MATRIX METALLOPROTEINASE-3 DOWN REGULATION AND CELL MIGRATION INHIBITION IN HUMAN PTERYGIUM FIBROBLASTS BY
MITOMYCIN-C, CURCUMIN & FIBRIN GLUE
Ferdian Ramadhan1, Mirza Febri1, Muhammad Abdurrauf1, Jamaluddin2, Ismi Zuhria3, Luki Indriaswati3, Evelyn Komaratih3
1 Ophthalmology resident, Faculty of Medicine, Universitas Airlangga, Surabaya – Indonesia 2 Medical Staff in Rumah Sakit Mata Masyarakat, Surabaya- Indonesia
3 Ophthalmologist, Faculty of Medicine, Universitas Airlangga, Surabaya – Indonesia Corresponding author :
Evelyn Komaratih ([email protected]) ABSTRACT
Objective: Determination of matrix metalloproteinase-3 (MMP-3) expression and fibroblast migration in human pterygium fibroblasts (HPF) following administration of curcumin, mitomycin-C (MMC) and fibrin glue (FG) inhibitory effect as an alternative adjuvant therapy for pterygium.
Methods : Human pterygium fibroblasts (HPF) were used in this study. HPFs were obtained from pterygium excision and primary cultured, and then divided into 4 groups: untreated, curcumin, MMC and FG. MMP-3 expression was analyzed using immunocytochemistry at 48 h, followed by intensity measurement using ImageJ software. Mobility was measured by stratifying fibroblast cultures at 0, 24, and 48 h of confluency and then assessed using ImageJ software. Results between groups were analyzed using ANOVA tests and post hoc tests.
Results: Curcumin at a dose of 100 mol/mL decreased the expression of MMP-3 (2205.84 ± 86.1), 200 mol/mL (1002.51 ± 25.22) and FG (1131.55 ± 17.71) compared to control (4703.49 ± 108.9) (p < 0.001 ) while the mobility of curcumin at 200 mol/mL dose was 178.67 ± 2.85 (24H), 88.83 ± 1.48 (48H) and 180.4 ± 2.56 (24 H), 72.45 ± 1.25 (48 H) in the FG group, respectively compared with control 141.59 ± 3.12 (24 H); 32.46 ± 1.25 (48 H) (p<0.05), curcumin and fibrin glue have the effect of reducing MMP-3 expression and inhibiting HPF migration.
Conclusion: Curcumin and fibrin glue can reduce the expression of MMP-3 and inhibit the migration of HPF cells.
Keywords: Curcumin, Mitomycin C, Fibrin Glue, Human Pterygium Fibroblast, MMP-3
INTRODUCTION
Pterygium is a degenerative and inflammatory disease of the conjunctiva with the clinical appearance of triangular fibrovascular tissue on the nasal side of the conjunctiva leading to the limbus. Risk factors associated with the development of pterygium include ultraviolet (UV) exposure, viral infections such as human papillomavirus, herpes simplex virus, and cytomegalovirus. Risk factors for pterygium in regions with dry climates, such as the tropics near the equator, with increased UV exposure, making Indonesia one of the countries with high incidence and recurrence rates. 1,2,3
The prevalence of pterygium worldwide was staggering (ranging from 7% to 15%) until the term pterygium band was known, indicating the region with the highest prevalence of pterygium. According to the 2010 Indonesian Basic Health Study, the prevalence of pterygium in Indonesia reached 3.2% in two eyes and 1.9% in one eye among all eye disease cases detected. 1,3
The relatively high recurrence rate after pterygium resection is another issue that clinicians must face. The incidence of postoperative recurrence varied from 3.8% to 89%.
The pathophysiology of recurrence is not fully understood, but the most likely causes are genetic factors, repeated exposure to risk factors, and surgical technique. The bare sclera technique was the first to be adopted and is still quite high in practice in Indonesia as a modality for the treatment of pterygium, but it is positively associated with a high recurrence rate (up to 24-89%), so treatment modification uses Conjunctival autografts or add adjuvant therapy such as the antimetabolites 5-fluorouracil (5-FU) or mitomycin-C (MMC) as antimitotic and antiapoptotic keratinocytes and myofibroblasts.The use of antimetabolites was successful in reducing the recurrence rate of pterygium but some serious complications became a new obstacle such as corneal edema, corneal perforation, and scleral calcification.1,4,5
Recent studies have shown that curcumin, which is a polyphenol derivative from turmeric, has pharmacological properties as antioxidants, antiapoptotic and antiproliferative properties. The inhibition of curcumin on Matrix Metalloproteinase (MMP) expressed from Human Pterygium Fibroblasts (HPF), especially MMP-3, the blocking effect of curcumin on Reactive Oxidative Species of fibroblasts as a result of UV exposure, has become a promising adjuvant therapy for the prevention of pterygium recurrence in future. 6,7,8
Fibrin Glue (FG) is a blood-derived product that is used as a biological adhesive and was first introduced in 1909. Fibrin glue acts by imitating the final step of the coagulation cascade when soluble fibrinogen is activated by thrombin. (Daponte et al., 2019). In the field of ophthalmology, it is an alternative therapy option that is often used in pterygium cases to prevent postoperative recurrence, accelerate wound healing by releasing anti-inflammatory cytokines. FG forms a seal on the wound after pterygium excision to accelerate the wound healing process, minimize recurrence, and prevent more postoperative complications. 9,10
In this study, the cell culture of HPF fibroblasts treated with curcumin intervention was performed in vitro to evaluate MMP-3 expression and fibroblast migration, divided into four groups, namely the HPF group cultured on growth medium as a negative control, The HPF group was cultured on growth medium with MMC intervention, the HPF group was cultured on growth medium with curcumin intervention as a positive control, and the HPF group was cultured on growth medium with fibrin glue (FG) intervention.
MATERIALS AND METHODS Reagents
Curcumin from Sigma-Aldrich® (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), trypsin, fetal bovine serum (FBS), PBS, penicillin, and streptomycin were purchased from Gibco-BRL (Carlsbad, CA, USA). Annexin V-FITC was purchased from BD
Biosciences (San Jose, CA, USA), MMP-3 polyclonal primary antibody was purchased from BIOSS
Preparation of curcumin
Curcumin with a molecular weight of 368.38 daltons was dissolved at 0.05% DMSO to make broth solutions at concentrations of 5, 50, 100, and 200 mmol/L stored at 2-8 oC.
Complete media DMEM was added to dilute the curcumin to the desired concentration then sonicated and filtered.
Fibrin Glue
Fibrin glue is derived from graded centrifugation of peripheral blood with anticoagulants. A total of 40 mL of peripheral blood become taken from the cubital vein the use of a sterile syringe containing CPDA in a ratio of 9:1 aseptically. Blood was positioned right into a sterile 10 mL centrifuge tube. The blood was then centrifuged at 3000 rpm for 15 minutes to pay attention the plasma. The plasma element changed into stored in a sterile centrifuge tube and stored at -20oC for 24 hours, then centrifuged at 4oC, 3000 rpm for 15 mins. After centrifugation, the top 2/3 of plasma was stored as a good deal as 10 mL for the instruction of the fibrinogen element and 1/3 of the plasma which was PRP become stored in sterile micro-tubes to be prepared as fabric for thrombin manufacturing. The upper 2/3 of the plasma turned into then added with 95% ethanol 1 ml and then incubated at 4oC for 30 minutes. then centrifuged at 4oC at 3000 rpm for 15 minutes. The supernatant turned into discarded and the sediment became used as an element of fibrinogen. Thrombin changed into made by blending the PRP with 10% CaCl2 0.05 ml. Fibrin glue is made via mixing fibrinogen and thrombin in a ratio of 1:1.11,12
Culture and passage of HPF
Pterygium tissue was taken in patients who had signed the informed consent.
Pterygium sterile in the Surgical Installation of the Rumah Sakit Mata Masyarakat, Surabaya,
Indonesia. Local anesthesia with subconjunctival injection of 2% lidocaine was performed before removal of the pterygium tissue. A sterile cloth was placed on the operating area and the eyeball was disinfected using 5% povidone iodine and then rinsed with Balance Salt Solution (BSS). Pterygium tissue retrieval was carried out according to the protocol developed by Li, et. al (2001).6
Figure 1. Pterygium excision technique with bare sclera, administration of lidocaine 2%
before tissue excision (left) and pterygium excision using a pterygium spoon (right)
After the pterygium tissue was removed, the top of the specimen was taken about 2 mm from the edge and then cut into small pieces (< 0.5 mm3), washed using Phosphate Buffer Saline (PBS), then given RBC Lysis Buffer for 5 minutes and placed on a 100 mm culture dish. The preparations were then dripped with 1 mL of Dulbecco's Modified Eagle's Medium (DMEM) containing 15% Fetal Bovine Serum (FBS), and Penicillin Streptomycin placed overnight in an incubator at 37OC, with 95% humidity and 5% carbon dioxide (CO2) content. The medium was replaced daily with fresh culture medium + FBS. When the cells began to reach 80% confluency, FBS was reduced to 10% concentration and then sub-passed.
P3-7 cultures were used in this study.
Figure 2. Primary culture on pterygium tissue showed fibroblast growth with a confluence rate of 90-100% on day 14, the cells were ready to be given treatment.
Cell’s characteristics and Vimentin staining
The confluent cells were transferred to wellplate 24 with a density of 2.5x 104 wells/plate and then allowed to remain until the cells were attached to the bottom of the plate and then washed 3 times with PBS for 5 minutes, then washed with PBS Triton-X 100 0.1%
for 1 minute. Cells were then incubated with 1% bovine serum albumin (BSA) for 30 minutes at room temperature. The BSA solution was then discarded and incubated with primary antibody for one night and then washed again with PBS. Cells were then incubated with Goat Anti Rabbit IgG & LTIRTC (AB 6718)-Vimentin and then washed with PBS and incubated with DAPI 1: 1000 for 5 minutes at room temperature. Cells were then photographed with a fluorescence microscope at 40x magnification.
Measurement of MMP-3 by Immunofluorescence Staining
Cells that were already confluent were harvested and planted on well plate 24 for 24- 48 hours. Then the wells were divided into untreated, with the intervention of MMC 0.2 and 0.4 mg/mL, curcumin 100 and 200 g/mL and fibrin glue. Untreated wells only contained cells + medium, MMC 0.2 and 0.4 mg/mL were given for 5 minutes to cells with medium, then washed once with PBS, curcumin was given at doses of 100 and 200 mol/L + medium, and fibrin glue was given until clots formed then removed and washed once with PBS. Each well
was then added 3-4% formaldehyde for 15 minutes and then washed with PBS for 2x then the cells were ready to be tested by immunocytochemistry.
The wellplate was then washed with PBS for 5 minutes three times, then washed with PBS Triton-X 100 0.1% for 5 minutes and incubated with 1% bovine sodium albumin (BSA) for 30 minutes at room temperature. The fluid was discarded and then incubated with MMP-3 Rabbit Polyclonal Antibody Bioss bs-0431R ® primary antibody for one night at 4o C. Then the antibody was washed with PBS, incubated with MMP-3 secondary antibody 1:1000 for 1 hour at room temperature. The wellplate was washed again with PBS for 5 minutes and incubated with DAPI 1:1000 for 5 minutes. The results were read with a fluorescent microscope at 40x magnification. Expression levels were analyzed using ImageJ software and expressed in corrected total cell fluorescence (CTCF) determined using the formula: Integrated Density - (Area of selected cell x Mean fluorescence of background readings).2
Cell Migration and in vitro scratch wound assay
Confluent pterygium fibroblasts were implanted on wellplate 24 with a density of 5x 104 cells per well and incubated for 24 hours to achieve 70-80% confluence. Wells were divided into untreated, with the intervention of MMC 0.4 mg/mL, curcumin 200 g/mL and fibrin glue.
The cells that had been given the intervention were then scratched perpendicular to the plate using a yellow tip 200 µL micropipette and photographed using an Olympus CKX53 microscope with a magnification of 40x. Migration distance is measured with imageJ.
Statistical analysis.
Comparative data between inhibition of fibroblast migration with the interventions and comparison of MMP-3 expression were tested using the one-way Anova test and Tukey PostHoc test is carried out to determine the significant difference between one group and
another. The p value is considered significant if the p value is < 0.05. All statistical data was processed using SPSS 26.0 software.
Results
Expression of Matrix Metalloproteinase-3 (MMP-3) on Mitomycin-C, Curcumin & Fibrin Glue
The results of analysis showed that the highest expression of MMP-3 was in the untreated group, then the lowest was in the MMC 0.4 mg/mL. MMP-3 expression of curcumin showed lower MMP-3 expression than negative control whereas 200 mol/L curcumin showed lower MMP-3 suppression than 100 mol/L curcumin, 200 mol/L curcumin had a stronger MMP-3 suppression effect not much different from fibrin glue.
One way Anova test with Posthoc Tukey analysis showed the comparison of all interventions p < 0.05 so that the comparison of MMP-3 expression in all samples was statistically significant.
Mean Std.
Deviation Minimum Maximum
K1 4703.49f 108.98 4532.22 4892.56
p<0.001
MMC 0.2 mg/mL 593.99b 24.40 546.72 641.32
MMC 0.4 mg/mL 270.60a 15.88 245.41 288.32
Curcumin 100 µmol/L 2205.84e 86.10 2072.31 2303.21
Curcumin 200 µmol/L 1002.51c 25.22 973.42 1034.45
Fibrin Glue 1131.55d 17.71 1113.56 1152.36
Table 1. MMP-3 expression in human pterygium fibroblasts by intervention
Figure 3. Pterygium fibroblast cells stained with MMP-3 antibody and DAPI to assess the expression of MMP-3 in pterygium fibroblast tissue in comparison without intervention and with intervention MMC 0.2 mg/mL, MMC 0.4 mg/mL, curcumin 100 mol/L, curcumin 200 mol/L and fibrin glue (Inverted Fluorescence Microscope, 400x magnification).
Inhibition of Fibroblast Migration in Mitomycin-C, Curcumin & Fibrin Glue
The migration rate showed that the area that migrated the fastest was in the untreated group, and the slowest migration rate was in the MMC group of 0.4 mg/mL at 48 hours. The
curcumin and fibrin glue groups had less significant differences at 24 hours and curcumin 200 mol/L had slower migration inhibition at 48 hours when compared to fibrin glue (figure 4).
Statistical test using Oneway Anova with Post Hoc Tukey showed a significant comparison (p < 0.05) in all intervention groups. This shows that the comparison of the migration rate of HPF after stratching with and without intervention is statistically significant (p<0.001).
Table 2. Migration rate of human pterygium fibroblast cells after stratching within 48 hours with intervention
Mean SD Minimum Maximum
Untreated 0H 221.33h 3.57 216.23 225.34
Untreated 24H 141.59e 3.12 137.32 147.22
Untreated 48H 32.46a 1.25 30.28 34.35
MMC 0.4 0H 233.60j 2.87 230.38 237.32
MMC 0.4 24H 210.34g 2.57 206.52 215.08
MMC 0.4 48H 132.05d 1.65 129.88 135.06
Curcumin 200 0H 228.89i 3.75 222.99 233.87 Curcumin 200
24H
178.67f 2.85 174.34 183.21
Curcumin 200
48H 88.83c 1.48 85.28 91.21
Fibrin Glue 0H 231.62i,j 3.17 227.16 235.88 Fibrin Glue 24H 180.40f 2.56 175.32 183.52
Fibrin Glue 48H 72.45b 1.25 70.77 74.67
Figure 4. Comparison of pterygium fibroblast cells without intervention and intervention. MMC 0.2 mg/mL, MMC 0.4 mg/mL, curcumin 100 mol/L, curcumin 200 mol/L and fibrin glue were scanned to assess cell migration at 0, 24 and 48 hours. Yellow line as the scratch border and red line as fibroblast migration.
Discussion
Pterygium is an ocular surface disorder that has capability to cause vision loss. This disease is due to numerous methods which incorporates cell proliferation, anti inflammatory processes, fibrosis, angiogenesis and degradation of the extracellular matrix. Pterygium additionally exhibits several tumor-like features including invasion, metaplasia of epithelial cells, presence of oncogenic viruses, inactivation of tumor suppressor genes, and absence of
pathogenesis of pterygium and its recurrence after excision is still no longer completely understood. identification of medication capable of suppressing the proliferative system could have a huge effect on the treatment of pterygium. 2,13
Matrix metalloproteinases (MMP) are zinc-dependent endopeptides capable of degrading all components of the Extracellular Matrix (ECM) in connective tissue. MMPs have an crucial function in pterygium formation because of the fact excessive fibroblast proliferation and invasion happens at the apex of the pterygium with destruction of the corneal stroma and basal lamina. Pterygium cells will produce MMP which dissolves the basal lamina and triggers the growth of stromal fibroblasts.2,13 Overexpression of MMP on basal limbal epithelial cells is the primary reason of pterygium development. ECM modulation through MMP occurs in the early phase of pterygium invasion, numerous studies have proven a sturdy affiliation among MMP and tumor development/invasion. Invasion of pterygium lesions will growth MMP activity. 13,14,15,16,17,18
Pterygium will result in activation of fibroblast tissue, specifically on the top of the pterygium, ensuing in cleavage of fibrillar collagen within the basal lamina, particularly because of MMP-1 and MMP-3. Degradation of the Extracellular Matrix (ECM) results in the release of stimulating cytokines which includes VEGF and bFGF that cause angiogenesis and MMP-3 has a major effect on angiogenesis because it triggers angiostatin. MMP-3 additionally has large outcomes due to the fact it may activate pro-MMPs such as MMP-1, MMP-9 and MMP-13 so that they play a role in cell migration, proteolysis and angiogenesis.2,13,18 In this study, it become proven that the highest MMP-3 expression came from the untreated group, while the lowest expression came from the 0.4 mg/mL MMC group, followed by the 0.2 mg/mL MMC group. The curcumin intervention also had a suppressive effect on MMP-3 that was more than 50% while compared to the untreated group or even more in the 200 mol/L curcumin group. The suppressive impact of fibrin glue is
much like that of curcumin, so it may be concluded that in this study curcumin and fibrin glue have been able to exert a suppressive effect on the expression of MMP-3 in pterygium fibroblast tissue.
Hwang B.M et al., (2013) who carried out a study the use of curcumin on skin fibroblast cells to prevent photoageing said that ROS are the basis for overexpression of UVB-modulated MMP-3. Curcumin has a robust antioxidant effect that may suppress ROS by means of suppressing the effects of NF-κB and AP-1 DNA binding activity. UVB induces phosphorylation of the Janus Kinase Pathway and p38 and curcumin has been shown to regulate the activation of NF-B and AP-1 through the MAPK signaling pathway.18
The migration rate showed that the area that migrated the quickest was in the untreated group, and the slowest migration rate was in the MMC group of 0.4 mg/mL at 48 hours. The curcumin and fibrin glue groups had much less substantial differences at 24 hours and curcumin 200 mol/L had slower migration inhibition at 48 hours whilst compared to fibrin glue (table 2). This indicates that the comparison of the migration rate of HPF after the stretch with and without intervention is statistically significant. The scratching method was done by scratching the petri base on the monolayer HPF cell culture using the tip of a yellow micropipette (200 µL) in a vertically perpendicular position on the petri meridian. This method is an easy and effective step to trigger inflammation.19 This is in accordance with the outcomes of a study conducted by Komaratih et al wherein there was migration of fibroblast cells in the tenon conjunctival tissue model that was scratched with the conclusion the less the rate of migration of tenon's fibroblast cells to the wound place, the decrease the opportunity of extracellular matrix manufacturing resulting in fibrosis in that place.11
Kim et al's study confirmed the relationship among MMP expression and activity on pterygium fibroblast migration and its suppressive effect on bevacizumab and cyclosporine A (CSA). The fibroblast migration ratio was assessed by scratching the wound on confluent
pterygium fibroblasts to assess the recovery migration process which confirmed the migration speed suppression of MMP-3 and MMP-13 after bevacizumab and/or CsA intervention.11 Chung et al, conducted an in vitro trial on cardiac fibroblasts and stated that curcumin had a higher inhibitory impact on fibroblast migration when compared to the control group and the MTT assay showed that the curcumin group also had a decrease proliferative effect than the control group, which is in line with the study. This indicates that the rate of migration in the curcumin group might be slower when compared to the control group. This occurs most probably due to the effect by suppressing the mediators including TGF-β, MMP and other profibrotic mediators via the NF-κB pathway.20
FG significantly inhibits collagen synthesis and induces collagen degradation in human tenons, in which the wound healing manner is regulated by inflammation. A decrease in the amount of collagen will eliminate the anti inflammatory response and fibrin glue substantially inhibits collagen in the injured tenon model and is able to reduce wound formation and contractures. 21 In line with this study, which showed that in addition to reduce MMP-3 expression, FG was also able to reduce the speed of HPF migration although it was not as significant as the standard for adjuvant pterygium treatment, particularly MMC.
Therefore, our findings suggest that curcumin and fibrin glue suppress the proliferation of pterygium by inhibiting HPF migration and suppressing MMP-3 expression.
So it is closer that fibrin glue and curcumin have potential as adjuvant therapy for MMC replacement in the management of pterygium. Based on our finding, this is the first study to analyze the inhibition of the expression of MMP-3 by HPFs by curcumin and fibrin glue.
However, additional studies on other wound healing mechanisms are warranted to fully determine and understand the effects.
Acknowledgements
This study was funded by Ministry of Research and Technology, Indonesia
Ethical Standard
Ethical approval was obtained from Health Research Ethics Committee, Universitas Airlangga School of Medicine, Surabaya Indonesia. (No: 131/EC/KEPK/FKUA/2021). All Procedures were performed with ethical standards.
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