不同频率的振动训练对大鼠早期膝骨关节炎关节软骨的修复作用及其JNK/NF-κB、SOX9机制*

目的: 探讨不同频率的振动训练对大鼠早期膝骨关节炎关节软骨的修复作用及其JNK/NF-κB、SOX9的机制。方法: 成年雄性SD大鼠48只,随机分为6组(n=8):模型对照组(MC组),高频振动1组(GP₁组,频率60 Hz),高频振动2组(GP₂组,频率40 Hz)、中频振动组(ZP组,频率20 Hz)和低频振动组(DP组,频率10 Hz),正常对照组(NC组)。除正常组外,各组大鼠经第1、4、7日双后腿膝关节腔注射2%木瓜蛋白酶溶液和L-半胱氨酸混合液6周后建立早期膝骨关节炎模型后,对振动组大鼠双膝进行4周,每天 40 min,振幅2~5 mm,每周振动5 d的训练。4周后,检测双膝关节股骨外侧髁关节软骨HE染色、番红O染色和Mankin评分形态学观察,股骨内侧髁关节软骨RT-qPCR检测JNK、NF-κB p65、SOX9 mRNA,Western blot 检测JNK、NF-κB p65、SOX9蛋白表达。结果: 与NC比较,其余各组Mankin评分均显著增高(P＜0.01),与MC相比,各振动组Mankin评分均显著降低(P＜0.05),其JNK、NF-κB p65mRNA和蛋白质表达显著降低(P＜0.01),SOX9mRNA和蛋白质表达显著升高(P＜0.01);与高频组相比,低频组的Mankin评分,JNK、NF-κB p65mRNA和蛋白质表达均显著降低(P＜0.05,P＜0.01),而SOX9mRNA和蛋白质表达则显著升高(P＜0.05,P＜0.01)。结论: 不同频率的振动训练对早期膝骨关节炎关节软骨可表现出不同程度的修复效应,低频振动训练软骨修复优于高频振动。可能通过下调关节软骨JNK/NF-κB表达,提高SOX9活性来调控胶原合成。     

Pseudolaric acid B ameliorates synovial inflammation and vessel formation by stabilizing PPARγ to inhibit NF-κB signalling pathway

Synovial macrophage polarization and inflammation are essential for osteoarthritis (OA) development, yet the molecular mechanisms and regulation responsible for the pathogenesis are still poorly understood. Here, we report that pseudolaric acid B (PAB) attenuated articular cartilage degeneration and synovitis during OA. PAB, a diterpene acid, specifically inhibited NF-κB signalling and reduced the production of pro-inflammatory cytokines, which further decreased M1 polarization and vessel formation. We further provide in vivo and in vitro evidences that PAB suppressed NF-κB signalling by stabilizing PPARγ. Using PPARγ antagonist could abolish anti-inflammatory effect of PAB and rescue the activation of NF-κB signalling during OA. Our findings identify a previously unrecognized role of PAB in the regulation of OA and provide mechanisms by which PAB regulates NF-κB signalling through PPARγ, which further suggest targeting synovial inflammation or inhibiting vessel formation at early stage could be an effective preventive strategy for OA.     

Adiponectin increases MMP-3 expression in human chondrocytes through AdipoR1 signaling pathway

Articular adipose tissue is a ubiquitous component of human joints, and adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. The adiponectin is significantly higher in synovial fluid of patients with osteoarthritis and rheumatoid arthritis. Matrix metalloproteinases (MMP)-3 may contribute to the breakdown of articular cartilage during arthritis. We investigated the signaling pathway involved in MMP-3 caused by adiponectin in human chondrocytes. Adiponectin increased the secretion of MMP-3 in cultured human chondrocytes, as shown by qPCR, Western blot, and ELISA analysis. Adiponectin-mediated MMP-3 expression was attenuated by AdipoR1 but not AdipoR2 siRNA. Pretreatment with 5'-AMP-activated protein kinase (AMPK) inhibitor (araA and compound C), p38 inhibitor (SB203580), and NF-κB inhibitor (PDTC and TPCK) also inhibited the potentiating action of adiponectin. Activations of p38, AMPK, and NF-κB pathways after adiponectin treatment were demonstrated. Taken together, our results provide evidence that adiponectin acts through AdipoR1 to activate p38 and AMPK, resulting in the activations of NF-κB on the MMP-3 promoter and contribute cartilage destruction during arthritis.     

Classical NF-kappaB activation negatively regulates noncanonical NF-kappaB-dependent CXCL12 expression

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.     

Immunohistochemical localization of osteogenetic protein (OP-1) and its receptors in rabbit articular cartilage.

We assessed the distribution and relative immunohistochemical staining intensity of the bone morphogenetic protein-7, osteogenic protein-1 (OP-1), in its pro- and mature forms, and four of its receptors, type I (ALK-2, ALK-3, and ALK-6) and type II in normal adolescent New Zealand White rabbit articular cartilage. Expression of the protein and its receptors was also examined in cartilage from joints that had been previously subjected to cartilage matrix degradation. Pro-OP-1 was moderately expressed in chondrocytes of the superficial, middle, and deep cartilage zones and in the osteocytes. The expression of mature OP-1 was similar, with the exception of less staining in the superficial zone of cartilage. Expression of these two forms of OP-1 was enhanced in the middle and deep cartilage zones after catabolic challenge. The type I receptor, ALK-6, displayed the strongest staining of the receptors in both cartilage and bone, whereas ALK-2 displayed the weakest staining. No differences were observed in the receptor staining levels after catabolic challenge. This study shows that OP-1 and its receptors have been identified in rabbit articular cartilage and bone, suggesting a possible role for this pathway in cartilage and bone homeostasis.     

Mechanical stress protects against osteoarthritis via regulation of the AMPK/NF-κB signaling pathway

Mechanical stress plays a key role in regulating cartilage degradation in osteoarthritis (OA). The aim of this study was to evaluate the effects and mechanisms of mechanical stress on articular cartilage. A total of 80 male Sprague-Dawley rats were randomly divided into eight groups (n = 10 for each group): control group (CG), OA group (OAG), and CG or OAG subjected to low-, moderate-, or high-intensity treadmill exercise (CL, CM, CH, OAL, OAM, and OAH, respectively). Chondrocytes were obtained from the knee joints of rats; they were cultured on Bioflex 6-well culture plates and subjected to different durations of cyclic tensile strain (CTS) with or without exposure to interleukin-1β (IL-1β). The results of the histological score, immunohistochemistry, enzyme-linked immunosorbent assay, and western-blot analyses indicated that there were no differences between CM and CG, but OAM showed therapeutic effects compared with OAG. However, CH and OAH experienced more cartilage damage than CG and OAG, respectively. CTS had no therapeutic effects on collagen II of normal chondrocytes, which is consistent with findings after treadmill exercise. However, CTS for 4 hr could alleviate the chondrocyte damage induced by IL-1β by activating AMP-activated protein kinase (AMPK) phosphorylation and suppressing nuclear translocation of nuclear factor (NF)-κB p65. Our findings indicate that mechanical stress had no therapeutic effects on normal articular cartilage and chondrocytes; mechanical stress only caused damage with excessive stimulation. Still, moderate biomechanical stress could reduce sensitization to the inflammatory response of articular cartilage and chondrocytes through the AMPK/NF-κB signaling pathway.     

CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Has Anti-Aging Effects That Protect Articular Cartilage from Age-Related Degenerative Changes

To examine the role of connective tissue growth factor CCN2/CTGF (CCN2) in the maintenance of the articular cartilaginous phenotype, we analyzed knee joints from aging transgenic mice (TG) overexpressing CCN2 driven by the Col2a1 promoter. Knee joints from 3-, 14-, 40-, and 60-day-old and 5-, 12-, 18-, 21-, and 24-month-old littermates were analyzed. Ccn2-LacZ transgene expression in articular cartilage was followed by X-gal staining until 5 months of age. Overexpression of CCN2 protein was confirmed through all ages in TG articular cartilage and in growth plates. Radiographic analysis of knee joints showed a narrowing joint space and other features of osteoarthritis in 50% of WT, but not in any of the TG mice. Transgenic articular cartilage showed enhanced toluidine blue and safranin-O staining as well as chondrocyte proliferation but reduced staining for type X and I collagen and MMP-13 as compared with those parameters for WT cartilage. Staining for aggrecan neoepitope, a marker of aggrecan degradation in WT articular cartilage, increased at 5 and 12 months, but disappeared at 24 months due to loss of cartilage; whereas it was reduced in TG articular cartilage after 12 months. Expression of cartilage genes and MMPs under cyclic tension stress (CTS) was measured by using primary cultures of chondrocytes obtained from wild-type (WT) rib cartilage and TG or WT epiphyseal cartilage. CTS applied to primary cultures of mock-transfected rib chondrocytes from WT cartilage and WT epiphyseal cartilage induced expression of Col1a1, ColXa1, Mmp-13, and Mmp-9 mRNAs; however, their levels were not affected in CCN2-overexpressing chondrocytes and TG epiphyseal cartilage. In conclusion, cartilage-specific overexpression of CCN2 during the developmental and growth periods reduced age-related changes in articular cartilage. Thus CCN2 may play a role as an anti-aging factor by stabilizing articular cartilage.     

Honokiol: an effective inhibitor of tumor necrosis factor-α-induced up-regulation of inflammatory cytokine and chemokine production in human synovial fibroblasts

In this study, we investigated the mechanisms underlying the anti-inflammatory effects of honokiol in tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis synovial fibroblasts (RASFs). RASFs pre-treated with honokiol (0-20 μM) were stimulated with TNF-α (20 ng/ml). The levels of prostaglandin E2 (PGE2), nitric oxide (NO), soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor-β1 (TGF-β1), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and Griess assay. In addition, protein expression levels of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and phosphorylated Akt, nuclear factor kappa B (NFκB), and extracellular signal-regulated kinase (ERK)1/2 were determined by western blot. The expression of NFκB-p65 was assessed by immunocytochemical analysis. TNF-α treatment significantly up-regulated the levels of PGE2, NO, sICAM-1, TGF-β1, MCP-1, and MIP-1α in the supernatants of RASFs, increased the protein expression of COX-2, iNOS, and induced phosphorylation of Akt, IκB-α, NFκB, and ERK1/2 in RASFs. TNF-α-induced expression of these molecules was inhibited in a dose-dependent manner by pre-treatment with honokiol. The inhibitory effect of honokiol on NFκB-p65 activity was also confirmed by immunocytochemical analysis. In conclusion, honokiol is a potential inhibitor of TNF-α-induced expression of inflammatory factors in RASFs, which holds promise as a potential anti-inflammatory drug.     

Exacerbation of arthritis by IL-1 in rat joints previously injured by peptidoglycan-polysaccharide

The arthropathic activity of mouse recombinant IL-1 (mrIL-1) after intraarticular (i.a.) injection into rat ankles was investigated. Nanogram quantities of either mrIL-1 alpha or mrIL-1 beta induced an acute transient arthritis. Arthritis induced by i.a. mrIL-1 developed more rapidly and was more severe in ankles previously injured by i.a. injection of group A streptococcal peptidoglycan-polysaccharide (PG-APS) fragments. In addition, a protracted pain response, as judged by severe limping, occurred 60 to 90 min after mrIL-1 injection into joints previously injured by PG-APS or 4 to 6 h after mrIL-1 injection into naive joints. The severity of arthritis was related to the mrIL-1 dose. Arthropathic activity of mrIL-1 alpha was neutralized by goat anti-mouse IL-1 alpha IgG, and the activity of both the alpha and beta preparations was heat labile. Repeated episodes of acute inflammation were induced by repeated i.a. injection of mrIL-1. In naive ankles this led to chronic synovitis without histologic evidence of erosions. However, in joints previously injured by PG-APS, repeated mrIL-1 injection induced a more severe chronic synovitis with a 50% incidence of early pannus formation and limited marginal erosions of cartilage and subchondral bone. Thus, mrIL-1 induces an acute exacerbation of arthritis in joints previously injured by PG-APS and repeated exposure of these joints to mrIL-1 promotes chronic erosive synovitis. These studies provide evidence for an in vivo function of IL-1 and are consistent with its role as one of the mediators in the local regulation of inflammation in recurrences of arthritis induced by bacterial cell wall polymers.     

Eosinophils attenuate arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway

Rheumatoid arthritis (RA) represents a type of autoimmune disease that mainly affect the joints due to persistent synovitis. Eosinophils were Th2 effector cells that have been shown to have anti-inflammatory role recently. In this study, we aimed to investigate the effects of eosinophils transfer on arthritis and underlying mechanisms. DBA/1 mice were induced with collagen-induced arthritis (CIA) and treated with purified eosinophils at different time points. We showed that eosinophils transfer attenuated arthritis in CIA mice. Meanwhile, TNF-α, IL-6, IL-12 and iNOS levels were decreased whereas TGF-β, IL-10, IL-13 and Arg1 levels were increased after eosinophil transfer. In vitro stimulation of bone marrow-derived macrophage (BMDM) with LPS and IFN-γ induced high expression of CD68, iNOS, TNF-α, IL-6, and IL-12, while treatment with eosinophils downregulated their expression levels. Furthermore, high levels of p-IκB and p-P38 expression in BMDM induced by LPS and IFN-γ could be suppressed by eosinophil treatment, and a P38 or IκB inhibitor accelerated the effect of eosinophils on macrophage polarization. Our results demonstrate that eosinophils exert anti-inflammatory effects in arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway.     

Macrophage-induced expression and release of matrix metalloproteinase 1 and 3 by human preadipocytes is mediated by IL-1β via activation of MAPK signaling

Obesity is associated with a chronic low‐grade inflammation and increased macrophage infiltration in adipose tissue. Matrix metalloproteinases (MMPs) are involved in adipose tissue remodeling and inflammatory responses in obesity. This study investigated whether macrophage‐derived factors modulate expression and secretion of MMP1 and MMP3 in human preadipocytes. The potential mediators and signaling pathways were also explored. MMP1 and MMP3 were primarily expressed and secreted by preadipocytes and dramatically reduced post‐differentiation. Preadipocytes were incubated with RPMI 1640 medium (control) or THP‐1 macrophage‐conditioned (MC) medium (25% and 100%) for 24 h. MC medium markedly increased mRNA levels of MMP1 (up to 122‐fold) and MMP3 (up to 59‐fold), as well as protein release of MMP1 (up to 378‐fold) and MMP3 (up to 10‐fold) in a dose‐dependent manner. Treatment with IL‐1β or TNFα, the major products of macrophages, also induced MMP1 and MMP3 secretion by preadipocytes. Neutralizing IL‐1β abolished the induction of MMP1 and MMP3 in preadipocytes by MC medium while the effects of TNFα neutralization were modest. Furthermore, MC medium or IL‐1β led to the phosphorylation of p38, ERK and JNK MAPKs. Inhibition of p38, ERK and JNK reversed the stimulatory effects of MC or IL‐1β on MMP1 and MMP3 production. MC medium and IL‐1β also activated NF‐κB p65 whereas reduced IκBα protein expression in preadipocytes. These results suggest that macrophage accumulation in adipose tissue has a central role in stimulating MMP1 and MMP3 production by preadipocytes, and this is partially mediated by IL‐1β via activation of the MAPK and NF‐κB signaling pathways. J. Cell. Physiol. 226: 2869–2880, 2011. © 2011 Wiley‐Liss, Inc.     

Blockade of TGF-β-activated kinase 1 prevents advanced glycation end products-induced inflammatory response in macrophages

Advanced glycation end products (AGEs), inflammatory-activated macrophages are essential in the initiation and progression of diabetic nephropathy (DN). TGF-β-activated kinase 1 (TAK1) plays a vital role in innate immune responses and inflammation. However, little information has been available about the effects of AGEs on the regulation of TAK1 expression and underlying mechanisms in AGEs-stimulated macrophage activation. We hypothesized TAK1 signal pathway in AGEs conditions could be a vital factor contributing to macrophage activation and inflammation. Thus, in the present study, we used bone marrow-derived macrophages (BMMs) to explore the functional role and potential mechanisms of TAK1 pathway under AGEs conditions. Results indicated that TAK1 played important roles in AGEs-induced mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B protein (NF-κB) activation, which regulated the production of monocyte chemo-attractant protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-α) in AGEs-stimulated macrophages. The results also suggested that TAK1 inhibitor (5Z-7-oxozeaenol) could inhibit AGEs-induced macrophage activation to down-regulate inflammatory cytokine production via MAPKs and NF-κB pathways, indicating that 5Z-7-oxozeaenol might be an immunoregulatory agent against AGEs-stimulated inflammatory response in DN.     

G-protein coupled receptor agonists mediate Neu1 sialidase and matrix metalloproteinase-9 cross-talk to induce transactivation of TOLL-like receptors and cellular signaling

The mechanism(s) behind GPCR transactivation of TLR receptors independent of TLR ligands is unknown. Here, GPCR agonists bombesin, bradykinin, lysophosphatidic acid (LPA), cholesterol, angiotensin-1 and -2, but not thrombin induce Neu1 activity in live macrophage cell lines and primary bone marrow macrophage cells from wild-type (WT) mice but not from Neu1-deficient mice. Using immunocytochemistry and NFκB-dependent secretory alkaline phosphatase (SEAP) analyses, bombesin induced NFκB activation in BMC-2 and RAW-blue macrophage cells, which was inhibited by MyD88 homodimerization inhibitor, Tamiflu, galardin, piperazine and anti-MMP-9 antibody. Bombesin receptor, neuromedin B (NMBR), forms a complex with TLR4 and MMP9. Silencing MMP9 mRNA using siRNA transfection of RAW-blue macrophage cells markedly reduced Neu1 activity associated with bombesin-, bradykinin- and LPA-treated cells to the untreated controls. These findings uncover a molecular organizational GPCR signaling platform to potentiate Neu1 and MMP-9 cross-talk on the cell surface that is essential for the transactivation of TLR receptors and subsequent cellular signaling.     

Pentraxin 3 regulated by miR-224-5p modulates macrophage reprogramming and exacerbates osteoarthritis associated synovitis by targeting CD32

Emerging evidence has shown an imbalance in M1/M2 macrophage polarization to play an essential role in osteoarthritis (OA) progression. However, the underlying mechanistic basis for this polarization is unknown. RNA sequencing of OA M1-polarized macrophages found highly expressed levels of pentraxin 3 (PTX3), suggesting a role for PTX3 in OA occurrence and development. Herein, PTX3 was found to be increased in the synovium and articular cartilage of OA patients and OA mice. Intra-articular injection of PTX3 aggravated, while PTX3 neutralization reversed synovitis and cartilage degeneration. No metabolic disorder or proteoglycan loss were observed in cartilage explants when treated with PTX3 alone. However, cartilage explants exhibited an OA phenotype when treated with culture supernatants of macrophages stimulated with PTX3, suggesting that PTX3 did not have a direct effect on chondrocytes. Therefore, the OA anti-chondrogenic effects of PTX3 are primarily mediated through macrophages. Mechanistically, PTX3 was upregulated by miR-224-5p deficiency, which activated the p65/NF-κB pathway to promote M1 macrophage polarization by targeting CD32. CD32 was expressed by macrophages, that when stimulated with PTX3, secreted abundant pro-inflammation cytokines that induced severe articular cartilage damage. The paracrine interaction between macrophages and chondrocytes produced a feedback loop that enhanced synovitis and cartilage damage. The findings of this study identified a functional pathway important to OA development. Blockade of this pathway and PTX3 may prevent and treat OA.Subject terms: Osteoarthritis, Extracellular signalling molecules     

Seomae mugwort and jaceosidin attenuate osteoarthritic cartilage damage by blocking IκB degradation in mice

Seomae mugwort, a Korean native variety of Artemisia argyi, exhibits physiological effects against various diseases. However, its effects on osteoarthritis (OA) are unclear. In this study, a Seomae mugwort extract prevented cartilage destruction in an OA mouse model. In vitro and ex vivo analyses revealed that the extract suppressed MMP3, MMP13, ADAMTS4 and ADAMTS5 expression induced by IL‐1β, IL‐6 and TNF‐α and inhibited the loss of extracellular sulphated proteoglycans. In vivo analysis revealed that oral administration of the extract suppressed DMM‐induced cartilage destruction. We identified jaceosidin in Seomae mugwort and showed that this compound decreased MMP3, MMP13, ADAMTS4 and ADAMTS5 expression levels, similar to the action of the Seomae mugwort extract in cultured chondrocytes. Interestingly, jaceosidin and eupatilin combined had similar effects to Seomae mugwort in the DMM‐induced OA model. Induction of IκB degradation by IL‐1β was blocked by the extract and jaceosidin, whereas JNK phosphorylation was only suppressed by the extract. These results suggest that the Seomae mugwort extract and jaceosidin can attenuate cartilage destruction by suppressing MMPs, ADAMTS4/5 and the nuclear factor‐κB signalling pathway by blocking IκB degradation. Thus, the findings support the potential application of Seomae mugwort, and particularly jaceosidin, as natural therapeutics for OA.     

Molecular mechanisms of the inhibitory effects of bovine lactoferrin on lipopolysaccharide-mediated osteoclastogenesis

Lactoferrin (LF) is an important modulator of the immune response and inflammation. It has also been implicated in the regulation of bone tissue. In our previous study we demonstrated that bovine LF (bLF) reduces LPS-induced bone resorption through a reduction of TNF-α production in vivo. However, it was not known how bLF inhibits LPS-mediated TNF-α and RANKL (receptor activator of nuclear factor κB ligand) production in osteoblasts. In this study we show that bLF impairs LPS-mediated TNF-α and RANKL production. bLF inhibited LPS-mediated osteoclastogenesis via osteoblasts in a co-culture system. Furthermore, bLF pretreatment inhibited LPS-induced NFκB DNA binding activity as well as IκBα and IKKβ (IκB kinase β) phosphorylation. MAP kinase activation was also inhibited by bLF pretreatment. However, bLF pretreatment failed to block the degradation of IRAK1 (interleukin-1 receptor-associated kinase 1), which is an essential event after its activation. Remarkably, we found that bLF pretreatment inhibited LPS-mediated Lys-63-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6). We also found that bLF is mainly endocytosed through LRP1 (lipoprotein receptor-related protein-1) and intracellular distributed bLF binds to endogenous TRAF6. In addition, bLF inhibited IL-1β- and flagellin-induced TRAF6-dependent activation of the NFκB signaling pathway. Collectively, our findings demonstrate that bLF inhibits NFκB and MAP kinase activation, which play critical roles in chronic inflammatory disease by interfering with the TRAF6 polyubiquitination process. Thus, bLF could be a potent therapeutic agent for inflammatory diseases associated with bone destruction, such as periodontitis and rheumatoid arthritis.