Limits...
(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: 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.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.

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

The effect of LLDT-8 on key proteins of the downstream signaling pathways of RANKL. RAW264.7 cells were treated with LLDT-8 (0 and 50 nM, respectively) for 4 hours, then induced by culture medium supplemented with 50 ng/ml RANKL for 0, 10, 30 and 60 min, respectively. The protein levels of p-IκB, p-P38, p-JNK, p-ERK and p-Akt were detected by western blotting. (A) Immunoblots of the key proteins. RANKL significantly increased the levels of phosphorylation of I-κB, P38, JNK, ERK and Akt in a short time. (B) Relative expression of the key proteins. Compared with the control group, LLDT-8 markedly inhibited the expression of p-IκB induced by RANKL. However, it had little effect on the expression of p-P38、p-JNK、p-ERK and p-Akt induced by RANKL. **p < 0.01, ***p < 0.001, compared to the control group (0 nM) at the same time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4379732&req=5

Fig7: The effect of LLDT-8 on key proteins of the downstream signaling pathways of RANKL. RAW264.7 cells were treated with LLDT-8 (0 and 50 nM, respectively) for 4 hours, then induced by culture medium supplemented with 50 ng/ml RANKL for 0, 10, 30 and 60 min, respectively. The protein levels of p-IκB, p-P38, p-JNK, p-ERK and p-Akt were detected by western blotting. (A) Immunoblots of the key proteins. RANKL significantly increased the levels of phosphorylation of I-κB, P38, JNK, ERK and Akt in a short time. (B) Relative expression of the key proteins. Compared with the control group, LLDT-8 markedly inhibited the expression of p-IκB induced by RANKL. However, it had little effect on the expression of p-P38、p-JNK、p-ERK and p-Akt induced by RANKL. **p < 0.01, ***p < 0.001, compared to the control group (0 nM) at the same time point.

Mentions: To reveal the mechanism of LLDT-8 on inhibiting osteoclastogenesis, we tested the effect of LLDT-8 on the downstream signaling pathways of RANKL by Western blotting. RANKL significantly increased the levels of phosphorylation of I-κB, P38, JNK, ERK and Akt in a short time (Figure 7A). Compared with the control group, LLDT-8 markedly inhibited the expression of p-IκB induced by RANKL. However, it had little effect on the expression of p-P38、p-JNK、p-ERK and p-Akt induced by RANKL (Figure 7B).Figure 7


(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)

The effect of LLDT-8 on key proteins of the downstream signaling pathways of RANKL. RAW264.7 cells were treated with LLDT-8 (0 and 50 nM, respectively) for 4 hours, then induced by culture medium supplemented with 50 ng/ml RANKL for 0, 10, 30 and 60 min, respectively. The protein levels of p-IκB, p-P38, p-JNK, p-ERK and p-Akt were detected by western blotting. (A) Immunoblots of the key proteins. RANKL significantly increased the levels of phosphorylation of I-κB, P38, JNK, ERK and Akt in a short time. (B) Relative expression of the key proteins. Compared with the control group, LLDT-8 markedly inhibited the expression of p-IκB induced by RANKL. However, it had little effect on the expression of p-P38、p-JNK、p-ERK and p-Akt induced by RANKL. **p < 0.01, ***p < 0.001, compared to the control group (0 nM) at the same time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4379732&req=5

Fig7: The effect of LLDT-8 on key proteins of the downstream signaling pathways of RANKL. RAW264.7 cells were treated with LLDT-8 (0 and 50 nM, respectively) for 4 hours, then induced by culture medium supplemented with 50 ng/ml RANKL for 0, 10, 30 and 60 min, respectively. The protein levels of p-IκB, p-P38, p-JNK, p-ERK and p-Akt were detected by western blotting. (A) Immunoblots of the key proteins. RANKL significantly increased the levels of phosphorylation of I-κB, P38, JNK, ERK and Akt in a short time. (B) Relative expression of the key proteins. Compared with the control group, LLDT-8 markedly inhibited the expression of p-IκB induced by RANKL. However, it had little effect on the expression of p-P38、p-JNK、p-ERK and p-Akt induced by RANKL. **p < 0.01, ***p < 0.001, compared to the control group (0 nM) at the same time point.
Mentions: To reveal the mechanism of LLDT-8 on inhibiting osteoclastogenesis, we tested the effect of LLDT-8 on the downstream signaling pathways of RANKL by Western blotting. RANKL significantly increased the levels of phosphorylation of I-κB, P38, JNK, ERK and Akt in a short time (Figure 7A). Compared with the control group, LLDT-8 markedly inhibited the expression of p-IκB induced by RANKL. However, it had little effect on the expression of p-P38、p-JNK、p-ERK and p-Akt induced by RANKL (Figure 7B).Figure 7

Bottom Line: 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.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.

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