Ferulic Acid Treats Gastric Ulcer via Suppressing Oxidative Stress and Inflammation

(1) Background: The aim of the present study was to evaluate the gastroprotective potential of ferulic acid (FA) on indomethacin-induced gastric ulcers in rats with macroscopic and microscopic examinations along with biochemical assays. (2) Methods: After 24 h starvation, the ulcer was induced in male Sprague-Dawley rats by subcutaneous indomethacin (25 mg/kg) injection. Fifteen minutes after ulcer induction, rats were treated with either tween 80 or FA. FA was given by oral gavage at 100 mg/kg, 250 mg/kg, and 500 mg/kg. In the fourth hour, rats were euthanized and collected gastric samples were evaluated macroscopically and microscopically. Antioxidant parameters including malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and inflammatory parameters comprising of myeloperoxidase (MPO), Tumor Necrosis Factor (TNF)-α, Interleukin (IL)-1β, IL-6 and Nuclear Factor Kappa-B (NF-κB) p65 levels were also determined. (3) Results: Indomethacin injection significantly increased the macroscopic and microscopic scores. In addition, it increased the gastric MDA, MPO, TNF-α, IL-1β, IL-6, and NF-κB p65 levels but reduced SOD and GSH content. Treatment with FA significantly improved the gastric injury macroscopically and microscopically. Moreover, FA displayed a marked decrease in the gastric levels of MDA, MPO, TNF-α, IL-1β, IL-6, and NF-κB p65 and a significant increase in SOD and GSH compared to the INDO group. Ultimately, 250 mg/kg FA was determined as the most effective dose. (4) Conclusion: Our results revealed that FA has a gastroprotective effect against indomethacin-induced gastric ulcers in rats due to its antioxidant and anti-inflammatory properties. As a result, FA may be a potential treatment choice for gastric ulcers.


Introduction
Gastric ulcer is a digestive system disease characterized by a lesion of the gastric tissue's mucosal integrity from the muscularis mucosa to the submucosa or deeper, affecting 5-10% of the world's population [1]. Under normal circumstances, mucus and bicarbonate (HCO 3 − ), which function as a protective layer, are released by mucosal cells to neutralize hydrochloric acid (HCl) [2]. The etiology of gastric ulcers is unknown, but the main mechanism underlying its development is an imbalance between protective (tight junctions between epithelial cells, microcirculation, HCO 3 − production, mucus secretion, adequate level of prostaglandins and nitric oxide) and aggressive factors (increased gastric acid secretion, pepsin activity, bile salt secretion, ethanol) [3]. Gastric ulcer is a polyetiological and polypathogenetic disease because of the various mechanisms underlying ulceration [4].
NSAIDs contain analgesic, antipyretic, and anti-inflammatory properties and are among the most extensively used medications for the treatment of inflammatory illnesses such as fever, acute or chronic pain, and rheumatic diseases across the world [5]. However,

Macroscopic Evaluation
The severity of macroscopic lesions developed was assessed as previously reported [19] using the following semiquantitative scale. This scale was applied for macroscopic scoring by two observers unaware of the experimental protocol and determined by measuring the length of each lesion along its greatest diameter, scoring from 0 to 3 as follows: 0: normal mucosa; 1: 1-4 small petechiae; 2: 5 or more petechiae or hemorrhagic streaks up to 4 mm long; and 3: erosion of more than 5 mm or confluent hemorrhages.

Estimation of Biochemical Parameters in Gastric Tissue
The stomach tissue samples from all the groups were collected, and parts of the stomach samples were cut into small pieces and then homogenized to obtain the supernatant. The gastric homogenate was used for the assessment of the following biochemical parameters: To assess gastric oxidative stress, MDA, GSH, and CAT levels in the gastric tissue were measured. Malondialdehyde (MDA) content in gastric tissues was tested to obtain quantified byproducts of membrane lipid peroxidation through assessing the formation of thiobarbituric acid-reactive substances as described before. When MDA is heated with TBA under acid circumstances, it forms a pink-colored substance. Then, a maximum absorbance of 532 nm was detected by a spectrophotometer (Shimadzu, UV-2600/UV-VIS/Spectrophotometer) and the results were expressed as nanomoles of MDA per gram of gastric tissue (nmol/g tissue).
Gastric enzymatic antioxidant enzymes were determined. The SOD test uses xanthine and xanthine oxidase to produce superoxide radicals, which react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to produce a red formazan dye. The degree of inhibition of this reaction is then used to determine the SOD activity. The concentrations of SOD in the stomach tissue homogenate were measured using a commercial kit (Relassay, Turkey) and a colorimetric autoanalyzer (Mindray, BS-400). The results are reported as the quantity of SOD in U/mL. Non-enzymatic antioxidant activity was also determined. GSH was measured using a spectrophotometric approach based on a modified Ellman procedure. Shortly, the supernatant was collected after centrifugation and added to a combination of 2 mL of 0.3 mol/l Na 2 HPO 4 -2H 2 O solution and 0.2 mL of dithiobis-nitrobenzoate solution (0.4 mg/mL in 1% sodium citrate). A spectrophotometer (Shimadzu, UV-2600/UV-VIS/Spectrophotometer) was used to determine the absorbance of the combination at 412 nm. GSH levels were expressed in micromoles per gram of tissue (µmol/g).

Measurement of MPO Activity and Gastric Inflammatory Mediators' Levels
To assess gastric inflammation, MPO activity and TNF-α, IL-1β, IL-6, NF-κB p65 levels in the gastric tissue were measured. The enzymatic activity of MPO was measured in gastric ulcer homogenized tissue. The H 2 O 2 -dependent oxidation of o-dianisidine 2HCl was used to measure MPO activity, which is a marker of neutrophil accumulation. The quantity of MPO present that causes changes in absorbance of 1.0 unit/min at 460 nm and 37 • C, expressed in units per g of tissue, was determined as one unit of enzyme activity. MPO activity was quantified spectrophotometrically according to the reaction. The activity of MPO was measured in units per gram of tissue (U/g). The concentrations of the proinflammatory cytokines TNF-α, IL-1β, IL-6 and NF-κB p65 in the homogenate of gastric tissue were performed using a specific rat enzyme-linked immunosorbent assay (ELISA) kit (Elabscience, Houston, TX, USA) according to the manufacturer's instructions. The optical density (OD) was 450 nm and it was measured spectrophotometrically (ELx800 Microplate Reader, Biotek, Winooski, VT, USA). The results of TNF-α, IL-1β, IL-6, and NF-κB were expressed in picograms per millimeter (pg/mL).

Histological Evaluation
Tissue samples were fixed in 10% formalin and processed for routine paraffin embedding for histological observations. Tissue sections (approximately 5 µm thick) were prepared using a rotary microtome (Rotary, Thermo Scientific, Waltham, MA, USA) and stained with hematoxylin and eosin to assess histological changes by light microscopy (Zeiss Axio Scope.A1 AX10), and photographs were taken (Zeiss AxioCam MRc 5). Specimens were graded by light microscopy according to a previously reported grading method [20] that comprises the assessment of epithelial desquamation, mucosal hemorrhage, glandular damage, and inflammatory cell infiltration on a scale of 0 to 3 (0: none, 1: mild, 2: moderate, and 3: severe) for each criterion.

Scanning Electron Microscopy (SEM)
Tissue samples were also evaluated by scanning electron microscopy. They were immersed in 2.5% glutaraldehyde (pH 7.2) and processed for scanning electron microscopy and examined under a scanning electron microscope (Thermo Fischer, Quattro S). Basal lamina and surface epithelium integrity were examined under a scanning electron microscope (Thermo Fischer, Quattro S).

Statistical Analysis
Data are expressed as mean ± standard error (SE). Statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison tests or Student's t-test. A p-value of <0.05 was taken to indicate statistical significance. The data were analyzed using GraphPad Prism software, version 8 (GraphPad Software, San Diego, CA, USA).

Macroscopic and Microscopic Scores
As shown in Figure 1A, administration of indomethacin at a single dosage of 25 mg/kg resulted in the production of gastric injury and caused a significant increase (2.167 ± 0.167, p < 0.001) in macroscopic score as compared to the control group (0.125). There were remarkable confluent hemorrhages, red-black bleeding areas, and petechial lesions in the stomachs of the indomethacin group while treated only with the vehicle. The rats in the control group did not show gastric mucosal lesions and had a normal anatomical appearance. The anti-ulcer effect of both doses of FA on indomethacin-induced gastric damage was macroscopically determined in rats. Compared with the indomethacin group, treatment with all doses of FA (100 mg/kg, 250 mg/kg, 500 mg/kg) revealed a decrease in hyperemia, hemorrhage, and ulceration. In macroscopic scoring, it was determined that the scores had a significantly higher in the FA100 group (1.63 ± 0.38, p < 0.01) compared to the control group (0.125). Notably, 250 mg/kg FA (1.75 ± 0.25; p < 0.05) significantly decreased the macroscopic scores (1.75 ± 0.25; p < 0.05) in comparison to the INDO group. These results showed that 250 mg/kg FA had a significant treating effect against the gastric injury caused by indomethacin. The gastroprotective effects of FA on stomach structural changes caused by indomethacin administration were also assessed using a light microscope to evaluate the histological examination of gastric tissues. In histological evaluation, the normal structure of the stomach was observed in the control group ( Figure 1B). On the other hand, in the INDO group, indomethacin administration caused severe gastric tissue damage, and histological changes including the marked presence of surface epithelial damage, mucosal hemorrhage, and inflammatory cell infiltration were determined. According to the microscopic score evaluation of gastric lesions, it was determined that the INDO group (6.125 ± 0.295) had a significantly higher score compared to the control group (0.429 ± 0.202 p < 0.001). In contrast, both doses of FA showed a decrease in the histological damage caused by indomethacin and repaired gastric mucosa integrity, decreasing the mucosal hemorrhage and infiltration of inflammatory cells. It was determined that the microscopic score significantly decreased in the FA100, FA250, and FA500 groups (4 ± 0.423; p < 0.01, 2.286 ± 0.395; p < 0.001, 2.571 ± 0.528; p < 0.001, respectively) compared to the INDO group. In the control group, the normal structure of the gastric mucosa was observed; nevertheless, oral administration of FA also showed lower histological changes.

Effect of FA on MPO and Gastric Oxidative Stress Markers Levels
MPO enzyme activity in gastric tissue was measured as a marker of neutrophil infiltration. The activity of MPO after administration of indomethacin led to an increase in the INDO group (68.23 ± 4.25 U/g) compared to the control group (45.22 ± 4.11 U/g; p < 0.05). Additionally, for all doses of FA (100 mg/kg, 250 mg/kg, 500 mg/kg), it was observed that there was a significant reduction in the levels of gastric MPO levels (respectively, 51.20 ± 5.96 U/g; p < 0.05, 52.91 ± 5.11 U/g; p < 0.05, 45, 76 ± 6.51 U/g; p < 0.05) (Figure 2A). Figure 2B shows the lipid peroxidation marker, malondialdehyde (MDA), and antioxidant activity in gastric tissue samples. The gastric MDA level in the indomethacin group (27.66 ± 4.023 nmol/g), was characterized by a significant increase compared to the control group (13.91 ± 1.033 nmol/g; p < 0,05), as a marker of lipid peroxidation and tissue damage. While the reduction with the 100 mg/kg dose did not reach statistical significance (20.18 ± 2.856 nmol/g), the MDA levels significantly decreased with both the 250 mg/kg The gastroprotective effects of FA on stomach structural changes caused by indomethacin administration were also assessed using a light microscope to evaluate the histological examination of gastric tissues. In histological evaluation, the normal structure of the stomach was observed in the control group ( Figure 1B). On the other hand, in the INDO group, indomethacin administration caused severe gastric tissue damage, and histological changes including the marked presence of surface epithelial damage, mucosal hemorrhage, and inflammatory cell infiltration were determined. According to the microscopic score evaluation of gastric lesions, it was determined that the INDO group (6.125 ± 0.295) had a significantly higher score compared to the control group (0.429 ± 0.202 p < 0.001). In contrast, both doses of FA showed a decrease in the histological damage caused by indomethacin and repaired gastric mucosa integrity, decreasing the mucosal hemorrhage and infiltration of inflammatory cells. It was determined that the microscopic score signifi-Life 2023, 13, 388 6 of 15 cantly decreased in the FA100, FA250, and FA500 groups (4 ± 0.423; p < 0.01, 2.286 ± 0.395; p < 0.001, 2.571 ± 0.528; p < 0.001, respectively) compared to the INDO group. In the control group, the normal structure of the gastric mucosa was observed; nevertheless, oral administration of FA also showed lower histological changes.

Effect of FA on Gastric Inflammatory Mediators' Levels
Indomethacin administration caused a dramatic increase levels of TNF-α, a key proinflammatory cytokine involved in the formation of the gastric ulcer in the INDO group (1.409 ± 19.66 pg/mL), compared to the control group (1.016 ± 65.54 pg/mL; p < 0.001). TNFα levels (1.357 ± 41.93 pg/mL) in the FA100 group were not different from the INDO group. On the other hand, doses of 250 mg/kg and 500 mg/kg FA (respectively, 1.086 ± 32.18 pg/mL; p < 0.001, 1.194 ± 23.49 pg/mL; p < 0.001) significantly decreased the level of TNF-α ( Figure 3A).
Similarly, significant increase in the level of gastric IL-6 was indicated in the INDO group (400.9 ± 17.71 pg/mL) when compared to the control group (246.4 ± 40.93 pg/mL; p  Figure 2B shows the lipid peroxidation marker, malondialdehyde (MDA), and antioxidant activity in gastric tissue samples. The gastric MDA level in the indomethacin group (27.66 ± 4.023 nmol/g), was characterized by a significant increase compared to the control group (13.91 ± 1.033 nmol/g; p < 0,05), as a marker of lipid peroxidation and tissue damage. While the reduction with the 100 mg/kg dose did not reach statistical significance (20.18 ± 2.856 nmol/g), the MDA levels significantly decreased with both the 250 mg/kg and 500 mg/kg FA treatments (17.1 ± 2.257 nmol/g; p < 0.05, 16.42 ± 1.81 nmol/g; p < 0.05, respectively).
The scavenging SOD activity of superoxide radicals was lower in the INDO group (75.70 ± 5.18 U/mL) compared to the control group (152.3 ± 44.63 U/mL; p < 0.05). A Life 2023, 13, 388 7 of 15 marked increase in SOD enzymatic activity was observed in the rats treated with all doses of FA compared to the INDO group (106.7 ± 14.17 U/mL; p < 0.05, 122 ± 16.20 U/mL; p < 0.01, 124 ± 21.03 U/mL; p < 0.05) ( Figure 2D). The most improvement in GSH content and SOD activity occurred by 250 mg/kg FA.

Histological Evaluation
Under light microscopy, epithelial desquamation, mucosal hemorrhage, glandular damage, and inflammatory cell infiltration parameters were assessed. Normal morphology with surface epithelium and gastric glands was present in the control group ( Figure  4A). On the other hand, indomethacin injection caused severe gastric tissue damage and apparent histopathological changes, including severe desquamation of surface epithelium, hemorrhage, glandular degeneration, and inflammatory cell infiltration ( Figure  4B,C). Desquamation of surface epithelium, degeneration of the gastric gland, and inflammatory cell infiltration in the mucosa were obviously visible in the 100 mg/kg FA ( Figure  4D). Mucosal degeneration, hemorrhage, and inflammatory cell infiltration were also detected in the FA500 group ( Figure 4F). On the other hand, 250 mg/kg FA treatment atten- Similarly, significant increase in the level of gastric IL-6 was indicated in the INDO group (400.9 ± 17.71 pg/mL) when compared to the control group (246.4 ± 40.93 pg/mL; p < 0.01). IL-6 levels of FA100 (384 ± 40.8) and FA500 (359.2 ± 46.55) groups were similar with the INDO group. There was a notable reduction only in the group treated with 250 mg/kg FA when compared with the INDO group (278.3 ± 38.21 pg/mL; p < 0.01) ( Figure 3B).
NF-κB p65 levels in the INDO group measured significantly higher (224.7 ± 12.34 pg/mL) than in the control group (159 ± 13.31 pg/mL); p < 0.05). Only 250 mg/kg FA treatment decreased NF-κB levels significantly (156.5 ± 18.40 pg/mL; p < 0.05). These results are in line with the evaluation of TNF-α, IL-6 and MPO where the 250 mg/kg dose of FA showed the greatest reduction in the inflammation process ( Figure 3D).

Histological Evaluation
Under light microscopy, epithelial desquamation, mucosal hemorrhage, glandular damage, and inflammatory cell infiltration parameters were assessed. Normal morphology with surface epithelium and gastric glands was present in the control group ( Figure 4A). On the other hand, indomethacin injection caused severe gastric tissue damage and apparent histopathological changes, including severe desquamation of surface epithelium, hemorrhage, glandular degeneration, and inflammatory cell infiltration ( Figure 4B,C). Desquamation of surface epithelium, degeneration of the gastric gland, and inflammatory cell infiltration in the mucosa were obviously visible in the 100 mg/kg FA ( Figure 4D). Mucosal degeneration, hemorrhage, and inflammatory cell infiltration were also detected in the FA500 group ( Figure 4F). On the other hand, 250 mg/kg FA treatment attenuated damage induced by indomethacin and decreased markedly the infiltration of neutrophils and hemorrhage ( Figure 4E). In addition, the FA250 group had a slight degeneration of the mucosa compared to the control group ( Figure 1B).  Figure 4E). In addition, the FA250 group had a slight degeneration of the mucosa compared to the control group ( Figure 1B). Stomach samples were also examined for surface epithelium integrity under the scanning electron microscope. The control group had steady surface epithelium ( Figure  5A,B), while the surface epithelium was degenerated in the INDO group ( Figure 5C,D). The damage to the surface epithelium caused by indomethacin improved in the FA250 group ( Figure 3G,H), whereas the FA100 (Figure 5E,F) and FA500 ( Figure 5I,J) groups were not different from the INDO group  Stomach samples were also examined for surface epithelium integrity under the scanning electron microscope. The control group had steady surface epithelium ( Figure 5A,B), while the surface epithelium was degenerated in the INDO group ( Figure 5C,D). The damage to the surface epithelium caused by indomethacin improved in the FA250 group ( Figure 5G,H), whereas the FA100 (Figure 5E,F) and FA500 ( Figure 5I,J) groups were not different from the INDO group.

Discussion
In the present study, for the first time, we investigated the gastroprotective effects of FA (100 mg/kg, 250 mg/kg, and 500 mg/kg) on the indomethacin-induced gastric ulcer model in terms of biochemical and inflammatory parameters in rats. We showed that FA has antioxidant, anti-inflammatory, and anti-ulcer effects.
Due to the blocking of COX activity, indomethacin decreases the prostaglandin level. Additionally, it decreases mucus and HCO 3 − secretion and mucosal blood flow. These effects also significantly contribute to changes in microvascular structures [21]. We used a semiquantitative scale for macroscopic scoring depending on the evaluation of the hemorrhages and petechial lesions in the gastric injury induced by indomethacin. In this context, ulceration and dark red-black bleeding areas were detected in the gastric mucosa of rats with gastric damage induced by indomethacin. All three doses of FA treatment improved gastric mucosal damage and lowered the macroscopic score. The 250 mg/kg dose of FA has shown a considerable decrease in damage score. This considerable increase in macroscopic score following indomethacin treatment might be attributed to an increase in ROS and inhibition of prostaglandin synthesis inhibition. FA attenuates macroscopic damage scores probably due to its antioxidant effects.
The MPO enzyme is localized in phagocytic cells and causes excessive production of ROS. It is a marker of regulation of neutrophil infiltration, which catalyzes the production of highly reactive HOCl from H 2 O 2 , which subsequently causes inflammation [22]. It was previously reported that indomethacin-induced mucosal damage induces an increase in mucosal MPO levels, suggesting that neutrophil infiltration plays a role in indomethacininduced stomach injury [23]. Treatment with FA reduces tissue MPO activity in animals with nephrotoxicity [24]. In the current study, an increase in MPO activity was detected in the gastric tissues of rats with gastric damage induced by indomethacin. This finding indicates a considerable influx of neutrophils into the mucosa in response to the subcutaneous administration of indomethacin in rats, which is consistent with the earlier research. Compared to the INDO group, increased gastric MPO activity decreased with FA administration, which indicates that FA, as an anti-ulcer agent, acts by diminishing neutrophil adherence.
During the production of ATP through mitochondrial respiration, the oxygen molecule is reduced to water and ROS are produced [25]. The imbalance between free radical formation and scavenging capacity results in oxidative stress, which plays a role in the pathogenesis of gastric ulcers. These ROS cause damage to gastric tissue by damaging membranes and cellular macromolecules such as lipids, proteins, carbohydrates, and nucleic acids [26]. MDA is a lipid peroxidation product that is the most widely used indicator for determining oxidative stress in cell and tissue damage [27]. NSAIDs, including indomethacin, increase oxidative stress by enhancing lipid peroxidation and thus cause stomach damage [28]. Furthermore, it was shown that FA treatment reduced MDA levels in a streptozotocin-induced diabetes model [29]. In this study, tissue MDA levels were dramatically higher in gastric damage with ulcer induction compared with the control group. On the contrary, treatment with 250 mg/kg and 500 mg/kg doses of FA significantly diminished high MDA levels in ulcerated gastric tissue. FA's capacity to scavenge ROS results in decreased levels of lipid oxidation, therefore helping to reduce oxidative gastrointestinal damage caused by ROS.
The body defends against the harmful effects of constantly produced ROS with the help of endogen antioxidants [30]. GSH is an endogenous antioxidant that helps preserve mucosal integrity and protect the gastric mucosa from free radical-induced tissue damage [31]. Based on previous research, indomethacin administration reduces GSH levels in stomach tissue [32]. In our study, GSH depleted in the gastric tissues of rats with gastric ulcers and FA treatment increased GSH levels, and the most effective dose was determined to be 250 mg/kg. Inhibition of GSH consumption in the stomach may constitute an important defense mechanism against oxidative stress-related gastric ulcers. Similar to our results, FA treatment prevents the depletion of tissue GSH levels in a hepatotoxicity model [33].
As one of the antioxidant enzymes that contributes to the enzymatic defense mechanisms, SOD protects the stomach tissue against damage by converting highly reactive O2-to less-reactive H 2 O 2 as the first line of defense against ROS [31]. We know that a decrease in SOD expression occurs in gastric mucosal tissue after indomethacin administration [34]. FA treatment has been shown to increase tissue SOD levels in models of hepatotoxicity [35]. While indomethacin-induced stomach injury resulted in a drop in SOD levels, the antioxidant property of FA treatment improved SOD levels compared to the INDO group, as the most effective dose was 250 mg/kg. The reduction in SOD levels caused by indomethacin is consistent with prior research findings. Activating antioxidant mechanisms involving SOD in gastric tissues contributes to the preservation of structural and functional mucosal integrity against indomethacin [36]. At the same time, the FA significantly reduced the lipid peroxidation level in stomach tissue when compared to the INDO group, which indicates that the FA may exert its gastroprotective effect via an antioxidant mechanism.
Ferulic acid has the structural components of the 3-methoxy and 4-hydroxyl groups on the benzene ring, as well as the carboxylic acid group. These components either stabilize the resulting phenoxyl radical intermediate or even inhibit the free radical chain reaction [37]. In addition, ferulic acid increases the antioxidant enzyme activity, including SOD, glutathione peroxidase, and catalase. Thus, the cytoprotective effects of FA may be related to its structure and ability to improve antioxidant enzyme activity [38]. In the study, improvements in the anti-oxidant enzyme SOD and endogenous GSH levels were compatible with FA's anti-oxidant properties.
Inflammation is a complex response to tissue damage that involves immune cells secreting proinflammatory cytokines, including TNF-α, IL-1β, and IL-6. The secretion of proinflammatory mediators and activation of the NF-κB signaling pathway play a critical role in the pathogenesis of gastric ulcers [39]. TNF-α is a proinflammatory cytokine released primarily by activated macrophages and it is considered a marker to assess gastric ulcer severity, closely related to the acute phase of inflammation and the degree of ulceration [40]. TNF-α and IL-1β have a synergistic effect and are associated with the acute phase of inflammation and the severity of a gastric ulcer [41]. In addition, IL-6 stimulates lymphocytes, macrophages, and neutrophils in the inflammatory region and triggers the oxidative pathway responsible for tissue damage during gastric ulcer. As suggested before, IL-6 levels increase in indomethacin-induced ulcers [42]. In this study, TNF-α, IL-1β, and IL-6 levels were higher in the INDO group compared to the control group. Compared to the INDO group, 250 mg/kg FA and 500 mg/kg FA doses significantly reduced TNF-α levels. Furthermore, only 250 mg/kg of FA treatment decreased tissue IL-6 levels compared to the INDO group, and FA had no significant effect on IL-1β levels. In addition, FA treatment has been shown to reduce serum TNF-α levels in a mouse model of high-fat diet-induced obesity [43] and serum IL-6 levels in formaldehyde-induced hepatotoxicity [44]. However, this is the first time that the inhibitory effects of FA treatment on gastric TNF-α and IL-6 levels have been demonstrated in an indomethacin-induced ulcer.
NF-κB, a crucial transcription factor, plays a vital role in the immune and inflammatory processes because it regulates the expression of various proinflammatory factors. There are four transcript variants of NF-κB encoding different isoforms, namely, p65, p105, p50, and p52 [45]. In resting conditions, NF-κB is retained in the cytosol as an inactive dimer bound to the NF-κB inhibitor (IκB) protein. When NF-κB is activated, the inhibitory complex IB is phosphorylated and degraded, which is controlled by the IB kinase (IKK), allowing the release of the NF-κB p65. The NF-κB p65 subunit is then translocated to the nucleus, where it stimulates the production of various inflammatory mediators [46]. Indomethacin activates NF-κB and induces the expression of several inflammatory genes, including TNFα, IL-1β, and IL-6 [47]. The NF-κB signaling pathway is associated with the pathogenesis and progression of gastric ulcer formation, and its inhibition has protective functions in the development of gastric ulcers [48]. NF-κB p65 levels were measured in gastric tissue and NF-κB p65 was significantly higher in the INDO group than in the control group. Treatment with FA decreased NF-κB p65 expression compared to the INDO group. It was determined that 250 mg/kg FA revealed a substantial reduction in the gastric level of NF-κB p65 compared to the INDO group. Similar to our results, FA treatment has been shown to diminish NF-κB p65 expression in lipopolysacharide-induced acute kidney injury [49]. As a result, FA lowered the indomethacin-induced gastric ulcer damage by decreasing the proinflammatory cytokine levels via the NF-κB signal pathway. NF-κB may play a crucial role in protecting the stomach against gastric injury by regulating the expression of proinflammatory parameters [47]. Extracts obtained from Lithraea molleoides [50], Baccharis dracunculifolia [51], and Pachira glabra leaves [52], all of which contain ferulic acid, have been shown to have gastroprotective and anti-ulcer effects. It has been shown that Pachira glabra leaves also exert their effects on ethanol-induced gastric ulcer damage by reducing the levels of NF-κB and COX-2.
Indomethacin injection causes erosive and ulcerative gastric lesions with histopathological findings [53]. In our study, the control group exhibited normal morphology, but the indomethacin-induced group had a significantly higher score with severe damage in gastric gland morphology, local hemorrhage, and inflammatory cell infiltration in the mucosa. Three FA groups significantly decreased microscopic scoring compared to the INDO group. In addition, scanning electron microscopy data showed that the gastric tissues in the control group reflected the normal topography, while in the INDO group, there was damage to the surface epithelium and deterioration in the basal lamina structure in line with the previous findings [54]. The epithelial cells reflected equivalent to normal topography in scanning electron microscopy at a dose of 250 mg/kg FA, which provided a remarkable decrease in microscopic scoring against the damage caused by indomethacin.

Conclusions
This study is the first to report that FA protects the gastric mucosa against indomethacininduced gastric mucosal damage. The protective effect of FA against gastric damage is related to its reducing effect on indomethacin-induced oxidative stress and inflammation. FA exhibits its gastroprotective effects via inhibiting neutrophil infiltration, suppressing lipid peroxidation, and modulating the antioxidant defense mechanisms. Moreover, it defends the stomach mucosa and plays a role in the structural integrity of the mucosa by inhibiting the NF-κB transcription factor, reducing damage at the tissue level. As a result, treatment with FA, especially the 250 mg/kg FA dose, has a marked gastroprotective effect against indomethacin-induced gastric ulcers. By means of its anti-ulcer properties derived from its antioxidant and anti-inflammatory activities, FA could be a promising therapeutic candidate for indomethacin-induced gastric ulcers.  Institutional Review Board Statement: All the experimental procedures were carried out following the guidelines for the care and handling of laboratory animals as recommended by the National Institutes of Health (NIH 85-23). The animal study protocol was approved by the Local Ethics Committee for Animal Experiments of Acibadem Mehmet Ali Aydinlar University, Istanbul (protocol code ACU-HADYEK 2020/26).

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.