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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

LLDT-8 inhibited secretion of IL-1β, IL-6 and IL-21, but promoted the secretion of IL-10 in the supernatants of SFMCs from synovial fluid of RA patients. SFMCs were isolated from synovial fluid of RA patients and cultured in the presence of anti-human CD3 (0.4 μg/ml) and LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 48 hours in incubator. The cell supernatants were harvested and the levels of the proteins were detected by ELISA. (A) The levels of IL-1β in the supernatants of the four groups. (B) The levels of IL-6 in the supernatants of the four groups. (C) The levels of IL-21 in the supernatants of the four groups. (D) The level of IL-23 in the supernatants of the four groups. (E) The levels of IL-10 in the supernatants of the four groups. **p < 0.01, ***p < 0.001, compared to the control group (0 nM).
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Fig4: LLDT-8 inhibited secretion of IL-1β, IL-6 and IL-21, but promoted the secretion of IL-10 in the supernatants of SFMCs from synovial fluid of RA patients. SFMCs were isolated from synovial fluid of RA patients and cultured in the presence of anti-human CD3 (0.4 μg/ml) and LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 48 hours in incubator. The cell supernatants were harvested and the levels of the proteins were detected by ELISA. (A) The levels of IL-1β in the supernatants of the four groups. (B) The levels of IL-6 in the supernatants of the four groups. (C) The levels of IL-21 in the supernatants of the four groups. (D) The level of IL-23 in the supernatants of the four groups. (E) The levels of IL-10 in the supernatants of the four groups. **p < 0.01, ***p < 0.001, compared to the control group (0 nM).

Mentions: Besides, we also detected the effect of LLDT-8 on the secretion of cytokines in SFMCs from RA patients, and found that LLDT-8 reduced the levels of IL-1β, IL-6, and IL-21 in the supernatants of SFMCs (Figure 4A-C). However, we did not find any distinct effect of LLDT-8 on the secretion of IL-23 (Figure 4D). We also found that LLDT-8 increased the level of IL-10 in the cell supernatants (Figure 4E). Above all, the results demonstrated that LLDT-8 could inhibite SFMCs secreting IL-1β, IL-6 and IL-21, but promoted IL-10 secretion in SFMCs.Figure 4


(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)

LLDT-8 inhibited secretion of IL-1β, IL-6 and IL-21, but promoted the secretion of IL-10 in the supernatants of SFMCs from synovial fluid of RA patients. SFMCs were isolated from synovial fluid of RA patients and cultured in the presence of anti-human CD3 (0.4 μg/ml) and LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 48 hours in incubator. The cell supernatants were harvested and the levels of the proteins were detected by ELISA. (A) The levels of IL-1β in the supernatants of the four groups. (B) The levels of IL-6 in the supernatants of the four groups. (C) The levels of IL-21 in the supernatants of the four groups. (D) The level of IL-23 in the supernatants of the four groups. (E) The levels of IL-10 in the supernatants of the four groups. **p < 0.01, ***p < 0.001, compared to the control group (0 nM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4379732&req=5

Fig4: LLDT-8 inhibited secretion of IL-1β, IL-6 and IL-21, but promoted the secretion of IL-10 in the supernatants of SFMCs from synovial fluid of RA patients. SFMCs were isolated from synovial fluid of RA patients and cultured in the presence of anti-human CD3 (0.4 μg/ml) and LLDT-8 (0, 12.5, 25 and 50 nM, respectively) for 48 hours in incubator. The cell supernatants were harvested and the levels of the proteins were detected by ELISA. (A) The levels of IL-1β in the supernatants of the four groups. (B) The levels of IL-6 in the supernatants of the four groups. (C) The levels of IL-21 in the supernatants of the four groups. (D) The level of IL-23 in the supernatants of the four groups. (E) The levels of IL-10 in the supernatants of the four groups. **p < 0.01, ***p < 0.001, compared to the control group (0 nM).
Mentions: Besides, we also detected the effect of LLDT-8 on the secretion of cytokines in SFMCs from RA patients, and found that LLDT-8 reduced the levels of IL-1β, IL-6, and IL-21 in the supernatants of SFMCs (Figure 4A-C). However, we did not find any distinct effect of LLDT-8 on the secretion of IL-23 (Figure 4D). We also found that LLDT-8 increased the level of IL-10 in the cell supernatants (Figure 4E). Above all, the results demonstrated that LLDT-8 could inhibite SFMCs secreting IL-1β, IL-6 and IL-21, but promoted IL-10 secretion in SFMCs.Figure 4

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