286 © 2024 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Ibn Al-Haitham Journal for Pure and Applied Sciences Journal homepage: jih.uobaghdad.edu.iq PISSN: 1609-4042, EISSN: 2521-3407 IHJPAS. 2024, 37(4) Study of Collagenase-3 Levels in Rheumatoid Arthritis Patients with and without Type 2 Diabetes Mellitus Nadia Abdul-Kareem Abdul-Zahra1* and Zainab M. Al-Rubaei2 1,2 Department of Chemistry, College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received: 24 May 2023 Accepted: 9 July 2023 Published: 20 October 2024 doi.org/10.30526/37.4.3524 Abstract This research sought early signs of type 2 diabetes mellitus (T2DM) in rheumatoid arthritis (RA) patients. The current research's objectives are to identify collagenase-3 (CL-3) in RA patients with and without T2DM as a disease complication, compare these findings to those of a control group, and ascertain whether or not CL-3 correlates with all of the parameters that were examined in each group. In the present research, (150) participants all between the ages of 30 and 50 were divided into three groups: (G1) control (N=50), (G2) RA (N=50), and (G3) RA with T2DM (N=50). Collagenase-3, glycated hemoglobin (HbA1c), erythrocyte sedimentaion (ESR), anti-cyclic citrullinated peptide (anti-CCP), and rheumatoid factor (RF) were all measured in this experiment. Data revealed that (G2) and (G3) had considerably greater RF, anti-CCP, and ESR levels than (G1) in comparison. Compared to G2, there was a very obvious increase in G3. The results of the HbA1c test showed that (G2) increased non-significantly compared to (G1), but (G3) was much more than (G2) and (G1). The levels of CL-3 in (G2) and (G3) were found to be significantly higher than in controls (G1) in these groups. A well-related biomarker with these patients was shown by CL-3, demonstrating a strong positive or negative association with all metrics for all groups. This suggests that the best medicine and therapy will be available for these patients. These results suggest that CL-3 might be a biochemical diagnostic for RA patients' early diagnosis of diabetes. Keywords: Collagenase-3, rheumatoid arthritis, rheumatoid factor, type 2 diabetes mellitus. 1. Introduction Rheumatoid arthritis, often known as RA, has been connected to abnormalities in glucose metabolism, most notably insulin resistance, which may lead to type 2 diabetes (T2DM) [1]. This connection has been shown in a few different cases. The research found that patients with RA had a greater risk of acquiring diabetes, but the researchers were unable to determine whether or not those who already had T2DM had an increased risk of developing RA. One of the purposes of the research as a result is to investigate the possibility of an occurrence in patients who have T2DM [2]. The development of tolerance to self-antigens is facilitated by regulatory T cells, https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0009-0006-6985-9392 mailto:Nadia.abd.mltd2@hiue.edu.iq https://orcid.org/0000-0002-2742-3068 mailto:Zainab.m.m@ihcoedu.uobaghdad.edu.iq IHJPAS. 2024, 37(4) 287 while the pro-inflammatory response to infections is mediated by Th17 cells. The balance between Treg and Th17 cells is disrupted in RA as well as other inflammatory disorders. Because of a rise in the amount of glucose that is consumed and the number of glycolytic pathways, the metabolic state of RA undergoes a transition from a low-energy state to a high- energy state [3]. In human beings, the matrix metalloproteinase-13 (MMP-13) gene [4] is responsible for encoding the collagenase-3 (CL-3) enzyme. It is a member of the MMP family, which stands for matrix metalloproteinases [5]. The MMP-13 has a molecular weight of 54 kDa, according to the calculations [6]. While the catalytic domain of CL-3 is ineffectual on its own, the hemopexin domain of CL-3 is involved in the degradation of collagen. Is expressed in the skeleton during the whole of embryonic development because it serves as a prerequisite for the reorganization of the collagen matrix, which is essential to stimulate bone mineralization. Pathological diseases, such as human carcinomas, RA, and osteoarthritis (OA), are examples of those in which it is significantly overexpressed [7,8]. It is hypothesized that the collagenase subfamily of MMP plays a part in the articular cartilage degradation that is characteristic of RA. Since CL-3 has a preference for collagen type II, it is a feasible candidate in the process of articular cartilage turnover and a prospective therapeutic target [9,10]. 2. Materials and Methods Everyone who took part in this study was between the ages of 30 and 50, and they were randomly assigned to one of three groups: (G1) control (N=50), (G2) RA (N=50), or (G3) RA with T2DM (N=50). There are a total of 150 people taking part in this study. Rheumatologists who have recently diagnosed (before to patients commencing treatment) patients at the teaching hospitals located in Al-Yarmuk and Baghdad during October 2022 and January 2022. The person interviews with each patient were carried out using a recently developed questionnaire format that contained a detailed account of the medical history of each patient. The interviews were conducted in this manner all the way through. The tests that were varied out of the biochemical examinations were rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and CL-3 where a sample of serum was used. Also the test erythrocyte sedimentation rate (ESR) and glycated hemoglobin (HbA1c) that a sample of whole blood was used to get an accurate reading. To analyze the data, the statistical program known as Statistical Packages for Social Sciences- version 21 was used. The mean value as well as the standard deviation were utilized to present a condensed overview of the data. The student's t-test, which is used to determine the degree of separation between two independent means, was used to explore the significance of the difference in mean between a variety of "quantitative data" kinds. In cases where the P-value was less than 0.05 or was otherwise comparable to that value, it was decided that statistical significance existed. Highly significant (HS) expressed at p ≤ 0.01. 3. Results and Discussion Rheumatoid arthritis may be a chronic, multisystemic, autoimmune, and inflammatory condition that can produce substantial functional impairment and depressive feelings. The performance of routine duties and activities at work may be adversely affected as a result of these changes, which may eventually influence life quality [11]. IHJPAS. 2024, 37(4) 288 Serum levels of anti-CCP and CL-3 were tested in three groups: G1 represented the control group, G2 represented patients with RA, and G3 represented patients with RA who also had T2DM as a consequence. When compared to group (G1), the serum RF levels in groups (G2) and (G3) were significantly greater than those seen in group (G1), as shown by the data in Table 1. In addition, a statistically significant higher quantity was recorded in (G3) in comparison to what was seen in (G2). Table 1. Levels of RF, ESR, and HbA1c in the studied groups. Parameter mean± SD p-value G1 (n=50) G2 (n=50) G3 (n=50) G1& G2 G1& G3 G2& G3 RF (IU/mL) 0.95±0,60 3.57±0,76 3.69±0,73 HS HS NS ESR (mm/hr) 12.33±4,21 34.37±8,36 45.10±7,43 HS HS HS HbA1c (%) 4.87±0.46 5.14±0.59 7.82±0.97 NS HS HS **HS: High significantly at the 0.01 level. The RF autoantibodies are generated locally by B cells in lymphoid follicles and germinal center-like structures that arise in inflamed RA synovial. These autoantibodies directly bind to the "fraction crystallizable" (Fc) component of accumulated IgG. Studies have shown that RF testing in individuals with RA has a sensitivity of up to 90% and an accuracy ranging from 48 to 92%. According to Table 1, groups (G2) and (G3) revealed a significant increase in ESR levels compared to group (G1). Results agree with another study [12]. When examining a variety of clinical diseases, including RA, an elevated ESR might be of assistance [13]. In RA patients, the ESR is often use as a monitoring tool for inflammation even though it is not a diagnostic tool for the underlying cause of the disease. It is the result of the ESR strategy, which is uncomplicated, achievable, applicable, and inexpensive in addition to having great therapeutic significance. An elevated ESR may be a result of inflammatory responses or the breakdown of tissue in the body, described in previous study [14]. The levels of HbA1c were also established as a part of this inquiry that was carried out. The results of the HbA1c test showed that the rise in (G2) was not statistically significant when compared to the increase in (G1), but the increase in (G3) was statistically significant when compared to both (G2) and (G1). The results of the vast majority of high-quality research point to a relationship between RA and the onset of T2DM. The potential relationship between RA and incident T2DM may be cause by the inflammatory responses that are triggered when both diseases occur. Both T2DM and RA are strongly linked to persistent inflammation throughout the body. Moreover, RA has been linked to the production of pro- inflammatory cytokines such as TNF-α and IL-6, which lead to systemic inflammation and insulin resistance. Insulin resistance, in turn, contributes to T2DM by blocking insulin action [15]. In addition, the immune cells in the liver produce proinflammatory cytokines in response to the inflammatory mediators that are generated by the adipose tissue. The development of RA has been linked to a wide variety of cytokines that promote inflammation [16]. In individuals with RA, impaired glucose metabolism has been linked to both clinical symptoms that are difficult to control and a considerably increased risk of developing T2DM [17]. IHJPAS. 2024, 37(4) 289 The CL-3 and anti-CCP levels are presented for all of the groups that are the focus of this inquiry in Table 2. In Table 1, patients with RA (G2) and patients with T2DM (G3) exhibited anti-CCP levels that were significantly greater than those of the controls (G1). In addition, in comparison to G2, it was found that G3 had a substantially large increase. Table 2. Levels of anti-CCP and CL-3 in the studied groups. parameter mean± SD p-value G1 (n=50) G2 (n=50) G3 (n=50) G1& G2 G1& G3 G2& G3 Anti-CCP (U/mL) 0.31±0.09 0.61±0.52 0.74±0.15 HS HS HS CL-3 (ρg/mL) 963.99±149.49 3037.75±1452.64 4013.49±2181.23 HS HS HS **HS: High significantly at the 0.01 level. The peculiar amino acid citrulline is one of the antigenic determinants that may be bound by autoantibodies that are directed against anti-CCP. Although it is possible to praduce amino acids by post-translational modification, the amino acid is not incorporated into the protein during the synthesis process itself. This is because post-translational modification is a process that occurs after translation has taken place. This is because the amino acid is not produced until after the synthesis of the protein has taken place. Citrullination is a process that involves the hydrolysis of the positively charged NH2– group that is present in the amino acid arginine into a neutral oxygen group. This process is catalyzed by enzymes. This method is also known as deamination in certain circles. People who suffer from RA are more likely to develop autoantibodies because these antibodies can detect a particular oxygen group included inside peptidyl citrulline. Citrulline residues, which are a key component of antigenic determinants such as these, are one of the antigenic determinants that may be identified by RA antibodies. Anti-CCP testing is particularly helpful in the diagnosis of RA [18] due to its high specificity early on in the progression of the disease as well as its ability to identify those who are likely to suffer from significant sickness and damage that cannot be repaired. Both of these qualities are important in determining who will ultimately be affected by RA. Both of these symptoms may be seen in a patient who is at an early stage of the disease's development [19]. The data that are presented in Table 2 for CL-3 illustrate that G3 has risen in a manner that is statistically significant when compared to the prior value. This is shown by the comparison of G3 to G2 and G1, which are both displayed in the Table. In addition to this, it was discovered that G2 had significantly improved in contrast to G1's performance. People who have T2DM have an increased risk of experiencing a rapid deterioration of the knee cartilage [20], which is now the issue that attracts the most attention. In particular, MMPs are anticipated to play a significant role in the process of cartilage disintegration during the induction of degenerative cartilage alterations that occur as a result of OA [21]. This is because MMPs are known to have a significant role in the progression of OA. This is because osteoarthritis is known to produce changes to the cartilage, which may result in discomfort and stiffness in the joints. The MMPs are the primary proteolytic enzymes of these proteins; they can destroy type II fibrillar collagen, in addition to collagen types IX, XI, and VI, as well as extra secondary collagens [22,23]. They also have the IHJPAS. 2024, 37(4) 290 potential to cleave additional secondary collagens. It is plausible to infer that this is the case given that the majority of cartilage's extracellular matrix is made up of proteoglycans, which are made up of collagen. In addition to this, MMP-13 was shown to be present in the synovial tissue of persons who had either OA or RA [24]. It has been shown that CL-3 is capable of degrading types I, II, and III of collagen, in addition to cartilage proteoglycan aggrecans. The characterization of CL-3 by the use of biochemical methods showed that it participates in a wide range of actions that are aimed at the components of connective tissue. Because CL-3 prefers to degrade collagen type II of hyaline cartilage rather than CL-3, which degrades this substrate more efficiently, one is tempted to speculate that CL-3 is an essential component of the cellular machinery that is responsible for the turnover of articular cartilage. This speculation is based on the fact that CL-3 prefers to degrade collagen type II of hyaline cartilage. This hypothesis is supported by the observation that CL-3 shows a strong preference for degrading type II collagen in hyaline cartilage [25]. As a direct consequence of this finding, this molecule would subsequently be brought to light as a potential therapeutic target for the treatment of cartilage degradation. A new glimmer of hope for the treatment of OA emerged not too long ago in the shape of an inhibition of CL-3, which was reported by Li and colleagues [26]. In the antibody- induced arthritis paradigm, the pharmacological lowering of CL-3 did not result in decreased arthritis; however, it did result in decreased arthritis in the collagen-induced arthritis model and the severe mixed immunodeficiency mouse implantation model [27]. Recent study has focused on using a model of arthritis called the K/BxN sera-transfer model to explore the part that CL-3 plays in the illness. This model is known as the K/BxN sera-transfer model. In the K/BxN paradigm, arthritis develops on its own in mice that express both the transgene-encoded KRN T-cell receptor and the IAg7 major histocompatibility complex class II allele [28,29]. This occurs because the KRN T-cell receptor acts as a trigger for the development of arthritis in these animals. This is because it is believed that the KRN T-cell receptor is involved in the development of arthritis. This is because the KRN T-cell receptor is important for the development of arthritis. Animals that are deficient in CL-3 have a reduced risk of inflammatory conditions and joint damage [30, 31]. This occurs as a result of an increase in the expression of CL-3 in C57BL/6 mice throughout the K/BxN serum-induced arthritis. There is an increase in the expression of CL-3 as the K/BxN serum-induced arthritis progresses in C57BL/6 mice. This increase in expression may be seen throughout the arthritis. Collagenase3 is the most effective collagenase when it comes to type II collagen, which is the primary structural collagen that is present in articular cartilage. This is the case since type II collagen is the primary constituent of articular cartilage. A diverse network of signal transduction pathways is activated when powerful pro-inflammatory stimuli such as IL-1 and OSM are present [32]. Research has demonstrated that a broad array of pro-inflammatory stimuli all use the same pathways to cause inflammation. These routes, when activated in conjunction with one another, have a synergistic effect that, when applied to chondrocytes, significantly boosts the synthesis of collagenase-3 [33]. Although we have just recently reported a role for the AP-1-binding factor activating transcription factor 3 (ATF3) in selectively mediating CL-3 expression that was nonetheless dependent on AP-1 [34]. It is essential to keep in mind that the cFos/cJun activator protein-1 (AP-1) transcription factor complex is required for the expression of CL-3. In addition, we have shown in the past that the signaling pathways for signal transducer and activator of transcription (STAT)3, phosphatidylinositol-3' kinase (PI3K/Akt), and protein kinase C (PKC) are of the utmost significance [35]. However, the identification of the downstream signaling components that it influences is unknown, as are the likely sites of cross-talk. Although it has IHJPAS. 2024, 37(4) 291 been shown that the atypical PKC isoform PKC may alter the induction of CL-3 in response to IL-1+OSM stimulation, it is unclear which components of the signaling pathway it impacts. This is even though it has been shown that the identification of the downstream signaling components that it affects is unknown, as proven by the fact that this is the case [36]. In Table 3, the findings of an analysis that was carried out on each of the groups that were examined to estimate the coefficient of association (r) between CL-3 and the other parameters are shown. This analysis was carried out to identify the relationship between CL-3 and the other parameters. Table 3. Relationship coefficient between CL-3 and other parameters for the studied groups. Parameters Relationship coefficient-r G1 G2 G3 RF (IU/mL) 0.04 -0.11 0.12 ESR (mm/hr) 0.33 -0.11 0.19 HbA1c (%) 0.02 0.22 0.33 Anti-CCP (U/mL) -0.07 0.35 0.17 The present study showed a negative relationship between CL-3 levels and RF in G2 (r2= -0.11). A positive relationship was found in G1 and G3 (r1=0.04, r3= 0.12) as shown in Figure 1. Collagenase-3 levels and ESR in G2 had a poor connection, according to the research (r2=0.11). While G1 and G3 showed a positive association (r1=0.33, r3=0.19), which is seen in Figure 2. Figure 1. Relationship between collagenase-3 and RF. Study revealed a positive relationship between CL-3 levels and HbA1c in all studied groups as shown in Figure 3. The results also showed an inversely correlated relationship between collagenase-3 levels and anti-CCP in G2 and G3 (r2= 0.35, r3= 0.17). In contrast, G1 showed a negative relationship (r1= -0.07), as seen in Figure 4. The suppression of cartilage development and increased chondrocyte death may be the underlying mechanism for CL-3's impacts on diabetic RA articular cartilage. However, further research is needed to fully understand the processes. IHJPAS. 2024, 37(4) 292 Figure 2. Relationship between collagenase-3 and ESR. Figure 3. Relationship between collagenase-3& HbA1C. Figure 4. Relationship between collagenase-3 and Anti-CCP. 4. Conclusion This study provides solid evidence that CL-3 contributes to an increase in inflammation and joint degradation in RA models by demonstrating a significant shift (either an increase or a decrease) in CL-3 and anti-CCP levels. According to evidence that demonstrates it is essential IHJPAS. 2024, 37(4) 293 for a protracted inflammatory response that takes place in the effector phase of arthritis, CL-3 may have a job in managing the inflammatory response in addition to damaging cartilage. This information suggests that CL-3 may have a role in controlling the inflammatory response. These results showed that these features have the potential to be use as a biochemical diagnostic for the early diagnosis of diabetes in RA patients. In addition to this, a strong positive or negative association between CL-3 and all parameters for all groups revealed a good connected biomarker with these patients, suggesting that it would be possible to employ the most effective medicine and treatment. Acknowledgment The authors thank the Department of Chemistry, College of Education for Pure Science (Ibn Al–Haitham), University of Baghdad for research approval. Conflict of Interest The authors declare that they have no conflicts of interest. Funding No founding. Ethical Clearance This work has been approved by the Scientific Committee at the University of Baghdad/ College of Education for Pure Science (Ibn Al–Haitham), which is consistent with the instructions of the Iraqi Ministry of Health and Environment (No. 15765 on 4/10/2022). References 1. Smolen, J.S.; Aletaha D.; Barton A.; Burmester G.R.; Emery, P.; Firestein G.S. Rheumatoid Arthritis. Nature Review Disease Primers 2018, 4(1), 18001. https://doi.org/10.1038/nrdp.2018.1. 2. Omran, R.H.; Zahra'a, A.A.; Alrawi, A.A. Evaluation of Some New Cytokines in Rheumatoid Arthritis. Journal of the Faculty of Medicine Baghdad 2022, 64(3), 159-162. https://doi.org/10.32007/jfacmedbagdad.6431963. 3. Lestarini, A.; Aryastuti, A.A.; Witari, N.P.; Sutarka, I.W.; Wardani, N.W.; Hastuti, P.; Sadewa, A.H. MCP-1 Serum Levels Were Higher in Patient with Diabetic Nephropathy among Balinese. Indian Journal of Public Health 2020, 11(1), 1351. https://www.researchgate.net/profile/Asri-Lestarini/publication/342688022. 4. Mengshol, J.A.; Mix, K.S.; Brinckerhoff C.E. Matrix Metalloproteinases as Therapeutic Targets in Arthritic Diseases: Bull’s-Eye or Missing the Mark. Arthritis and Rheumatism 2002, 46(1), 13–20. https://doi.org/10.1002/art.497. 5. Cui, N.; Hu, M.; Khalil, R.A. Chapter One-Biochemical and Biological Attributes of Matrix Metalloproteinases. Progress in Molecular Biology and Translational Science 2017, 147(1), 1–73. https://doi.org/10.1016/bs.pmbts.2017.02.005. 6. Schieir, O.; Tosevski, C.; Glazier, R.H.; Hogg-Johnson, S.; Badley, E.M. Incident Myocardial Infarction Associated aith Major Types of Arthritis in the General Population: A Systematic Review and Meta-Analysis. Annals of the Rheumatic Diseases 2017, 76(8), 1404–1396. https://doi.org/10.1136/annrheumdis-2016-210275. 7. Al-Ameri, M.A.; Al-Rubaei, Z. Study of Tumor Necrosis Factor-β (Lymphotoxin) and Cell Death Receptor (TNFR-2) in Rhumatiod Arthritis with Type 2 Diabetes Mellitus. Pakistan Journal of Medical and Health Sciences 2022, 16(6), 468–468. https://doi.org/10.53350/pjmhs22166468. https://doi.org/10.1038/nrdp.2018.1. https://doi.org/10.32007/jfacmedbagdad.6431963 https://www.researchgate.net/profile/Asri-Lestarini/publication/342688022 https://doi.org/10.1002/art.497 https://doi.org/10.1016/bs.pmbts.2017.02.005 https://doi.org/10.1136/annrheumdis-2016-210275 https://doi.org/10.53350/pjmhs22166468 IHJPAS. 2024, 37(4) 294 8. Stahle-Backdahl, M.; Sandstedt, B.; Bruce, K.; Lindahl, A.; Jimenez, M.G.; Vega, J.A.; López-Otín C. Collagenase-3 (MMP-13) Is Expressed During Human Fetalossification and Re-Expressed in Postnatal Bone Remodeling and in Rheumatoid Arthritis. Laboratory Investigation 1997, 76(5), 717– 728. https://europepmc.org/article/med/9166290. 9. Boneva, B.; Ralchev, N.; Ganova, P.; Tchorbanov, A.; Mihaylova, N. CollagenaseInduced Mouse Model of Osteoarthritis—A Thorough Flow Cytometry Analysis. Life 2022, 12(11), 1938. https://doi.org/10.3390/life12111938. 10. Xin, X.; Tan, Q.; Li, F.; Chen, Z.; Zhang, K.; Li, F.; Yang, B.; Xing, Z.; Zhou, F.; Tian, Y.; Lv, Y. Potential Value of Matrix Metalloproteinase-13 as a Biomarker for Osteoarthritis. Frontiers in Surgery 2021, 8(1), 528. https://doi.org/10.3389/fsurg.2021.750047. 11. Hussein, W.A. The Quality of Life in Patients with Rheumatoid Arthritis in Baghdad, 2017: A Cross- Sectional Study. International Journal of Medical Research and Health Sciences 2017, 6(11), 20-34. https://www.indianjournals.com/ijor.aspx?target=ijor:ijmrhs&volume. 12. Kang, E.H.; Ha, Y.J.; Lee, Y.J. Autoantibody Biomarkers in Rheumatic Diseases. International Journal of Molecular Sciences 2020, 21(4), 1382. https://doi.org/10.3390/ijms21041382. 13. Nazar, L.; Eiman, A.A. Study of IL-33 and IL-1R4 in Iraqi Rheumatoid Arthritis Female Patient's with and without Dyslipidemia Prone to Atherosclerosis. Ibn AL-Haitham Journal For Pure and Applied Sciences 2019, 32(1), 48–56. https://doi.org/10.30526/32.1.1920. 14. Shapiro, S.C. Biomarkers in Rheumatoid Arthritis. Cureus 2021, 13(5), e15063. https://doi.org/10.7759/cureus.15063. 15. Lee, Y.H.; Song, G.G. Causal Association between Rheumatoid Arthritis with the Increased Risk of Type 2 Diabetes: A Mendelian Randomization Analysis. Journal of Rheumatic Diseases 2019, 26(2), 131-136. https://doi.org/10.4078/jrd.2019.26.2.131. 16. Tian, Z.; Mclaughlin, J.; Verma, A.; Chinoy, H.; Heald, A. H. The Relationship between Rheumatoid Arthritis and Diabetes Mellitus: A Systematic Review and Metaanalysis. Cardiovascular Endocrinology and Metabolism 2021, 10(2), 125. https://doi.org/10.1097/XCE.0000000000000244. 17. Ahmad, R.; Zgair, A. Immunological and Biological Manifestation of Rheumatoid Arthritis Patient in Iraq. Indian Journal of Forensic Medicine and Toxicology 2021, 15(4), 1344-1350. https://pdfs.semanticscholar.org/66ac/c786bd5ad9db2bb642a. 18. Iwasaki, T.; Nakabo, S.; Terao, C.; Murakami, K.; Nakashima, R.; Hashimoto, M.; Imura, Y.; Yukawa, N.; Yoshifuji, H.; Miura, Y.; Yurugi, K.; Maekawa, T.; van Delft, M. A. M.; Trouw, L.A.; Fujii, T.; Mimori, T.; Ohmura, K. Long-Term Follow-Up of Patients with Anti-Cyclic Citrullinated Peptide Antibody-Positive Connective Tissue Disease: A Retrospective Observational Study Including Information on the HLA-DRB1 Allele and Citrullination Dependency. Arthritis Research and Therapy 2020, 22(1), 1-9. https://doi.org/10.1186/s13075-020-02351-4. 19. Khidhir, R.M.; Al-Jubouri, R.H. The Study of Tempromandibularjoint Disorders and Anti-Cyclic Citrullinated Peptide Antibodies in Serum and Saliva of Patients with Rheumatoid Arthritis. Journal of Baghdad College of Dentistry 2013, 25(Special Is), 67-71. https://www.jbcd.uobaghdad.edu.iq/index.php/jbcd/article/view/201. 20. Haydar, A.A.; Abdullah, I.R. The Impact of Therapies as Monotherapy with Combined Therapy on Novel and Traditional Biomarkers in Patients with Rheumatoid Arthritis. Zanco Journal of Medical Sciences 2020, 24(3), 325-332. https://doi.org/10.15218/zjms.2020.038. 21. Oliveira, T.H.C.; Marques, P.E.; Proost, P.; Teixeira, M.M.M. Neutrophils: A Cornerstone of Liver Ischemia and Reperfusion Injury. Laboratory Investigation 2018, 98(1), 51–62. https://doi.org/10.1038/labinvest.2017.90. 22. Jabłońska-Trypuć, A.; Matejczyk, M.; Rosochacki, S. Matrix Metalloproteinases (Mmps), The Main Extracellular Matrix (ECM) Enzymes in Collagen Degradation, as a Target for Anticancer Drugs. Journal of Enzyme Inhibition and Medicinal Chemistry 2016, 31(1), 177–183. https://doi.org/10.3109/14756366.2016.1161620. https://europepmc.org/article/med/9166290 https://doi.org/10.3390/life12111938 https://doi.org/10.3389/fsurg.2021.750047 https://www.indianjournals.com/ijor.aspx?target=ijor:ijmrhs&volume https://doi.org/10.3390/ijms21041382 https://doi.org/10.30526/32.1.1920 https://doi.org/10.7759/cureus.15063. https://doi.org/10.4078/jrd.2019.26.2.131 https://doi.org/10.1097/XCE.0000000000000244 https://pdfs.semanticscholar.org/66ac/c786bd5ad9db2bb642a https://doi.org/10.1186/s13075-020-02351-4 https://www.jbcd.uobaghdad.edu.iq/index.php/jbcd/article/view/201 https://doi.org/10.15218/zjms.2020.038 https://doi.org/10.1038/labinvest.2017.90 https://doi.org/10.3109/14756366.2016.1161620 IHJPAS. 2024, 37(4) 295 23. Luo, S.; Li, W.; Wu, W.; Shi, Q. Elevated Expression of MMP8 and MMP9 Contributes to Diabetic Osteoarthritis Progression in a Rat Model. Journal of Orthopaedic Surgery and Research 2021, 16(1),1-9. https://doi.org/10.1186/s13018-021-02208-9. 24. Wilkinson, D.J.; Falconer, A.M.; Wright, H.L.; Lin, H.; Yamamoto, K.; Cheung, K.; Charlton, S.H.; Arques, M.D.C.; Janciauskiene, S.; Refaie, R.; Rankin, K.S. Matrix Metalloproteinase‐13 is Fully Activated by Neutrophil Elastase and Inactivates Its Serpin Inhibitor, Alpha‐1 Antitrypsin: Implications for Osteoarthritis. The FEBS journal 2022, 289(1), 121-139. https://doi.org/10.1111/febs.16127. 25. Fosang, A.J.; Last, K.; Knauper, V.; Murphy, G.; Neame, P.J. Degradation of Cartilage Aggrecan by Collagenase-3 (MMP-13). FEBS Letters 1996, 380(1-2), 17-20. https://doi.org/10.1016/0014- 5793(95)01539-6. 26. Li, N.G.; Shi, Z.H.; Tang, Y.P.; Wang, Z.J.; Song, S.L.; Qian, L.H.; Qian, D.W.; Duan, J.A. New Hope for the Treatment of Osteoarthritis Through Selectiveinhibition of MMP-13. Current Medicinal Chemistry 2011, 18(7), 977–1001. https://doi.org/10.2174/092986711794940905. 27. Nasr, M.H.; Hassan, B.A.R.; Othman, N.; Karuppannan, M.; Abdulaziz, N.B.; Mohammed, A.H.; Alsarani, M.A.; Eskembaji, M.H.; Aman, A.M.; Othman, G. Prevalence of Vitamin D Deficiency between Type 2 Diabetes Mellitus Patients and Non-Diabetics in the Arab Gulf. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2022, 15(1), 647-657. https://doi.org/10.2147/DMSO.S350626. 28. Kouskoff, V.; Korganow, AS.; Duchatelle, V.; Degott, C.; Benoist, C.; Mathis, D. Organ-Specific Disease Provoked by Systemic Autoimmunity. Cell 1996, 87(5), 811–822. https://doi.org/10.1016/S0092-8674(00)81989-3. 29. Castrogiovanni, P.; Di Rosa, M.; Ravalli, S.; Castorina, A.; Guglielmino, C.; Imbesi, R.; Vecchio, M.; Drago, F.; Szychlinska, M.A.; Musumeci, G. Moderate Physical Activity As a Prevention Method for Knee Osteoarthritis and the Role of Synoviocytes as Biological Key. International Journal of Molecular Sciences 2019, 20(3), 511. https://doi.org/10.3390/ijms20030511. 30. Hu, Q.; Ecker, M. Overview of MMP-13 as a promising target for the treatment of osteoarthritis. International Journal of Molecular Sciences 2021, 22(4), 1742. https://doi.org/10.3390/ijms22041742. 31. Meehan, R.T.; Regan, E.A.; Hoffman, E.D.; Wolf, M.L.; Gill, M.T.; Crooks, J.L.; Parmar, P.J.; Scheuring, R.A.; Hill, J.C.; Pacheco, K.A.; Knight, V. Synovial Fluid Cytokines, Chemokines and MMP Levels in Osteoarthritis Patients with Knee Pain Display a Profile Similar to Many Rheumatoid Arthritis Patients. Journal of Clinical Medicine 2021, 10(21), 5027. https://doi.org/10.3390/jcm10215027. 32. Chan, C.M.; Macdonald, C.D.; Litherland, G.J.; Wilkinson, D.J.; Skelton, A.; EuropeFinner, G.N.; Rowan, A.D. Cytokine-Induced MMP13 Expression In Human Chondrocytes is Dependent on Activating Transcription Factor 3 (ATF3) Regulation. Journal of Biological Chemistry 2017, 292(5), 1625-1636. https://doi.org/10.1074/jbc.M116.756601. 33. Rowan, A. D.; Young, D. A.; Collagenase Gene Regulation by Pro-Inflammatory Cytokines in Cartilage. Frontiers in Bioscience-Landmark 2007, 12(1), 536-550. https://doi.org/10.2741/2080. 34. Catterall, J.B.; Carrere, S.; Koshy, P.J.T.; Degnan, B.A.; Shingleton, W.D.; Brinckerhoff, C.E.; Rowan, A.D. Synergistic Induction of Matrix Metalloproteinase 1 by Interleukin‐1α and Oncostatin M in Human Chondrocytes Involves Signal Transducer and Activator of Transcription and Activator Protein 1 Transcription Factors Via a Novel Mechanism. Arthritis and Rheumatism 2001, 44(10), 2296-2310. https://doi.org/10.1002/1529-0131(200110)44:10%3C2296::AID-ART392%3E3.0.CO;2-9. 35. Litherland, G.J.; Elias, M.S.; Hui, W.; Macdonald, C.D.; Catterall, J.B.; Barter, M.J.; Rowan, A.D. Protein Kinase C Isoforms Ζ and Ι Mediate Collagenase Expression and Cartilage Destruction Via STAT3-and ERK-Dependent C-Fos Induction. Journal of Biological Chemistry 2010, 285(29), 22414-22425. https://doi.org/10.1074/jbc.M110.120121%20. https://doi.org/10.1186/s13018-021-02208-9 https://doi.org/10.1111/febs.16127 https://doi.org/10.1016/0014-5793(95)01539-6 https://doi.org/10.1016/0014-5793(95)01539-6 https://doi.org/10.2174/092986711794940905 https://doi.org/10.2147/DMSO.S350626 https://doi.org/10.1016/S0092-8674(00)81989-3 https://doi.org/10.3390/ijms20030511 https://doi.org/10.3390/ijms22041742 https://doi.org/10.3390/jcm10215027 https://doi.org/10.1074/jbc.M116.756601 https://doi.org/10.2741/2080. https://doi.org/10.1002/1529-0131(200110)44:10%3C2296::AID-ART392%3E3.0.CO;2-9 https://doi.org/10.1074/jbc.M110.120121 IHJPAS. 2024, 37(4) 296 36. Litherland, G.J.; Dixon, C.; Lakey, R.L.; Robson, T.; Jones, D.; Young, D.A.; Rowan, A.D. Synergistic Collagenase Expression and Cartilage Collagenolysis are Phosphatidylinositol 3-Kinase/ Aktsignaling-Dependent. Journal of Biological Chemistry 2008, 283(29), 14221-14229. https://doi.org/10.1074/jbc.M710136200. https://doi.org/10.1074/jbc.M710136200