
==== Front
J Med Life
J Med Life
JMedLife
Journal of Medicine and Life
1844-122X
1844-3117
Carol Davila University Press Romania

JMedLife-17-574
10.25122/jml-2023-0544
Original Article
Caffeic acid phenethyl ester attenuates Enterococcus faecalis infection in vivo: antioxidants and NF-κB have a protective role against stomach damage
Al-Ghamdi Abdulaziz Yahya 1 *
1 Biology Department, Faculty of Science, Al-Baha University, Al-Baha, Saudi Arabia
* Corresponding author Abdulaziz Yahya Al-Ghamdi Biology Department, Faculty of Science, Al-Baha University, Al-Baha, Saudi Arabia E-mail: drazizghamdi2008@gmail.com
6 2024
17 6 574581
31 12 2023
16 4 2024
© 2024 by the authors.
2024
https://creativecommons.org/licenses/by/4.0/ This open access article is published and licensed by the Journal of Medicine and Life under Creative Commons Attribution 4.0 International (CC BY 4.0). http://creativecommons.org/licenses/by/4.0
The mammalian gastrointestinal tract hosts a significant microbial symbiont community, an intriguing feature of this complex organ system. This study aimed to investigate the anti-inflammatory, antioxidant, and protective effects of caffeic acid phenethyl ester (CAPE) against Enterococcus faecalis infection in the stomach at a dose of 106 CFU in Swiss mice. A total of 30 mice were randomly assigned to three groups of ten mice each. Group I was the negative control, Group II was infected orally with E. faecalis for 18 days, and Group III was infected with E. faecalis and treated with CAPE orally at a daily dose of 4 mg/kg for 18 days. We assessed the antioxidant activities of stomach homogenate and the immunohistochemical expressions of the transcription factor nuclear factor kappa B (NF-κB) and proliferating cell nuclear antigen (PCNA). Histopathological examination was performed on the stomachs of all mice. Group II had decreased levels of antioxidant activity and positive expressions of NF-κB and PCNA. Histological observations revealed an increase in mucosal and glandular thickness compared with Group I. Group III, treated with CAPE, showed a significant increase in antioxidant activities and a significant decrease in NF-κB and PCNA immunoreactivities compared with Group II. In addition, Group III showed restoration of the normal thickness of the non-glandular and glandular parts of the stomach. Our results revealed that E. faecalis infection has damaging effects on the stomach and proved that CAPE has promising protective, anti-inflammatory, and antioxidant effects against E. faecalis. Further studies may investigate the potential therapeutic effects of CAPE against E. faecalis infection.

Enterococcus faecalis
CAPE
NF-κB
PCNA
antioxidant
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pmcINTRODUCTION

Enterococcus faecalis is a Gram-positive, facultative anaerobic bacterium found in various environments, including the gastrointestinal tract of humans and animals, soil, water, and fermented foods [1,2]. Despite its commensal nature, E. faecalis is responsible for many infections, such as urinary tract infections, endocarditis, and surgical site infections [3]. Its adaptability, intrinsic resistance to antibiotics, and ability to acquire exogenous resistance genes through horizontal gene transfer pose significant challenges to its clinical management [4,5].

E. faecalis is a leading cause of nosocomial infections, particularly in immunocompromised patients or those undergoing invasive procedures. The development of multidrug-resistant strains further complicates treatment options, necessitating a multifaceted approach to managing E. faecalis infections [6,7]. Owing to its intrinsic and acquired resistance mechanisms, E. faecalis has become less susceptible to many commonly used antibiotics, including β-lactams, aminoglycosides, and vancomycin [8]. Therefore, a deeper understanding of E. faecalis and the development of novel therapeutic strategies are needed to improve patient outcomes [9,10].

An important active ingredient in honeybee propolis extract, caffeic acid phenethyl ester (CAPE) has long been used in traditional medicine [11]. Research has demonstrated that CAPE exhibits a range of beneficial properties, including anti-inflammatory, immunomodulatory, antineoplastic, antioxidant, and wound-healing effects [12,13]. The inflammatory response is initiated by releasing chemical mediators from damaged tissues and migrating cells. These mediators include biogenic amines, eicosanoids (metabolites of arachidonic acid), platelet-activating factors, cytokines like interleukins and tumor necrosis factor-α (TNF-α), and reactive oxygen species [14]. These compounds are produced by inflammatory cells, including mast cells, endothelial cells, macrophages, monocytes, lymphocytes, and polymorphonuclear leukocytes (neutrophils, eosinophils, and basophils) [15]. CAPE suppresses the inflammatory process by preventing the production of chemokines, cytokines, T-cell proliferation, and lymphokines. In particular, CAPE is a strong and selective inhibitor of activating the transcription factor nuclear factor kappa B (NF-κB), which may be the molecular basis for its various anti-inflammatory and immunomodulatory effects [16,17].

NF-κB is involved in numerous physiological processes, such as inflammation, cell division, and immunological responses [18]. It facilitates the transcription of several cytokines, enzymes, chemokines, antiapoptotic factors, and cell growth factors [19]. Both in vitro and in vivo studies have shown that CAPE, at micromolar concentrations, exhibits various biological activities, including the selective inhibition of NF-κB and the suppression of the lipoxygenase pathway of arachidonic acid metabolism during inflammation [14]. CAPE inhibits NF-κB activation triggered by reactive oxygen species (ROS)-generating agents in human histiocytic and coronary artery endothelial cells [20]. It achieves this by blocking the interaction between NF-κB proteins and DNA rather than preventing the degradation of inhibitor κB α. [21]. The inhibition of ROS suppression of NF-κB activation and the direct inhibition of iNOS catalytic activity are most likely caused by the anti-inflammatory effect of CAPE [22].

This study aimed to highlight the anti-inflammatory and protective role of CAPE against acute inflammation caused by E. faecalis. We assessed various inflammatory and biochemical markers in stomach tissue homogenates, including antioxidants and enzymes. It also aimed to analyze NF-κB, with a specific focus on proliferating cell nuclear antigen (PCNA) as an important inflammatory marker. This study may help diagnose and monitor inflammatory conditions.

MATERIAL AND METHODS

Animals

Healthy adult Swiss albino male mice (22–25 g) were obtained from King Abdulaziz University, Jeddah, Saudi Arabia. Mice were acclimatized for 5 days at the Faculty of Science, Al-Baha University, under adapted temperature, kept in a 12-h light-dark cycle, and maintained on a standard diet and water ad libitum. The study followed the animal handling guidelines of the Ethical Committee and Scientific Research, University of Al-Baha.

Enterococcus faecalis

Ten mice were used to determine the number of viable Enterococcus faecalis bacteria. We performed gastric gavage with 0.5 ml of diluent from a frozen overnight culture, using a Perfektum stainless-steel feeding tube (Popper & Sons), aiming to inoculate each mouse with 106 colony-forming units (CFUs). The weight and clinical condition of the mice were monitored every 2–3 days. Each mouse received E. faecalis (106 CFU/mouse) three times a week for three weeks. The number of viable E. faecalis bacteria was determined in mouse feces using the drop plate method [23]. Fecal samples were dissolved in sterile phosphate-buffered saline (PBS), and 10 µL of each serial dilution was plated three times as drops onto Enterococcagar to detect E. faecalis. Petri dishes were incubated aerobically at 37 °C for two days. Colonies displaying typical morphology on Enterococcagar differential media were counted, averaged, and multiplied by the dilution factor, accounting for the weight of the fecal sample.

CAPE and lethality study

To create a solution with a 15 mg/ml concentration, CAPE was dissolved in a droplet of dimethyl sulfoxide (DMSO) (Sigma). According to their intended usage, different substance concentrations were made in PBS. The mice were divided into four groups of ten each and were given serialized CAPE concentrations. The number of live mice was counted each day. The procedure was continued following ethical standards for 30 days. Following the injection of DMSO, a series of ten mice tests showed no significant effect, and the vehicle was considered appropriate for administration.

Experimental design

Mice were randomly distributed into three groups of ten mice per group. Group I was kept as the negative control. Group II was injected orally with E. faecalis at a dose of 106 CFU in 0.5 ml of saline every 2 days for 18 days and did not receive any treatment. Group III was injected orally with E. faecalis at a dose of 106 CFU in 0.5 ml of saline and treated orally daily with 4 mg/kg of CAPE, starting from day 1, concurrent with E. faecalis 106 CFU in 0.5 ml of saline, for 18 days, after a high level of stool colonization in mice was established. At 24 h after the last treatment, the stomach was removed, cleaned, and prepared to assess the levels of antioxidants superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA). The remaining stomach was kept in 10% formalin for immunohistochemical and histopathological assessment.

Determination of SOD, GST, GSH-Px, and MDA activity

The stomach tissue was cut into small pieces, washed with PBS, and ground in a homogenization buffer (0.05 M Tris-HCl pH 7.9, 25% glycerol, 0.1 mM EDTA, and 0.32 M (NH4)2SO4) containing a protease inhibitor tablet (Roche). The tissue lysates were then homogenized on ice using an HG-15D homogenizer (Witeg Labortechnik). The solution was sonicated for 15 seconds in an ice bath to avoid overheating. Next, it was centrifuged for 5 min at 12,000 r.p.m. and 4 °C. After isolating the supernatant and storing it at −80 °C, the homogenate’s SOD, GST, and GSH-Px activity was measured by determining the amount of protein in each sample. GST activity was assessed by reacting the GSH−SH group with 1-chloro-2,4-dinitrobenzene, while GSH-Px activity was measured by monitoring the reduction of t-butyl hydroperoxide with nicotinamide adenine dinucleotide phosphate. All measurements were performed using a Spectro 24RS visible spectrophotometer (Labomed) with a temperature-controlled cuvette holder at 19 °C, and enzyme activities were expressed in U/g. The following wavelengths were used in the analysis: 480 nm for SOD, 340 nm for GST, and 412 nm for GSH-Px, as described previously [24]. Malondialdehyde (MDA) is a primary byproduct of lipid peroxidation. It reacts with thiobarbituric acid, along with other byproducts, to form a colored complex that has a maximum absorbance of 535 nm. This absorbance represents the color produced by all thiobarbituric acid-reactive substances [25].

Immunohistochemical examination of NF-κB

Immunohistochemistry was used to assess the expression of NF-κB in all mouse groups. After rinsing in PBS, the prepared sections were covered for 1.5 h in 1: 100 mouse anti-NF-κB p65 antibodies (Sigma-Aldrich). The sections were then washed and treated for 30 min with a secondary antibody tagged with a poly-horseradish peroxidase enzyme (Sigma-Aldrich). The slides were treated with freshly prepared 3,3′ diaminobenzidine tetrahydrochloride solution for 5 min, washed and treated with Mayer’s hematoxylin, dehydrated in graded alcohol (50%, 70%, 90%, and 100%), cleared with xylene, and mounted using a nonaqueous permanent mounting medium, as described previously [26].

Immunohistochemical examination of PCNA

Each mouse stomach was removed, and 4mm slices were cut and put on silane-coated slides. The sections were deparaffinized with 100% xylene and rehydrated through a graded ethanol series. For antigen retrieval, the sections were immersed in tris-buffered saline (TBS) with a pH of 6 and heated in a microwave oven at 750 W. After cooling to room temperature, the sections were treated with MCM7 monoclonal mouse anti-human primary antibodies (Thermo Fisher Scientific) and PCNA monoclonal mouse anti-human antibodies (Thermo Fisher Scientific) at a dilution of 1:2.000 for 1 h. Then, the sections were treated with Dako Envision following washing in TBS. Antibody expression was visualized using diaminobenzidine and counterstained with Mayer’s hematoxylin [27].

Histopathological examination of the stomach

The remaining stomach tissue was sectioned at 5 µm thickness after being dried, fixed in paraffin, and preserved in 10% paraformaldehyde. The sections were stained with hematoxylin and eosin (H&E). Morphological changes were assessed under an Eclipse 80i microscope (Nikon), and images were recorded using a DS-Fi1 digital microscope camera (Nikon).

Sample size

Sample size calculation was based on previous equivalent studies [28]. Using G*power v.3.1.9.5 to calculate the sample size based on an effect size of 1.6254, a two-tailed test, an α error of 0.05, and a power of 90.0%, the sample size was determined as ten in each group.

Computer-assisted digital image analysis (digital morphometric study)

Slides were photographed using an MVV5000CL digital eyepiece with a 5.0 M pixel sensor installed on a MEIJI MX5200L microscope using 20× and 40× objectives. The resulting 20× images were analyzed on an Intel Core i7-based computer using Fiji ImageJ v.1.51r (NIH). To measure the percentage of the staining surface area, the color deconvolution 2 plugin was used. Five random fields from each tissue specimen were analyzed, as described previously [29].

Statistical analysis

Data were analyzed using GraphPad Prism 9 (GraphPad Software). Numerical data were analyzed for normality using the Shapiro–Wilk test and presented as mean ± SD. One-way analysis of variance (ANOVA) and Tukey’s test were used to compare parametric data. A P value of ≤0.05 was considered statistically significant.

RESULTS

Enterococcus faecalis infection and the effects of CAPE on the activities of SOD, GST, GSH-Px, and MDA

To assess the impact of E. faecalis infection, we measured the activities of superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the stomach tissues. Group II, infected with E. faecalis, showed significantly decreased levels of SOD, GST, and GSH-Px compared to the control Group I. The significant changes observed in the parameters were dose-dependent at 106 E. faecalis inoculated orally. SOD levels were 36.17 ± 2.87 U/g in Group II versus 67.49 ± 3.18 U/g in Group I (P < 0.001). GST and GSH-Px levels in Group II were 37.11 ± 2.67 U/g and 36.13 ± 1.41 U/g, respectively, compared to 76.50 ± 1.98 U/g and 75.59 ± 1.67 U/g in Group I (P < 0.001). Conversely, MDA levels were significantly higher in Group II (15.92 ± 0.42 nmol/mg) compared to Group I (9.47 ± 0.32 nmol/mg; P < 0.001). The findings showed that oral treatment with CAPE at a dose of 4 mg/kg in Group III elicited a significant increase in SOD, GST, and GSH-Px levels and a decrease in MDA, especially when compared with Group II (P < 0.001). Thus, treatment with CAPE at the chosen dose of 4 mg/kg enhanced the elimination of ROS in the mouse stomach (Table 1).

Table 1 SOD, GST, GSH, and MDA levels across different groups

Parameter	Group I (control)	Group II
(infected with E. faecalis)	Group III (infected with E. faecalis
and treated with CAPE	P value	
SOD (U/g)	67.49 ± 3.18	36.17 ± 2.87***	66.84 ± 3.82 ###	<0.0001	
GST (U/g)	76.50 ± 1.98	37.11 ± 2.67***	74.78 ± 1.19###	<0.0001	
GSH (U/g)	75.59 ± 1.67	36.13 ± 1.41***	75.10 ± 1.38###	<0.0001	
MDA (U/g)	9.47 ± 0.32	15.92 ± 0.42***	9.89 ± 0.89###	<0.0001	
Data expressed as mean ± SD. One-way ANOVA followed by post-hoc Tukey’s test. *P < 0.05 vs. Group I. #P < 0.05 vs. Group II.

Immunohistochemical expression of NF-κB

Regarding the immunohistochemical expression of NF-κB, stained sections of stomachs from Group I exhibited normal appearance, with a negative expression of NF-κB in the non-glandular and glandular parts. In Group II, the stomach showed increased expression of NF-κB in the non-glandular and glandular parts, whereas, in Group III, there was a marked reduction in NF-κB immune reactivity in the non-glandular and glandular parts of the stomach (Figure 1). We observed a significant increase in the size of the NF-κB-positive area in Group II compared to the control group and a significant decrease in the size of the NF-κB-positive area in Group III compared to Group II (Table 2).

Figure 1 Immunohistochemical staining of NF-κB among mice in different study groups, in the non-glandular forestomach (upper row) and glandular stomach (lower row). The control group shows minimal to no NF-κB immune reactivity in the non-glandular and glandular parts. Group II, infected with Enterococcus faecalis at a dose of 106 CFU shows increased NF-κB immune reactivity in both stomach parts. Group III, treated with CAPE, shows a marked reduction in NF-κB immune reactivity in the non-glandular and glandular parts of the stomach. Original magnification 100×.

Table 2 Comparison of NF-κB immunohistochemical staining among mice in different groups. Data expressed as percentage of NF-κB-positive area.

  Stomach part	Group I	Group II	Group III	P value	
Non-glandular	2.41 ± 1.69	19.71 ± 7.57*	5.58 ± 2.65#	0.0002	
Glandular	2.66 ± 3.55	18.97 ± 6.82*	6.19 ± 5.76#	0.0014	
Data expressed as mean ± SD. One-way ANOVA followed by post-hoc Tukey’s test. *P < 0.05 vs. Group I. #P < 0.05 vs. Group II.

PCNA immunoreactivity examination

The immunohistochemical expression of PCNA showed no immunoreactivity in the control group. In Group II, there was significant immunoreactivity in both stomach parts, and in Group III, mild immunoreactivity was observed in the non-glandular and glandular parts of the stomach (Figures 2 and 3). There was a significant increase in the PCNA-positive area in Group II compared with the control group and a significant decrease in the PCNA-positive area in Group III compared with Group II (Table 3).

Figure 2 Immunohistochemical staining of PCNA among mice in different study groups, in the non-glandular forestomach (upper row) and glandular stomach (lower row). The control group shows minimal to no PCNA immune reactivity in the non-glandular and glandular parts. Group II, infected with Enterococcus faecalis at a dose of 106 CFU shows increased PCNA immune reactivity in both stomach parts. Group III, treated with CAPE, shows a marked reduction in PCNA immune reactivity in the non-glandular and glandular parts of the stomach. Original magnification 100×.

Figure 3 Comparison of NF-κB and PCNA immunohistochemical staining among mice in different groups. A, NF-κB IHC staining area percentage. B, PCNA IHC staining area percentage.

Table 3 Comparison of PCNA immunohistochemical staining among mice in different groups. Data expressed as percentage of PCNA-positive area.

Stomach part	Group I	Group II	Group III	P value	
Non-glandular	3.78 ± 2.56	29.07 ± 15.24*	4.89 ± 1.72#	0.0011	
Glandular	4.15 ± 2.79	29.66 ± 7.79*	5.80 ± 3.78#	<0.0001	
Data expressed as mean ± SD. One-way ANOVA followed by post-hoc Tukey’s test. *P < 0.05 vs. Group I. #P < 0.05 vs. Group II.

Histopathological results (light microscopy examination of H&E-stained sections)

The H&E-stained stomach sections from the control group showed normal histological structure characterized by a stratified squamous keratinized epithelium, with an underlying submucosa and muscularis in the non-glandular part and a fundus showing normal histological structure of the gastric glands in the glandular part. In contrast, Group II exhibited increased mucosal and glandular thickness compared to the control. However, Group III had nearly complete restoration of normal thickness in both the non-glandular and glandular parts of the stomach (Figure 4).

Figure 4 Histopathological examination (H&E staining) among mice in different study groups, in the non-glandular forestomach (upper row) and glandular stomach (lower row). The control group shows normal histological structure of stratified squamous keratinized epithelium with underlying submucosa and muscularis in the non-glandular part, and the fundus shows normal histological structure of gastric glands in the glandular part. Group II, infected with Enterococcus faecalis at a dose of 106 CFU, shows increased mucosal and glandular thickness. Group III, treated with CAPE, shows almost complete restoration of the normal thickness of the non-glandular and glandular parts of the stomach. Black arrow, epithelium; red arrow, submucosa; blue arrow, muscles; green arrow, gastric glands. Original magnification 100×.

DISCUSSION

The human microbiome refers to the vast array of microorganisms that inhabit different body cavities, ranging from the skin and lungs to the vagina and, most prominently, the gastrointestinal tract. The gut microbiota, in particular, is recognized for its extensive diversity and abundance of microbial species [30]. Changes in the gut microbiota composition have been implicated in various diseases, including inflammatory bowel disease and colorectal cancer [31]. Therefore, an analysis to clarify the role of the microbiota in each stage of these diseases is required. New diagnostic techniques and potential therapies can be developed in response to changes in the microbiota of the gastrointestinal tract.

E. faecalis is an opportunistic pathogen that can translocate across the mucosal barrier to cause systemic infections [32]. It is primarily described as a core commensal member of the human gut, present in more than 90% of bacterial isolates [33]. In this study, E. faecalis administered orally at a dose of 106 CFU resulted in a significant reduction of the antioxidant activity of SOD, GST, and GSH-Px compared with the control group (Table 1). Oxygen is abundant in Earth's atmosphere and plays a critical role in supporting diverse life forms. However, as pointed out by Markkanen [34], excessive reactive oxygen species (ROS) can chemically modify various macromolecules like RNA, DNA, proteins, and lipids through oxidation. As a result, the structure and function of these macromolecules are affected, leading to cell toxicity. According to Fasnacht and Polacek [35], ROS can be produced in bacteria and other organisms either internally due to aerobic metabolism or externally due to local exposure to elevated levels of oxidative agents. Cells can withstand low concentrations of ROS and have systems in place to combat oxidation.

According to Dryden et al., at low concentrations, ROS function as signaling molecules in regulating a variety of cellular functions, including quorum sensing, biofilm formation, and bacterial self-destruction [36]. Oxidative stress occurs when there is an imbalance between the quantity of ROS and the body’s capacity to remove them. This has been demonstrated by our results, which are consistent with those of Berghoff and Klug [37]. Three naturally occurring ROS species, namely superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (HO•), show different reactivities and have major relevance in aerobic environments and oxidative stress, leading to decreased SOD, GST, and GSH-Px levels and increased MDA levels, as demonstrated by the results in Group II. These enzymes are balanced with ROS and occasionally inhibit the elevated levels of ROS. Moreover, Huycke et al. [38] found that E. faecalis produces substantial quantities of extracellular superoxide (O2−) and derivative ROS, such as H2O2 and hydroxyl radicals, through the autoxidation of membrane-associated demethylmenaquinone. These oxidants may have an important role in decreasing antioxidant levels, as suggested by the results of this study.

In contrast, Group III, which was infected with E. faecalis and treated with CAPE at a dose of 4 mg/kg, showed increased antioxidant enzyme expression and significantly improved SOD, GST, and GSH-Px levels compared with Group II (Table 1), with levels close to normal. These results are consistent with those of Pérez et al. [39]. They are also in accordance with the studies of Wang et al., Chen et al., and Wang et al. [40-42], which demonstrated the strong antioxidant properties of CAPE by quenching the 2,2-diphenyl-1-picrylhydrazyl radical and lowering the formation of the superoxide anion that results from the autoxidation of β-mercaptoethanol. In addition to its strong cytoprotective and antigenotoxic antilipoperoxidative potential against oxidative damage, CAPE also inhibits the activity of xanthine oxidase.

A growing body of research indicates that NF-κB is essential for controlling inflammation and the immune system, both of which are involved in the etiology of neurodegenerative diseases [43]. Pro-inflammatory and anti-inflammatory cytokines are released as a result of the innate and adaptive immune systems being activated during the inflammatory response. These cytokines are important for the resolution of inflammation [44]. The parenchyma of the brain also exhibits these systemic inflammatory reactions, also referred to as neuroinflammation. Patients with various neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and frontotemporal dementia, have specific inflammatory signatures in their brains and bloodstream [45]. Interestingly, CAPE may be a relevant therapeutic agent for controlling NF-κB signaling and reducing neuroinflammation in these disorders [22].

In this study, Group II exhibited increased NF-κB immune reactivity, whereas the CAPE-treated Group III showed a significant reduction in NF-κB immune reactivity in the non-glandular and glandular parts of the stomach. The control group showed minimal to no NF-κB immune reactivity (Figure 1 and Table 2). Similar results were obtained by Zou and Shankar [46], who found that E. faecalis with macrophages instead of internalized bacteria had an important role in NF-κB activation and subsequent cytokine expression. This implies that the cell surface has a major role in the pathogen-associated molecular patterns phagocytes identify during enterococcal infections. The results of Group III are also consistent with those of Natarajan et al., Lee et al., and Sun et al. [47-49], who analyzed for the first time the molecular mechanisms through which CAPE prevents NF-κB activation.

CAPE has been found to completely block the activation of NF-κB through TNF-α in a dose- and time-dependent manner. Moreover, CAPE prevents NF-κB activation caused by okadaic acid, ceramide, hydrogen peroxide, phorbol ester, and other inflammatory substances. Reduction agents, on the other hand, reverse the inhibitory effects of CAPE, suggesting that sulfhydryl groups have a critical role in NF-κB activation. Moreover, CAPE inhibits the p65 subunit of NF-κB from moving to the nucleus, but it has no discernible effect on the TNF-α-induced degradation of IκBα. However, it delays IκBα resynthesis. Therefore, our findings support the hypothesis that CAPE inhibits p65 translocation and IκBα degradation in human chondrocytes to prevent NF-κB activation. PCNA, which is involved in DNA replication and repair, is an indicator of DNA damage [22].

Our finding that Group II (infected with E. faecalis) showed significant PCNA immunoreactivity is consistent with that of Kaplan et al. [50], who found that the increased detection of PCNA could be a secondary effect of cytokines originating from both inflammatory cells and keratinocytes. Thus, the immunodetection of PCNA by current immunohistochemical methods in the non-glandular and glandular parts of the stomach was sufficient to be used as an indicator of infection.

In contrast, in the CAPE-treated group, the histochemical and immunohistochemical analysis revealed that CAPE treatment significantly reduced the number of PCNA-positive cells, consistent with the results of Mir et al. [51], who found that CAPE led to the inhibition of PCNA expression in glioma cell proliferation and could prove to be an effective adjunct to therapies.

Lastly, the histopathological examination of stomachs was conducted across different study groups. The fact that Group II had an increase in mucosal and glandular thickness and Group III had an almost comprehensive restoration of the normal thickness of the non-glandular and glandular parts of the stomach demonstrates the potent effect of CAPE in protecting the stomach against infection, damage, and inflammation induced by E. faecalis.

CONCLUSION

The results of this study suggest that CAPE has protective effects against E. faecalis infection of the stomach and that it could be potentially used in controlling inflammation and protecting from stomach injury in experimental models, as evidenced by its ability to increase the levels of antioxidant markers and reduce the inflammatory mediator NF-κB. The results also suggest that, alongside other markers, PCNA may serve as a promising diagnostic and prognostic marker in future assessments. Finally, these results highlight the diverse biological and pharmacological properties of CAPE, its capability to be a functional coadjuvant for preventing infection-mediated inflammation or gastrointestinal diseases, and its potential applications in human clinical trials as an antibacterial agent. Although these results shed some light on the processes underlying the benefits of CAPE, further research of the underlying molecular pathways and signaling cascades will improve our comprehension of the compound’s anti-inflammatory and protective qualities.

Conflict of interest

The author declares no conflicts of interest.

Ethical approval

The study was approved by the Ethical and Scientific Research Committee of the University of Al-Baha (approval no. 44125197, 6 June 2023).

Data availability

Further data are available from the author upon reasonable request.

Personal thanks

I would like to acknowledge the assistance of Dr. O.A. Elkashty at the Faculty of Dentistry, Mansoura University, in the histopathological part of this study.

Authorship

AYA was responsible for conceptualization, methodology, data collection and analysis, and writing and editing the manuscript.
==== Refs
1 Anderson AC Jonas D Huber I Karygianni L Wölber J Hellwig E Enterococcus faecalis from Food, Clinical Specimens, and Oral Sites: Prevalence of Virulence Factors in Association with Biofilm Formation Front Microbiol 2016 6 1534 10.3389/fmicb.2015.01534 26793174
2 García-Solache M Rice LB The Enterococcus: a Model of Adaptability to Its Environment Clin Microbiol Rev 2019 Jan 30 32 2 e00058 18 10.1128/CMR.00058-18 30700430
3 Codelia-Anjum A Lerner LB Elterman D Zorn KC Bhojani N Chughtai B Enterococcal Urinary Tract Infections: A Review of the Pathogenicity, Epidemiology, and Treatment Antibiotics (Basel) 2023 Apr 19 12 4 778 10.3390/antibiotics12040778 37107140
4 Grudlewska-Buda K Skowron K Bauza-Kaszewska J Budzyńska A Wiktorczyk-Kapischke N Wilk M Assessment of antibiotic resistance and biofilm formation of Enterococcus species isolated from different pig farm environments in Poland BMC Microbiol 2023 Mar 30 23 1 89 10.1186/s12866-023-02834-9 36997857
5 Munita JM Arias CA Mechanisms of Antibiotic Resistance Microbiol Spectr 2016 Apr 4 2 10.1128/microbiolspec.VMBF-0016-2015
6 Fiore E Van Tyne D Gilmore MS Pathogenicity of Enterococci Microbiol Spectr 2019 Jul 7 4 10.1128/microbiolspec.GPP3-0053-2018
7 Arias CA Contreras GA Murray BE Management of multidrug-resistant enterococcal infections Clin Microbiol Infect 2010 Jun 16 6 555 562 10.1111/j.1469-0691.2010.03214.x 20569266
8 Jahansepas A Aghazadeh M Rezaee MA Hasani A Sharifi Y Aghazadeh T Occurrence of Enterococcus faecalis and Enterococcus faecium in Various Clinical Infections: Detection of Their Drug Resistance and Virulence Determinants Microb Drug Resist 2018 Jan/Feb 24 1 76 82 10.1089/mdr.2017.0049 28525287
9 Deng Z Lin B Liu F Zhao W Role of Enterococcus faecalis in refractory apical periodontitis: from pathogenicity to host cell response J Oral Microbiol 2023 Mar 1 15 1 2184924 10.1080/20002297.2023.2184924 36891193
10 Beganovic M Luther MK Rice LB Arias CA Rybak MJ LaPlante KL A Review of Combination Antimicrobial Therapy for Enterococcus faecalis Bloodstream Infections and Infective Endocarditis Clin Infect Dis 2018 Jul 2 67 2 303 309 10.1093/cid/ciy064 29390132
11 Taysi S Algburi FS Taysi ME Caglayan C Caffeic acid phenethyl ester: A review on its pharmacological importance, and its association with free radicals, COVID-19, and radiotherapy Phytother Res 2023 Mar 37 3 1115 1135 10.1002/ptr.7707 36562210
12 Olgierd B Kamila Ż Anna B Emilia M The Pluripotent Activities of Caffeic Acid Phenethyl Ester Molecules 2021 Mar 2 26 5 1335 10.3390/molecules26051335 33801469
13 Sun W Xie W Huang D Cui Y Yue J He Q Caffeic acid phenethyl ester attenuates osteoarthritis progression by activating NRF2/HO-1 and inhibiting the NF-κB signaling pathway Int J Mol Med 2022 Nov 50 5 134 10.3892/ijmm.2022.5190 36102306
14 Armutcu F Akyol S Ustunsoy S Turan FF Therapeutic potential of caffeic acid phenethyl ester and its anti-inflammatory and immunomodulatory effects (Review) Exp Ther Med 2015 May 9 5 1582 1588 10.3892/etm.2015.2346 26136862
15 Kany S Vollrath JT Relja B Cytokines in Inflammatory Disease Int J Mol Sci 2019 Nov 28 20 23 6008 10.3390/ijms20236008 31795299
16 Wang LC Chu KH Liang YC Lin YL Chiang BL Caffeic acid phenethyl ester inhibits nuclear factor-kappaB and protein kinase B signalling pathways and induces caspase-3 expression in primary human CD4+ T cells Clin Exp Immunol 2010 May 160 2 223 232 10.1111/j.1365-2249.2009.04067.x 20059479
17 Mapesa JO Waldschmitt N Schmoeller I Blume C Hofmann T Mahungu S Catechols in caffeic acid phenethyl ester are essential for inhibition of TNF-mediated IP-10 expression through NF-κB-dependent but HO-1-and p38-independent mechanisms in mouse intestinal epithelial cells Mol Nutr Food Res 2011 Dec 55 12 1850 1861 10.1002/mnfr.201100105 22038897
18 Mulero MC Huxford T Ghosh G NF-κB, IκB, and IKK: Integral Components of Immune System Signaling Adv Exp Med Biol 2019 1172 207 226 10.1007/978-981-13-9367-9_10 31628658
19 Liu T Zhang L Joo D Sun SC NF-κB signaling in inflammation Signal Transduct Target Ther 2017 2 17023 10.1038/sigtrans.2017.23 29158945
20 Silva H Lopes NMF Cardiovascular Effects of Caffeic Acid and Its Derivatives: A Comprehensive Review Front Physiol 2020 Nov 27 11 595516 10.3389/fphys.2020.595516 33343392
21 Gupta SC Sundaram C Reuter S Aggarwal BB Inhibiting NF-κB activation by small molecules as a therapeutic strategy Biochim Biophys Acta 2010 Oct-Dec 1799 10-12 775 787 10.1016/j.bbagrm.2010.05.004 20493977
22 Pérez R Burgos V Marín V Camins A Olloquequi J González-Chavarría I Caffeic Acid Phenethyl Ester (CAPE): Biosynthesis, Derivatives and Formulations with Neuroprotective Activities Antioxidants (Basel) 2023 Jul 27 12 8 1500 10.3390/antiox12081500 37627495
23 Makusheva Y Goncharova E Bets V Korel A Arzhanova E Litvinova E Restoration of Lactobacillus johnsonii and Enterococcus faecalis Caused the Elimination of Tritrichomonas sp in a Model of Antibiotic-Induced Dysbiosis Int J Mol Sci 2024 May 7 25 10 5090 10.3390/ijms25105090 38791132
24 Sayed AA Ferulsinaic Acid Modulates SOD, GSH, and Antioxidant Enzymes in Diabetic Kidney Evid Based Complement Alternat Med 2012 2012 580104 10.1155/2012/580104 22991571
25 Janero DR Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury Free Radic Biol Med 1990 9 6 515 540 10.1016/0891-5849(90)90131-2 2079232
26 Sivakumar M Yoithapprabhunath TR Nirmal RM Veeravarmal V Dineshshankar J Amsaveni R Immunohistochemical analysis of Nuclear Factor-kappa B (NF-κB) between follicular and plexiform ameloblastomas: A pilot study J Oral Maxillofac Pathol 2020 Sep-Dec 24 3 466 471 10.4103/jomfp.JOMFP_150_20 33967482
27 Shamloo N Taghavi N Ahmadi S Shalpoush S Immunohistochemical analysis of proliferating cell nuclear antigen and minichromosome maintenance complex component 7 in benign and malignant salivary gland tumors Dent Res J (Isfahan) 2022 Feb 28 19 17 10.4103/1735-3327.338780 35308440
28 Faul F Erdfelder E Lang AG Buchner A G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences Behav Res Methods 2007 May 39 2 175 191 10.3758/bf03193146 17695343
29 Sánchez-Jaramillo EA Gasca-Lozano LE Vera-Cruz JM Hernández-Ortega LD Salazar-Montes AM Automated Computer-Assisted Image Analysis for the Fast Quantification of Kidney Fibrosis Biology (Basel) 2022 Aug 17 11 8 1227 10.3390/biology11081227 36009854
30 Hou K Wu ZX Chen XY Wang JQ Zhang D Xiao C Microbiota in health and diseases Signal Transduct Target Ther 2022 Apr 23 7 1 135 10.1038/s41392-022-00974-4 35461318
31 Quaglio AEV Grillo TG De Oliveira ECS Di Stasi LC Sassaki LY Gut microbiota, inflammatory bowel disease and colorectal cancer World J Gastroenterol 2022 Aug 14 28 30 4053 4060 10.3748/wjg.v28.i30.4053 36157114
32 Bolocan AS Upadrasta A Bettio PHA Clooney AG Draper LA Ross RP Evaluation of Phage Therapy in the Context of Enterococcus faecalis and Its Associated Diseases Viruses 2019 Apr 20 11 4 366 10.3390/v11040366 31010053
33 EFSA Panel on Animal Health and Welfare (AHAW)Nielsen SS Bicout DJ Calistri P Canali E Drewe JA Garin-Bastuji B Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): antimicrobial-resistant Enterococcus faecalis in poultry EFSA J 2022 Feb 21 20 2 e07127 10.2903/j.efsa.2022.7127 35228848
34 Markkanen E Not breathing is not an option: How to deal with oxidative DNA damage DNA Repair (Amst) 2017 Nov 59 82 105 10.1016/j.dnarep.2017.09.007 28963982
35 Fasnacht M Polacek N Oxidative Stress in Bacteria and the Central Dogma of Molecular Biology Front Mol Biosci 2021 May 10 8 671037 10.3389/fmolb.2021.671037 34041267
36 Dryden MS Cooke J Salib RJ Holding RE Biggs T Salamat AA Reactive oxygen: A novel antimicrobial mechanism for targeting biofilm-associated infection J Glob Antimicrob Resist 2017 Mar 8 186 191 10.1016/j.jgar.2016.12.006 28213334
37 Berghoff B Klug G Hess WR Marchfelder A Small RNAs with a Role in the Oxidative Stress Response of Bacteria Regulatory RNAs in Prokaryotes 2012 Vienna Springer Vienna 1 14 10.1007/978-3-7091-0218-3_1
38 Huycke MM Abrams V Moore DR Enterococcus faecalis produces extracellular superoxide and hydrogen peroxide that damages colonic epithelial cell DNA Carcinogenesis 2002 Mar 23 3 529 536 10.1093/carcin/23.3.529 11895869
39 Pérez R Burgos V Marín V Camins A Olloquequi J González-Chavarría I Caffeic Acid Phenethyl Ester (CAPE): Biosynthesis, Derivatives and Formulations with Neuroprotective Activities Antioxidants (Basel) 2023 Jul 27 12 8 1500 10.3390/antiox12081500 37627495
40 Wang T Chen L Wu W Long Y Wang R Potential cytoprotection: antioxidant defence by caffeic acid phenethyl ester against free radical-induced damage of lipids, DNA, and proteins Can J Physiol Pharmacol 2008 May 86 5 279 287 10.1139/y08-029 18432289
41 Chen YJ Huang AC Chang HH Liao HF Jiang CM Lai LY Caffeic acid phenethyl ester, an antioxidant from propolis, protects peripheral blood mononuclear cells of competitive cyclists against hyperthermal stress J Food Sci 2009 Aug 74 6 H162 H167 10.1111/j.1750-3841.2009.01199.x 19723200
42 Wang X Stavchansky S Kerwin SM Bowman PD Structure-activity relationships in the cytoprotective effect of caffeic acid phenethyl ester (CAPE) and fluorinated derivatives: effects on heme oxygenase-1 induction and antioxidant activities Eur J Pharmacol 2010 Jun 10 635 1-3 16 22 10.1016/j.ejphar.2010.02.034 20226179
43 Guo Q Jin Y Chen X Ye X Shen X Lin M NF-κB in biology and targeted therapy: new insights and translational implications Sig Transduct Target Ther 2024 Mar 4 9 153 1 37 10.1038/s41392-024-01757-9
44 Harvanová G Duranková S Bernasovská J The role of cytokines and chemokines in the inflammatory response Alergol Pol 2023 10 3 210 219 10.5114/pja.2023.131708
45 Zhang W Xiao D Mao Q Xia H Role of neuroinflammation in neurodegeneration development Signal Transduct Target Ther 2023 Jul 12 8 1 267 10.1038/s41392-023-01486-5 37433768
46 Zou J Shankar N Roles of TLR/MyD88/MAPK/NF-κB Signaling Pathways in the Regulation of Phagocytosis and Proinflammatory Cytokine Expression in Response to E faecalis Infection PLoS One 2015 Aug 28 10 8 e0136947 10.1371/journal.pone.0136947 26317438
47 Natarajan K Singh S Burke TR Jr Grunberger D Aggarwal BB Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B Proc Natl Acad Sci U S A 1996 Aug 20 93 17 9090 9095 10.1073/pnas.93.17.9090 8799159
48 Lee Y Shin DH Kim JH Hong S Choi D Kim YJ Caffeic acid phenethyl ester-mediated Nrf2 activation and IkappaB kinase inhibition are involved in NFkappaB inhibitory effect: structural analysis for NFkappaB inhibition Eur J Pharmacol 2010 Sep 15 643 1 21 28 10.1016/j.ejphar.2010.06.016 20599928
49 Sun W Xie W Huang D Cui Y Yue J He Q Caffeic acid phenethyl ester attenuates osteoarthritis progression by activating NRF2/HO-1 and inhibiting the NF-κB signaling pathway Int J Mol Med 2022 Nov 50 5 134 10.3892/ijmm.2022.5190 36102306
50 Kaplan I Vered M Moskona D Buchner A Dayan D An immunohistochemical study of p53 and PCNA in inflammatory papillary hyperplasia of the palate: a dilemma of interpretation Oral Dis 1998 Sep 4 3 194 199 10.1111/j.1601-0825.1998.tb00278.x 9972170
51 Mir AH Iqbal MK Banday MZ Balkhi HM Haq E Combination of Caffeic Acid Phenethyl Ester and Crocin Realign Potential Molecular Markers in U87-MG Glioma Cells Curr Ther Res Clin Exp 2023 Feb 14 98 100695 10.1016/j.curtheres.2023.100695 36936719
