The Effect Of Topical Application Of Methotrexate(MTX) In Preventing Intraarticular Scar Adhesion Via Endoplasmic Reticulum Stress Signaling Pathway

Objective:To study the effect of topical application of MTX inducing apoptosis of the fibroblast and preventing scar adhesion after knee surgery, and discuss the possible mechanism.Methods:Eighteen mature male New Zealand white rabbits were used for this study. The rabbits were randomly and equally divided into 3 groups:a 1 mg/ml MTX group, a 2mg/ml group and a control (saline) group. Approximately 10 mm × 10 mm of the cortical bone was removed in each rabbit from both sides of the right femoral condyle to expose the underneath cancellous bone. MTX (1 mg/ml and 2 mg/ml) or saline was applied to the cancellous bone for 10 min. After 4 weeks, all animals were sacrificed. Histological analysis, fibroblast counting and CHOP expression of the scar tissue were used to evaluate the effect of MTX on preventing scar adhesion after knee surgery. The fibroblasts were isolated from the intraarticular adhesion scar and cultured. Fibroblasts were cultured in vitro, after dealing with different concentrations of methotrexate overnight, CCK-8 was used to explore the cell viability and Hoechst 33342 staining was used to test the apoptosis of the fibroblasts. Western-blot was used to test the expression of ER stress-related and apoptosis-related proteins, including cleaved-PARP, CHOP, Bax and Bcl-2.Results:The fibroblasts in the 1 mg/ml and 2 mg/ml group of MTX were significantly fewer than the control group (P<0.05), and the 2 mg/ml group is also fewer than that of the 1mg/ml group (P<0.05). The collagen density of the intraarticular tissue in the experimental group was obviously lower than in the control group, and there is also a decrease from 1mg/ml to 2mg/ml, the results of Image Pro Plus proved these (P<0.05). Immunohistochemistry showed that the expression of CHOP was increased in a dose-dependent manner (P<0.05). These result suggested that that MTX could prevent scar adhesion, at the same time we found that 2mg/ml MTX has a better effect than lmg/ml MTX. The results of CHOP expression in scar tissue were also illustrated that ER stress was activated. In vivo exprinments, MTX could decrease the cell viablity of fibroblast in a dose-dependent manner, Hoechst 33342 staining showed us that MTX could induce apoptosis of the fibroblast. Results of western-blot showed that the expression of cleaved-PARP, CHOP and bax were up-regulated after dealt with MTX for different durations, but the Bcl-2 was down-regulated. These results showed that MTX could decrease the cell viability and induce the apotosis, the endoplasmic reticulum stress signaling pathway was involved.Conclusion:MTX could prevent intraarticular scar adhesion, and the endoplasmic reticulum stress signaling pathway was involved.