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(5R)-5-Hydroxytriptolide (LLDT-8) inhibits osteoclastogenesis via RANKL/RANK/OPG signaling pathway.

Shen Y, Jiang T, Wang R, He S, Guo M, Zuo J, He D - BMC Complement Altern Med (2015)

Bottom Line: The levels of interleukin (IL) 1β, IL-6, IL-10, IL-21 and IL-23 in the supernatants of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were assayed by ELISA.Tartaric acid phosphatase (TRAP) staining was used to identify the osteoclast-like cells derived from RAW264.7.LLDT-8 inhibited IL-1β, IL-6, IL-21 and IL-23 secretion, but promoted the secretion of IL-10 in the supernatants of PBMCs and SFMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Shanghai Guanghua Hospital of Integrated Traditional and Western Medicine, Shanghai, 200052, China. shenyi_740@hotmail.com.

ABSTRACT

Background: The aim of this study was to investigate the regulative activity of (5R)-5-hydroxytriptolide (LLDT-8) on receptor activator of nuclear factor κ-B ligand (RANKL)/receptor activator of nuclear factor κ-B (RANK)/Osteoprotegerin (OPG) system in rheumatoid arthritis (RA) and its anti-osteoclastogenesis mechanism.

Methods: The expression of OPG, RANK and RANKL in CD3(+) T leukomonocytes in both peripheral blood and synovial fluid of RA patients was evaluated by flow cytometry. The levels of interleukin (IL) 1β, IL-6, IL-10, IL-21 and IL-23 in the supernatants of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were assayed by ELISA. Tartaric acid phosphatase (TRAP) staining was used to identify the osteoclast-like cells derived from RAW264.7. Western blotting analysis was used to check the downstream molecules of RANKL.

Results: LLDT-8 increased the rate of OPG expression in CD3(+) T leukomonocytes in peripheral blood as well as the ratio of OPG/RANKL in both peripheral blood and synovial fluid. LLDT-8 inhibited IL-1β, IL-6, IL-21 and IL-23 secretion, but promoted the secretion of IL-10 in the supernatants of PBMCs and SFMCs. In addition, LLDT-8 decreased the number of TRAP-positive cells derived from RAW264.7 in the presence of RANKL and M-CSF. Furthermore, LLDT-8 also inhibited the expression of p-IκB, a key regulator of RANKL signaling pathway.

Conclusions: LLDT-8 exerts its anti-osteoclastogenesis effect in RA probably through regulating RANKL/RANK/OPG system and its downstream signaling pathway as well as cytokine productions.

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Related in: MedlinePlus

Cell morphology of RAW264.7. RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with various concentrations of LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 6 days, then the cells were fixed and stained with TRAP kit on day 1, 2, 3 and 6. The cell morphology were observed using light microscopy, The number of TRAP-positive staining multinuclear cells increased gradually since 72 h (A), and reached the top amount at day 6 (B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (C). TRAP-positive cells were not observed in the culture medium without RANKL and M-CSF (D). (A-C) 400-fold amplification, bar = 25 μm; (D) 200-fold amplification, bar = 50 μm.
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Fig5: Cell morphology of RAW264.7. RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with various concentrations of LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 6 days, then the cells were fixed and stained with TRAP kit on day 1, 2, 3 and 6. The cell morphology were observed using light microscopy, The number of TRAP-positive staining multinuclear cells increased gradually since 72 h (A), and reached the top amount at day 6 (B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (C). TRAP-positive cells were not observed in the culture medium without RANKL and M-CSF (D). (A-C) 400-fold amplification, bar = 25 μm; (D) 200-fold amplification, bar = 50 μm.

Mentions: To detect the effect of LLDT-8 on RANKL-induced osteoclastogenesis, RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with or without various concentrations of LLDT-8 for 6 days. The total number of TRAP-positive cells in each well was counted. We found that the cells showed enlarged volume and irregular shape, and the staining was burgundy. The number of osteoclasts increased gradually since 72 h (Figure 5A), and reached its top amount at day 6 (Figure 5B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (Figure 5C). The cells without RANKL and M-CSF inducing, were unable to transform into TRAP-positive osteoclasts (Figure 5D). In comparison with the non-treated group, LLDT-8 reduced the number of TRAP-positive osteoclasts at 48 h (Figure 6B), and significantly decreased the number of TRAP-positive osteoclasts from the 3rd day to the 6th day (Figure 6C & D). We did not observe any significant difference between LLDT-8-treated groups and the non-treated group at 24 h (Figure 6A).Figure 5


(5R)-5-Hydroxytriptolide (LLDT-8) inhibits osteoclastogenesis via RANKL/RANK/OPG signaling pathway.

Shen Y, Jiang T, Wang R, He S, Guo M, Zuo J, He D - BMC Complement Altern Med (2015)

Cell morphology of RAW264.7. RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with various concentrations of LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 6 days, then the cells were fixed and stained with TRAP kit on day 1, 2, 3 and 6. The cell morphology were observed using light microscopy, The number of TRAP-positive staining multinuclear cells increased gradually since 72 h (A), and reached the top amount at day 6 (B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (C). TRAP-positive cells were not observed in the culture medium without RANKL and M-CSF (D). (A-C) 400-fold amplification, bar = 25 μm; (D) 200-fold amplification, bar = 50 μm.
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Related In: Results  -  Collection

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Fig5: Cell morphology of RAW264.7. RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with various concentrations of LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 6 days, then the cells were fixed and stained with TRAP kit on day 1, 2, 3 and 6. The cell morphology were observed using light microscopy, The number of TRAP-positive staining multinuclear cells increased gradually since 72 h (A), and reached the top amount at day 6 (B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (C). TRAP-positive cells were not observed in the culture medium without RANKL and M-CSF (D). (A-C) 400-fold amplification, bar = 25 μm; (D) 200-fold amplification, bar = 50 μm.
Mentions: To detect the effect of LLDT-8 on RANKL-induced osteoclastogenesis, RAW264.7 cells were cultured in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with or without various concentrations of LLDT-8 for 6 days. The total number of TRAP-positive cells in each well was counted. We found that the cells showed enlarged volume and irregular shape, and the staining was burgundy. The number of osteoclasts increased gradually since 72 h (Figure 5A), and reached its top amount at day 6 (Figure 5B). A small number of TRAP-positive cells were observed at day 6 in the presence of RANKL (50 ng/ml) and M-CSF (50 ng/ml) with 50 nM LLDT-8 (Figure 5C). The cells without RANKL and M-CSF inducing, were unable to transform into TRAP-positive osteoclasts (Figure 5D). In comparison with the non-treated group, LLDT-8 reduced the number of TRAP-positive osteoclasts at 48 h (Figure 6B), and significantly decreased the number of TRAP-positive osteoclasts from the 3rd day to the 6th day (Figure 6C & D). We did not observe any significant difference between LLDT-8-treated groups and the non-treated group at 24 h (Figure 6A).Figure 5

Bottom Line: The levels of interleukin (IL) 1β, IL-6, IL-10, IL-21 and IL-23 in the supernatants of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were assayed by ELISA.Tartaric acid phosphatase (TRAP) staining was used to identify the osteoclast-like cells derived from RAW264.7.LLDT-8 inhibited IL-1β, IL-6, IL-21 and IL-23 secretion, but promoted the secretion of IL-10 in the supernatants of PBMCs and SFMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Shanghai Guanghua Hospital of Integrated Traditional and Western Medicine, Shanghai, 200052, China. shenyi_740@hotmail.com.

ABSTRACT

Background: The aim of this study was to investigate the regulative activity of (5R)-5-hydroxytriptolide (LLDT-8) on receptor activator of nuclear factor κ-B ligand (RANKL)/receptor activator of nuclear factor κ-B (RANK)/Osteoprotegerin (OPG) system in rheumatoid arthritis (RA) and its anti-osteoclastogenesis mechanism.

Methods: The expression of OPG, RANK and RANKL in CD3(+) T leukomonocytes in both peripheral blood and synovial fluid of RA patients was evaluated by flow cytometry. The levels of interleukin (IL) 1β, IL-6, IL-10, IL-21 and IL-23 in the supernatants of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were assayed by ELISA. Tartaric acid phosphatase (TRAP) staining was used to identify the osteoclast-like cells derived from RAW264.7. Western blotting analysis was used to check the downstream molecules of RANKL.

Results: LLDT-8 increased the rate of OPG expression in CD3(+) T leukomonocytes in peripheral blood as well as the ratio of OPG/RANKL in both peripheral blood and synovial fluid. LLDT-8 inhibited IL-1β, IL-6, IL-21 and IL-23 secretion, but promoted the secretion of IL-10 in the supernatants of PBMCs and SFMCs. In addition, LLDT-8 decreased the number of TRAP-positive cells derived from RAW264.7 in the presence of RANKL and M-CSF. Furthermore, LLDT-8 also inhibited the expression of p-IκB, a key regulator of RANKL signaling pathway.

Conclusions: LLDT-8 exerts its anti-osteoclastogenesis effect in RA probably through regulating RANKL/RANK/OPG system and its downstream signaling pathway as well as cytokine productions.

Show MeSH
Related in: MedlinePlus