雷公藤内酯醇调控miR-155抑制类风湿性关节炎患者单核细胞促炎反应

Triptolide inhibits the inflammatory response of monocytes from rheumatoid arthritis patients by regulating miR-155

目的探讨雷公藤内酯醇(TPT)能否通过调控miR-155抑制脂多糖(LPS)刺激的类风湿性关节炎(RA)患者单核细胞炎症反应。方法分离RA患者外周血单个核细胞(PBMC),CD14+磁珠分选单核细胞,LPS刺激24 h后用于实验。ELISA检测不同浓度TPT作用下,单核细胞TNF-α和IL-6的表达;实时定量PCR(qRT-PCR)检测TPT干预前后单核细胞miR-155的表达;LipofectamineTM2000脂质体分别转染miR-155 mimic和对照24 h,TPT再干预24 h,通过ELISA检测单核细胞TNF-α和IL-6表达,Western blot法检测单核细胞细胞因子信号抑制蛋白1(SOCS1)、含SH2肌醇磷酸酯酶1(SHIP-1)的表达。结果 TPT抑制LPS刺激的RA患者外周血单核细胞促炎细胞因子和miR-155的表达。过表达miR-155明显逆转TPT抑制的单核细胞促炎细胞因子的表达。TPT上调RA患者单核细胞SOCS1和SHIP-1表达,但过表达miR-155能逆转上调的SHIP-1而不影响SOCS1的表达。结论 TPT抑制miR-155表达而释放其靶标SHIP-1,从而下调LPS诱导的RA患者单核细胞炎症反应。

Objective To explore the anti-inflammatory effect of triptolide （TPT） by regulating miR-155 in monocytes pre-stimulated by lipopolysaccharide （LPS） from rheumatoid arthritis （RA） patients. Methods Monocytes were isolated by CD14＋ magnetic beads from peripheral blood mononuclear cell~ （PBMCs） of RA and stimulated by LPS for 24 hours. The levels of tumor-necrosis factor-a （TNF-a） and interleukin 6 （IL-6） in monocytes were detected by ELISA and the expression of miR-155 was measured by real-time quantitative PCR （qRT-PCR） in monocytes before and after the treatment of TPT at different concentrations. MiR-155 mimic and negative control were respectively transfected into the LPS-stimulated monocytes by LipofectamineTM2000. Twenty-four hours later, the monocytes were treated with or without TPT for another 24 hours. TNF-a and IL-6 expressions in the cell culture supernatants were detected by ELISA and the expressions of suppressor of cytokine signaling-1 （ SOCS1 ） and Src homology 2 domain-containing inositol 5-phosphatase 1 （ SHIP-1 ） were tested by Western blotting. Resalts TPT suppressed the expressions of TNF-a, IL-6 and miR-155 in LPS-stimulated peripheral blood monocytes from RA patients. Over-expression of miR-155 significantly reversed the down-regulation of TNF-a and IL-6 by TPT in monocytes. TPT up-regulated the expressions of SOCS1 and SHIP-1 in monocytes, but over-expressed miR-155 antagonized the effect of TPT on SHIP-1 while the expression of SOCS1 was not affected. Conclusion TPT suppressed the expression of miR-155 and up-regulated the release of SHIP-1, thus inhibiting the inflammatory response in the LPS-stimulated monocytes of RA patients.