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Chinese Journal of Digestive Diseases 2002; 3; 75–78

Effect of Helicobacter pylori infection on cyclooxygenase-2 expression in gastric antral mucosa

Hong LU, Xiao Yu CHEN, Wen Zhong LIU, Yan Shen PENG & Shu Dong XIAO

Department of Gastroenterology, Renji Hospital, Shanghai Second Medical University, Shanghai Institute of Digestive Disease, Shanghai, China

OBJECTIVE: Helicobacter pylori infection is a majoretiological cause of chronic gastritis. Induciblecyclooxygenase (COX-2) is an important regulator ofmucosal inflammation. Recent studies indicate thatexpression of COX-2 may contribute to gastro-intestinal carcinogenesis. The aim of this study was toinvestigate the effects of H. pylori infection and eradi-cation therapy on COX-2 expression in gastric antralmucosa.

METHODS: Antral biopsies were taken from 46H. pylori-infected patients, who also had chronic gas-tritis, both before and after anti-H. pylori treatment.The COX-2 protein was stained by using immunohis-tochemical methods and COX-2 expression was quan-tified as the percentage of epithelial cells expressingCOX-2. Gastritis and H. pylori infection status weregraded according to the Sydney system.

RESULTS: Cyclooxygenase-2 expression was detectedin the cytoplasm of gastric antral epithelial cellsboth before and after the eradication of H. pylori.Cyclooxygenase-2 expression in mucosa with H. pyloriinfection was compared with the corresponding mucosaafter successful H. pylori eradication (20.1 ± 13.1%vs 13.8 ± 5.9%; P < 0.05). At the same time, COX-2expression in H. pylori-infected mucosa wascompared with the normal controls (18.0 ± 14.1% vs12.3 ± 4.6%, P < 0.05). Expression of COX-2 wascorrelated with the degree of chronic inflammation(r = 0.78, P < 0.05).

CONCLUSIONS: Our results showed that H. pyloriinfection leads to gastric mucosal overexpression ofCOX-2 protein, suggesting that the enzyme isinvolved in H. pylori-related gastric pathology inhumans.

KEY WORDS: chronic gastritis, cyclooxygenase-2, Helicobacter pylori.

INTRODUCTION

Cyclooxygenase (COX) is the rate-limiting enzymerequired for the conversion of arachidonic acid toprostaglandins (PG). Two cyclooxygenase isoformshave been identified, which are referred to as COX-1and COX-2. Under normal circumstances, COX-1 is

produced constitutively in normal tissue, whereasCOX-2 is inducible in macrophages, fibroblasts andother cell types involved in inflammation.1 Expressionof COX-2 is induced by phorbol esters and pro-inflammatory cytokines, including interleukin-1β(IL-1β) and tumor necrosis factor-α (TNF-α). Recentstudies indicate that inducible COX-2 plays animportant role in gastrointestinal carcinogenesis.2,3

Helicobacter pylori infection is a major cause of chronicactive gastritis and has been associated with gastricadenocarcinoma and MALToma. The present studywas designed to investigate the effects of H. pyloriinfection on COX-2 expression in gastric antralmucosa.

Correspondence to: Dr Wen Zhong LIU, Department of Gastroenterology, Renji Hospital, Shanghai Second Medical University, Shanghai Institute of Digestive Disease, 145 Shandong Zhong Road, Shanghai 200001, China. Email: liuwzmd@online.sh.cn

Originally published in: Chin J Dig 2001; 21: 287–9.

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Chinese Journal of Digestive Diseases 3, 75–78

MATERIALS AND METHODS

Patients

All patients underwent upper gastrointestinal endo-scopy at the Endoscopy Center of Renji Hospital,Shanghai (affiliated with Shanghai Second MedicalUniversity), between 1997 and 1998. The study groupcomprised 32 men and 14 women (age range 16–64years). During endoscopy, three biopsy specimenswere taken from both the antrum and the corpus. Ofthese, one specimen from each region of the stomachwas used for rapid urease testing, the others were fixedin 10% formalin and stained with: (i) hematoxylinand eosin (H&E) for histological examinations; and(ii) Giemsa stain for H. pylori assessment. Helicobacterpylori assessment was performed by using the rapidurease test and histopathological analyses. Twenty-seven patients with superficial chronic gastritis and 19with mild atrophic gastritis, all of whom testedpositive for H. pylori in both endoscopic and histo-pathological investigations, were selected for study.

All patients were given a 7-day course of bismuth-based triple therapy: 240 mg colloidal bismuth sub-citrate (CBS) plus two antibiotics. Endoscopy andbiopsy were performed again 4–6 weeks after thecessation of therapy. Helicobacter pylori was consideredto be eradicated if the rapid urease test was negativeand microorganisms were absent during histopatho-logical examinations.

Ten H. pylori-negative patients (five men and fivewomen; age range 16–38 years) with endoscopicallyand histologically confirmed normal gastric mucosawere selected as the control group.

None of patients in this study had been taking non-steroidal anti-inflammatory drugs (NSAIDs) or gluco-corticoids regularly before endoscopy was performed.

Histological assessment

All biopsy specimens for histological examinationwere fixed in 10% formalin and embedded inparaffin, then cut into sequential 5-mm sections. Alltissue sections were stained with H&E for histologicalexamination and with Giemsa stain for H. pyloriassessment. Immunostaining was only performed ongastric biopsies from the antrum.

The severity of chronic gastritis, atrophy and intestinalmetaplasia, as well as H. pylori density, were evaluatedand graded on a four-point scale (absent or normal,mild abnormality, moderate abnormality and severe

abnormality) according to the Sydney system for thehistological grading of gastritis.

Immunohistochemistry

The COX-2 polyclonal antisera and the avidin–biotin–peroxidase complex (ABC) kit were produced by theSanta Cruz Corporation, USA. Tissue sections weredewaxed in xylene and rehydrated through gradedalcohol to distilled water. Antigen retrieval wasperformed by microwave treatment in sodium citratebuffer (10 mmol/L, pH 6.0) and then cooled to roomtemperature. Subsequently, they were immersed in1% H2O2 in methanol for 15 min and then immersedin normal monkey serum for 15 min to block non-specific binding sites. After extra normal serumremoval, slides were incubated with goat polyclonalimmunoglobulin G specific for human COX-1/COX-2in a dilution of 1 : 100 overnight at 4°C. The sectionswere thereafter treated with biotinylated secondaryantibodies at 37°C for 60 min. Sections were thenstained with the standard ABC methods and visual-ized using 3,3′-diaminobenzidine (DAB). The sectionswere counterstained with hematoxylin.

Evaluation of immunostaining

Immunohistochemical staining for COX-2 wasassessed by counting the number of positive- andnegative-staining cells on the surface epithelial cellsand the foveolae and propria glands of the gastricmucosa of each biopsy specimen. The number ofpositive-staining cells was expressed as a percentage ofthe total number of cells. The pathologist did notknow the H. pylori status of the patient. Positive cellshad cytoplasmic and perinuclear brownish staining.Only blue nuclear staining was recognized as negative.From the 10 fields counted, the average percentage ofpositive cells was graded semiquantitatively.

Statistical analysis

Statistical significance was calculated by usingStudent’s t-test and the Spearman signed rank test.Statistical significance was set at P < 0.05.

RESULTS

COX-2 protein expression in gastric antral mucosa

Stained COX-2 protein was detected in the cytoplasmof surface epithelial cells and propria glands in theantral mucosa of samples from the normal groupboth before and after eradication of H. pylori. Positivecells were mainly located in the neck of the foveolae

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Chinese Journal of Digestive Diseases 3, 75–78

and superficial mucosa. Almost all intestinal-typeepithelium stained positive for COX-2 protein. Inter-stitial cells, including monocytes, endothelium andsmooth muscle fibers also displayed sporadic COX-2protein positive staining.

COX-2 protein expression and H. pylori infection

The mean percentage of cells staining for COX-2 wassignificantly higher in H. pylori-infected mucosacompared with normal controls (18.0 ± 14.1% vs12.3 ± 4.6%, P < 0.05). Successful eradication ofH. pylori was achieved in 32 of 46 (70%) patients aftertriple therapy. The mean percentage of cells stainingfor COX-2 before treatment was 20.1 ± 13.1%,decreasing to 13.8 ± 5.9% after successful H. pylorieradication (P < 0.05). There was no significant differ-ence between normal controls and patients aftersuccessful H. pylori eradication (13.8 ± 5.9% vs12.3 ± 4.6%; P > 0.05). The mean percentage of cellsstaining for COX-2 was 17.8 ± 10.1% in 14 patients inwhom H. pylori infection remained after treatment.There was no significant difference between this groupand the before-treatment group (P > 0.05).

COX-2 immunostaining and chronic inflammation

Cyclooxygenase-2 protein expression was significantlycorrelated with the severity of chronic inflammation(r = 0.78, P < 0.05). However, no correlation wasfound between COX-2 protein expression, the densityof H. pylori colonization and the activity of gastritis(P > 0.05).

DISCUSSION

Several studies have demonstrated that COX-2 proteinexpression is significantly increased in gastric andcolonic adenocarcinoma.4,5 Studies using an animalmodel of human familial adenomatous polyposis(FAP) have shown that elevated levels of COX-2mRNA and protein expression were also observed inthe epithelial cells of colonic adenomas. Both NSAIDsand specific inhibitors of COX-2 may reduce the sizeand number of adenomas, and may play a pro-phylactic role in the adenoma–carcinoma sequence,suggesting that COX-2 may play an important role inearly gastrointestinal carcinogenesis.6,7 This effect hasalso been demonstrated in humans.8

Helicobacter pylori infection is the major cause ofgastroduodenal diseases, including chronic activegastritis, peptic ulcers and gastric malignancy (bothadenocarcinoma and MALToma). The InternationalAgency for Cancer Research has classified H. pylori as

a class I carcinogen, a definite cause of gastric cancerin humans, but with a mechanism of carcinogenesisthat remains unclear.

In the present study, elevation of COX-2 proteinexpression was observed in epithelial cells, monocytesof the normal controls and intestinal-type epithelialcells both before and after eradication of H. pylori.Expression of COX-2 protein was significantlyincreased in the epithelial cells of H. pylori-infectedmucosa and was closely associated with the severity ofchronic gastritis. Recently, Zarrilli et al. demonstratedthat COX-2 mRNA expression was significantlyincreased in H. pylori-infected mucosa compared withH. pylori-negative mucosa.9 How H. pylori infectioninduces the expression of COX-2 protein in gastricepithelial cells remains unknown. Romano et al.found that COX-2 mRNA and prostaglandin E2 levelsincreased fivefold and threefold, respectively, 24 hafter incubation of MKN-28 cells with broth culturefiltrates from an H. pylori strain.10 These results suggestthat COX-2 expression is induced by H. pylori and itsbacterial toxins or inflammatory cytokines, or by aparacrine signaling pathway between macrophagesand the neighboring epithelial cells.11

It remains unclear how COX-2 protein overexpressioncontributes to carcinogenesis. Studies in animalmodels found that high levels of COX-2 proteinincreased prostaglandin E2 biosynthesis, which couldinduce cell proliferation and stimulate the overexpres-sion of bcl-2 protein and therefore inhibit cellapoptosis.12 Loss of balance during proliferation andapoptosis may also promote the progression oftumors.13 Helicobacter pylori infection can induceproliferation in gastric epithelial cells.14 Severalstudies have demonstrated that there is a high levelof proliferation in gastric epithelial cells, but nocorresponding change in apoptosis in H. pylori infec-tion.14–16 The COX-2 protein may play an importantrole in the multi-step progression from H. pylori-related gastritis to precancerous lesion to gastriccancer. This supports a possible role for specificCOX-2 inhibitors as chemoprophylactic agents in theprevention of gastrointestinal carcinogenesis.

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