose striatin group (1,500 mg/kg BW, p = 0.007), which also demonstrated improved epithelial thickness, reduced ulcer area, and lower inflammation scores. Lower doses did not produce significant changes in ITF levels.

Conclusion:

Adjunctive striatin, particularly at higher doses, enhances gastric mucosal healing primarily through epithelial restitution rather than angiogenesis. These findings suggest potential adjunctive benefits, although further studies are needed to establish optimal dosing and clinical relevance.

1 Introduction

Aspirin is a major cause of gastric mucosal injury because cyclooxygenase inhibition reduces prostaglandin-mediated epithelial and microvascular protection (Vane and Botting, 2003). The resulting ischemia, oxidative stress, and epithelial necrosis initiate leukocyte recruitment and cytokine release, amplifying mucosal damage. Gastric ulcer healing then progresses through tightly coordinated phases of epithelial regeneration, granulation tissue formation, angiogenesis, and matrix remodeling, culminating in scar restoration. These events are regulated by interconnected growth factor, cytokine, and transcription factor signaling networks (Vane and Botting, 2003; Treuting et al., 2018; Braga Emidio et al., 2020).

Taken together, current evidence suggests that effective gastric ulcer healing requires more than luminal acid suppression. Although proton pump inhibitors such as omeprazole remain central to the management of NSAID-related gastropathy, their therapeutic effect is primarily limited to reducing gastric acidity and stabilizing the intraluminal environment. Acid suppression alone does not directly activate epithelial restitution, growth factor signaling, or late-phase mucosal remodeling, processes that are critical for complete structural and functional recovery of the gastric barrier (Bjarnason et al., 2018). With increasing reports of long-term PPI-associated adverse effects, there is growing interest in therapeutic strategies that target endogenous regenerative pathways to achieve more complete mucosal recovery. Consequently, there is increasing interest in adjunctive strategies that can biologically complement acid suppression by actively engaging endogenous regenerative pathways.

Striatin, a standardized bioactive protein fraction derived from Channa striata, has emerged as a promising regenerative adjunct due to its documented anti-inflammatory, antioxidant, and wound-healing properties. Unlike conventional gastroprotective agents, striatin targets epithelial repair biology by modulating trefoil factor signaling and supporting cellular migration, barrier restitution, and tissue remodeling (Rahayu et al., 2016; Hapsari and Tjandrawinata, 2025; Yulizal et al., 2020). Importantly, gastric healing is a temporally structured process in which angiogenic mediators such as vascular endothelial growth factor (VEGF) dominate the early repair phase, whereas epithelial restitution markers such as intestinal trefoil factor (ITF) become more relevant during the later stages of mucosal recovery (Thim and May 2022). However, the extent to which a dose-escalated regenerative adjunct can differentially influence these temporally distinct healing pathways when combined with acid suppression remains insufficiently characterized.

Therefore, this study was designed to evaluate whether escalating doses of striatin, administered as an adjunct to omeprazole, can shift the gastric healing phenotype beyond acid suppression alone in an aspirin-induced gastric injury model. By integrating biochemical markers representing early angiogenic response (VEGF) and late epithelial restitution (ITF) with histopathological and macroscopic healing indices, this study aims to clarify not only whether combination therapy improves outcomes but also how it modulates the biological trajectory of gastric mucosal repair.

Although previous studies have demonstrated the gastroprotective effects of Channa striata and its derivatives as monotherapy, the potential benefit of combining striatin with proton pump inhibitors has not been well established. In particular, it remains unclear whether such combination therapy can enhance mucosal healing beyond acid suppression via distinct biological pathways, including angiogenesis and epithelial restitution. Therefore, this study specifically evaluates the adjunctive role of dose-escalated striatin combined with omeprazole using integrated biochemical and histopathological outcomes.

2 Materials and methods2.1 Study design

This research used a controlled experimental laboratory design to evaluate whether combining omeprazole with graded doses of striatin provides greater mucosal protection and repair than omeprazole alone in aspirin-induced gastric injury. The research was conducted at the Laboratory for Animal Experimentation, Faculty of Medicine, Universitas Diponegoro, Semarang, Indonesia, using 30 male Wistar rats assigned to a pre-test–post-test control group model. Animals were randomly allocated into six groups: a healthy control group (K0), a negative control group (K−), a positive control group (K+) receiving omeprazole monotherapy (20 mg/kg BW), and three treatment groups (P1, P2, and P3) receiving a combination of omeprazole (20 mg/kg BW) with striatin at doses of 500, 1,000, and 1,500 mg/kg BW, respectively. Animals were randomly allocated using a computer-generated randomization sequence. All procedures were conducted under standardized conditions, and investigators performing the assessments were blinded to treatment allocation to minimize observational bias.

2.2 Animals and ethical approval

Male Rattus norvegicus Wistar rats (8–12 weeks old; 200–300 g) were used in this study. Animals were housed in a controlled environment maintained at 22 °C–24 °C with 70%–75% relative humidity and a 12-h light–dark cycle. Rats were accommodated in sanitized polypropylene group cages (50 × 40 × 20 cm; three rats per cage) with corn cob bedding and were provided standard BR-594 chow and water ad libitum. Animals were acclimatized for 7 days before experimentation to reduce physiological stress. All procedures for animal handling, housing, and welfare followed the ARRIVE 2.0 guidelines and international standards for laboratory animal care. Ethical approval was obtained from the Health Research Ethics Committee of the Faculty of Medicine, Universitas Diponegoro/RSUP Dr. Kariadi Semarang (Ethical Clearance No.: 110/EC/KEPK/FK-Undip/V/2025). Sample size was determined using power analysis to achieve sufficient statistical power while adhering to the principles of the 3Rs (replacement, reduction, and refinement).

2.3 Gastric injury induction and treatment procedures

Gastric injury was induced using oral aspirin, which induces mucosal erosions through prostaglandin inhibition and direct epithelial irritation. Prior to induction, rats were fasted for 24 h with free access to water. Aspirin was administered orally at a dose of 450 mg/kg body weight, prepared by suspending the powder in 0.5% CMC. The aspirin suspension was delivered once daily via intragastric gavage for 18 consecutive days to all rats except those in the healthy control group. Animals were observed throughout the induction period to verify the successful establishment of the gastric injury model before initiating treatment.

After aspirin induction, the animals proceeded to the treatment phase and received either omeprazole alone or omeprazole in combination with increasing doses of striatin. The negative control group received aspirin induction without therapeutic intervention, whereas the positive control group was administered omeprazole monotherapy at a dose of 20 mg/kg body weight. All treatments were delivered once daily via intragastric gavage for 14 days (day 26 to day 39) to ensure consistent and controlled dosing.

Omeprazole was prepared at a dose of 20 mg/kg BW by suspending the powdered formulation in 0.5% CMC, and the suspension was administered orally. Striatin, supplied by Dexa Laboratories of Biomolecular Sciences (DLBS; Bekasi, West Java, Indonesia), was obtained from Channa striata fillets through water-based extraction followed by fractional purification, concentration, and stabilization with sodium alginate, resulting in a standardized bioactive protein fraction produced under Good Manufacturing Practice conditions. Striatin was administered at doses of 500, 1,000, and 1,500 mg/kg BW, prepared in 0.5% CMC, and co-administered with omeprazole throughout the 14-day treatment period. All administrations were performed via oral gavage to maintain dosing precision and uniformity.

2.4 Blood and gastric tissue collection

Blood samples were collected from the retro-orbital vein. Before the intervention phase, baseline serum sampling was performed in all rats on day 22 for VEGF measurement and day 24 for ITF measurement. VEGF was re-measured on day 27 (early treatment phase; day 2 of intervention) to capture early angiogenic response, while ITF was re-measured on day 39 (end of treatment) to reflect late epithelial restitution. Approximately 100 µL of serum was collected for each assay at every time point. Gastric tissue was obtained from all groups on day 39, 24 h after the final intervention dose.

2.5 Biochemical analysis

Levels of VEGF and ITF were quantified by sandwich ELISA, with all samples analyzed in duplicate according to the manufacturer’s instructions. Blood was centrifuged at 1000 g to separate serum, which was then stored at −20 °C to −80 °C until analysis to prevent degradation from repeated freeze–thaw cycles. Calibrators were prepared through serial dilution to generate an eight-point standard curve. For each assay, 100 µL of standards or samples was added to the wells and incubated at 37 °C, followed by sequential incubation with detection reagents A and B, substrate addition, and termination of the reaction. Absorbance was measured at 450 nm using a microplate spectrophotometer.

2.6 Macroscopic evaluation of gastric ulcer area

Following euthanasia, stomachs were dissected, opened along the greater curvature, gently rinsed with saline, and positioned flat for macroscopic examination. Ulcerative lesions were identified as distinct areas of hemorrhage or necrosis. Digital images were obtained under standardized lighting conditions by examiners blinded to treatment allocation. Gastric ulcer severity was assessed using a standardized scoring scale based on lesion number and diameter: 0 = no lesion; 1 = edematous/erythematous mucosa; 2 = 1–5 small lesions; 3 = 5 small or 1 moderate lesion (3–4 mm); 4 = ≥2 moderate lesions or 1 large lesion (>4 mm); and 5 = perforated ulcer. Macroscopic evaluations were performed by an expert anatomical pathologist, with representative findings shown in Figure 1.

Macroscopic evaluation of gastric ulcers. Black arrows indicate ulceration; blue arrows indicate mucosal erythema.

2.7 Histological assessment and epithelial thickness

Histopathological slide preparation was performed according to the standardized protocol established in the dose-finding phase. Gastric tissue samples were fixed in buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Histopathological parameters were assessed, including gastric epithelial thickness, which was measured microscopically using a calibrated ocular micrometer across five representative fields per specimen. Epithelial thickness was also evaluated categorically in comparison with the healthy control group (K0) and classified into four categories: 25%–50%, 50%–75%, 75%–100%, or 100% of normal mucosal thickness. All histopathological evaluations were conducted by an expert anatomical pathologist blinded to treatment allocation, as illustrated in Figure 2. However, because a semi-quantitative scoring system was used, observer bias cannot be entirely excluded.

Gastric histopathological findings in each group (hematoxylin and eosin staining, ×100). Black arrows indicate inflammatory cell infiltration, and blue arrows indicate areas of gastric erosion.

2.8 Inflammation scoring

The inflammatory response was evaluated using a semi-quantitative scoring system based on the degree of inflammatory cell infiltration and the extent of mucosal disruption. Inflammation severity was classified into four categories: no infiltration, mild infiltration, moderate infiltration, and severe infiltration. Histopathological assessment was performed by an expert anatomical pathologist blinded to the treatment allocation to minimize assessment bias, as shown in Figure 2. The use of a semi-quantitative scoring system may introduce subjectivity despite blinding procedures.

2.9 Statistical analysis

Data were expressed as the mean ± standard deviation (SD) or median (Q1–Q3), as appropriate. Normality was assessed using the Shapiro–Wilk test. Pre- and post-intervention comparisons were analyzed using the paired t-test or Wilcoxon test, while between-group comparisons were analyzed using the independent t-test or Mann–Whitney test. For multiple group comparisons, one-way ANOVA with post-hoc testing was used for normally distributed data, and the Kruskal–Wallis test followed by the Mann–Whitney test was applied otherwise. Delta values were calculated to assess changes from baseline and were analyzed accordingly. Statistical significance was defined as p ≤ 0.05. No formal correction for multiple comparisons was applied; therefore, the results should be interpreted with caution due to the potential increased risk of type I error.

3 Results3.1 Characteristics of experimental animals

A total of thirty male Wistar rats were enrolled and allocated into six groups after a seven-day acclimatization period under controlled housing conditions. Baseline characteristics, including body weight and general condition, were comparable across groups, and no clinically relevant differences were observed prior to treatment administration.

3.2 Vascular endothelial growth factor

Serum VEGF concentrations varied across individuals but showed comparable baseline levels among groups. Only the positive control group exhibited a significant within-group increase after treatment, while the remaining groups did not demonstrate significant changes. Between-group comparisons after treatment showed no statistically significant differences (p > 0.05). These findings indicate that neither omeprazole monotherapy nor its combination with striatin significantly altered VEGF levels under the current experimental conditions. The complete VEGF results are presented in Table 1.

GroupVEGF preVEGF post (minimum–maximum)PΔVEGFK07.53 (6.01–12.69)4.50 (3.51–14.26)0.500†−4.02 (−4.65–8.25)K–11.65 (7.02–20.62)4.26 (6.01–35.91)0.274¶0.00 (−3.70–24.26)K+4.50 (2.53–10.61)13.21 (8.55–14.78)0.013¶*4.69 (2.60–10.22)P19.06 (3.02–14.26)10.09 (3.02–16.37)0.601¶2.11 (−7.59–11.76)P28.55 (1.57–13.73)6.51 (4.01–22.23)0.828¶0.00 (−7.22–12.14)P38.04 (2.53–13.21)15.31 (11.13–19.55)0.057¶7.27 (0.00–14.04)p0.187§0.144‡​0.199‡

Statistical analysis of VEGF levels before and after treatment across groups.

*significant (p < 0.05); † Wilcoxon; ¶ paired t-test; § one-way ANOVA; ‡ Kruskal–Wallis.

3.3 Intestinal trefoil factor

ITF concentrations increased following treatment, with a statistically significant increase observed only in the highest-dose combination group (1,500 mg/kg BW) compared to baseline (p = 0.007). Baseline ITF levels were comparable across groups, while no significant changes were observed in the lower-dose groups (500 and 1,000 mg/kg BW). Detailed values are presented in Table 2.

GroupITF preITF postpΔITFK010,539.0 ± 860.410,308.0 ± 1371.60.521¶−231.0 ± 734.3K–10,112.2 ± 595.110,454.8 ± 2052.00.766¶342.6 ± 2,400.9K+8831.2 ± 1144.59430.2 ± 1070.50.174¶599.0 ± 811.3P1a9165.4 ± 702.411,264.4 ± 2737.20.068¶2,099.0 ± 1,892.6P29293.6 ± 1674.59905.8 ± 994.70.448¶612.2 ± 1,630.86P3a,b,c,d8363.0 ± 1407.812,191.4 ± 1696.60.008¶*3,828.4 ± 1,740.6P0.055§0.089§​0.007§*

Statistical analysis of ITF levels before and after treatment across groups.

*significant (p < 0.05); paired t-test; § one-way ANOVA.

K0, normal control; K−, negative control; K+, positive control (omeprazole 20 mg/kg BW); P1, combination of omeprazole and striatin (500 mg/kg BW); P2, combination of omeprazole and striatin (1,000 mg/kg BW); P3, combination of omeprazole and striatin (1,500 mg/kg BW).

a

p < 0.05 by the post-hoc Mann–Whitney test compared to the K0 group.

b

p < 0.05 by the post-hoc Mann–Whitney test compared to the K− group.

c

p < 0.05 by the post-hoc Mann–Whitney test compared to the K+ group.

d

p < 0.05 by the post-hoc Mann–Whitney test compared to the P2 group.

3.4 Epithelial thickness

Aspirin-induced injury resulted in reduced epithelial thickness in the negative control group. In contrast, increased epithelial thickness was observed in all treatment groups. The highest-dose combination group (1,500 mg/kg BW) demonstrated the greatest epithelial thickness values among the treatment groups. Statistical analysis showed significant differences among groups (p < 0.05). These findings are summarized in Table 3.

GroupMedian (minimum–maximum)pK0587.95 (580.97–637.32)<0.001K–a189.30 (163.7–211.36)​K+a,b381.51 (350.89–388.14)​P1a,b,c487.61 (423.11–536.97)​P2a,b,c529.32 (506.76–552.29)​P3b,c,d,e603.28 (563.92–606.82)​

Statistical analysis of epithelial thickness.

K0, normal control; K−, negative control; K+, positive control (omeprazole 20 mg/kg BW); P1, combination of omeprazole and striatin (500 mg/kg BW); P2, combination of omeprazole and striatin (1,000 mg/kg BW); P3, combination of omeprazole and striatin (1,500 mg/kg BW).

a

p < 0.05 by the post-hoc Mann–Whitney test compared to the K0 group.

b

p < 0.05 by the post-hoc Mann–Whitney test compared to the K− group.

c

p < 0.05 by the post-hoc Mann–Whitney test compared to the K+ group.

d

p < 0.05 by the post-hoc Mann–Whitney test compared to the P1 group.

e

p < 0.05 by the post-hoc Mann–Whitney test compared to the P2 group.

3.5 Categorical distribution of epithelial thickness

When epithelial thickness was analyzed categorically, the healthy control group predominantly showed full-thickness mucosa, whereas the aspirin-induced group demonstrated substantial epithelial loss. The intermediate- and high-dose combination groups (1,000 and 1,500 mg/kg BW) showed a higher proportion of samples in the upper thickness categories. Overall differences among groups were statistically significant (p < 0.05), with several significant pairwise comparisons observed. The categorical distribution is shown in Table 4.

Group100%75%–100%50%–75%25%–50%pK05 (100%)000<0.001*K–a004 (80%)1 (20%)​K+a02 (40%)3 (60%)0​P1a,b03 (60%)2 (40%)0​P2a,b,c05 (100%)00​P3a,b,c05 (100%)00​

Categorical distribution of epithelial thickness.

K0, normal control; K−, negative control; K+, positive control (omeprazole 20 mg/kg BW); P1, combination of omeprazole and striatin (500 mg/kg BW); P2, combination of omeprazole and striatin (1,000 mg/kg BW); P3, combination of omeprazole and striatin (1,500 mg/kg BW).

a

p < 0.05 by the post-hoc Mann–Whitney test compared to the K0 group.

b

p < 0.05 by the post-hoc Mann–Whitney test compared to the K− group.

c

p < 0.05 by the post-hoc Mann–Whitney test compared to the K+ group.

3.6 Inflammatory score of gastric mucosa

Inflammatory infiltration was absent in the healthy control group but present in the aspirin-induced group. Lower inflammation scores were observed in the treatment groups; however, no statistically significant differences were found between treatment groups. The overall comparison across groups was statistically significant (p < 0.05), primarily due to differences between the healthy control group and the other groups. Details are provided in Table 5.

GroupNo inflammationMild inflammationpK05 (100%)0<0.001*K–a05 (100%)​K+a05 (100%)​P1a05 (100%)​P2a1 (20%)4 (80%)​P3a05 (100%)​

Inflammation degree across groups.

K0, normal control; K−, negative control; K+, positive control (omeprazole 20 mg/kg BW); P1, combination of omeprazole and striatin (500 mg/kg BW); P2, combination of omeprazole and striatin (1,000 mg/kg BW); P3, combination of omeprazole and striatin (1,500 mg/kg BW); a p < 0.05 by the post-hoc Mann–Whitney test compared to the K0 group.

3.7 Gastric ulcer area

Macroscopic evaluation showed no ulceration in the healthy control group, whereas the aspirin-induced group exhibited evident gastric lesions. Reduced ulceration was observed in the treatment groups. No macroscopic lesions were detected in the highest-dose striatin combination group (1,500 mg/kg BW). Statistical analysis demonstrated significant overall differences among groups (p < 0.05), with multiple significant pairwise comparisons, particularly between the aspirin-induced group and all treatment groups. Full ulcer grading appears in Table 6.

GroupNo lesionEdema–erythema1–5 small lesions≥5 small or 1 medium≥2 medium or 1 largepK05 (100%)0000<0.001*K–a0002 (40%)3 (60%)​K+a,b003 (60%)2 (40%)0​P1a,b,c03 (60%)2 (40%)00​P2a,b,c05 (100%)000​P3b,c,d,e5 (100%)0000​

Ulcer area distribution across groups.

K0, normal control; K−, negative control; K+, positive control (omeprazole 20 mg/kg BW); P1, combination of omeprazole and striatin (500 mg/kg BW); P2, combination of omeprazole and striatin (1,000 mg/kg BW); P3, combination of omeprazole and striatin (1,500 mg/kg BW).

a

p < 0.05 by the post-hoc Mann–Whitney test compared to the K0 group.

b

p < 0.05 by the post-hoc Mann–Whitney test compared to the K− group.

c

p < 0.05 by the post-hoc Mann–Whitney test compared to the K+ group.

d

p < 0.05 by the post-hoc Mann–Whitney test compared to the P1 group.

e

p < 0.05 by the post-hoc Mann–Whitney test compared to the P2 group.

4 Discussion4.1 Interpretation of findings

VEGF analysis revealed a significant increase only in the omeprazole monotherapy group (K+), whereas the combination groups showed no substantial change and delta VEGF values remained non-significant. This pattern is consistent with the well-characterized temporal kinetics of VEGF, an angiogenic mediator that is rapidly induced via hypoxia-driven HIF-1α signaling immediately following mucosal injury. Because serum VEGF was measured on day 2 of treatment, the sampling window likely captured a post-peak intermediate phase of the angiogenic response. VEGF expression typically reaches its maximal elevation within the first 6–48 h after tissue insult; moreover, cessation of aspirin reduces the hypoxic stimulus for further VEGF transcription, and early luminal stabilization from omeprazole or striatin may have already diminished mucosal stress (Braga Emidio et al., 2020; Rahayu et al., 2016).

ITF analysis demonstrated a significant increase only in the highest-dose combination group (P3; 1,500 mg/kg BW). This suggests a potential threshold effect rather than a clear dose–response relationship. Given that ITF was measured on day 14, corresponding to the later phase of mucosal healing, this finding supports its role in epithelial restitution. However, the absence of significant changes in the lower-dose groups indicates that higher doses may be required to elicit measurable biological effects (Braga Emidio et al., 2020; Rahayu et al., 2016).

Integration of biomarker data with histopathological findings suggests that the highest-dose combination group (P3; 1,500 mg/kg BW) was associated with greater epithelial thickness, smaller ulcer areas, and lower inflammatory infiltration than the other groups. These findings are consistent with a more advanced stage of mucosal recovery; however, a direct causal relationship between biomarker changes and histological outcomes cannot be established (Hapsari and Tjandrawinata, 2025; Ali Khan et al., 2014).

4.2 Comparison with previous studies

Aspirin-induced ulcers are typically marked by epithelial loss, increased neutrophil infiltration, and delayed mucosal repair due to the suppression of protective prostaglandins and disruption of growth factor pathways (Bjarnason et al., 2018; Rahayu et al., 2016). Prior studies have shown that PPI therapy promotes ulcer healing but does not fully restore mucosal integrity, particularly when oxidative stress and cytokine imbalance persist (Hapsari and Tjandrawinata, 2025). The present findings are generally consistent with previous studies demonstrating that Channa striata extracts reduce inflammatory infiltration and support mucosal repair (Hapsari and Tjandrawinata, 2025; Yulizal et al., 2020; Mabrok and Mohamed, 2019). Similar patterns of epithelial restoration have been reported in experimental ulcer models