Methotrexate combined with cyclophosphamide could synergistically inhibit the expression of receptor activator of nuclear factor-κB ligand induced by interleukin-6 in fibroblast-like synoviocytes of rheumatoid arthritis / 中华风湿病学杂志

ABSTRACT

Objective:To study the expression of receptor activator of nuclear factor-κB ligand (RANKL) induced by interleukin-6 (IL-6) in fibroblast-like synoviocytes of rheumatoid arthritis (RA-FLS) and its signal pathway, to clarify the synergistic mechanism of methotrexate (MTX) combined with cyclophosphamide (CTX) in inhibiting RA bone erosion.Methods:The synovial tissues of 6 patients with active RA were collected, and RA-FLS were cultured in vitro. IL-6/sIL-6R and drug intervention were given successively. All RA-FLS were divided into five groups, including blank control group, IL-6/sIL-6R group, CTX group, MTX group and MTX+CTX group. The expression levels of RANKL and signal pathway protein in RA-FLS were detected by real-time quantitative polymerase chain reaction (qPCR), Western blotting and cell-based enzyme-linked im-munosorbent assay (ELISA). Single factor analysis of variance was used to compare in the comparison of multiple groups, LSD- t test or Dunnett's T3 test was used in the comparison of two groups, and 2×2 factorial analysis of var-iance was used in the interaction of two drugs. Results:① There were statistically significant differences in the mRNA and protein levels of RANKL in each group ( F=26.246, P<0.01; F=4.565, P=0.023). The highest level of RANKL mRNA (2.14±0.40) and protein (2.33±0.39) was found in IL-6/sIL-6R group, and the lowest level of RANKL mRNA (0.10±0.08) and protein (0.75±0.21) in MTX+CTX group. ② The difference of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) level in each group was statistically significant ( F=18.151, P<0.01). The level of p-STAT3 (0.328±0.073) was the highest in IL-6/sIL-6R group and the lowest (0.178±0.022) in MTX+CTX group. There was no significant difference in STAT3 in each group ( F=3.173, P=0.051). ③ MTX combined with CTX had interaction on the expression of RANKL mRNA and protein ( F=33.932, P<0.01; F=16.265, P<0.01), and had interaction on the expression of p-STAT3 ( F=16.477, P=0.01), but had no interaction on the expression of STAT3 ( F=0.471, P=0.500). Conclusion:IL-6 can up-regulate the expression of RANKL in RA-FLS through JAK2/STAT3 signal pathway. MTX combined with CTX can down-regulate the expression of RANKL by inhibiting the phosphorylation of STAT3, and this combination is synergistic.