Serum matrix metalloproteinase-3 levels monitor the therapeutic efficacy in Syrian patients with rheumatoid arthritis

Abstract

Background

The plans for the successful treatment of rheumatoid arthritis (RA) seek to attain low disease activity or reach clinical remission.

Objective

Our study aimed to compare the serum MMP-3 levels with predictors of response to therapy of rheumatoid arthritis in Syrian patients and explore its worth as a new valuable biomarker for RA therapy outcomes in daily practice.

Methods

Serum samples were gathered from 43 RA patients at diagnosis and 12 weeks of therapy. Related clinical and laboratory tests were estimated, levels of serum MMP-3 were measured by ELISA method and the disease activity was assessed using disease activity scores in 28 joints with an erythrocyte sedimentation rate (DAS28-ESR) before and after therapy.

Results

The mean of Serum MMP-3 levels significantly decreased (322.3 ± 43.83 ng/ml) after therapy (12 weeks) in RA patients compared to its mean at baseline (486.49 ± 34.5 ng/ml). There wasn't a statistically significant difference in the mean of MMP-3 levels before and after therapy (P = 0.137) in non-responder patients. Patients who showed a good response (N = 38) presented higher MMP-3 levels at first which subsequently decreased significantly at the 12-week follow-up (P < 0.05). Also, there was a statistically significant difference in MMP-3 levels between the two groups of patients after therapy (P = 0.002). To differentiate between RA patients who responded to therapy and who did not, our results found that the cut-off value of serum MMP-3 was 317.8 ng/ml (sensitivity was 80%, specificity was 73%, AUC was 0.818, 95% CI: 1.114–112.5; P = 0.045) and the best cut-off value of DAS28-ESR was 5.325 (sensitivity 100%, specificity 100%, AUC = 100%,95% CI: 15.2 to 47203.8).

Conclusion

serum MMP-3 can be added as a novel and valuable biomarker in estimating the therapeutic response in RA patients, but it isn't better than DAS28-ESR.

1. Introduction

Rheumatoid arthritis (RA) is an autoimmune disease recognized by long-lasting inflammation of the synovial membrane leading to cartilage devastation and local bone erosions [[1], [2], [3]]. If the disease is incompetently controlled, it will lead to permanent joint injury and significant functional incapacity [4]. RA therapy aims to accomplish full remission or at least significantly minor disease activity within 6 months to stop irreversible outcomes [5,6]. The specific treatment of RA depends on conventional synthetic disease-modifying antirheumatic drugs called (csDMARDs), targeted synthetic DMARDs, and biological DMARDs (bDMARDs) [7,8]. It is observed that about 20–40% of RA patients do not achieve a successful response or have no response to TNF-α antagonists [9]. According to the European League Against Rheumatism (EULAR) recommendations, it would require at least 3 months of treatment to assess the responsiveness [8]. If RA patients are disabled to obtain remission or do not show improvement within 3 months, adjunctive treatment will be considered [10]. The disease activity score in 28 joints (DAS28) and C-reactive protein (CRP) are commonly measured to appreciate the therapeutic response in rheumatoid arthritis patients [7,11]. The variation in the therapeutic response in RA patients offers a strong signal that suggests that the disease is triggered by one or more different biochemical pathways. Therefore, this study has shed light on essential biomarkers that can predict the therapeutic response to RA treatment [12].

Matrix metalloproteinase-3 (MMP-3) identified as stromelysin-1 is a proteolytic enzyme. It plays an essential role in both cartilage and joint erosion in rheumatoid arthritis. It mediates the breaking down of many extracellular components such as matrix proteins and proteoglycans, collagens (types III, IV, V, IX, and XI), and energizing other pro-MMPs such as pro-MMP-1, pro-MMP-7, and pro- MMP-9 [6,13,14]. In RA, MMP-3 is released into the blood after being locally formed in inflamed joints [15,16]. The synovial membrane extremely expresses high levels of MMP-3 suggesting that MMP-3 is the main protease of articular cartilage devastation [10,17,18]. The focal cells that produce MMP-3 are chondrocytes, synovial fibroblasts, and monocytes as a result of the effect of inflammatory cytokines [14]. It has been proposed that levels of serum MMP-3 correlate with levels formed in the synovium [19,20]. In adult rheumatoid arthritis (RA), MMP levels are increased in both the synovial fluid and serum and are associated with joint damage [21]. Several studies indicated that increased serum MMP-3 levels have been shown to correlate with the grade of synovial inflammation and disease activity [17,[21], [22], [23]].

A few revisions have found that serum MMP-3 could be used as an additional marker for evaluating the clinical therapeutic efficacy in RA [19,24,25]. One study confirmed that the treatments which achieve both DAS28 remission and dominance of MMP-3 levels lead to improved outcomes compared with treatments that accomplish either of these goals alone [17]. Another study estimated the effects of the administration of rituximab on serum MMP-3 levels in patients with active RA not giving a reply to anti-TNF-a therapy [26]. A few studies have also reported the predictive value of MMP-3 in therapeutic response only to infliximab in treated RA patients with active disease [22]. In addition, the amelioration of MMP-3 levels foretells the remission stage in RA patients treated with adalimumab [27]. The point of novelty of our current study is that MMP-3 serum levels can be used for monitoring the efficacy of therapy for RA in responsive newly diagnosed patients in the early stage of the disease before taking therapy and reaching to irreversible damage. Our research investigated the effect of different drugs used to treat rheumatoid arthritis (Methotrexate alone and its combination with other DMARDs) on MMP-3 levels, and the comparison between their levels and therapeutic response indicators (DAS28, CRP).

2. Materials and methods

Our study was a cross-sectional study. It was achieved in a cohort of 43 (7 male, 36 female) patients with an early diagnosis of RA (ACR/EULAR 2010 criteria for RA 2010) and untreated during the period from January 2020 to April 2021. Attendance was taken from the Rheumatology Clinics in Damascus city hospitals: Almoujtahed Hospital and Al-Mowasat Hospital. The duration of symptoms had been less than two years before diagnosis.

2.1. Ethical approval

Our research was made in agreement with the Helsinki Declaration and agreed upon by the Ethics Commission (No.3/February 26, 2020). Written informed consent was acquired and each subject was adequately informed about the research aim.

Inclusion Criteria included: Only newly diagnosed with RA untreated patients and their ages from 18 to 65 years were involved in our research.

2.2. Exclusion criteria

Progressive patients with severe deformity of joints, presence of additional autoimmune illnesses such as Sjogren's syndrome, and systemic lupus erythematosus (SLE). Patients having liver and kidney diseases also were excluded from the study.

2.3. Laboratory and clinical tests

The following tests were ordered and completed for all patients: Complete blood count (CBC), C-reactive protein (CRP) and Rheumatoid factor (RF) by latex method, erythrocyte sedimentation rate (ESR) using the Westergren method, and anti-cyclic citrullinated peptide antibodies (ACPA) using Enzyme-Linked Immuno-Sorbent Assay (ELISA) method (Bio-System company).

2.4. Evaluation of disease activity

Disease activity score 28 (DAS28) was estimated based on swollen joint count evaluated on 28 joints (SJC28) and tender joint count assessed on 28 joints (TJC28), the patient's global assessment called (general-visual analog scale (VAS): 0–100 mm) (VAS) with zero = best, 100 = worst] then (DAS28-ESR) was calculated as the following:

$$\mathrm{D}\mathrm{A}\mathrm{S}28-\mathrm{E}\mathrm{S}\mathrm{R}=0.56\sqrt{}(\mathrm{T}\mathrm{J}\mathrm{C}28)+0.28\sqrt{}(\mathrm{S}\mathrm{J}\mathrm{C}28)+0.70\mathrm{Ln}(\mathrm{E}\mathrm{S}\mathrm{R})+0.014(\mathrm{G}\mathrm{H})\text{.}$$

2.5. Rheumatoid arthritis activity was explained as follows

Reach remission when DAS28 ≤ 2.6, disease activity is low when (2.6 < DAS28 ≤ 3.2), disease activity is moderate when (3.2 < DAS28 ≤ 5.1), and high disease activity when (DAS28 > 5.1).

The patients’ samples were gathered (3 ml of blood) and centrifuged at 1980 g within 20 min. All the serum was separated and frozen immediately at −20C until examination. Blood samples were collected at two stages in our study. The first stage was when diagnosis with rheumatoid arthritis was confirmed, and the second stage was at 12 weeks after treatment. Follow-up assessments included laboratory tests, parameters of disease activity, and the concentrations of MMP-3 were measured at both two periods.

2.6. The principle of the MMP-3 assay

MMP-3 was measured based on the sandwich of enzyme-linked immunosorbent assay method (ELISA) by the commercial kit (Aviva Systems Biology, Catalog No201-12-0908, USA) with an intra-assay coefficient of variation percent (CV%): 4.7% and inter-assay: 8.3%. An antibody used in the kit was both biotinylated and specific for MMP3 and has been pre-coated onto a 96-well plate. We added standards and the serum samples in the wells, then incubated the plate. A biotinylated antibody was added, incubated, and followed by washing. The kit used Avidin-Peroxidase Conjugate which was then appended, incubated, and the unbound conjugate was washed away. TMB substrate is added to form an enzymatic reaction. It is catalyzed by HRP creating a blue color product that changes into yellow after adding another solution called acidic stop. The density of yellow coloring taken through the absorbance at 450 nm was quantitatively relative to the amount of serum MMP3. The curve of standards was formed by staining the absorbance of each standard value against MMP-3 values. The detection range was 78–5000 pg/ml, the limit of detection (LOD) < 33 pg/ml. Results were expressed as ng/ml.

2.7. Statistical analysis

Statistical analysis was accomplished using the program called Statistical Package for the Social Sciences (SPSS) version 25. Microsoft Excel 2019 was also used. Data were calculated as mean ± SD for quantitative and median and percentiles for quantitative non-parametric measures. The Shapiro-Wilk test was done for the normality of the distribution of continuous variables. Wilcoxon's rank sum test was used to evaluate the serum MMP-3 before and after therapy. Mann- Whitney test for two independent groups was designed to assess the differences between groups. Chi-square test was completed for the evaluation of variables including categorical data. The ROC curve (receiver operating characteristic) was used to determine the standard limit of MMP-3 and assess its sensitivity and specificity in differentiation between RA patients. In our study, positive predictive value (PPV) = TP/(TP + FP) X100), and negative productive value (NPV) = TN/(TN + FN) X 100) were measured. P-value<0.05 was considered significant statistically.

3. Results

3.1. Characteristics of RA participants

Our research comprised of 43 RA patients (7 males and 36 females); their ages ranged between 30 and 65 with a mean of 48.71 ± 10.45 years, and the duration of symptoms ranged from 3 to 24 months with a mean of 8.05 ± 3.63 months. Also, 32 (74%) RA patients were RF positive and 35 (81%) were ACPA positive. 38 (88.4%) of RA patients had a good response. Joint erosions were seen in seven patients (16.3%) of RA patients. Table 1 shows the characteristics related to RA patients in terms of gender, age groups, Joint erosions, and response to therapy.

Table 1.

Demographic characteristics of RA patients (N = 43).

Variables	classification	N (%)
Sex	male	(15.6%)7
	Female	38 (84.4%)
Age groups	<40	(15.6%)7
	40–50	16 (35.6%)
	≥50	22 (48.9%)
Therapeutic response	Responder	38 (88.4%)
	Non-Responder	5 (11.6%)
Joints deformities	Yes	7 (16.2%)
	No	6 (83.7%)3
The used drug	MTX or Leflunomide	24 (55.8%)
	MTX with SSZ	11 (25.6%)
	MTX with Infliximab	8 (18.6%)

MTX: Methotrexate, SSZ: Sulfasalazine.

3.2. Distribution of RA patients according to therapy and disease activity grade

For the used treatment: RA patients were divided into several subgroups. The first group included 24 (55.8%) RA patients who received csDMARDs monotherapy: Methotrexate (MTX) (its dose was 12.5–25 mg/week) or leflunomide (10–20 mg/day), with an oral pulse of prednisolone (5 mg/day). The second group involved 11 (25.6%) RA patients treated with a combination of csDMARD (MTX) and sulfasalazine (SSZ) (2 g/day). The third group included 8 (18.6%) RA patients who required anti-TNF-α (infliximab, 3 mg/kg) with Methotrexate. Most of the RA patients had taken nonsteroidal anti-inflammatory drugs (NSAIDs) for at least 8 weeks before registration into our study (Table 1).

According to DAS2- ESR, 23 (53.48%) of RA patients had the highest disease activity, 18 (41.86%) had a modest activity of the disease, and 2 patients (4.6%) had little disease activity at diagnosis. After treatment, 15 (34.88%) of RA patients had remission, 7 (16.27%) of RA patients had low disease activity, 12 (34.88%) were in moderate disease activity and 9 (13.97%) had high disease activity (Fig. 1).

Fig. 1.

Categorical distribution of disease activity status at baseline and 12 weeks of therapy in 43 RA.

3.3. The study variables in RA patients before and after therapy

Our study presented a significant difference (p < 0.05) in the study variables (RF, ESR, CRP, DAS28, SJC, TJC, and patient global VAS score) before and after therapy in RA patients participating in our research (Table 2).

Table 2.

Clinical and laboratory characteristics of RA patients at baseline and after therapy.

Variables	Baseline		After therapy		P-value
	Mean ± SD	Median	Mean ± SD	Median
RF (IU/ml)	201.05 ± 215.4	113	105.6 ± 115.7	65	0.000b
CRP (mg/dl)	23.10 ± 29.5	10.1	5.95 ± 9.48	1.37	0.000b
ESR (mm/h)	50.49 ± 22.4	49	29.02 ± 20.4	20	0.000b
DAS28-ESR	5.29 ± 0.9	5.25	4.08 ± 1.04	4.01	0.000a
TJC	9.7 ± 4.9	10.0	5.56 ± 3.1	5.0	0.000b
SJC	5.53 ± 2.3	6.0	2.74 ± 1.68	3.0	0.000b
VAS (%)	0.67 ± 0.21	0.70	0.43 ± 0.21	0.40	0.000b

RF: Rheumatoid factor, CRP: C-reactive protein, ESR: erythrocyte sedimentation rate, TJC: tender joint count, SJC: swollen joint count, VAS: visual analogue scale of pain.

^aPaired Samples Test.

^bWilcoxon Signed Ranks Test.

3.4. Comparison of the serum MMP-3 levels in RA patients before and after therapy

Our result exposed that the (mean ± SD) of sMMP-3 levels was (486.49 ± 34.5 ng/ml) at diagnosis, (322.3 ± 43.83 ng/ml) after treatment (12 weeks), with a significant statistical difference (Wilcoxon Signed P = 0.0001) in serum MMP-3 levels at two points. Fig. 2 shows serum levels of MMP-3 in rheumatoid arthritis patients (N = 43) at baseline and after therapy).

Fig. 2.

Serum MMP-3 level in rheumatoid arthritis patients (N = 43) at baseline and after therapy in all RA patients (N = 43).

sMMP-3 levels decreased significantly in three RA patients’ groups after therapy according to the kind of drug. They fell from (485.84 ± 34.3 to 330.20 ± 84.7 ng/ml) in a group receiving MTX or LEF alone (P < 0.0001), (476.27 ± 33.1 ng/ml to 298.75 ± 82.9 ng/ml) in a group receiving MTX plus SSZ (P < 0.0001) and (502.51 ± 32.6 to 330.97 ± 92.8 ng/ml) in a group receiving MTX plus Infliximab (P = 0.017) as revealed in (Fig. 3).

Fig. 3.

Serum MMP-3 level in rheumatoid arthritis patients at baseline and after therapy based on the used drug.

3.5. sMMP-3 levels according to the therapeutic response

Our results showed that there was a statistically significant difference in serum MMP-3 (p = 0.0001) between the two study moments (before and after 12 weeks). No significant difference was found in serum MMP-3 levels in patients who did not respond to treatment between the two study points (0.137). The results also indicated that there is no real significant difference in MMP-3 concentrations mean before treatment between patients who responded to treatment and those who did not respond (P = 0.191). But there is a statistically significant difference in levels of serum MMP-3 between RA patients who responded to treatment and those who did not after treatment (P = 0.002) (Table 3).

Table 3.

Comparison of serum level of MMP-3 between the responsive and non-responsive groups at baseline and after therapy.

	(ng/ml) levels MMP-3		P-value
	At baseline	After therapy
	N = 38	N = 38	0.0001b
Responsive RA group	Mean ± SD	Mean ± SD
	484.35 ± 35.52	308.59 ± 74.33
	Median	Median
	493.47	311.13
Non-responsive RA group	N = 5	N = 5	0.137a
	Mean ± SD	Mean ± SD
	502.77 ± 11.19	426.5 ± 95.35
	Median	Median
	496.3	466.63
P−value	0.191c	0.002d

^aPaired Samples Test.

^bWilcoxon Signed Ranks Test.

^cIndependent Samples Test.

^dMann-Whitney Test.

3.6. Analysis of sensitivity and specificity of MMP-3, CRP, and, DAS28 after therapy

The ROC curve analysis showed that the sMMP-3 cut-off <317.8 ng/ml predicts therapeutic response in RA patient subclasses. The Area under the curve (AUC) was 0.818 (95% CI: 0.626–1, P = 0.022) to discriminate between RA patients who responded and who those did not (Table 4, Fig. 4 (a–c)). Our results pointed out that 14 RA patients were MMP-3 positive (4 true positive, 10 false positive) and non-responsive to therapy, and 29 negative cases (28 true negative, 1 false negative) were responsive to therapy. Also, it was found that the odds ratio was 13.51 which is statistically significant (P = 0.045) (Table 5).

Table 4.

Area under the curve (AUC) for MMP-3 level and DAS 28-ESR in RA patients in order to therapeutic response.

Test Result Variable(s)	AUC	Cut-off	Std. Error	Asymptotic Sig	Asymptotic 95% Confidence Interval
					Lower Bound	Upper Bound
CRP (mg/dl)	0.762	–	0.118	0.060	0.530	0.994
MMP-3 (ng/ml)	0.818	317.8	0.098	0.022	0.626	1.000
DAS28-ESR	1.00	5.325	0.000	0.000	1.000	1.000

Fig. 4.

(a–c): Receiver operating characteristic (ROC) curve of: a). CRP level, b). MMP-3 level, and c). DAS28-ESR for classification RA patients' groups according to therapeutic response.

Table 5.

The Intersection table for the distribution of RA patients according to positive or negative of serum MMP-3 after therapy.

Serum MMP-3	Non-responsive	responsive	All patients
Positive (non-responsive)	4	10	14
Negative (responsive)	1	28	29
All patients	5	38	43
Sensitivity: 80% Specificity: 73.6% PPV: 28.5% NPV: 96.5%
Odd Ratio (OR) Value & 95% CI & P-value: 11.2, (1.114 to 112.5), P = 0.045
DAS28-ESR	Non-responsive	responsive	All patients
Positive (non-responsive)	5	0	5
Negative (responsive)	0	38	38
All patients	5	38	43
Sensitivity: 100% Specificity: 100% PPV: 100% NPV: 100%
Odd Ratio (OR) Value & 95% CI & P-value: 847.0, (15.2 to 47203.8), P = 0.001

PPV: Positive predictive value, NPV: Negative predictive value.

ROC curve analysis for CRP level was used to discriminate between the two groups of RA patients according to the therapeutic response. Our result showed that ROC curve was unimportant and misleading (P = 0.060, AUC was 0.762) (Table 4, Fig. 4 (a–c)).

The ROC curve also exposed that the cut-off of DAS28 after therapy was (5.325). The AUC was: 1 (95% C.I., 1- 1; P = 0.001), (Table 4, figures [4 (a-c)]). The results showed that 5 people were diagnosed as non-responsive to treatment according to the cut-off of DAS28, all of them were true positive cases without false positives, and 38 were all negative cases, all true negative and without any false negative cases, with an accuracy rate of 100%. It was found that the Odd Ratio (OR) Value was 847.0 which is statistically significant (0.001) (Table 5).

4. Discussion

In rheumatoid arthritis, the main purpose of therapy is to preserve lower remaining disease activity or obtain clinical remission. Because the DAS28 score is one of the greatest procedures for assessing RA activity, rheumatologists measure DAS28 regularly and use it as one of the explanations for the effect of treatments [28,29]. MMP-3 is an imperative protease enzyme that is concerned responsible for joint destruction and plays an important role in the pathogenetic mechanism of rheumatoid arthritis [30]. Our study measured the serum levels of MMP-3 rather than measuring synovial MMP-3 because of the difficulty in obtaining the synovial samples that are not usually reliable in the clinical diagnosis of the disease. Also, MMP-3 is locally produced in RA in the inflamed joint and released into the bloodstream. It has been suggested that serum MMP-3 levels associate with levels formed by the synovium, and thus mirror the level of activity of rheumatoid synovitis [17,24,31]. Two procedures are used to measure serum levels of MMP-3, and these calibrate either the activity of MMP-3 using a substrate or the total MMP-3 protein. Only act-MMP-3 can cleave ECM proteins, while the level of the latent form only reflects the possibility of MMP-3 in degrading proteins. Thus, act-MMP3 may provide other information beyond total MMP-3, like the activation of pro-MMP3. Monitoring act-MMP-3 levels is restricted by the current commercial tests, which cannot distinguish the pro-form and act-form MMP-3. Also, the antibody only revealed act-MMP-3 and not pro-MMP-3, obviously assuring the specificity of the antibody [16].

The literature has indicated that the serum levels of MMP-3 correlated with RA activity, elevated in 62–80% of RA patients [32] reflecting the degree of synovitis [13,33]. Because a few studies designated the role of serum MMP-3 in RA therapy, our study has focused on evaluating its levels mean which can be used as a new interpreter and predictor of the therapeutic response in RA patients.

Our study found a reduction in the mean of serum MMP-3 levels (322.3 ± 43.83 ng/ml) after 12 weeks of therapy initiation compared to its levels mean at baseline (486.49 ± 34.5 ng/ml) (P < 0.05). The normal range of serum MMP-3 is (18–60 ng/ml for female) or (24–120 ng/ml for male) [34]. This increase in serum levels at diagnosis is due to the effect of activated inflammatory cytokines that induce synovial cells and neutrophils to increase the expression of MMP-3 [35]. It may be also due to the effect of fibroblasts and chondrocytes that lead to increased secretion of MMP-3 in the blood or the synovial fluid [36]. This confirms that increased levels of MMP-3 may mirror disruptive mechanisms in the joints, and indicate a bad prognosis in the early stage of the disease [31]. Our result is consistent with Kotani et al. study in Japan in 2012 that included (N = 12) RA patients [35] and Hattori et al. studies in Japan 2018 and 2019, they confirmed elevated levels of serum MMP-3 present in RA patient groups before therapy [17,27].

An important decrease in serum MMP-3 levels after treatment can be explained depending on the type of the applied drug. It is attributed to: either the toxicity of MTX as a mono-therapy and its role in reducing the number of fibroblasts, which in turn leads to increased production of MMP-3 [37], or as a result of the Leflunomide effect which directly disturbs the synovial tissue, leads to a decrease in the appearance of adhesion molecules and mineral proteases especially MMP-3, through its mechanism of inhibiting dihydroorotate dehydrogenase enzyme, and thus it reduces leaching Synovial tissue CD4 T cells being among the most sensitive to depletion of pyrimidines, and limits the inflammatory mechanism [38]. On the other hand, anti-TNF-a treatment such as Infliximab led to a decline in the serum MMP-3 levels, due to the central role of TNF-a in releasing protease enzymes, especially MMP-3 [16]. Our study is in agreement with the Shiozawa et al. study in Japan 2016 that included (N = 161) of RA patients who had been followed for 3 years with receiving methotrexate as monotherapy and found that (50.4%) of RA patients achieved clinical remission and showed that possibility of relying on serum MMP-3 for predicting the therapeutic response to MTX in RA patients [25]. In the Morota et al. study in Japan, 28 patients with RA received MTX as monotherapy after treatment for 12 weeks; it was found that the concentration of MMP-3 was significantly reduced in the group of RA patients, suggesting that the modified MMP-3 levels after treatment may play a substantial part in clinical response in RA patients [37]. Kotani et al. study in Japan showed also a decrease in 12 RA patients after therapy with Infliximab [35].

Our results revealed that there was no significant difference in MMP-3 concentration mean before and after treatment in non-responsive patients (P = 0.137). This may be due to several reasons: first, their duration of symptoms (>12 months) compared to (<12 months) in the RA responder group. Second, these patients received MTX monotherapy only. Third, two of them had returned to the hospital with joint erosions so the destructive progression was thought to be due to the potent effect of the MMPs family [13].

Patients with initially higher baseline in MMP-3 levels (n = 38) who showed a good response, presented a significant decrease during treatment (P < 0.05). In addition, there was a statistically significant difference in the levels of serum MMP-3 between the two groups of patients after treatment (P = 0.002). Our study converges the result with two studies: Rinaudo et al. study in 2019 and Tokai et al. in Japan 2018 and both of them showed that the decrease in MMP-3level after treatment was higher in RA patients who achieved remission, compared with RA patients who didn't [10,22]. This confirms the importance of assaying serum levels of MMP-3 as a specific biomarker for detecting therapy consequences in RA patients.

The ROC curve was used to differentiate between RA patients who responded to therapy and those who did not. Our results indicated that the cut-off of serum MMP-3 at 317.8 ng/ml had a sensitivity of 80% and specificity of 73% Compared to DAS28-ESR, the best cut-off value was 5.325 (sensitivity of 100%; specificity of 100%). Research results vary in determining the cut-off, sensitivity, and specificity of MMP-3 levels to distinguish between RA patient groups. Hattori et al. study noted that at the 113 ng/ml value of sMMP-3, the sensitivity and specificity respectively were (57.32%, 78.57%) in discriminating patients who achieved remission [17]. Another study done by Hattori et al. revealed that the limit value of MMP-3 was less than 76.7 ng/ml and (sensitivity: 47.27%, specificity: 83.05%, AUC: 63.73%) to reach remission [27]. In Tokai et al. study, the sensitivity was 81.7% and the specificity was 61.4% [10]. It can be said that the variance in these results is due to several different factors: the number of RA patients and their ages in the mentioned literature, the kits used for the MMP-3 assay, the applied therapy, and the period of patient follow-up.

5. Conclusion

The decrease in serum MMP-3 after treatment is an essential indicator of evaluating therapeutic response in rheumatoid arthritis It has an advantage over serum CRP, but it cannot replace DAS28-ESR.

5.1. Limitations and powerfulness of the study

The strength of our study is the use of the sensitivity ELISA method for MMP-3 concentrations which also, measured the early onset of RA before therapy in newly RA-diagnosed. There were some limitations of our study. First, the low sample size (n = 43) RA patients from two centers. Since no multi-center longitudinal study was applied, the present findings should be applied with caution to other populations. Second, limited follow-up checks, our results were based on data obtained at a single time point. Third, serum levels of MMP-3 were not evaluated according to sex although influenced by whether the patient is male or female. However, the overall results of the current study are generally consistent with many previous studies. It might be had been much more reliable if we had managed to avoid some limitations.

Author contribution statement

Rama Hussein: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Imad Aboukhamis: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.