Effect of Helicobacter pylori infection on gastric mucosal pathologic change and level of nitric oxide and nitric oxide synthase

Abstract

AIM: To investigate the level of nitric oxide (NO) and nitrous oxide synthase (NOS) enzyme and its effect on gastric mucosal pathologic change in patients infected with Helicobacter pylori (H pylori ), and to study the pathogenic mechanism of H pylori.

METHODS: The mucosal tissues of gastric antrum were taken by endoscopy, then their pathology, H pylori and anti-CagA-IgG were determined. Fifty H pylori positive cases and 35 H pylori negative cases were randomly chosen. Serum level of NO and NOS was detected.

RESULTS: One hundred and seven cases (71.33%) were anti-CagA-IgG positive in 150 H pylori positive cases. The positive rate was higher especially in those with pre-neoplastic diseases, such as atrophy, intestinal metaplasia and dysplasia. The level of NO and NOS in positive group was higher than that in negative group, and apparently lower in active gastritis than in pre-neoplastic diseases such as atrophy, intestinal metaplasia and dysplasia.

CONCLUSION: H pylori is closely related with chronic gastric diseases, and type I H pylori may be the real factor for H pylori-related gastric diseases. Infection with H pylori can induce elevation of NOS, which produces NO.

INTRODUCTION

There is evidence that Helicobacter pylori (H pylori ) is closely related with gastric carcinoma, and is considered as the first grade oncogene of gastric carcinoma by World Health Organization (WHO). H pylori infection correlates closely with gastric mucous pathology[1-4].

NO is a medium produced in vessel endothelial cells or smooth muscle cells by NOS[5,6]. As an inflammatory medium, NO plays an important role in the physical function and pathological process. Changes of NO in serum and tissue are related with damage to gastric mucosa and H pylori infection[7-11].

This study aimed to investigate the changes of NO, NOS and the pathological transformation of gastric mucosa in patients infected with H pylori.

MATERIALS AND METHODS

Patients

Two hundred and eighty-two patients with chronic gastric disease were enrolled in this study. H pylori was detected by both rapid urease test and real-time fluorescent quantitative PCR in these patients. Anti-CagA-IgG was detected in the H pylori positive patients, the serum samples were collected from 50 H pylori positive patients and 35 H pylori negative patients for detection of NO and NOS.

Real-time fluorescent quantitative PCR

Real-time fluorescent quantitative PCR was performed with PCR kit (Da’an Gene Diagnosis Center, Guangzhou). Fluorescence was detected with a type DA620 fluorescent detector.

Cag A H pylori-IgG

CagA H pylori IgG was detected according to the manufacturer’s instructions (Shanghai Jingying Biology Corporation).

Measurement of NO and NOS

Because NO could be converted into NO2- and NO3- in vivo, nitrate reductase was used to deoxidize NO₃^- into NO₂^-, and to determinate its concentration. NO and NOS were tested with the kits, (Nanjing Jiancheng Biology Corporation).

Statistical analysis

Data were presented as mean±SD and analyzed with SPSS software. Statistical analysis was performed using two-tailed Student’s t test and χ² test. P<0.05 was considered statistically significant.

RESULTS

Relationship between H pylori infection and pathology

Among the 282 cases, H pylori was found in 150 cases, (53.19%), including 38.54% (37/96) in chronic superficial gastritis group, 51.26% (61/119) in atrophic gastritis group, 73.17% (30/41) in intestinal metaplasia group, and 84.62% (22/26) in dysplasia group. The H pylori positive rate in atrophic gastritis group was higher than that in chronic superficial gastritis group (P<0.05), and significantly higher in intestinal metaplasia group and dysplasia group than that in chronic superficial gastritis group (P<0.01, Table 1).

Table 1.

H pylori positive rate in chronic gastric disease (%)

Group	n	H pylori positive	H pylori negative
CSG	96	37 (38.54)	59 (61.46)
CAG	119	61 (51.26)a	58 (48.74)
IM	41	30 (73.17)b	11 (26.83)
Dysplasia	26	22 (84.62)b	4 (15.38)
Total	282	150 (53.19)	132 (46.81)

^aP<0.05,

^bP<0.01 vs CSG.

Relationship between anti-CagA-IgG and pathology

The anti-CagA-IgG positive rate was 71.33% (107/150) in 150 H pylori positive patients, including 40.54% (15/37) in chronic superficial gastritis group, 75.41% (46/61) in atrophic gastritis group, 86.67% (26/30) in intestinal metaplasia group and 90.91% (20/22) in dysplasia group. The anti-CagA-IgG positive rate in chronic superficial gastritis group was significantly lower than that in the other three groups (Table 2).

Table 2.

Positive rate of anti-CagA in 150 H pylori positive patients (%)

Group	CSG	CAG	IM	Dysplasia
n	37	61	30	22
Anti-CagA Positive (%)	15 (40.54)b	46 (75.41)	26 (86.67)	20 (90.91)

^bP<0.01 vs Cag, IM, dysplasia.

Relationship between NO, NOS, and H pylori infection

The serum concentration of NO and NOS was 87.6±16.1 μmol/L and 51.4±13.3 μmol/L respectively in H pylori positive group, and 69.8±19.4 μmol/L and 35.2±13.3 μmol/L respectively in H pylori negative group (Table 3).

Table 3.

Serum concentration of No and NOS(mean±SD)

Group	n	NO(μmol/L)	NOS(μmol/L)
H pylori positive	50	87.6±16.1b	51.4±13.3b
H pylori negative	35	69.8±19.4	35.2±13.3

^bP<0.01 vs H pylori negative group.

Relationship between NO, NOS, and pathology

The serum concentration of NO in chronic superficial gastritis group was significantly lower than that in atrophic gastritis group, intestinal metaplasia group and dysplasia group (P<0.05, Table 4).

Table 4.

Serum concentration of NO in different pathological groups(μmol/L, mean±SD)

Group	H pylori positive		H pylori negative
	n	Concentration	n	Concentration
CSG	16	80.0±14.6a	11	62.2±16.9a
CAG	25	95.4±8.4	21	74.6±19.2
IM	12	91.2±13.9	4	75.5±27.7
Dysplasia	9	95.3±10.3	3	71.5±19.6

^aP<0.05 vs atrophic gastritis group, intestinal metaplasia group, and dysplasia group.

The serum concentration of NOS in chronic superficial gastritis group was significantly lower than that in atrophic gastritis group, intestinal metaplasia group and dysplasia group (P<0.05), but there was no significant difference among the four groups (Table 5).

Table 5.

Serum concentration of NOS in different pathological groups(μmol/L, mean±SD)

Group	H pylori positive		H pylori negative
	n	Concentration	n	Concentration
CSG	16	38.0±12.4a	11	31.7±9.4
CAG	25	57.6±8.3	21	35.4±13.0
IM	12	54.8±8.3	4	26.1±4.4
Dysplasia	9	59.6±9.4	3	46.1±22.9

^aP<0.05 vs atrophic gastritis group, intestinal metaplasia group, and dysplasia group.

DISCUSSION

H pylori infection plays a leading role in the pathogenesis of chronic gastritis. Furthermore, H pylori infection is also a high risk factor for the development of gastric cancer[12]. H pylori can destroy gastric mucosa, leading to inflammation of gastric mucosa and digestive symptoms.

Our study showed that the H pylori positive rate in chronic superficial gastritis group was 38.54%, suggesting that H pylori is related to inflammation of gastric mucosa. Other factors may be involved in inflammation of gastric mucosa, such as pH value, mucus, glycoprotein. But in atrophic gastritis group, intestinal metaplasia group, and dysplasia group, the H pylori positive rate was 51.26%, 73.17% and 84.62%, respectively, indicating that H pylori infection has a close relationship with gastric pre-neoplastic diseases, such as atrophy, intestinal metaplasia, and dysplasia.

It was reported that H pylori has two types. Type I H pylori possesses high virulence energy producing cytotoxin-associated protein A and vacuole toxin, which are responsible for inflammatory response of gastric epithelial cells, and promotes cell proliferation and apoptosis[13,14]. Therefore, type I H pylori has a close relationship with development of gastric pre-neoplastic diseases[15-18]. Our study showed that the pathological change of gastric mucosa was parallel with the anti-CagA-IgG positive rate. These observations support the hypothesis that type I H pylori infection is a high risk factor for the development of gastric pre-neoplastic diseases.

It has been proved that there are lots of NOS in smooth muscle cells and myenteric nerve plexus of stomach[19], which are induced to produce endogenic NO by cytotoxins of H pylori. Moreover, a high pH value is beneficial for anaerobes to colonize in the stomach, and can degrade nitrate of food into nitrite. NO is regarded as an important inflammatory medium, related with acute and chronic inflammatory responses[20-22]. But NO seems to have both beneficial and harmful effects on different stages of inflammation. In earlier period, NO can relieve mucosal inflammation and prevents cellular damage. However, it can prevent cellular apoptosis, induce mutation and contribute to the development of gastric pre-neoplastic diseases in later period[23].

In this study, the levels of NO and NOS in chronic superficial gastritis group were significantly lower than those in pre-neoplastic diseases groups, such as atrophic gastritis group, intestinal metaplasia group, and dysplasia group in H pylori positive patients, but the condition existed not only in H pylori positive group, but also in H pylori negative group, suggesting that the serum level of NO induced by H pylori may be related with pre-neoplastic diseases. In H pylori negative patients, the levels of NOS had no difference in every pathological group, but the levels of NO were significantly higher in gastric pre-neoplastic disease groups, showing that other ways may stimulate the producing of NO besides H pylori in pre-neoplastic diseases. However, we believe that NO plays an important role in the development of pre-neoplastic diseases.