Celecoxib inhibits Helicobacter pylori colonization-related factors

Abstract

AIM: To investigate the effect of celecoxib, a selective COX-2 inhibitor, on Helicobacter pylori (H. pylori) colonization-related factors and its mechanism.

METHODS: After co-incubation with celecoxib, morphology of H. pylori strain 26695 was observed under a transmission electron microscope. Flagella motility was assessed by stab agar motility test. Adherence of H. pylori to AGS cells was determined by enzyme linked immunosorbent assay. Levels of mRNA expression in flagellar genes (flaA, flaB), urease genes (ureA, ureB) and adhesin genes (babA, sabA, alpA, alpB, hpaA, hopZ) were measured by real-time polymerase chain reaction.

RESULTS: Separation and non-integrity of bacterial cell wall, rarefaction and asymmetry of cytoplasm, and even lysis of H. pylori were observed in the presence of celecoxib. When H. pylori strains were incubated in the presence of celecoxib, their flagellar motility and adherence to AGS cells were inhibited. The expression of ureA, ureB, babA, sabA, alpA, alpB, hpaA, hopZ was up-regulated while the expression of flaA, flaB was down-regulated in the presence of celecoxib.

CONCLUSION: Celecoxib inhibits flagellar motility and adherence of H. pylori to AGS cells, and destructs their normal structure in vitro.

Key Words: Helicobacter pylori; Celecoxib; Colonization; Ultrastructure

INTRODUCTION

About 30% of the population in developed countries and up to 90% of the population in developing countries are chronically infected with Helicobacter pylori (H. pylori)[1,2]. Non-steroidal anti-inflammatory drugs (NSAID) are the most commonly used drugs, on a world-wide scale, which are used by at least 30 million people[3]. NSAID and H. pylori infection are two major factors for gastric injuries. Subjects taking NSAID are often infected with H. pylori. However, whether these two factors exert synergistic or antagonistic actions on gastric mucosa is still controversial[4,5]. Data from a meta-analysis review have shown that the risk of peptic ulcer is approximately 60-fold higher in H. pylori positive subjects taking NSAID than in H. pylori negative subjects not taking NSAID[6]. Since both H. pylori and NSAID are responsible for mucosal damage, they can increase the risk of developing uncomplicated and complicated peptic ulcer. However, data from several studies do not always confirm such an assumption[5]. A large clinical trial demonstrated that eradication of H. pylori delays the healing of gastric ulcers in NSAID users after treatment with omeprazole[7], implying that H. pylori may protect individuals against NSAID-induced ulcer, possibly by stimulating mucosal prostaglandins and other protective factors.

Recent studies in vitro also suggested that aspirin and celecoxib, a selective COX-2 inhibitor, inhibit the growth of H. pylori and decrease the activity of urease and vacuolating cytotoxin in a dose-dependent manner[8-12], indicating that NSAID may antagonize injuries of gastric mucosa caused by H. pylori infection. Colonization of H. pylori in gastric mucosa is a prerequisite for pathogenicity and needs to have at least 4 basic characteristics: integrate helical shape, motility of flagella, specific binding to adhesin and its receptors, and urease activity that provides an appropriate microenvironment[13]. We hypothesize that NSAID and celecoxib may influence the pathogenicity of H. pylori in gastric mucosa injury by altering the colonization. Therefore, the aim of the present study was to investigate the effect of celecoxib on H. pylori colonization- related factors and its mechanism in vitro.

MATERIALS AND METHODS

Bacterial culture

H. pylori 26695 strain was cultured at 37°C in a microaerobic atmosphere containing 5% O₂, 85% N₂, and 10% CO₂ for 48 h on Colombia agar medium supplemented with 8% (v/v) defibrinated goat blood containing 0.02 mmol/L celecoxib or vehicle control (1/1000 DMSO).

Stab agar motility test

H. pylori strains were grown on Colombia agar medium for 48 h and then harvested into a brain heart infusion (37 g/L). After the concentration of bacteria was adjusted to 10⁸ CFU/mL, 10 μL was inoculated into a 0.3% agar Brucella broth medium containing 8% defibrinated goat blood using a sterile picker. Five days after incubation under microaerobic condition at 37°C, the halo diameter was measured.

Ultrastructural analysis

Forty-eight hours after exposure to 0.02 mmol/L celecoxib, H. pylori cells were collected and rinsed three times with 0.01 mol/L PBS, fixed in phosphate-buffer solution containing 2.5% glutaraldehyde at 4°C for 2 h. After centrifugation, pellets were embedded in 2% agar, fixed in 1% osmium tetraoxide (OsO4) at 4°C, and rinsed three times with 0.01 mol/L PBS. After dehydrated in a series of graded acetone at 4°C, specimens were embedded in Epon 812 (Emicron). The sample was cut into 90 nm-thick sections which were stained with uranyl acetate and lead citrate, and observed under a JEM1230 transmission electron microscope.

Adhesion of H. pylori to AGS cells

AGS cells (10⁴/well) were seeded in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum in a 96-well plate containing 5% CO₂ at 37°C for 20 h. H. pylori (10⁷ CFU/well) were pretreated with celecoxib at the concentrations of 0.01, 0.02 and 0.03 mmol/L. The plate was agitated at 60 r/min for 30 min at 37°C. Cultures were fixed with 1% paraformaldehyde. After washed with PBS, H. pylori cells were blocked with 5% bovine serum albumin (BSA) for 30 min, and incubated for 24 h with mouse monoclonal anti-H. pylori antibody (Santa cruz). After washed three times with PBS, goat anti-mouse IgG-HRP (Santa cruz) was added for 1 h. Binding was visualized by incubating with 100 μL TMB substrate for 30 min. Absorbance was read at 450 nm after 2 mol/L of sulphuric acid was added to terminate the reaction. Adherence of H. pylori to AGS cells was calculated according to the formula: [(A AGS cells with H. pylori -A AGS cells without H. pylori)/(A positive control -A negative control)] × 100. For positive control, only bacteria were added and allowed to adhere to the well. Wells containing neither AGS cells nor H. pylori were prepared as a negative control.

H. pylori RNA isolation and reverse transcription

Forty-eight hours after pretreatment with 0.02 mmol/L celecoxib, strains of H. pylori were rinsed with Tris-HCl and cleared with 1 mL of TRIzol. After 200 μL of chloroform was added, the sample was vigorously shaken and centrifuged. RNA in aqueous phase was precipitated with 0.5 mL of isopropanol. The pellet was washed with ethanol and dried. The RNA was resuspended in sterile water and quantified by UV absorbance. Total RNA (4 μg) treated with RO1 RNase-free DNase (Promega) to remove DNA was used for reverse transcription reaction. In brief, 1.5 μL of random primers was added, the samples were heated to 70°C for 5 min. Then, 10 μL of 5 × RT buffer, 2.5 μL of dNTPs, and 2 μL of M-MLV were added. cDNA synthesis reaction was performed for 60 min at 37°C and then at 70°C for 10 min. Aliquots of cDNA were stored at -70°C.

Real-time polymerase chain reaction (RT-PCR)

mRNA levels of flagellar genes (flaA, flaB), urease genes (ureA, ureB) and adhesin genes (babA, sabA, alpA, alpB, hpaA, hopZ) were measured by real-time PCR using the ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystems, Foster City, Calif). Specific primers and house-keeping gene 16SrRNA were designed with the aid of Primer Express 3.0 software (Applied Biosystem Perkin-Elmer) (Table 1). Real-time PCR was performed in a 25 μL reaction volume containing 2.5 μL of cDNA, 12.5 μL of SYBR green real time PCR master mix (Toyobo), 1 μL of sense and antisense primers (5 pmol/L), and 9 μL of DEPC water. PCR was carried out at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, at 61°C for 1 min. A further melting curve step analyzing the purity of PCR products was performed at 95°C for 15 s, at 61°C for 30 s, and at 96°C for 15 s. A standard curve was plotted using 10-fold serial dilution of each cDNA. mRNA level was expressed as the ratio of detected mRNA to 16S rRNA mRNA [detected mRNA (U/mL)/16S rRNA mRNA (U/mL) × 100 000]. PCR was carried out in quintuple using samples prepared at the same time.

Table 1 Primers and probes used in real-time quantitative PCR.

Gene	Primer (5’-3’)
flaA-F	ATTGGCGTGTTAGCAGAAGTGA
flaA-R	TGACTGGACCGCCACATC
flaB-F	ACATCATTGTGAGCGGTGTGA
flaB-R	GCCCCTAACCGCTCTCAAAT
ureA-F	GCTGGTGCGATTGGCTTTA
ureA-R	GGATAGCGACTTGCACATCGT
ureB-F	TCCTGATGGGACAAAACTCGTA
ureB-R	ACGGCTTTTTTGCCTTCGT
babA-F	TGCTCAGGGCAAGGGAATAA
babA-R	ATCGTGGTGGTTACGCTTTTG
sabA-F	GGTGTGCTGCAACAGACTCAA
sabA-R	CATAAGCTGTTGCGCCAAATT
alpA-F	GCACGATCGGTAGCCAGACT
alpA-R	ACACATTCCCCGCATTCAAG
alpB-F	ACGCTAAGAAACAGCCCTCAAC
alpB-R	TCATGCGTAACCCCACATCA
hpaA-F	GAGCGTGGTGGCTTTGTTAGT
hpaA-R	TCGCTAGCTGGATGGTAATTCA
hopZ -F	GCGCCGTTACTAGCATGATCA
hopZ-R	GAAATCTTTCGGCGCGTTT
16SrRNA-F	CCGCCTACGCGCTCTTTAC
16SrRNA-R	CTAACGAATAAGCACCGGCTAAC

PCR: Polymerase chain reaction.

Statistical analysis

All experiments were performed at least in triplicate. Data were presented as mean ± SD. Statistical analysis between sample and control was conducted by Student’s t-test using SPSS 11.0 software. P < 0.05 was considered statistically significant.

RESULTS

Effects of celecoxib on H. pylori motility

The halo diameter for the growth of H. pylori in the presence of celecoxib was 5.92 ± 1.20 mm after 5-d incubation, which was significantly smaller than that (8.21 ± 1.63 mm) of DMSO control (P < 0.05, Figure 1), indicating that the motility of H. pylori is decreased in the presence of celecoxib.

Figure 1 Stab agar motility tests showing the H. pylori motility. A: DMSO control (1/1000); B: Celecoxib (0.02 mmol/L).

Ultrastructural effects of celecoxib on H. pylori

Transmission electron microscopy demonstrated that both cytoplasmic and outer membranes of H. pylori were intact, the cytoplasm was well-distributed and the electron density was moderate in DMSO control. When incubated with 0.02 mmol/L of celecoxib, V- and U-shaped H. pylori were observed. The cell wall of H. pylori was attenuated with abscission, or even perforation but no integrity. Separation of the outer membrane from the cytoplasmic membrane (cell wall breakaway) and even cell lysis were observed. Rarefaction and asymmetry were observed in cytoplasm of H. pylori and the components of H. pylori cells disappeared and distributed abnormally (Figure 2).

Figure 2 Transmission electron microscopy (TEM). TEM showing rod-shaped H. pylori (A), well-distributed cytoplasm and moderate electron density (B), U-shaped (C, arrow) and V-shaped (D, arrow) H. pylori, non-integrity (E, arrow) and abscission (F, arrow) of H. pylori cell wall, outer membrane separated from the cytoplasmic membrane (G, arrow), decreased electron density in cytoplasm (H, arrow), and cell lysis (I) after treatment with celecoxib.

Effects of celecoxib on H. pylori adherence to AGS cells

Compared to the DMSO control (1/1000), celecoxib significantly inhibited the adherence of H. pylori to AGS cells in a dose-dependent manner (P < 0.05) (Figure 3).

Figure 3 Adhesion of H. pylori to AGS cells after treatment with celecoxib at different concentrations.

Effects of celecoxib on H. pylori flagellin, urease and adhesin gene expression

The mRNA expression levels in flagellar genes (flaA, flaB), urease genes (ureA, ureB) and adhesin genes (babA, sabA, alpA, alpB, hpaA, hopZ) were measured by real-time PCR. After treatment with 0.02 mmol/L celecoxib, the mRNA expression levels in flaA and flaB were lower than those in DMSO control (P < 0.05). However, the mRNA expression levels were higher in urease genes (ureA, ureB) and adhesin genes (babA, sabA, alpA, alpB, hpaA, hopZ) than in DMSO control (P < 0.05). The mRNA expression levels in the above genes increased or decreased 1.5-2.5 folds in the presence of celecoxib (Table 2).

Table 2 mRNA levels in H. pylori flagellin, urease and adhesin genes meausred by real-time quantitative PCR (mean ± SD).

Gene	Celecoxib (0.02 mmol/L)	DMSO control (1/1000)
flaA	23.08 ± 1.70a	51.08 ± 6.91
flaB	16.01 ± 0.04a	34.80 ± 7.13
ureA	19.61 ± 1.78a	7.65 ± 0.38
ureB	29.59 ± 5.31a	13.80 ± 1.63
babA	16.78 ± 0.91a	12.38 ± 0.38
sabA	49.00 ± 4.10a	22.55 ± 2.26
alpA	15.55 ± 0.78a	7.34 ± 0.20
alpB	14.07 ± 0.23a	8.95 ± 0.38
hpaA	123.98 ± 11.82a	57.15 ± 2.56
hopZ	100.25 ± 4.37a	45.54 ± 11.64

^aP < 0.05 vs DMSO control.

DISCUSSION

NSAID and H. pylori infection are the two main etiological factors for peptic ulcers. However, their role in the pathogenesis of gastric mucosal damage is still controversial[6]. It has been demonstrated that eradication of H. pylori can decrease the recurrence rate of peptic ulcer and its complications in chronic NSAID users[14], while their co-existence aggravating gastric mucosal damage has not been confirmed[4,5]. It was reported that the prostaglandin synthesis level in mucosa is significantly higher in H. pylori positive patients than in H. pylori negative patients[15,16], demonstrating that colonization of H. pylori reduces the inhibitory effect of NSAID on prostaglandin synthesis. In vitro studies further revealed that NSAID can inhibit the growth of H. pylori, and decrease the activity of urease and vacuolating cytotoxin[8-12], suggesting that NSAID may alter the pathogenicity of H. pylori in gastric mucosa injury when the two factors are co-existed in gastric mucosa.

H. pylori infection may persist for many years in the host and H. pylori colonization-related factors include its spiral shape, flagellar motility, urease and adhesin. Urease neutralizes the pH around H. pylori during exposure to the acidic lumen of stomach. The flagella and the spiral shape of H. pylori enable H. pylori strains to move and penetrate the mucin layer where they come into contact with gastric epithelial cells. Adherence of H. pylori to AGS cells is a crucial initial step in colonization[17,18], as non-adhering H. pylori strains would be washed away during peristalsis-mediated flushing of stomach. NSAID and celecoxib do not increase the colonization of H. pylori in gastric mucosa[19-23]. On the contrary, the incidence of H. pylori infection in patients taking NSAID is low[24,25], which may be partially explained by the fact that celecoxib can destruct the normal structure of H. pylori, and inhibit the motility of flagella, and the adherence of H. pylori to AGS cells and the activity of urease[12], which is consistent with the findings in our study.

Adhesin, exposed on the surface of H. pylori cells, facilitates interaction with host cellular receptors. The particularly more important adhesins of H. pylori are BabA, SabA, AlpA, AlpB, HpaA, HopZ[26-30]. Their content and expression under different environmental conditions are variable. In our in vitro study, celecoxib inhibited the adherence of H. pylori to AGS cells, but increased the mRNA expression levels in babA, sabA, alpA, alpB, hpaA, hopZ. Whether the increased mRNA expression in such genes is accompanied with an increased competent protein or just a compensatory increase in mRNA expression for the inhibition of H. pylori growth and adherence activity remains to be further studied. On the other hand, variable expression of cell receptors in a single host and genetic variability of receptor expression in different hosts make the adherence system very complex. Host receptor expression is up-regulated following H. pylori adherence[31]. In this study, the impaired adherence of H. pylori to AGS cells in the presence of celecoxib down-regulated the host receptor expression. In this condition, although the expression of H. pylori adhesins increases, the adherence of H. pylori to AGS cells may decrease.

Urease in H. pylori accounts for approximately 10% of the total bacterial protein pool[32]. Urease hydrolyzes urea and releases ammonia, which neutralizes acid, thus enabling survival and initial colonization. It has been shown that urease activity is essential for the initial bacterial colonization[33-35]. Anti-ulcer drug, ecabet, interferes with H. pylori colonization by inhibiting urease activity[36]. In the present study, celecoxib inhibited the urease activity in a dose-dependent manner, suggesting that it may further influence H. pylori colonization.

Urease is composed of two structural subunits, UreA and UreB. Urease gene clusters include ure A, B, C, D, E, F, G, H, I, with ureA and ureB being the structural genes. ureC and ureD are located before the structural genes. ureI, ureE, ureF, urgG and ureH are auxiliary genes. These genes and the structural genes are necessary for urease activity[37]. Urease is a metal enzyme possessing nickel and its activity depends on the two Ni²⁺ inserted into its 6 active sites. The insertion process is accomplished by proteins encoded by auxiliary genes in a urease gene cluster. At present, a variety of identified proteins can regulate the activity of urease by influencing nickel ions. Besides proteins, different ion concentrations also accommodate urease activity[38]. Urease inhibitors can be generally classified into active site-directed (substrate-like) and mechanism-directed inhibitors. Since active site-directed inhibitors bridge the two paramagnetic nickel ions in the active site of urease, the octahedral nickel ions and the amino acid residues in the active site-directed inhibitors are in an orientation similar to those of the urease substrate, the mechanism-directed inhibitors are designed to interfere with the urease’s catalysis mechanism leading to enzyme inactivation. In the present study, celecoxib inhibited the urease activity in H. pylori, but increased the mRNA expression levels in ureA and ureB. The mechanism still remains unclear. Further studies are needed to determine whether alterations occur at protein translation or modification level or some other mechanisms are involved.

The motility of H. pylori is considered another colonization factor. Less motile strains are less able to colonize or survive in the host than fully motile strains. It has been demonstrated that the degree of the motility of H. pylori strains is correlated with the degree of infectivity in gnotobiotic piglets. The most motile strains have a 100% infection rate, while the least motile strains have an infection rate of only 17%[39]. Strains without flagella or flagellar mutant strains cannot colonize the gastric mucosa, thus losing their pathogenicity. The flagella consist mainly of the flagellins, FlaA and FlaB. Both genes coding for these flagellins are necessary for the full motility of H. pylori. Elimination of flaB yields normal-looking flagella that retain some functions and propel about 60% of the bacteria[40,41]. Elimination of flaA yields truncated flagella that only slightly move the bacteria. Elimination of both flagellins results in aflagellated immobile bacteria[41]. It was reported that NSAID inhibit the movement of Proteus vulgaris, Proteus mirablis, Providencia rettgeri, Providencia stuartiii and Burkholderia cepacia in a dose- dependent manner[42], and prevent emergence of Escherichia coli flagella by inhibiting flagellin synthesis[43]. In this study, celecoxib inhibited the motility of H. pylori and decreased the mRNA expression in flaA and flaB.

The relation between the degree of H. pylori motility, cytokine response levels and the severity of disease has been extensively studied[44,45]. The H. pylori motility levels are correlated with IL-8 induction[44]. Kurihara[45] also found that the degree of H. pylori motility is low in strains isolated from remnant gastritis, which is distinct from chronic gastritis, peptic ulceration or gastric cancer, indicating that the type and phase of H. pylori-related diseases dictate the selective pressure for maintenance of high H. pylori motility levels. Further study is needed to demonstrate whether celecoxib prevents the progress of H. pylori-related diseases by inhibiting H. pylori motility.

Besides the flagella, the shape of H. pylori strains makes them possible to penetrate the mucin layer where they come into contact with the gastric epithelial cells. In the present study, transmission electron microscopy showed that celecoxib could impair the formation of H. pylori, break the bacterial outer membrane, and destruct its structure. Since the spiral shape of H. pylori is one of the important virulence factors, celecoxib-related morphological changes may have an impact on the progress of H. pylori-induced diseases.

Gastric carcinoma is the forth most common cancer and the second leading cause of cancer-related deaths worldwide. The high mortality is largely attributed to the huge number of at-risk individuals. Chemoprevention appears to be the most promising approach in reducing the incidence and mortality of H. pylori-related gastric cancer. WHO defined H. pylori as a risk factor for gastric carcinoma and classified H. pylori strains as group I carcinogen in 1994[46]. The prevalence of H. pylori infection increases with age[47], and 50% of NSAID users are over 60-year old. NSAID contribute to the chemoprevention of gastric cancer and prevention of lymphatic metastasis by inhibiting angiogenesis and inducing apoptosis of epithelial cells through the COX-dependent and independent pathway. It has been shown that long-term intake of NSAID and aspirin can significantly reduce the incidence of non-cardial gastric cancer in a dose-dependent manner[48]. The results of our study further suggest that celecoxib can reduce H. pylori colonization, thus attenuating the pathogenesis in gastric mucosa. Although regular use of aspirin can prevent gastric cancer, it may be disadvantageous for populations with a lower risk of gastric cancer. Those with a high risk of gastric cancer can use celecoxib, a selective COX-2 inhibitor with few gastrointestinal side-effects.

COMMENTS

Background

Use of non-steroidal anti-inflammatory drugs (NSAID) and Helicobacter pylori (H. pylori) infection are the two main etiological factors for gastric injuries. Subjects taking NSAID are often co-infected with H. pylori, but the interaction between NSAID taking and infection with H. pylori remains unclear. Data from clinical and epidemiological studies are still controversial.

Research frontiers

The relation between NSAID and H. pylori in the pathogenesis of gastric mucosal damage is still controversial. A number of studies have shown that it is not simply additive, synergistic or antagonistic. There may be complex interactions between them which affect the pathogenicity of each other.

Innovations and breakthroughs

NSAID, as a harmful factor for gastric mucosal barrier, may be expected to increase the colonization of H. pylori in gastric mucosa. However, evidence from epidemiological studies indicates a lower prevalence of H. pylori infection in patients taking NSAID, which may partially be explained by the fact that celecoxib destructs the normal structure of H. pylori, and inhibits the flagellar motility, the adherence of H. pylori to AGS cells and the urease activity, as observed in this study.

Applications

Colonization of H. pylori is a crucial initial step in the pathogenesis of H. pylori in gastric mucosa. The present study suggested that celecoxib could reduce the colonization of H. pylori, thus attenuating the pathogenicity in gastric mucosa.

Terminology

SYBR green teal-time polymerase chain reaction (PCR): a quantitative PCR method for determination of the cope number of PCR templates such as DNA or cDNA in a PCR reaction. SYBR green: A dye that binds to the minor groove of double stranded DNA. When SYBR green dye binds to double stranded DNA, the intensity of fluorescent emissions increases. As more double stranded amplicons are produced, SYBR green dye signals increase.

Peer review

The study described the effect of celecoxib on H. pylori. The study is well- designed. The experimental data are sufficient to support its conclusion.