The evaluation of MMP-2/TIMP-1 ratio in peptic ulcer and its association with refractory helicobacter pylori infection

doi:10.21203/rs.3.rs-2593628/v1

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The evaluation of MMP-2/TIMP-1 ratio in peptic ulcer and its association with refractory helicobacter pylori infection

https://doi.org/10.21203/rs.3.rs-2593628/v1

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published 21 Aug, 2023

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Background

Helicobacter pylori (H.pylori) is one of the leading causes of peptic ulcers, and its treatment is a worldwide challenge. Matrix metalloproteinases and their inhibitors influence the development and healing of peptic ulcers. This study aimed to evaluate the ratios of matrix metalloproteinase-2 (MMP-2) to tissue inhibitor of metalloproteinase-1 (TIMP-1) in patients with a peptic ulcer that are sensitive or resistant to H. pylori treatment and compare them with healthy individuals.

Methods

In this study, 95 patients were included and divided into two groups sensitive (41 patients) and resistant to treatment (54 patients). The results were compared with a control group of twenty participants with normal endoscopy. After obtaining written informed consent, five ml of venous blood was taken to determine their serum MMP-2 and TIMP-1 levels using an enzyme-linked immunosorbent assay.

Results

In patients with H. pylori-induced peptic ulcers, the MMP-2/TIMP-1 ratio was significantly higher than those of the healthy controls (P < 0.05). MMP-2 level was associated with patients’ response to treatment (P < 0.05). The MMP-2/TIMP-1 ratio and the chance for refractory H. pylori-induced peptic ulcer disease were higher in patients with simultaneous gastric and duodenal ulcers (P < 0.05).

Conclusion

It seems that peptic ulcer disease caused by infection with H. pylori increases the MMP-2/TIMP-1 ratio in patients with peptic ulcers. However, it might not be a good predictor of refractory H. pylori-induced peptic ulcer disease.

MMP-2

TIMP-1

Peptic ulcer

Helicobacter pylori infection

Peptic ulcer disease (PUD) is a prevalent condition in the digestive system which usually occurs in the stomach or duodenum due to the damage caused by peptic acid (1). Helicobacter pylori (H. pylori) and non-steroidal anti-inflammatory drugs (NSAIDs) are the leading cause of PUD. However, only a limited number of H. pylori-positive patients or NSAID consumers become afflicted with PUD (2, 3). Diagnostic tests for detecting active H. pylori infection include endoscopy, urea breath test, and stool antigen test. A stool antigen test is usually used to primarily detect H. pylori infection and confirm its eradication (4, 5). H. pylori infection is common in Iran and its prevalence has been reported to be between 36% and 90% in different geographic areas (6).

Due to the increase in resistance to antibiotics and failure of treatment in almost 20% of the patient, the first attempt to eradicate H. pylori has become a challenge. The first attempt at H. pylori eradication fails in almost 20% of the patients. Hence, a confirmation test for the eradication of H. pylori is essential at the end of the course of H. pylori eradication therapy. The stool antigen test is an efficient and common tool for this purpose (7).

Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes that degrade the basement membrane (BM) and extracellular matrix (ECM) components (8). Tissue inhibitors of metalloproteinases (TIMPs) are endogenous and naturally occurring inhibitors of MMPs, which inhibit their actions by creating noncovalent complexes with them. Four members of TIMPs, TIMP-1, -2, -3, and − 4, have been characterized. The equilibrium between MMPs and TIMPs regulates extracellular matrix turnover and keeps tissue homeostasis. Variations in the equilibrium between MMPs and TIMPs are associated with the pathogenesis of different diseases. TIMP-1 is a potent inhibitor of many MMPs. On the other hand, it also controls many biological processes, such as cell growth, proliferation, and apoptosis, through binding to undisclosed receptors (9).

Although some studies have reported a decrease in MMP-2 and TIMP-1 levels in H. pylori infection patients, others have reported quite the opposite (10, 11). That is what makes the association between refractory H. pylori infection and the levels of MMP-2 and TIMP-1 unclear. Therefore, this study intended to investigate the MMP-2/TIMP-1 ratio in patients with PUD and determine its relationship with refractory H. pylori infection.

2.1 study design and Patients

In this study, 95 patients referring to the Gastroenterology Clinic were investigated. The patients who entered the study were diagnosed with PUD and H. pylori infection based on their endoscopy and biopsy results. Before entering the study, written informed consent was obtained from each of the patients. A checklist was used to record demographic information including age, sex, and endoscopy results.

The participants then underwent standard quadruple therapy for H. pylori infection with 14 days of omeprazole (20 mg twice a day), metronidazole (500 mg twice a day), clarithromycin (500 mg twice a day), and bismuth subsalicylate (two tablets, four times a day). In the end, a stool antigen test was performed 8–12 weeks after the end of therapy, for confirmation of H. pylori eradication.

Based on the results obtained from the stool antigen test, 54 patients who still tested positive were allocated to the resistant group and 41 patients who tested negative were assigned to the sensitive group. In addition, 20 individuals with normal endoscopy and negative H. pylori test were included as the control group. Participants with chronic PUD, pneumonia, bronchiectasis and drug history of NSAIDs or malignancies were all excluded from the study.

To evaluate the serum MMP-2 and TIMP-1 levels in the three study groups 5 ml of blood sample was taken from each of the participants. After keeping the blood samples for 20 minutes at room temperature, samples were centrifuged at 2500–3000 rpm for 10–15 minutes to separate the serum. The samples were then transferred to a refrigerator and stored at -80°C until use. Finally, the samples were analyzed for serum MMP-2 and TIMP-1 levels using ELISA (Enzyme-Linked Immunosorbent Assay) kits according to the manufacturer’s protocol, The present study was approved by the Research Ethics Committee of the University of Medical Sciences with the approval code of IR.ARUMS.REC.1399.081.

2.2 Statistical analyses

The obtained data were first coded and then fed into SPSS Software, Version 26. Then various descriptive and inferential statistical tools such as t-test, Mann–Whitney U test, ANOVA, post hoc test of Tukey, Kruskal–Wallis test, Chi-square test, and Fisher’s exact test were used to analyze the data. The significance level was set at 0.05 for all tests.

3.1 Demographic features and endoscopy findings of the enrolled population

The current study included 115 individuals (70 males and 45 females). The mean age of the patients was 52.23 ± 9.91 years. Regarding endoscopy findings and response to treatment, significant differences were observed among the three study groups with gastropathy, bulbopathy, and gastrobulbopathy (P < 0.05) (Table 1).

Table 1

The distribution of demographic features and endoscopy findings in the study groups
Variables		Patients		Control	P-value
Variables		Sensitive (n = 41)	Resistant (n = 54)	Control	P-value
Gender	Male	23 (20%)	35 (30.4%)	12 (10.4%)	0.68
Gender	Female	18 (15.7%)	19 (16.5%)	8 (7%)	0.68
Age (Years)	< 40	2 (1.7%)	6 (5.2%)	2 (1.7%)	0.44
	40–54	17 (14.8%)	29 (25.2%)	10 (8.7%)
	55–70	22 (19.1%)	19 (16.5%)	8 (7%)
Alcohol	Yes	6 (5.2%)	1 (0.9%)	4 (3.5%)	0.02
Alcohol	No	35 (30.4%)	53 (46.1%)	16 (13.9%)	0.02
smoking	Yes	8 (7%)	11 (9.6%)	0 (0%)	0.09
smoking	No	33 (28.7%)	43 (37.4%)	20 (17.4%)	0.09
Hookah	Yes	3 (2.6%)	6 (5.2%)	0 (0%)	0.28
Hookah	No	38 (33%)	48 (41.7%)	20 (17.4%)	0.28
Endoscopic findings	Gastropathy	17 (17.9)	22 (23.1%)	-	0.005
	Bulbopathy	7 (7.4%)	6 (6.3%)	-
	Gastrobulbopathy	17 (17.9%)	26 (27.4%)	-

3.2 Comparison of MMP-2 and TIMP-1 ratios with demographic features and endoscopy findings

In Table 2, mean MMP-2 and TIMP-1 levels and MMP-2/TIMP-1 ratios and their relationship with other variables of the study are presented. The results revealed a significant relationship between the mean MMP-2, TIMP-1, MMP-2/TIMP-1 ratio and the patients’ endoscopic findings (P < 0.05).

Table 2

Analysis of mean MMP-2 and TIMP-1 levels and their relationship with demographic features and endoscopy findings
Variables		MMP-2 (ng/ml)	P-value	TIMP-1 (ng/ml)	P-value	MMP-2/TIMP-1 ratios	P-value
Age (Years)	< 40	740.90 ± 250.34	0.54	204.81 ± 80.42	0.76	3.34 ± 1.17	0.40
	40–54	632.12 ± 274.25		184.77 ± 70.50		3.77 ± 2.01
	55–70	648.27 ± 285.99		189.56 ± 89.70		4.24 ± 3.01
Alcohol	Yes	449.27 ± 265.70	0.02	182.27 ± 75.44	0.77	2.70 ± 1.97	0.052
Alcohol	No	669.53 ± 280.07	0.02	189.23 ± 80.29	0.77	4.06 ± 2.46	0.052
smoking	Yes	811.84 ± 234.72	0.003	194.89 ± 73.42	0.68	5.06 ± 2.08	0.01
smoking	No	616.13 ± 284.11	0.003	187.31 ± 81.1	0.68	3.71 ± 2.45	0.01
Hookah	Yes	820.11 ± 265.43	0.07	200.72 ± 71.94	0.61	5.19 ± 2.47	0.14
Hookah	No	633.89 ± 283.12	0.07	187.53 ± 80.39	0.61	3.83 ± 2.42	0.14
Endoscopic findings	Gastropathy	695.25 ± 215.56	< 0.001	195.46 ± 62.70	0.04	3.78 ± 1.64	0.002
	Bulbopathy	642.67 ± 205.42		229.10 ± 92.56		3.83 ± 2.43
	Gastrobulbopathy	863.50 ± 202.06		188.63 ± 71.05		5.56 ± 3.04
	Control	633 ± 155.49		243.77 ± 92.44		3.33 ± 1.65
MMP: Matrix metalloproteinase; TIMP: Tissue inhibitor of matrix metalloproteinase

3.3 Comparison of MMP-2/TIMP-1 ratios in the study groups

The mean MMP-2 level was 741.93 ± 218.16 ng/ml in the patient group and 204.50 ± 33.80 ng/ml in the control group. The mean TIMP-1 level for these two groups was 205.67 ± 77.18 ng/ml and 107.31 ± 9.27 ng/ml, respectively. Furthermore, the mean MMP-2/TMP-1 ratio in these two groups was 4.36 ± 2.48 ng/ml and 1.92 ± 0.39 ng/ml, respectively. The results of the analyses indicated that the patient and control groups were significantly different in terms of mean MMP-2 level, TIMP-1 level, and MMP-2/TIMP-1 ratio (Fig. 1) (P < 0.001).

The mean MMP-2 level in the sensitive group was 677.34 ± 177.28 ng/ml and in the resistant group was 790.96 ± 234.56 ng/ml. The mean TIMP-1 levels for these two groups were 220.49 ± 88.99 ng/ml and 194.42 ± 65.49 ng/ml, respectively. Also, the mean MMP-2/TIMP-1 ratios in the two groups were 4.07 ± 2.01 ng/ml and 4.57 ± 2.79 ng/ml, respectively. The results of the analyses revealed that the two groups were significantly different in terms of mean MMP-2 levels (P < 0.05). However, as regards the mean TIMP-1 level and MMP-2/TIMP-1 ratio, no significant differences were observed between these two groups (P > 0.05) (Fig. 2).

H. pylori infection is among the leading causes of PUD. Research has revealed that eradicating H. pylori in the affected patients not only cures PUD but also improves the related clinical symptoms. PUD heals following the formation of granulation tissue and repair of the injured tissue in the base of the ulcer. The balance between MMPs and TIMPs affects peptic ulcer formation and healing processes. A higher proportion of MMPs increases proteolysis in ECM and causes ulcers, while a higher proportion of TIMPs brings about better protection for ECM, decreases proteolysis, and heals the ulcer (12, 13).

The few studies on the effects of MMP-2 and TIMP-1 on patients with H. pylori-induced PUD show controversial results. In 2004, Antonio et al. conducted a study investigating the variations of MMP-2, MMP-3, MMP-13, and their inhibitor (TIMP-1) in a mouse model with an acetic acid-induced ulcer. They observed that the variations in the levels of MMPs and TIMP-1 were similar during the healing process of the ulcer; their levels increased rapidly after the injury occurrence, reached the highest after one week, and constantly reduced through the last phase of tissue repair. However, quantitative analyses indicated a relative increase in the expression of TIMP-1 in comparison with MMPs during the first week, while after four weeks, the expression of MMPs was found to be relatively higher (14). Our study assessed MMP-2 and TIMP-1 levels and their ratio only once, and an increase in MMP-2, TIMP-1, and their ratio was detected (P < 0.001). Furthermore, we observed that in patients with coincident gastric and duodenal ulcers, the MMP-2 and MMP-2/TIMP-1 ratios were higher than the other ulcers (P < 0.05), which might be due to the higher rate of ECM degradation and the healing process in the presence of multiple ulcers.

In a study by Rautelin et al., the increase of MMPs in response to H. pylori-induced gastritis was investigated. MMP-2, MMP-7, MMP-8, MMP-9, NE, MPO, and TIMP-1 levels were examined. The results indicated that in H. pylori-positive patients with gastritis, the serum levels of myeloperoxidase (MPO), neutrophil elastase (NE), collagenase-2 (MMP-8), and gelatinase B (MMP-9) increased significantly. Also, gelatinase A (MMP-2) and TIMP-1 levels were decreased significantly compared to controls with negative H. pylori infection. As regards the serum level of matrilysin-1 (MMP-7), the two groups revealed no significant difference (11). Their study's sample size was smaller than ours (44 vs. 95 patients, respectively), which can be suggestive of the lower precision of the results obtained in their study. Contrary to the findings reported in the mentioned study, our study indicated that MMP-2 and TIMP-1 levels and MMP-2/TIMP-1 ratio significantly increased in patients with H. pylori-induced PUD. Studies show higher levels of TIMP-1 in H. pylori infection-induced PUD than other reasons, such as NSAIDs (12). These findings prevail the efficacy of these factors’ measurements in patients with PUD for differentiating the cause of PUDs.

In the study groups in our research, variations in the site of peptic ulcer in endoscopy were also significant. The highest level of resistance to treatment was observed in the coincident gastric and duodenal ulcers, then gastric ulcer, and duodenal ulcer, respectively; and finally, the lowest level of resistance was observed in the control group. It indicates that in a patient with multiple sites of PUD, a robust treatment method should be chosen because there might be a higher chance for refractory H. pylori-induced PUD.

In our study, the levels of MMP-2, TIMP-1, and their ratio have been investigated in refractory H. pylori-induced PUD for the first time. It is demonstrated that there is a significant increase in the MMP-2 level, but the TIMP-1 level and MMP-2/TIMP-1 ratio both show insignificant results. Based on the existing data, higher levels of MMP-2 in the absence of other diseases can be used as a predictive factor for the existence of refractory H. pylori-induced PUD. However, the MMP-2/TIMP-1 ratio might not be used as a refractory H. pylori-induced PUD marker.

It is suggested that future studies should be working on comparing endoscopy-based histopathologic findings and serum levels of these molecules. By taking deeper biopsies of PUD sites and normal sites, comparing the MMPs and TIMPs levels between different types and places of ulcers, and comparing these factors with the duration of symptoms because maybe their levels may contribute to the duration of PUD incidents as is seen in previous studies in mouse models (14). In this study, we investigated MMP-2 and TIMP-1 levels in serum which is less accurate compared to the histologic evaluation, and inspected MMP-2 and TIMP-1 levels after the first course of treatment which could offer adequate information if it was assessed twice in the study and compared with each other.

H. pylori-induced PUD can increase the MMP-2/TIMP-1 ratio, but it might not be a good predictor of refractory H. pylori-induced PUD. However, the increased MMP-2 level in the absence of other diseases can be used as a predictive marker for refractory H. pylori-induced PUD.

H. pylori: Helicobacter pylori

MMP: Matrix metalloproteinase

TIMP: Tissue inhibitor of metalloproteinase

PUD: Peptic ulcer disease

NSAIDs: Non-steroidal anti-inflammatory drugs

BM: Basement membrane

ECM: Extracellular matrix

ELISA: Enzyme-Linked Immunosorbent Assay

MPO: Myeloperoxidase

NE: Neutrophil elastase

Ethics approval and consent to participate

This study was approved by the Research Ethics Committee of the Ardabil University of Medical Sciences (IR.ARUMS.REC.1399.081). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all subjects according to the instructions of Ethics Committee of the Ardabil University of Medical Sciences.

Consent for publication

not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interest to disclose.

Funding

This article was prepared without any support or funding and

Authors' contributions

MN drafted, checked, and revised the manuscript; ES drafted the work or substantively revised it; NF analyzed, and interpreted the data; SM drafted and designed the work; SP drafted the manuscript.

Acknowledgments

Not applicable.

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No competing interests reported.

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Journal Publication

published 21 Aug, 2023

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Editorial decision: Major revision
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You are reading this latest preprint version

The evaluation of MMP-2/TIMP-1 ratio in peptic ulcer and its association with refractory helicobacter pylori infection

Status:

Journal Publication

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Methods

2.1 study design and Patients

2.2 Statistical analyses

Results

3.1 Demographic features and endoscopy findings of the enrolled population

3.2 Comparison of MMP-2 and TIMP-1 ratios with demographic features and endoscopy findings

3.3 Comparison of MMP-2/TIMP-1 ratios in the study groups

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1