1. Introduction
Gastric ulcer (GU) is a painful lesion of the gastric mucosa in the upper abdomen that can lead to more serious symptoms including internal hemorrhage, stenosis, perforation and pyloric obstruction. As a serious health problem affecting 4% of the population throughout the world [
1], GU is a complex multicausal disease induced by multiple factors, including Helicobacter pylori infection, nonsteroidal anti-inflammatory drugs (NSAIDs), stress, alcohol, damaged barrier effect of the gastric mucosa, excess secretion of gastric acid, etc. [
2,
3]. The restoration of protective layers of the gastric mucosa and decreasing gastric acid secretion are important for minimizing the tissue damage in treating GU. However, the conventional medications in GU treatment, including proton pump inhibitors (PPIs), histamine H2 receptor blockers, Helicobacter pylori eradication therapy and antacids [
4], may result in high relapse incidence and various side effects such as functional mucosal changes, intestinal microflora disorders, diarrhea and arrhythmia. Detecting efficient natural agents against GU may offer a chance to avoid the side effects of conventional therapies [
5]. Ginsenosides are the principal active ingredients in Panax ginseng C. A. Meyer and are mainly responsible for its various pharmacological activities including protective effects against gastric ulcer [
6,
7,
8]. Protopanaxatriol (PPT) is an aglycone of protopanaxatriol-type ginsenosides (Re, Rg1, Rg4, Rg6, Rh4, Rh1, Rg2, etc.), which were reported to possess wound-healing, anti-inflammation, immune-stimulatory and antioxidant activities in vivo in rats and mice [
9,
10,
11]. Protopanaxatriol-type ginsenosides could be metabolized to PPT both in the stomach (acid hydrolysis) and in the gastrointestinal tract (bacterial hydrolysis) after oral administration [
12,
13,
14]. In addition, PPT is not only a metabolite in body but also a natural compound that could be prepared by acid hydrolysis, alkaline hydrolysis or microbial transformation [
15]. PPT has shown anti-oxidation and anti-inflammation activities [
16,
17]; however, there was no literature on the therapeutic effect of PPT on GU.
Metabolomics, defined as the comprehensive qualitative and quantitative analysis of all small molecules (metabolites) present in a biosystem [
18], is a branch of “omics” that involves the identification and quantification of metabolites and chemical footprints of cellular regulatory processes in different biological species [
19] to monitor the dynamic changes in endogenous metabolites and systematically link metabolic pathways and disease processes as well as elucidate the mechanisms of action (MOA) of drugs [
20]. Network pharmacology, first proposed by Hopkins in 2008, emphasizes ‘multi-compounds, multi-target, and multi-disease’ rather than ‘a-drug, a-gene, a-disease’ drug action patterns and appropriately adheres to the holistic approach of herbal medicines [
21,
22], which is used to explore the complex relationships among proteins, diseases and drugs [
23]. A strategy of integrating these two approaches offers the opportunity to elucidate the molecular mechanism by connecting active ingredients, targets, endogenous metabolites and pathways, which may compensate each other for upstream molecular mechanisms and pathophysiological data.
In the current study, an acetic acid-induced gastric ulcer model, one of the representative experimental GU models [
24,
25], was used to investigate the therapeutic effect of PPT for treating GU. We also developed an integrated strategy combining metabolomics and network pharmacology to elucidate the molecular mechanism of PPT’s anti-GU effects; then, we used molecular docking to show the high affinities between key targets and PPT. The results shed light on the complicated pathogenesis of GU and suggested a potential natural anti-GU agent and strategies.
3. Discussion
Gastric ulcer (GU) is an ulcerative lesion in the surface of stomach mucosa, which is caused by various invasive factors especially the gastric acid secretion. Conventional medications to treat GU have shown various side effects and high relapse incidence. Thus, an effective antiulcer drug with few side effects is needed. PPT as an important human microbial metabolite of ginsenosides has shown good anti-oxidation and anti-inflammation activities. In this study, we have evaluated the therapeutic effect of PPT on GU.
GU is one of the major gastrointestinal disorders, which often occur due to an imbalance between offensive factors and defensive factors [
26]. The offensive factors mainly include gastric acid secretion, pepsin, proinflammatory cytokines (such as IL-6, TNF-α, ET-1, IL-2, IL-1β), oxidative stress (such as MDA, SOD, NOS2, GSH-Px) and platelet activating factor (PAF), and the defensive factors mainly include gastric mucus, gastric mucosa, EGF and EGFR, NO, NOS and prostaglandins (PGs). In addition, various evidence has indicated that both inflammation and oxidative stress played an important role in GU. ET-1, IL-6 and TNF-α were vital pro-inflammatory cytokines in GU formation. ET-1 could contract the vessels, reduce the blood supply of gastric tissue and cause local hypoxia and acidosis [
27,
28]. IL-6 could induce tissue damage by initiating neutrophil accumulation, inducing a transcriptional inflammatory response and producing noxious products, reactive oxygen radicals and the lysosomal enzymes responsible for tissue damage in GU [
29,
30]. TNF-α could elicit an acute inflammatory reaction accompanied by neutrophil infiltration in gastric mucosa and also regulate apoptotic cell death in the gastric mucosa [
31]. SOD could convert superoxide to hydrogen peroxide, and MDA is the final product of lipid peroxidation that causes loss of membrane integrity. Both SOD and MDA were reported to play important roles in the formation and development of the ulcer [
32]. EGF is a ligand of EGFR that reduces gastric acid secretion and promotes the epithelial cell repair. EGF can aid the healing of ulcers when secreted in the gastrointestinal tract, increase the blood flow of the basic gastrointestinal mucosa and regulate the immune system of the digestive tract [
33]. EGFR is important for the proliferation and differentiation of gastric mucosal cells, and its expression increases during early stages of gastric ulcer healing [
34]. Thus, the proinflammatory cytokines including IL-6 and TNF-α and reactive oxygen species including MDA and SOD, together with ET-1, EGF and EGFR, were used in this study to evaluate the anti-GU effect of PPT. In the acetic acid-induced GU model, SOD, EGF and EGFR were down-regulated, while ET-1, IL-6, MDA and TNF-α were up-regulated. The intervention of PPT treatment re-regulated these factors to normal levels and significantly reduced the ulcer areas in a dose-dependent manner, suggesting a remarkable gastroprotective effect of PPT.
Metabolomics and network pharmacology analysis were carried out to identify the pharmacological effect from the aspects of potential biomarkers, metabolic pathways, targets, respectively. Firstly, metabolomics was used to decipher the dynamic changes of endogenous metabolites for assessing drug effects systematically. In our study, pattern recognition with multivariate statistical analysis suggested that the metabolic profiles of the acetic acid-induced GU rats were markedly different from that of the normal control rats, while the PPT-treated GU treatment groups had similar metabolomic profiles as the normal control group. We identified 26 potential metabolites involving 7 metabolic pathways. Secondly, network pharmacology at the molecular level was carried out to explain the relationships between the latent metabolites and pathways. The PPI network was constructed, the bub genes were screened out, and the component-target-pathway network indicated that PPT was closely related with 45 target genes and 16 pathways. Finally, an integrated analysis of metabolomics and network pharmacology was performed, and the ‘compound-reaction-enzyme-gene’ network was established: 16 biomarkers, 3 targets and 3 metabolic pathways were screened out that were considered to be closely related to the therapeutic effect of PPT on GU.
Two of these three pathways were lipid metabolism, including linoleic acid metabolism and sphingolipid metabolism, and one was arachidonic acid metabolism. Alteration in the metabolism of lipid was reported to be disturbed in GU [
35]. Phosphatidylcholines (PCs) as the most abundant phospholipid in the cell and are directly related to cell proliferation, also play a vital role in lipid metabolism. Elevated level of 12,13-EpOME and decreased level of linoleic acid and PC (O-16:0/2:0) were observed in GU rats and restored by PPT as compared with the controls, suggesting PPT might relieve GU by stimulating linoleic acid metabolism, in accordance with previous studies [
36]. Sphingolipid metabolism was also reported to participate in the occurrence and development of ulcer [
37]. Sphingomyelins (SMs) are a group of common mammalian cell membrane sphingolipids that maintain cellular stability and regulate signal transduction. SMs including SM (d18:0/22:0), SM (d18:0/20:0), SM (d18:0/20:2 (11Z,14Z)), SM(d17:1/24:1(15Z)), SM(d18:1/14:0), SM (d18:1/24:1(15Z)) were down-regulated in the GU group. Phytosphingosine can prevent the acute ulcer formation while sphinganine participates in regulating the cell growth, adhesion, migration, death and inflammation process [
38,
39]. The abovementioned metabolites were re-regulated to normal levels after PPT treatment in GU rats, which highlights the involvement of sphingolipid metabolism in GU. In the arachidonic acid metabolism, arachidonic acid is one constituent of the cell membrane, which can be hydrolyzed, released or metabolized to various bioactive substances such as prostaglandins or 19(S)-HETE [
40]. The levels of 19(S)-HETE, arachidonic acid and prostaglandin F2a elevated in the GU model group. Phosphatidylethanolamines (PEs) are a kind of gastricsurface active phospholipids which are synthesized and secreted in the gastric surface cells [
41]. In the model group, increased levels of PE(22:1(13Z)/16:0), PE(20:3(8Z,11Z,14Z)/P-18:1(9Z)) and LysoPC (18:1(9Z)) were observed. They were re-regulated after PPT treatment indicating that the imbalance of arachidonic acid metabolism in GU can be reversed by PPT.
Three targets including CYP2C9, CYP3A4 and PIK3CA were suggested as the potential therapeutic targets. CYPs are members of the cytochrome P450 families, which are mainly expressed in the gastrointestinal tract and human liver. CYPs can convert numerous exogenous compounds, such as most therapeutic agents, to less toxic components [
42,
43]. CYP2C9 and CYP3A4 are two major CYP enzymes which are vital determinants for the oral bioavailability of drugs. CYP2C9 plays a key role in the oxidative metabolism of up to 15% drugs that undergo phase I metabolism while this enzyme was also closely related to non-aspirin NSAID-related gastrointestinal bleeding [
44,
45,
46]. CYP3A4 is a dominant CYP enzyme highly expressed in both liver and extra-hepatic tissues. It metabolizes around 60% of currently used drugs [
47,
48]. Omeprazole and glycyrrhizin are the drugs that widely used to treat peptic ulcer. Co-administration of omeprazole and glycyrrhizin has shown better therapeutic effect which was believed to through inducing the activity of CYP3A4 [
49]. Since the significant role of CYP3A4-mediated metabolism in PPIs clearance in humans, it is important to gain a better insights of the drug–drug interaction potential of PPIs with CYP3A4 inhibitors or inductors [
50]. Patients with GU have high risk of developing gastric cancer [
51]. Phosphatidylinostitol 3-kinase catalytic subunit (PIK3CA) can decrease the invasive capacity of gastric cancer cells in vitro [
52]. PIK3CA amplification could activate the PI3K/Akt pathway in gastric cancer resulting in the poor survival rates of gastric cancer patients [
53]. Microsomal cytochrome P450 also played an important role in the metabolism of PPT. It was reported that the oxidized metabolites of PPT were formed via combinatorial metabolism including both colonic microflora and the cytochrome P450 enzymes [
54]. Both chemical inhibition and human recombinant P450 isoform assays indicated that CYP3A4 was the predominant isozyme responsible for the oxygenation metabolism of PPT ginsenosides [
55]. PPT was metabolized to a pair of epimers (namely, M1-1 and M1-2), and CYP3A4 was the predominant isoform involved in the oxidative metabolism of M1-1 and M1-2 [
56].
Cytochrom enzymes including CYP2CA and CYP3A4 are very important superfamily of biotransformation enzymes that are involved in oxidative metabolism of a wide variety of endogenous and exogenous compounds such as drugs. Numerous people in many countries have taken ginseng or its derived products, however, little is known about the interactions between ginseng and prescription drugs. Therefore, it is necessary to evaluate whether ginseng and its active components exhibit the potential to exert influence on metabolic enzymes. Thus, it is meaningful to explore the relationship between PPT and P450 enzymes. Actually, the complex relationship among PPT, biomarkers, targets and metabolic pathways were found based on metabolomics study combined with network pharmacology study. That is, some metabolites had been re-regulated after PPT binding to CYP3A4 and CYP2CA. Just as shown in
Figure 10, PPT could indirectly regulate the metabolites such as linoleic acid and 12,13-EpOME by acting on the CYP2CA and CYP3A4. Although we did not perform the CYP induction assays, but previous studies showed that PPT exhibited moderate inhibition against CYP2C9 with an IC50 of 33.7 ± 2.7 μM, and inhibitory effect against CYP3A4 with an IC50 of 7.1 ± 0.9 μM [
57]. The activity of cytochrome P450 enzymes generated reactive metabolites and free radicals which in turn bind to macromolecules, caused membrane lipid peroxidation, and increased cellular toxicity. PPT might also inhibit the activities of cytochrome P450 enzymes in order to attain dynamic homeostasis and reduce cellular toxicity [
58].
Taken together, the three key target genes play important roles in the occurrence and development of gastric ulcer. Molecular docking also verified the high affinities of PPT with these key targets, while CYP3A4/PPT has the lowest binding energy.
Our findings showed that PPT could exert anti-GU effect similar to that of omeprazol. Though they showed similar effect, the mechanism of them were not the same. Omeprazol is belonging to proton pump inhibitors (PPIs), which could inhibit H+, K+-ATPase (proton pump), reduce the H+ secretion by parietal cells, effectively decrease gastric acid secretion and exert anti-GU effect. As for PPT, our study based on integrated metabolomics and network pharmacology showed that PPT could play a therapeutic effect on GU by regulating three key targets, 16 biomarkers and 3 metabolomic pathways. Both PPT and omeprazol showed good anti-GU effect, so they may be used together in patients. Though the mechanism of PPT and omeprazol were different, it is not sure whether there would be some potentially drug interactions after combination of them. Ginsenosides always showed synergistic effect by increasing efficacy and decreasing toxicity when combined with other drugs. For example, ginsenoside Rg3 can enhance drug efficacy and reduce drug-induced toxicity when used together with first-line chemotherapy drugs in clinical [
59], ginsenoside compound K could enhance the efficacy of cisplatin in lung cancer cells with low toxicity and minimal side effects [
60]. So we speculated that PPT may increase efficacy and decrease toxicity when combined with omeprazol against GU, which would be studied further in the future.
However, we are aware that our research may have some limitations. For instance, it would have been better if the experiments had been set up as days 1, 3, 5 and 7 and the data had been collected on each of these days. Future studies could improve the experimental design and collect time-course data.