Sinomenine down-regulated RANKL-induced

<div><p>NF-κB <b>activation</b>. </p> <p>RAW264.7 cells, stably or transiently expressing NF-κB-luc reporter gene construct, were stimulated with RANKL for 8 h to study the effects of SIN on (A) stable or (B) transient NF-κB transcription. In another experiment, RAW264.7 cells were pretreated with SIN for 30 min and stimulated with 100 ng/ml RANKL for 30 min to (C) perform Western blot analysis of nuclear p65 and IκBα or (D) to observe p65 translocation to nuclear by fluorescence image. (C) The translocation of p65 by Western blot was expressed by dividing the nuclear p65 density with cytoplamic p65 density, the fold change of IκBα was normalized to β-actin. Densitometric quantification and statistical analysis include the results from 3 independent experiments. *P<0.05; **P<0.01 (compared with RANKL group); # P<0.05, # #P<0.01 (compared with control group).</p></div

Sinomenine Suppresses Osteoclast Formation and <i>Mycobacterium tuberculosis</i> H37Ra-Induced Bone Loss by Modulating RANKL Signaling Pathways

<div><p>Receptor activator of NF-κB ligand (RANKL) is essential for osteoclastogenesis. Targeting RANKL signaling pathways has been an encouraging strategy for treating lytic bone diseases such as osteoporosis and rheumatoid arthritis (RA). Sinomenine (SIN), derived from Chinese medicinal plant <i>Sinomenioum</i><i>acutum</i>, is an active compound to treat RA, but its effect on osteoclasts has been hitherto unknown. In the present study, SIN was found to ameliorate <i>M. tuberculosis</i> H37Ra (Mt)-induced bone loss in rats with a decreased serum level of TRACP5b and RANKL, and an increased level of osteoprotegerin (OPG). <i>In vitro</i> study also showed that SIN could inhibit RANKL-induced osteoclast formation and bone resorption. The osteoclastic specific marker genes induced by RANKL including c-Src, MMP-9, TRACP were inhibited by SIN in a dose dependent manner. Signal transduction studies showed that SIN could obviously reduce the expression of RANK adaptor molecule TRAF6 and down-regulate RANKL-induced NF-κB activation. It decreased the RANKL-induced p38, JNK posphorylation but not ERK1/2 posphorylation. SIN could also reduce RANKL-mediated calcium influx which is associated with TRAF6/c-Src complex. Finally, SIN suppressed RANKL induced AP-1 and NFAT transcription, as well as the gene expression of NFATc1 and AP-1 components (Fra-1, Fra-2, c-Fos). The protein expression of c-Fos and TRAF6 were also inhibited by SIN after RANKL stimulation. Taken together, SIN could attenuate osteoclast formation and Mt-induced bone loss by mediating RANKL signaling pathways.</p> </div

Sinomenine ameliorated Mt-induced arthritis and bone loss in rats.

<p>SD rat was injected with 2 mg heat-killed M. tuberculosis H37Ra (Mt) in 200 µl mineral oil subcutaneously at the base of the tail at day 0 to induce arthritis (n=8). SIN was administrated intraperitoneally at a dose of 80 mg/kg/day. Another anti-rheumatic drug, TWP, was administrated orally at a dose of 2.5 mg/kg/day. Rats in control group were not immunized with Mt. (A) Arthritic edema was indicated by measuring the swelling volume of the hind paw. At day 28, rats were killed and the hind lambs were stocked in 10% formalin to obtain (B) the 2-D image of hind lamb and (C) the bone parameters by Micro-CT. (D) Serum prepared from rat abdominal aorta blood was used to measure RANKL, OPG, RANKL/OPG ratio, TRACP5b (showing the osteoclast activity), ALP activity (showing the osteoblast activity). All the bars are mean±SEM of a representative experiment. *P<0.05; **P<0.01 (compared with model group); # P<0.05, # #P<0.01 (compared with control group).</p

Sinomenine reduced intracellular calcium influx.

<p>RAW264.7 cells were incubated with RANKL (100 ng/ml) in the presence or absence of SIN (0.5 mM) for 72 h. For Ca²⁺ measurement, cells were incubated with Fluo-3/AM for 30 min in serum-free DMEM followed by confocal analysis. (A) Intracellular calcium was illustrated by green fluorescence (Fluo-3/AM). (B) Each line represented the fluorescence intensity of one cell. The fluorescence intensity in each group was recorded within 200s and (C) statistically analyzed at time point of 200s. P<0.05; **P<0.01 (compared with RANKL group); # P<0.05, # #P<0.01 (compared with control group).</p

Sinomenine dose-dependently inhibited RANKL-induced osteoclast-specific genes and TRAF6.

<p>RAW264.7 cells were plated into 6-well plates, incubated with RANKL (100 ng/mL) and SIN for 24 h. Total RNA was extracted and real-time PCR was performed. Relative Quantification (RQ) was used to determine the fold-change of gene expression compared with the GAPDH control gene. (A) osteoclast-specific genes, (B) RANK and TRAF6 genes expression were investigated. RAW264.7 cells were incubated with SIN for 30 min and then stimulated with RANKL for 30 min, (C) TRAF6 protein expression was analyzed by Western blot. β-actin was used as loading control. Densitometric quantification and statistical analysis include the results from 3 independent experiments. *P<0.05; **P<0.01(compared with RANKL group); # P<0.05, # #P<0.01 (compared with control group).</p

Sinomenine inhibited RANKL-induced NFAT, AP-1 activation.

<p>RAW264.7 cells, transiently expressing NFAT-luc or AP-1-luc reporter gene construct were pre-treated with SIN for 30 min and followed by 100 ng/ml RANKL stimulation for 12 h. (A) NFAT and (B) AP-1 transcriptions are indicated by luciferase activity. (C) RAW264.7 cells were plated into 6-well plates, incubated with RANKL (100 ng/mL) and SIN for 24 h. Real-time PCR was performed to determine gene expressions of NFATc1 and AP-1 components (c-Fos, Fra-1, Fra-2). RAW264.7 cells were incubated with SIN for 30 min and then stimulated with RANKL for 30 min. (D) c-Fos protein expression was analyzed by Western blot. β-actin was used as loading control. Densitometric quantification and statistical analysis include the results from 3 independent experiments. P<0.05; **P<0.01(compared with RANKL group); # P<0.05, # #P<0.01 (compared with control group). (E) A schematic diagram shows a hypothetical model by which SIN inhibits osteoclast differentiation and function.</p