The Effect of Low- dose Methotrexate on Collagen and MMP-1 Expression by Fibroblast in Vitro

Background: Methotrexate(MTX) , an antimetabolite, antifolate drug, is commonly used for the treatment of  cancer, autoimmune diseases, psoriasis and ectopic pregnancy. Low dose MTX is recommended as a first-line drug by the European League Against Rheumatism (EULAR) and the American College of Rheumatology (ACR)  in the management of early and established RA.  Systemic Low dose regimen is also used in psoriatic patients who cannot be controlled with topical agents or phototherapy. Although, the side effects of the high dosage of MTX used in malignancies are well known and related to its antagonistic effect to folic acid, the underlying mechanism of liver and pulmonary fibrosis seen in patients on long- term, low dose MTX has not been established yet. It has been shown previously by our group that low-dose MTX increases the proliferation of the fibroblast. We hypothesized that low-dose MTX may increase collagen expression as well. In this study, the effect of low-dose MTX on collagen and MMP-1 expression by human skin fibroblast in vitro was observed. Methods: Human skin fibroblasts, harvested from skin samples obtained with informed consent from adult patients undergoing elective circumcision or reconstructive surgery, were treated with 50ng/ml of MTX for 24 hours. Cells were harvested at different time points after treatment. Protein analysis using Western blot was investigated to determine collagen and MMP-1 expression.  The quantities of the proteins were evaluated by densitometry using ImageJ64 software. MTT assay was performed to examine the viability of the fibroblasts. Each experiment was repeated at least three times and the results are presented as the mean ± the standard deviation. Data were analyzed with t test and P values<  0.05 were considered statistically  significant. Results: Collagen/β-actin ratio for control and treated group were 1.551± 0.45 and 0.375± 0.41 respectively, 72 hours after treatment( P value=0.05). MMP-1/β-actin ratio was 0.73± 0.11 for untreated and 1.457± 0.1 for treated group (P value=0.007). Different strains of fibroblast showed 50% to 80% reduction in collagen expression after treatment with 50ng/ml MTX compared to control group. On the other hand, MMP-1 expression showed two fold increases following treatment in comparison to the untreated group. MTT assay revealed no significant difference between control and treated group. Conclusions: The underlying mechanism of liver fibrosis seen in patients on long-term low-dose MTX cannot be explained by direct stimulatory effect of MTX on excessive collagen production by fibroblast. In contrary, our results suggest that low-dose MTX has potential to be used as an anti-fibrotic drug in fibroproliferative disorders such as hypertrophic scar and keloid. The specific mechanism by which MTX decreases collagen and increases MMP-1expression remains unknown.