Introduction
The histological definition of gastric ulcerative disease is mucous membrane injury that affects of the stomach superficial or deeper muscularis mucosa [
1,
2]. The epidemiological data has unveiled remarkable rates of incidence and prevalence across diverse geographical regions. It has been demonstrated that the worldwide occurrence of gastric ulcer amounted to approximately 8.1 million individuals, signifying a notable surge of 25.8% compared to the figures recorded in 1990 [
3]. The disease origin is complex and multifaceted, largely due to an imbalance between endogenous protective and aggressive factors to the stomach mucosa [
4]. Defense factors include mucus, cytoprotective prostaglandins, nitric oxide, bicarbonate, the endogenous antioxidant system, and a sufficient blood flow [
5]. The most important aggressive agents involve hydrochloric acid, pepsin, bile reflux, reactive oxygen species (ROS), reduced blood flow, and infection with
Helicobacter pylori [
6]. Additionally, exogenous aggressive factors including stress, alcoholism, smoking, nutritional deficiencies and the frequent and indiscriminate usage of non-steroidal anti-inflammatory drugs (NSAIDs) contribute to a large extent to the development of ulceration [
7].
Because of their acidic nature, NSAIDs cause primary mucosal irritation and secondary or indirect harm to the gastric mucosa by inhibiting the synthesis of gastro-protective thromboxane and prostaglandins [
8]. Aside from that, all of the previously mentioned noxious agents, including NSAIDs, might contribute to ulcer formation via producing various kinds of ROS, which increase the release of several inflammatory cytokines, including nuclear factor kappa (NF-κB) and tumor necrosis factor-α (TNF-α) [
9].
Gastric ulcer complications can cause gastroduodenal perforation, bleeding, and obstruction if they are not addressed or treated appropriately [
10]. Treatment often consists of anti-secretory medicines such as H2-receptor blockers and proton pump inhibitors, as well as antibiotics such as clarithromycin and/or metronidazole if an
H. pylori infection is demonstrated to be active by laboratory testing [
11]. Nonetheless, because of the bad responses and limited efficacy of currently available medications, managing stomach ulcers is one of the most difficult challenges, with a large economic impact on public health systems [
12]. Proton pump inhibitors have been linked to major side effects such as fractures, renal disease, greater vulnerability to certain infections, and calcium, magnesium, and vitamin B12 deficiency [
13]. Many nations have phased out the H2-blocker ranitidine due to the identification of above-the-limit amounts of the carcinogenic pollutant N-nitroso-dimethylamine (NDMA) [
14]. Furthermore, there is evidence that proton pump inhibitors can enhance the intestinal injury produced by NSAIDs by changing the microbial composition of the intestine [
15] and increase the risk of gastric cancer [
16]. Thus, traditional medicine may be of significant interest in the search for non-toxic, easily accessible, and economical anti-ulcer drug.
Various herbal medicines are prevalently employed to treat and/or prevent many diseases including gastric ulcers [
17‐
21]. The anti-ulcerogenic and gastroprotective potentials of natural products are attributed mostly to their diverse metabolic profile and antioxidant properties [
22‐
24]. Therefore, complementary treatments could be developed to mitigate the ulcerative diseases and inhibit recurrences with less side effects, higher efficacy, and affordability compared to synthetic drugs [
25].
Apium graveolens L. (Apiaceae, Umbelliferae), commonly known as celery, is an aromatic biennial herb indigenous to Southern Europe, Asia and Africa. It has long been consumed as medicine or food in the form of entire herb, leaves, stalks, seeds or seed [
26,
27]. The aerial parts extract as well as the essential oil have been demonstrated to exhibit a significant antiulcer effect [
27,
28]. The findings indicated that
A. graveolens has the ability to diminish gastric acid secretion and strengthen the defense factors of gastric mucosa. These anti-secretory and cytoprotective effects are probably mediated by its antioxidant properties through reduction of lipid peroxidation and elevation of gastric mucosal non-protein sulfhydryl groups [
28]. In addition to its anti-acid and softening effects [
27,
29]. Interestingly, a recent study has demonstrated that celery also exhibits bactericidal activity against
H. pylori [
30].
Seeds of
A. graveolens have been traditionally used as a flavoring agent or in treatment of gut diseases, urinary calculi, flatulence, gripping pain and visceral [
31,
32]. Moreover, previous pharmacological studies reported that the seeds revealed hypoglycemic [
33], bactericidal [
34], antifungal [
35], nematocidal, mosquitocidal [
36], anti-inflammatory [
37] and anti-hypertensive [
38] activities. Therefore, the present study was designed to investigate the phytochemical composition of the seeds of
A. graveolens through metabolomic profiling using liquid chromatography high-resolution electrospray ionization mass spectrometry (LC-HR-ESI–MS), along with in vivo evaluation of the anti-gastric ulcer potential of the seed extract in experimental animals. Furthermore, the pharmacological and biochemical results were substantiated by a histopathological assessment of the gastric tissues, and by an in silico network pharmacology study involving all identified metabolites, in a trial to determine the gene enrichment analysis and to understand the top biological pathway of the anti-gastric ulcer activity.
Discussion
Gastric ulcers continue to be a prevalent gastrointestinal disorder on a global scale. Indomethacin exhibits a greater propensity for ulcerogenesis compared to other NSAIDs. Henceforth, it is regarded as the preferred pharmaceutical agent for instigating harm to the gastric mucosa within an experimental milieu [
79]. The ingestion of NSAIDs leads to an increased ulcer index, as evidenced by the presence of distinct long hemorrhagic bands and petechial lesions with ulcerative inflammation [
80], Additionally, there is observed gastric degeneration and necrosis [
81], along with the death of epithelial cells and the occurrence of local or multiple hemorrhagic ulcers [
82]. The observed phenomenon can be ascribed to the upregulation of diverse inflammatory cytokines and chemokines that possess chemotactic properties towards leukocytes and other cells involved in inflammatory responses [
83]. Additionally, it can be partially attributed to the induction of oxidative stress [
80].
Pre-treatment with the seed extract of
A. graveolens exhibited a notable amelioration of gastric mucosal damage, as indicated by a significant decrease in the ulcer index, comparable to the preventive effect observed with famotidine (the reference drug). Our observations exhibited similarity to the outcomes of previous investigations, which have likewise exhibited the manifestation of gastric mucosal damage induced by indomethacin [
84,
85].
Stimulation of gastric mucosal damage occurs through the activation of ROS and cytokines, both through direct and indirect mechanisms [
86]. Experimental investigations have provided evidence suggesting that ROS play a significant role in the development of gastric ulcers induced by NSAIDs [
87]. The metabolism of NSAIDs through the action of peroxidase enzymes is accountable for the pro-oxidant properties exhibited by indomethacin in the gastric mucosa [
88]. Elevated levels of ROS have been observed to be linked with heightened lipid peroxidation of the membranes of gastric cells. This phenomenon is also accompanied by an increase in mucosal MDA levels, a decrease in the secretion of gastric mucus, and the occurrence of DNA damage [
89]. MDA is widely recognized as a biomarker that is intricately associated with the process of lipid peroxidation. Fascinatingly, within our research, the heightened concentration of MDA, which is linked to damage in the mucosal tissue, exhibited a significant decrease upon administration of
A. graveolens seed extract. In contrast, GSH plays a pivotal role as one of the primary cellular antioxidant mechanisms, ensuring the maintenance of the cell redox status [
90,
91]. The levels of GSH in gastric tissues exhibited a notable reduction subsequent to the administration of indomethacin. The observed decrease in GSH content could potentially be attributed to the generation of superoxide radicals, as these radicals have a tendency to deplete a significant quantity of endogenous antioxidant enzymes during scavenging [
82,
92]. The exacerbation of gastric mucosal damage can be attributed to the declined level of GSH, which leads to a rapid increase in hydrogen peroxide and lipid peroxides within the cells of the gastric mucosa. Hence, an increase in the expression of antioxidant enzymes and a rise in the gastric content of GSH may serve as a significant protective mechanism against gastric ulcers that are linked to oxidative stress [
93].
The remarkable gastroprotective activity of
A. graveolens seed extract might be attributed to its rich chemical profile, which was deduced through metabolomics profiling analysis using LC-HR-ESI–MS. This has led to the tentative identification of 18 different compounds. These constituents actually belong to various phytochemical classes, including coumarins, phenolic acids, and flavonoids, previously reported to have potent anti-inflammatory and antioxidant effects [
94‐
96]. They preserve the gastric GSH level by acting as scavenging free radicals instead of GSH and preventing lipid peroxidation, which can ultimately accelerate the healing of ulcers. Interestingly, phenolic acids as well as coumarins have also been shown to exhibit anti-ulcer and gastroprotective effects on experimental gastric acid ulcer models in rats [
97,
98]. These metabolites can protect the gastric mucosa via several mechanisms of action, such as enhanced mucus production, antioxidant and free-radical scavenging properties, antisecretory action, prevention of
Helicobacter pylori growth, and stimulation of the antioxidative defense enzyme activities [
99].
Gastric ulcer repair necessitates the restoration of epithelial structures and the underlying connective tissue, which involves cell proliferation and angiogenesis. Several growth factors have been linked to the healing of ulcers [
100]. VEGF is a key angiogenic element that promotes the creation of granulation tissue and new micro capillaries via angiogenesis, which speeds up the healing of gastric and duodenal ulcers [
101]. In our research, we discovered that indomethacin dramatically reduced VEGF expression in gastric tissues.
A. graveolens treated rats, on the contrary, have significantly enhanced VEGF expression in gastric regions, aiding the healing process.
NF-κB, a key transcription factor, exerts significant influence over various immune and inflammatory mechanisms, encompassing the intricate orchestration of gastric ulcer formation [
102]. NF-κB exhibits swift production upon cellular injury, encompassing the exposure to ROS. The initiation of NF-κB activation occurs through the degradation of the IκB-α protein, which is triggered by signals. The NF-κB complex, once activated, undergoes translocation from the cytoplasmic region to the nucleus. This migration enables the complex to generate transcription factors like TNF-α, IL-1β, and IL-6 [
79,
103]. The present study has demonstrated that the induction of gastric injury triggers the activation of the IKκB/NF-κB signal transduction pathway. This is evident from the significant elevation in levels of phosphorylated NF-κB p-65 protein in the gastric mucosa, when compared to the mucosa of rats in the control group. The extract exhibited significant reduction in the IKκB/NF-κB signaling pathway activated by indomethacin. This was evident from the notable decrease in levels of phosphorylated NF-κB p-65 protein in comparison to the ulcer group. The extract exerted inhibitory effects on the activation of NF-κB by impeding the phosphorylation process and subsequent degradation of IκB-α. Previous research has demonstrated that antioxidants, including polyphenolics, impede the activation of NF-κB by inhibiting the phosphorylation of IκB-α induced by signaling [
104]. The observed decrease in NF-κB p-65 concentration in rats pretreated with
A. graveolens could potentially be attributed to the plant extract's capacity to scavenge ROS [
105]. Additionally, it has been documented that the inhibition of NF-κB p65 hinders the expression of COX-2, a pro-inflammatory mediator involved in gastrointestinal damage [
106]. The inhibitory effects of
A. graveolens seed extract on COX-2 expression were observed, suggesting a potential correlation with the extract's gastroprotective properties. In this context, the inhibitory impacts of the botanical extract on the IKκB/NF-κB signaling pathway could potentially elucidate the reduced expression of COX-2 and the mitigation of gastric inflammation induced by indomethacin [
107].
Inflammatory cytokines, including TNF-α, IL-1β, and IL-6, are secreted as part of the immune response, resulting in the attraction of neutrophils and mononuclear cells to the lamina propria. In accordance with scientific observations, heightened levels of TNF-α, IL-1β, and IL-6 have been found to exhibit a clinical correlation with the magnitude of gastric inflammation. Having confirmed that numerous biological agents able to diminish the levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, could potentially exhibit effectiveness in the management of gastric ulceration [
108]. In this context, indomethacin induced a pronounced inflammatory response in the gastric mucosa, as evidenced by notable increases in the infiltration of inflammatory cells, along with elevated levels of TNF-α, IL-6, and IL-1β, when compared to the undisturbed gastric mucosa observed in the control rats. The suppressive effects of the
A. graveolens extract on the IKκB/NF-κB signaling pathway may be linked to its ability to reduce the levels of IL-1β, IL-6, and TNF-α, as well as its antioxidant properties [
107,
109]. The tested extract exhibited the ability to inhibit the infiltration of inflammatory cells in the gastric region, leading to significant reductions in the levels of TNF-α, IL-1β, and IL-6 within the mucosal tissue. These findings suggest that
A. graveolens plays a role in mitigating inflammation. These findings align with prior observations indicating that an excessive production of pro-inflammatory cytokines is causative of gastric mucosal injury and exacerbates the inflammation of the gastric mucosa [
79]. Therefore, by reducing TNF-α, IL-6, and IL-1β expression,
A. graveolens may be considered as an alternative agent for treating gastric ulcer patients.
IL-6 exerts its stimulatory effects on lymphocytes, macrophages, and neutrophils present at the site of inflammation. This stimulation subsequently triggers an overproduction of ROS and lysosomal enzymes. Consequently, these excessive levels of ROS and lysosomal enzymes contribute to the detrimental tissue damage observed in gastric ulcers. TNF-α exerts a significant influence on the development of gastric mucosal injury caused by indomethacin [
110]. This influential cytokine diminishes blood flow to the gastric mucosa and enhances the expression of gastrin and vascular endothelial growth factor genes within the gastric mucosa. Consequently, it impedes the natural healing process of ulcers [
111]. TNF-α, a cytokine, is responsible for facilitating the transcription of various adhesion molecules. This process ultimately results in the observed infiltration of inflammatory cells in rats treated with indomethacin. Moreover, the infiltrating leukocytes play a significant role in the production of ROS, which can further disrupt the oxidative balance. IL-1β, a cytokine with pro-inflammatory properties, plays a crucial role in the regulation of multiple genes associated with the inflammatory response and the consequent tissue damage. This includes the disruption of enterochromaffin-like cell function. Furthermore, it has been observed that IL-1β exerts an inhibitory effect on the proliferation of gastric epithelial cells [
112]. Remarkably, the extract derived from
A. graveolens exhibited a notable improvement in the infiltration of inflammatory cells, while also significantly reducing the heightened levels of TNF-α, IL-1β, and IL-6 in the gastric contents of rats treated with indomethacin.
In previous investigations, it was observed that the production of TNF-α has the potential to enhance the production of nitric oxide (NO) through the overexpression of iNOS in indomethacin-induced jejunoileitis [
113]. NO which is generated by iNOS, assumes a significant role in the process of ulcer formation. This is primarily achieved through the generation of peroxynitrite (ONOO-) and subsequent cellular toxicity, protein tyrosine nitration, production of hydroxyl radicals (HO.), and consequent tissue damage [
114]. The current investigation demonstrated that the administration of indomethacin resulted in an upregulation of iNOS expression within the gastric tissues. This observation potentially correlates with the upregulation of TNF-α synthesis within the gastric tissues. The extract exhibited a level of inhibition in the expression of iNOS that was comparable to that of famotidine. This inhibition effectively prevented the excessive release of NO, which is known to worsen gastric mucosal injury. As a result, the extract contributed to the improvement of ulcer healing.
Pharmacology networking study was carried out to investigate the chemical-biological interactions between the identified metabolites of A. graveolens L. seed extract and gastric ulcer. The pharmacology network explained target genes related to the identified metabolites to possess 672 interactions between the 18 identified metabolites and 379 genes, among which PTGS2, MMP2 and PTGS1 were the top annotated genes related to gastric ulcer. The pharmacology network determined the top KEGG pathways of A. graveolens L. The PPI network identified VEGFA, HIF1A, TP53, and EGFR as the genes with high interactions. The top KEGG biological pathway according to fold enrichment was the bladder cancer and the top signaling pathway was the VEGF signaling pathway. These results assure the ability of the Apium graveolens L. to influence the gastric ulcer by 17 genes and determined the biological pathways.
Taken together, this study is the first to highlight the role of Apium graveolens L. in indomethacin-induced gastric ulcer, and correlate it to IKκB/NF-κB p65/IL1β, IL-6, TNF-α/iNOS signaling pathways. Additionally, it presents scientific evidence regarding the potential gastroprotective activity exhibited by Apium graveolens L. Furthermore, it reinforces the significance of Apium graveolens L. as a promising natural supplement with anti-ulcerative properties. Therefore, the clinical significance of investigating natural products combating gastric ulcers resides in their potential to offer efficacious, secure, and readily available therapeutic alternatives for the prevention and management of gastric ulcers.