Synergic Effects of Curcumin, Licorice, and Endoscopic Photodynamic Therapy Against Helicobacter Pylori by Modulating the NF-Κb Signaling Pathway

Abstract

Background:

Helicobacter pylori (H. pylori) infection affects nearly half of the global population and is a significant contributor to gastroduodenal conditions, including gastric carcinoma. Research on herbal extracts has highlighted their antibacterial, antioxidant, and anti-inflammatory properties, indicating potential for novel, synergistic therapeutic approaches.

Objective:

To assess the synergistic antimicrobial effect of Curcumin, Licorice, and photodynamic therapy (PDT) against the H. pylori infection in a rat model by evaluating the NF-κB signaling pathway.

Materials and Methods:

Forty male rats (250-300 g) were divided into eight groups as follows: control, rats treated with 600 mg.kg^-1 of Curcumin and 200 mg.kg^-1 of Licorice, H. pylori-infected rats, H. pylori-infected rats treated with 600 mg.kg^-1 of Curcumin, H. pylori-infected rats treated with 200 mg.kg^-1 of Licorice, H. pylori-infected rats treated with 600 mg.kg^-1 of Curcumin and 200 mg.kg^-1 of Licorice, H. pylori-infected rats treated with PDT (based on 3′-sialyllactose [3SL]-conjugated, poly-l-lysine-based photosensitizer [p3SLP]), and H. pylori-infected rats treated with PDT combined with Curcumin and Licorice. The immunohistochemistry test was performed to investigate NF-κB p65 expression in gastric epithelial cells (GECs).

Results:

The average H. pylori colonization score was zero for the control and Curcumin + Licorice groups. However, the scores were 1.8, 1.0, 1.2, 0.8, 0.6, and 0.2 for the H. pylori, H. pylori + Curcumin, H. pylori + Licorice, H. pylori + Curcumin and Licorice, PDT alone treatment, and PDT + Curcumin and Licorice groups, respectively. Regarding H. pylori colonization and gastric inflammation scores, the treatment effect of PDT combined with Curcumin and Licorice was significantly greater than that of the other treatments (P<0.05). H. pylori infection significantly increased the NF-κB expression in GECs compared to the control group (20.6%±2.1% vs. 10.1%±2.2%, P=0.035). This expression was significantly reduced in the H. pylori + PDT combined with Curcumin and Licorice (8.8%±1.2%), the H. pylori + Curcumin (15.3%±2.4%), and the H. pylori + Licorice groups (16.4%±2.2%) compared to the H. pylori only group (P <0.05). The administration of Curcumin and Licorice alone did not alter baseline NF-κB expression in GECs (8.3%±1.8%, P=0.120).

Conclusion:

Our results demonstrated that combination treatment of PDT with Curcumin and Licorice may exert stronger anti-inflammatory effects by suppressing NF-κB p65 expression in GECs compared to each treatment alone. This combination could serve as a potential treatment option for future studies.

1. Background

Helicobacter pylori (H. pylori) infection is prevalent globally, affecting approximately half of the world’s population, with 90% of individuals in developing nations impacted by this gram-negative bacterium ( 1 ). The persistence of this infection is associated with the development of various gastroduodenal conditions, including peptic ulcers, chronic gastritis, and gastric carcinoma. The standard treatment protocol for H. pylori-associated gastroduodenal diseases typically involves a combination of amoxicillin, a proton pump inhibitor, and clarithromycin or metronidazole ( 2 , 3 ). The extensive use of antimicrobials has led to increased levels of antimicrobial resistance globally. It has been reported that 95% of H. pylori-infected patients exhibit resistance to metronidazole, and 25% are resistant to clarithromycin ( 4 ). Furthermore, the effectiveness of these therapeutic regimens in clinical practice is limited, primarily due to inappropriate use and associated side effects. These challenges have hindered the development of novel antibacterial agents and methods with improved efficacy, safety, and cost-effectiveness ( 5 , 6 ) Several studies investigating extracts from herbal agents have explored the in vitro susceptibility of H. pylori, showing the potential for discovering new treatments .

Licorice, scientifically known as Glycyrrhiza glabra Linn. (Leguminosae), has a long history of use ( 12 ), and was traditionally considered a primary treatment for ulcers until the introduction of cimetidine ( 13 , 14 ). Various preclinical studies have reported the effectiveness of Licorice in the treatment of peptic ulcers ( 15 - 17 ). It has been shown that glycyrrhetinic acid, the major metabolite of glycyrrhizin, can inhibit H. pylori activity. Flavonoids present in Licorice have been identified as active agents against H. pylori ( 18 ), and have demonstrated antiulcer properties. Additionally, Licorice extract has been deemed clinically safe and effective in patients with functional dyspepsia ( 19 ). Moreover, this extract has been found to be non-genotoxic in various in vitro genotoxicity tests, including bacterial reverse mutation, micronucleus tests, and chromosome aberration ( 20 ).

Curcumin, a yellow polyphenolic pigment found in the roots of the Curcuma longa plant, is a member of the Zingiberaceae family ( 21 ). Curcumin polyphenols possess potent antioxidant and anti-inflammatory properties and are potentially capable of modulating crucial signaling molecules, making them of great interest for research and pharmacological applications ( 22 ). Curcumin can inhibit nuclear factor-kappa B (NF-κB) activity, which regulates interleukin-8 (IL-8) production and blocks the mutagenic response in H. pylori bacteria ( 23 ). Regarding the growth inhibition properties of Curcumin against H. pylori, studies have shown that this agent can inhibit the growth of H. pylori in in vitro settings ( 24 , 25 ).

The main roles of Curcumin and Licorice in H. pylori infection are outlined in Figure 1. Both possess important properties, including anti-inflammatory, antioxidant, anticancer, and antimicrobial effects ( 23 , 26 , 27 ), which inhibit H. pylori-induced NF-κB, activation-induced cytidine deaminase (AICD), IL-8, matrix metalloproteinases-3 (MMP-3), and MMP-9 in host epithelial cells ( 28 , 29 ).

Figure 1.

The role of Curcumin and Licorice in H. pylori infection.

Photodynamic therapy (PDT) utilizes photosensitizers that, upon laser irradiation, produce reactive oxygen species (ROS), leading to the oxidation of biomolecules and irreversible damage to cells. This approach has gained interest as a viable method for eliminating pathogenic bacteria ( 30 , 31 ). A key advantage of PDT over traditional antibiotic treatments is its ability to be administered repeatedly without promoting drug resistance ( 32 ). For an effective PDT strategy targeting H. pylori, it is essential to use an appropriate photosensitizer that specifically targets H. pylori, minimizing potential damage to healthy cells ( 33 ). One outer membrane protein of H. pylori is sialic acid-binding adhesin (SabA), which selectively binds to the α-2,3-linked terminal sialic acid of sialyl-dimeric-Lewis X antigens found on gastric epithelial cells ( 34 , 35 ). Therefore, 3′-sialyllactose (3SL), which also features α-2,3-linked terminal sialic acid, can be used as an H. pylori-targeting agent. Notably, 3SL demonstrates high specificity for H. pylori, as its receptors are not expressed in mammalian cells ( 36 ). Im et al. ( 37 ) developed a poly-L-lysine-based photomedicine (p3SLP) conjugated with 3SL that can bind to H. pylori bacteria and act as a photosensitizer in an endoscopic PDT laser system. p3SLP is an orally administered novel photosensitizer and could represent an effective strategy for clinical endoscopic PDT of H. pylori.

As there are different mechanisms for treating infections, combining various techniques may provide additive or even synergistic therapeutic effects. The synergistic effects observed when combining different agents or methods primarily arise from their diverse mechanisms, including antibacterial, antioxidant, and anti-inflammatory properties. Due to these therapeutic advantages, combinations of drugs and methods have gained widespread acceptance and emerged as a primary alternative for treating infectious diseases ( 50 , 57 - 59 ).

2. Objectives

The objective of this research was to evaluate the synergistic antimicrobial impact of PDT in combination with Curcumin and Licorice against H. pylori infection in a rat model, with a focus on the NF-κB signaling pathway.

3. Materials and Methods

3.1. Animals and Experimental Groups

All experiments and procedures involving animals were approved by the Animal Experimentation Ethics Committee and the National Research Ethics Board (NO. SDTHEC2022012056). Forty male Sprague-Dawley rats, weighing 250-300 g, were divided into eight identical groups. The animals were housed in a temperature-controlled room maintained at 22-25 °C, with a 12-hour light/dark cycle.

The animal groups included: 1) Control: the rats received a standard saline solution (1 mL per rat) orally via an intragastric tube (IGT) twice a day with a 4-hour interval for three consecutive days. After 14 days, the rats were administered a standard saline solution (1 mL per rat) orally via IGT once daily for 7 days; 2) Curcumin+Licorice: the rats received 600 mg.kg^-1 of Curcumin and 200 mg.kg^-1 of Licorice extracts orally, administered with normal saline (2 mL per rat) via an IGT twice daily with a 4-hour interval, continuously for three consecutive days. After 14 days, the rats were given 600 mg.kg^-1 of Curcumin and 200 mg.kg^-1 of Licorice dissolved in 0.1% DMSO (2 mL per rat) orally via IGT once daily for 7 days; 3) H. pylori-infected rats: the animals were infected with H. pylori for three consecutive days; 4) H. pylori treatment with Curcumin (H. pylori + Curcumin): H. pylori-infected rats were treated with 600 mg.kg^-1 of Curcumin for 7 days, starting two weeks after infection; 5) H. pylori treatment with Licorice (H. pylori + Licorice): H. pylori-infected rats were treated with 200 mg.kg^-1 of Licorice for 7 days, starting two weeks after infection; 6) H. pylori treatment with Curcumin and Licorice (H. pylori + Curcumin and Licorice): H. pylori-infected rats were treated with 600 mg.kg^-1 of Curcumin and 200 mg.kg^-1 and Licorice for 7 days, starting two weeks after infection; 7) H. pylori treatment with PDT: H. pylori-infected rats were treated with PDT based on 3′-sialyllactose (3SL)-conjugated poly-L-lysine-based photosensitizer (p3SLP) once, two weeks after infection; and 8) H. pylori treatment with PDT combined with Curcumin and Licorice: H. pylori-infected rats were treated with PDT (once), after being treated with 600 mg.kg^-1 of Curcumin and 200 mg.kg^-1 and Licorice for 7 days, starting two weeks after infection.

3.2. Inoculation of H. Pylori Infection

The induction of H. pylori (strain ATCC 43504, Beinuo Life Science, Shanghai, China) infection was performed following the method described by Duangporn Werawatganon ( 42 ). The rats were pretreated with streptomycin suspended in tap water (5 mg. mL^-1) for three days. The animals were then infected with an H. pylori suspension (5×10⁸ to 5×10¹⁰ CFU.mL^-1; 1 mL per rat), administered via gavage twice daily with a 4-hour interval for three consecutive days.

3.3. Assessment of H. Pylori Infection

The presence of H. pylori infection in the animals was evaluated through histological examination. For this, the stomachs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) at room temperature for 24 hours. The tissue was then processed, embedded in paraffin, and cut into 5 µm-thick sections. The sections were stained with eosin and hematoxylin and microscopically examined for the presence of H. pylori infection. In cases where clarity was insufficient, H. pylori infection was further confirmed using Warthin-Starry staining.

A grading system was used to assess the level of bacterial colonization as follows: score 0 indicated no detectable bacteria; score 1 represented mild colonization in some gastric crypts; score 2 indicated mild colonization in most gastric crypts; score 3 reflected moderate colonization in all gastric crypts; and score 4 denoted dense colonization in some gastric crypts. The results were presented as bacterial colonization scores for each group. Additionally, gastric inflammation was classified according to the Sydney system ( 43 ), where infiltration of mononuclear and polymorphonuclear leukocytes in the gastric mucosa determined the inflammatory scores. Scores ranging from 0 to 3 corresponded to normal, mild, moderate, and marked histopathological changes, respectively.

3.4. In vivo Anti-H. Pylori Study

Following the method outlined by Im et al. ( 37 ), p3SLP, a PDT agent designed to target H. pylori, was synthesized using 3SLs, poly-L-lysine (PLL), and the photosensitizer PheoA. The multiple 3SL moieties were conjugated to PLL to enable effective binding to SabA, a protein found on the outer membrane of H. pylori. For treatment, p3SLP in distilled water (with a PheoA concentration of 2.5 μg.mL^-1) was administered two weeks post-infection. After 30 minutes, the subjects were irradiated with a fiber-coupled laser system (LaserLab, Korea) emitting 20 watts of near-infrared light (wavelength 1064 nm, beam diameter 8 mm). The laser operated in continuous mode for 30 minutes, with the laser tip stabilized via a feeding needle catheter.

Forty-eight hours after laser treatment, the rats were euthanized, and their stomachs were excised. The stomachs were opened along the gastrointestinal tract, and their internal contents were removed and rinsed with PBS. Cell membranes were permeabilized using 0.1% (v/v) Triton^TM X-100 (Sigma-Aldrich) at 4 °C for 5 minutes. The resulting suspension was serially diluted 10-fold in PBS, ranging from 10^-1 to 10^-5 of the original concentration. Ten microliters of each dilution were inoculated onto Brucella agar plates supplemented with Skirrow’s additives (2.5 IU. mL^-1 polymyxin B, 5 μg. mL^-1 trimethoprim lactate, and 10 μg. mL^-1 vancomycin) and 10% horse serum. The plates were incubated for 2–3 days at 37 °C in a gas mixture containing 5% O₂, 10% CO₂, and 85% N₂ to determine colony-forming units (CFUs).

3.5. Immunohistochemistry

The stomach sections were deparaffinized using xylene and gradually dehydrated in ethyl alcohol. Antigen retrieval was then performed by immersing the sections in a citric acid buffer (pH 6.0) and heating them in a microwave oven for 13 minutes. To block endogenous peroxidase activity and prevent nonspecific binding, the sections were treated with 3% hydrogen peroxide (Merck, Hohenbrunn, Germany) for 5 minutes, followed by 3% normal horse serum (Gibco, Carlsbad, CA, USA) for 20 minutes.

Following these steps, the sections were incubated with a polyclonal antibody against the p65 subunit of NF-κB, diluted 1:100, in a humidified chamber for 1 hour at room temperature. Next, the sections were incubated with biotinylated anti-immunoglobulin in a humidified chamber for 30 minutes. The reaction was visualized using diaminobenzidine as the substrate, followed by additional staining with hematoxylin. Under a light microscope, NF-κB p65 expression was predominantly cytoplasmic, with occasional positive nuclear staining observed throughout. Cells exhibiting a dark brown stain in their nuclei were identified as NF-κB p65 immunoreactive cells (IRCs). For quantification, one thousand gastric epithelial cells (GECs) were manually counted per rat under a 40X objective lens.

3.6. Statistical Analysis

The mean values of the data were analyzed using one-way analysis of variance (ANOVA), followed by Fisher’s Least Significant Difference (LSD) post hoc test. Correlations between the NF-κB p65 IRCs and H. pylori infection scores were evaluated using Pearson’s correlation. All statistical analyses were performed using SPSS version 22 (SPSS Inc., Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.

4. Results

4.1. Assessment Of H. Pylori Colonization and Histological Alterations

Histological examinations revealed H. pylori colonization in the H. pylori-related groups; however, no colonization was observed in the control and Curcumin + Licorice groups (Fig. 2). In contrast, the infected rats exhibited significant inflammatory infiltrates, characterized by numerous lymphocytes and neutrophilic granulocytes, along with mild chronic inflammation in the areas affected by H. pylori.

Figure 2.

Histological examinations. A) Control; B) H. pylori; C) H. pylori + Curcumin; D) H. pylori + Curcumin and Licorice; and E) H. pylori + PDT combined with Curcumin and Licorice groups.

As expected, there was a significant reduction in immune cell infiltration and H. pylori presence in the groups treated with PDT alone, as well as those receiving the combination of Curcumin and Licorice, and the PDT with Curcumin and Licorice group. Furthermore, no apparent damage was observed in the stomachs of healthy rats treated with the Curcumin and Licorice combination. Importantly, H&E-stained images of gastric tissue from the PDT and Curcumin and Licorice groups revealed no significant pathological alterations. Collectively, these findings suggest that p3SLP effectively targets H. pylori during PDT while minimizing undesirable side effects.

The levels of H. pylori colonization and gastric in-flammation scores are presented in Table 1. The average score for the control and Curcumin + Licorice groups was zero. However, the scores for H. pylori colonization were 1.8, 1.0, 1.2, and 0.8 for the H. pylori, H. pylori + Curcumin, H. pylori + Licorice, and H. pylori + Curcumin and Licorice groups, respectively. In the PDT alone and PDT + Curcumin and Licorice groups, these scores were further reduced to 0.6 and 0.2, respectively.

Table 1.

The level of H. pylori colonization and gastric inflammation scores.

Groups (n=5 for each group)	Level of H. pylori colonization					Score of gastric inflammation
	0	1	2	3	4	0	1	2	3
Control	5	-	-	-	-	5	-	-	-
Curcumin+ Licorice	5	-	-	-	-	5	-	-	-
H. pylori	-	2	2	1	-	-	3	2	-
H. pylori + Curcumin	1	3	1	-	-	1	3	1	-
H. pylori + Licorice	1	2	2	-	-	1	3	1	-
H. pylori + Curcumin and Licorice	2	2	1	-	-	2	2	1	-
H. pylori + PDT	3	1	1	-	-	2	2	1	-
H. pylori + PDT combined with Curcumin and Licorice	4	1	-	-	-	3	2	-	-

Similarly, the gastric inflammation scores in the H. pylori-related groups (H. pylori, H. pylori + Curcumin, H. pylori + Licorice, H. pylori + Curcumin and Licorice, H. pylori + PDT, and PDT + Curcumin and Licorice) were 1.4, 1.0, 1.0, 0.8, 0.8, and 0.4, respectively.

4.2. NF-Κb P65 Expression in the Investigated Groups

The mean ± standard deviation (SD) of NF-κB p65 IRCs (%) across all experimental groups, along with the corresponding statistical values, is illustrated in Figure 3. H. pylori infection significantly elevated NF-κB levels in GECs, with the proportion of IRCs notably increasing in the H. pylori group (20.6% ± 2.1%) compared to the control group (10.1% ± 2.2%, P = 0.005). Supplementation with Curcumin and Licorice reduced NF-κB levels in GECs (P < 0.03).

Figure 3.

Mean ± standard deviation (SD) of the NF-κB p65 immunoreactive cells (%) in all experimental groups. The “*” sign shows the significant difference with the control group (P<0.05). The “#” sign shows the significant difference H. Pylori without treatment group (P<0.05). The “**” “#” sign shows the significant difference between the groups determined by the bracket.

The findings indicated no significant difference in IRC values between the H. pylori + Curcumin and H. pylori + Licorice groups (P > 0.05). In the Curcumin + Licorice group, the baseline NF-κB levels in GECs remained unchanged. However, a significant reduction in IRCs was observed in the H. pylori + Curcumin and Licorice group compared to other H. pylori-infected groups (except the control and Curcumin + Licorice groups).

PDT alone for H. pylori led to a greater reduction in IRCs than treatment with Curcumin or Licorice alone or their combination. However, the difference between PDT alone and PDT combined with Curcumin and Licorice was not statistically significant (P = 0.174). Notably, the combination of PDT with Curcumin and Licorice resulted in the lowest IRCs among all H. pylori-treated groups, indicating a synergistic effect of the combined treatment methods.

4.3. Correlation Of NF-Κb P65 Expression with Scores of H. Pylori Colonization and Gastric Inflammation

The percentages of NF-κB p65 IRCs were plotted against H. pylori colonization and gastric inflammation scores for individual rats across all groups (Fig. 4). A strong positive correlation was observed between NF-κB levels and H. pylori colonization scores (R² = 0.840) as well as gastric inflammation scores (R² = 0.793).

Figure 4.

Correlation of the NF-κB p65 immunoreactive cell (%) with A) H. pylori colonization and B) gastric inflammation scores.

5. Discussion

In this study, we evaluated the effects of Curcumin, Licorice, and PDT individually, as well as their combined effects, on NF-κB, p65 levels, IRC scores, and H. pylori colonization in a rat model. NF-κB is a critical regulator of various cellular processes, particularly immune responses and inflammation ( 44 , 45 ). It functions as a dimeric complex composed of five mammalian Rel proteins: c-Rel, p65, p52/NF-κB2, p50/NF-κB1, and RelB, which can form diverse combinations. In quiescent cells, NF-κB is sequestered in the cytoplasm through its association with inhibitors (IκB). Activation of specific intracellular signaling pathways leads to the activation of the IκB kinase complex (IKK complex). The activated IKK complex phosphorylates IκB at specific amino acid residues, triggering the polyubiquitination of IκB ( 46 ). This process results in IκB degradation by the proteasome, allowing NF-κB to be released and activated. Subsequently, NF-κB translocates to the nucleus, where it modulates gene expression dependent on NF-κB activation.

NF-κB is known to upregulate genes encoding proinflammatory cytokines and chemokines, playing a significant role in H. pylori-induced gastric infla-mmation ( 47 ).

Sintara et al. ( 48 ) investigated the potential of Curcumin (administered at doses of 200 or 600 mg.kg^-1) in mitigating NF-κB p65 expression and macromolecular leakage in the gastric mucosa of 25 H. pylori rats. They found that NF-κB p65 expression in GECs and the leakage of macromolecules from the gastric mucosal microcirculation significantly increased in the H. pylori group compared to the control group. Specifically, the IRCs in the H. pylori and control groups were 16.02% and 10.72%, respectively (P = 0.004), and macromolecular leakage in the H. pylori and control groups was 15.41% and 10.69%, respectively. However, treatment with Curcumin significantly reduced both NF-κB p65 expression (IRCs and macromolecular leakage) compared to the H. pylori group. The results of our study are consistent with the findings of Sintara et al. ( 48 ).

In the present study, we found that Licorice could reduce the side effects of H. pylori by modulating NF-kB. Several studies have also reported the ameliorating effects of Licorice ( 29 , 49 ). For instance, Asha et al. ( 45 ) evaluated the anti-H. pylori effects of GutGards, a flavonoid-rich extract derived from Licorice, and explored its potential mechanisms. They conducted assays for DNA gyrase, protein synthesis, dihydrofolate reductase, and an anti-adhesion assay using a human gastric mucosal cell line to investigate GutGards’ anti-H. pylori mechanisms. The results showed that GutGards exhibited anti-H. pylori activity in both agar and microbroth dilution methods. Glabridin, the primary flavonoid constituent of GutGards, demonstrated notable activity against H. pylori and potentially inhibited DNA gyrase, protein synthesis, and dihydrofolate reductase. In another study, Frattaruolo et al. ( 29 ) investigated the chemical composition of three Licorice leaf extracts, obtained using maceration and ultrasound-assisted techniques. They identified three primary components: glabranin, pinocembrin, and licoflavanone. All extracts exhibited antioxidant properties, and they observed modulation of the NF-κB/mitogen-activated protein kinases (MAPK) pathway, which underlies these effects. This suggests the potential of Licorice as a new scaffold in anti-inflammatory drug research ( 50 ). Previous studies on native Glycyrrhiza glabra L. have identified a strong flavonoid with antioxidant and anti-inflammatory activities, pinocembrin, in leaf extracts . Pinocembrin, the principal component of the total extract recoverable from the methanol solution after column chromatography, is known for its antioxidant and anti-inflammatory properties. It can downregulate tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 1 beta (IL-1β) ( 53 ). These activities involve significant suppression of the phosphorylation and activation of extracellular signal-regulated kinase (ERK), p38MAPK, and c-Jun NH2-terminal kinase (JNK), as well as reduction in the phosphorylation of IκBα, which is directly implicated in the regulation of NF-κB, as demonstrated by experiments conducted in vitro and in vivo ( 54 ).

We used p3SLP as a photosensitizer for PDT in rats. As demonstrated in a previous study ( 37 ), this agent successfully binds to SabA, which is expressed on the outer membrane of H. pylori bacteria. This agent remains stable for up to 2 hours in the highly acidic environment of the stomach following oral administra-tion. Furthermore, p3SLP is highly soluble in water and exhibits increased photoactivity in the aqueous phase. p3SLP generates singlet oxygen more efficiently upon laser irradiation compared to its solid phase. Therefore, we diluted p3SLP in distilled water and administered it orally to rats as a photosensitizer for H. pylori in PDT treatment. In a study by Im et al. ( 37 ), the effects of PDT using the p3SLP photosensitizer on H. pylori were assessed in C57BL/6 mice. The researchers reported significant H. pylori -specific antibacterial effects of PDT, with no adverse effects on normal tissues, as indicated by gastrointestinal pathological analyses of the infected mice. Additionally, an anti-inflammatory response was noted at the infection site following p3SLP treatment. Fluorescence of p3SLP was detected in the stomach and duodenum immediately after administration. The study revealed that while p3SLP gradually migrated towards the tip of the intestine over time, a substantial amount remained in the stomach for up to 12 hours. By 24 hours post-oral inoculation, nearly all of the p3SLP had moved into the large intestine. Importantly, no fluorescence of p3SLP was found in major organs, suggesting negligible migration to these areas. This indicates that orally administered p3SLP is unlikely to be absorbed systemically and will be excreted through the large intestine ( 37 ). Overall, the study highlights the efficacy of anti- H. pylori PDT with p3SLP in infected mice and suggests that this approach has significant potential as an alternative to antibiotic-based therapies.

We combined Curcumin and Licorice observed higher therapeutic effects compared to each agent alone, regarding the level of H. pylori colonization, gastric inflammation, and IRCs. Combining PDT with these agents also resulted to a very high treatment effect, which can be considered a potential treatment for future research in humans. Numerous studies have demonstrated the synergistic potential of combining different agents in terms of antioxidant activity and inhibiting cellular damages ( 40 , 55 - 58 ). Parasramka and Gupta ( 56 ) examined the combination treatment efficiency of garcinol and Curcumin, on human pancreatic cancer cells (BxPC-3 and Panc-1). They observed that the combined use of garcinol and Curcumin had 2 to 10 times more treatment effect compared to individual application of these compounds. This suggests that the synergistic interaction between Curcumin and garcinol enhances their bioactivity, thus reducing the effective dose for each compound when used alone. In a study by Sharma et al. ( 58 ), the synergistic antioxidant and antibacterial effects of essential oils derived from six selected medicinal plants (Citronella java, Ocimum gratissimum L., Mentha longifolia L., Vitex negundo L., Callistemon lanceolatus Sweet, and Cymbopogon flexuosus) were investigated both individually and in combination with synthetic antioxidants and antibiotics. The antibacterial synergistic effects were assessed against three Gram-positive and two Gram-negative bacteria. The results revealed synergistic interactions among combinations of essential oils, leading to enhanced antioxidant and antibacterial activities.

It was reported that combination of Curcumin with various antimicrobials treatments against H. pylori, potentially enhancing eradication rates ( 59 ). For instance, the aqueous extract of Hibiscus sabdariffa L. was found to exhibit a significant synergistic effect when combined with clarithromycin and metronidazole against H. pylori isolates, resulting in a 40 and 46-fold reduction in dosage, respectively ( 60 ). Ranjbar and Mohammadi ( 61 ) investigated the synergistic effects of combining Curcumin and antibiotics in alleviating H. pylori in Balb/c mice. While no significant difference was noted between the groups of mice treated with Curcumin and antimicrobials separately, synergism was observed upon combining the drugs. This combination led to a reduction in the levels of gastrin, IFNγ, and certain enzymes involved in lipid peroxidation in the animals. Feliciano et al. ( 38 ) assessed the synergic effect of Curcuma extract with Antimicrobials against H. pylori isolates. They found that the combination Curcuma extract with metronidazole, levofloxacin, and rifampin exhibited a synergistic effect across all isolates.

There are notable disparities in inhibition concentration values in previous studies. These variations stem from differences in the chemical composition of the extract, which are influenced by factors such as the extraction methods, the evaluated doses, the methods for determining antimicrobial activity, and the stage of development and extraction of the product ( 62 ).

One primary limitation of our study was the restricted selection of doses for Curcumin and Licorice. Future investigations could explore additional doses to ascertain the optimal dosage for each agent. Another limitation of our study is that we used one type of photosensitizer PheoA, as well as one frequency and power of laser exposure. Other appropriate photosensitizer along with suitable laser irradiation, can be used in future investigations. We only evaluated the NF-κB signaling pathway in this study, as our main aim was to assess the combination effect of Curcumin, Licorice, and PDT on H. pylori treatment. Understanding the molecular interactions and signaling pathways could be a potential subject for future research. Furthermore, our technique could be a suitable candidate for the treatment of other infections. Therefore, assessing the synergistic effect of Curcumin and Licorice with other natural compounds and PDT could be investigated for their antibacterial and anti-inflammatory effects on various infectious diseases.

6. Conclusion

The current study revealed that H. pylori triggered NF-κB activation in GECs. Supplementation with Curcumin and Licorice may exert anti-inflammatory effects by suppressing NF-κB p65 expression in these cells. Moreover, PDT can effectively reduce H. pylori colonization, stomach inflammation, and IRCs. The combination of PDT with the plant extract demonstrated strong synergism in vivo, suggesting its potential use as a complementary treatment for H. pylori infection.