Anping Peng1, Xianzhang Huang1, Ruiping Liu1, Xiaoyun Wang2, Junhua Zhuang1. 1. Department of Laboratory Science, Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120, China. 2. Department of Gynecology, Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510120, China.
Abstract
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 cells (PBMCs) of RA and stimulated by LPS for 24 hours. The levels of tumor-necrosis factor-α (TNF-α) 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 Lipofectamine(TM)2000. Twenty-four hours later, the monocytes were treated with or without TPT for another 24 hours. TNF-α 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. RESULTS: TPT suppressed the expressions of TNF-α, 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-α 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.
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 cells (PBMCs) of RA and stimulated by LPS for 24 hours. The levels of tumor-necrosis factor-α (TNF-α) 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 Lipofectamine(TM)2000. Twenty-four hours later, the monocytes were treated with or without TPT for another 24 hours. TNF-α 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. RESULTS:TPT suppressed the expressions of TNF-α, IL-6 and miR-155 in LPS-stimulated peripheral blood monocytes from RApatients. Over-expression of miR-155 significantly reversed the down-regulation of TNF-α 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 RApatients.