Effects Of Sinomenine On RANKL-induced And LPS-induced Osteoclast And The Relative Mechanisms

The bone metabolic stability need the dynamic balance between osteoclasts (OCs)-mediated bone resorption and osteoblasts-mediated bone formation, both to maintain function balanced in healthy individuals, and balanced once broken, it may lead to bone diseases occurrence. Bone destruction caused by abnormal osteoclast activation, excessive bone destruction may lead to some of the common diseases, such as rheumatoid arthritis, osteoarthritis, osteomyelitis, periodontitis, etc. Medication for these diseases is quite limited, mainly dependent on glucocorticoids, immunosuppressants, antibiotics or non-steroidal anti-inflammatory drugs, but these drugs can not reverse bone erosion, and some even counterproductive. Osteoclast is the uniqueness cell responsible for bone resorption in vivo, the most direct therapeutic strategies targeting inhibition of osteoclast activation is considered to be the most effective to reverse the bone destruction, has become the hot research field.Osteoclasts derived from bone marrow monocyte-macrophage lineage cells in the body must rely on a number of incentives to induce differentiation and development of mature multinucleated osteoclasts. Currently,It considered the most direct activation of osteoclasts endogenous molecular is receptor activation of nuclear factor NF-κB ligand (RANKL). RANKL and osteoclast cell membrane receptor RANK combination raised the formation of a complex of TRAF6and c-Src to activated the downstream signaling pathways (NF-κB, MAPK and Ca2+channel). Next,these signal transduction pathways activate the nuclear transcription factors,such as NF-κB. AP-1and NFATcl, through increasing the expression of osteoclast-related genes, such as the V-ATPase d2, TRACP, MMP-9, integrin αVβ3and cathepsin K, to promote the osteoclast differentiation, enhance bone resorption activity, inhibit osteoclast apoptosis, and ultimately lead to excessive activationr of osteoclasts.Lipopolysaccharide (LPS) is considered to be the most important medium of chronic G-bacteria infection of bone loss.The induction of osteoclast activation is closely related to the mechanism include:inducing the expression of RANKL in the bone cells to the differentiation and activation of osteoclasts; stimulating macrophages to produce TNF-a, IL-1and IL-6and other inflammatory cytokines, and extending life cycle of osteoclasts.Sinomenine is the main active ingredient in the traditional anti-rheumatic medicine Sinomenioum acutum. It has obvious anti-rheumatic, anti-inflammatory, anti-immune pharmacological effects. Preparations in clinical exact effect has a variety of rheumatism. Although the research found to the sinomenine have the effect of reducing the extent of bone destruction, but still can not see whether the inhibition of osteoclast has been reported. In this study, sinomenine was used in RANKL and LPS-induced osteoclast system in vitro, and Mt-induced adjuvant arthritis bone destruction model and LPS-induced acute skull bone destruction model in vivo.Then, we investigated the regulation of osteoclasts and molecular mechanisms of sinomenine. The article mainly includes three parts:1. The effects of sinomenine on RANKL-induced osteoclasts;2. The mechanisms of sinomenine on RANKL-induced osteoclasts;3. The effects and mechanisms of sinomenine on LPS-induced RAW-OCLs.Methods:1) Examining the impact of sinomenine on the viability of RAW264.7cells in different times. Using RAW264.7cell-devised OCs system by RANKL in vitro, to investigate the effect of sinomenine on OCs formation and survival by TRACP staining; through the bone slices absorption experiment, to evaluate the effect of sinomenine on the resorptive function of OCs; using phalloidin-FITC staining to evaluate the destruction of sinomenine on actin ring structure of OCs; by Real-Time PCR, to detect the affection of sinomenine on the expression of osteoclast-related genes by RANKL-induced,such as TRACP, MMP-9, c-Src, Integrin β3and cathepsin K. Establish Mt-induced arthritis bone destruction SD rat model to evaluate the effects of sinomenine on the model animal ankle swelling, bone destruction and pathological changes, the activation of osteoclasts, bone parameters and cytokine levels in serum.2) In RANKL-induced RAW264.7cell system in vitro, the effects of sinomenine on nuclear transcription factor NF-κB, the AP-1and NFAT activation were detected by fluorescent reporter gene technology; further by Western blot and RT-PCR, to detect the effects of sinomenine on NF-κB signaling pathway (p65and iκB-a) and its upstream molecules (RANK and TRAF6), MAPK signaling pathway molecules (p38, ERK and JNK) phosphorylation, AP-1related molecules (c-Fos, Fra-1and Fra-2) as well as NFATcl expression regulation; to detect the nuclear translocation of p65as well as Ca2+influx by fluorescent assay. The detection of apoptosis in RANKL-induced OCs was through DNA Ladder analysis, Hochst33258staining evaluation, Caspase-3expression levels by Western blot and Caspase-3enzyme activity by colorimetric detection.3) Using RAW264.7cell-OCLs system by LPS induced in vitro, to investigate the effect of sinomenine on RAW-OCLs formation and survival by TRACP staining; through the bone slices absorption experiment, to evaluate the effect of sinomenine on the resorptive function of RAW-OCLs; using phalloidin-FITC staining to evaluate the destruction of sinomenine on actin ring structure of RAW-OCLs; utilization the ELISA to test the impact of sinomenine on the release of TNF-a in LPS-induced RAW-OCLs; by Real-Time PCR, to detect the affection of sinomenine on the expression of osteoclast-related genes in LPS-induced RAW264.7cells and RAW-OCLs, such as TRACP, MMP-9, c-Src, Integrin β3and cathepsin K. Establish LPS-induced acute skull bone destruction murine model to evaluate the effects of sinomenine on skull bone destruction, osteoclast activation and serum levels of TNF-a.The study of mechanisms:the effects of sinomenine on nuclear transcription factor NF-κB, the AP-1and NFAT activation were detected by fluorescent reporter gene technology. The effects of sinomenine on the molecule expression levels of NF-κB signaling pathway (cytoplasmic/nuclear protein p65and total protein κB-a, TLR4and TRAF6), MAPK signaling pathway (p38, ERK and JNK) and c-Fos molecules were detected by Western blot. The impact of sinomenine on the genes expression of upstream of NF-κB (TLR4and TRAF6), AP-1(c-Fos, Fra-1and Fra-2), and NFATcl in LPS-induced RAW264.7cells and RAW-OCLs was detected by Real-Time PCR; The detection of sinomenine on LPS-induced p65protein nuclear translocation as well as the impact of Ca2+influx were using by fluorescent assay.Results:1)Except1mM sinomenine intervention for5days (inhibition rate of approximately50%), sinomenine (0-1mM) had no significant effect on the viability of RAW264.7cells in5days.0.5mM and1mM sinomenine can significantly inhibit RANKL-induced OCs formation, survival and resorption in vitro.1mM sinomenine significantly destructed the actin ring structure of OCs in6h. Sinomenine has a dose-dependent inhibition of the genes expression of TRACP,MMP-9, c-Src, Integrin β3and cathepsin K in RANKL-induced RAW264.7cells.80mg/kg/day sinomenine blocking Mt-induced arthritic rat paw edema, reduce synovial sites of inflammation invasion and osteoclast activation, increased bone mineral density significantly increased bone parameters TMD, BMD, BV/TV, Tb.Th, Tb.N, reduce Tb.Sp, significantly reducing the content of serum RANKL, RANKL/OPG ratio and TRACP enzyme activity, the elevated OPG content and ALP activity.2) The mechanism study found that sinomenine had dose-dependent inhibition of RANKL-induced NF-κB, NFAT and AP-1reported gene expression. Sinomenine suppressed RANKL-induced NF-κB signaling pathway by inhibiting the degradation of IκB-α, translocation of p65into the nucleus and inhibition of the upstream molecules TRAF6expression. Sinomenine by selective inhibition of RANKL-induced JNK and p38phosphorylation and Ca2+influx, thereby inhibited the AP-1related molecules (c-Fos, Fra-1and Fra-2) and NFATcl gene expression and c-Fos protein expression.Sinomenine led DNA ladder in the RANKL-induced mature OCs, to0.5mM for 24h most significant. Sinomenine led to a large number of OCs nucleus typical fracture gathered and concentrated by Hochst33258staining, however the phenomenon was partially blocked by Ac-DEVD-CHO. It was found by Western blot sinomenine-induced activation of Caspase-3subunit expression, while through colorimetry method sinomenine significantly improve caspase-3enzyme activity of OCs, but can be completely blocked by Ac-DEVD-CHO3)0.5mM and1mM sinomenine significantly inhibit LPS-induced RAW-OCLs formation, survival and resorption in vitro.1mM sinomenine significantly undermine actin ring structure of OCLs within6h.. Sinomenine can significantly reduce TNF-a release of OCLs in a dose-dependent manner in vitro. Sinomenine showed a dose-dependent inhibition of TRACP, MMP-9, c-Src, Integrin β3and cathepsin K in LPS-induced RAW264.7cells and OCLs.100and50mg/kg sinomenine can significantly reduce the inflammation invasion area of LPS-induced mouse skull bone destruction model, osteoclast activation and TNF-a level in the serum.The mechanism study found that sinomenine inhibited LPS-induced NF-κB reporter gene expression in a dose-dependent manner. Sinomenine suppressed LPS-induced NF-κB signaling pathway via degradation of IκB-α, translocation of p65into the nucleus and inhibition of the upstream molecules TLR4and TRAF6expression. Sinomenine selective inhibited LPS-induced MAPK pathway via phosphorylation of p38and ERK. Sinomenine blocked LPS-induced RAW-OCLs Ca2+influx. These suggest that sinomenine may affect the downstream transcription factor AP-1and NFATc1. Further study found sinomenine significantly inhibited LPS-induced NFAT and AP-1reporter gene activation in a dose-dependent manner, inhibition of the related molecule c-Fos, Fra-1, Fra-2and NFATcl gene expression in RAW264.7cells and RAW-OCLs and inhibition of c-Fos protein expression levels in LPS-induced RAW264.7cells.Conclusion:1) Sinomenine suppressed RANKL-induced OCs formation and resorptive function in vitro.2) Sinomenine induced OCs apoptosis through activation of Caspase-3and destructs the actin ring structure of to inhibit OCs survival.3) Sinomenine attenuated OCs activation in Mt-induced arthritic rats, thereby reducing bone destruction and increasing bone density.4) suppressing upstream TRAF6, on the one hand,regulation of NF-κB signaling pathway inhibits nuclear transcription factor NF-κB activation; On the other hand, inhibition of phosphorylation of JNK and p38and Ca2+influx suppress the activation of AP-1and NFATc1; sinomenine final blocks OCs gene expression to inhibit activation of RANKL-induced OCs.5) Sinomenine inhibited LPS-induced RAW-OCLs formation, survival and resorptive function in vitro.6) Sinomenine blocked LPS-induced osteolysis in C57BL mouse calvarial model through inhibition of OCs activation and TNF-a.production.7) Sinomenine ultimately inhibited LPS-induced OCLs activation through regulation of upstream TLR4and TRAF6to affect NF-κB, MAPK signaling pathway and Ca2+influx, thereby suppressing the activation of NF-κB, AP-1and NFATc1, to result in blocking OCs related gene expression and the release of TNF-a.