Part 2:Acteoside Suppresses RANKL-Mediated Osteoclastogenesis By Inhibiting C-Fos Induction And NF- KB Pathway And Attenuating ROS Production
Mar 05, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
Seung-Youp Lee1,2., Keun-Soo Lee3.¤, Sea Hyun Yi2., Sung-Ho Kook, Jeong-Chae Lee22,3*
Acteoside Inhibits Bone Resorption by Macrophages Acteoside also prevented RANKL-mediated bone resorption in a dose-dependent manner, as measured by an in vitro model system (Fig. 3A). Bone resorption was significantly inhibited when BMMs were incubated with 1 mM acteoside (Fig. 3B). A 10 mM acteoside treatment almost completely attenuated RANKL-induced pit formation by BMMs. Similarly, acteoside decreased bone resorption in RANKL-stimulated RAW264.7 cells (Figs. S2A and B). The ability of acteoside to inhibit bone resorption depended on the timing of the treatment relative to RANKL stimulation. Acteoside (10 mM) added 4 days after RANKL stimulation did not reduce pit formation in BMMs, whereas it suppressed the number of osteoclasts formed (Fig. 3C). This different result was in part due to the pit area already formed after 4 days of RANKL stimulation formation (Fig. 3D).
Acteoside Down-Regulates Early RANKL Signaling Pathways
RANKL induces the activation of 3 well-known MAPKs and NF-kB in osteoclast precursors, and this activation is required for early osteoclast differentiation. To understand the possible mechanisms by which acteoside inhibits osteoclastogenesis, we investigated the effect of acteoside on MAPKs and NF-kB activation in macrophages. BMMs and RAW264.7 cells were pretreated with 10 mM acteoside for 2 h and then stimulated with 100 ng/ml RANKL for 30 min. MAPK phosphorylation was examined by Western blotting and immunometric analysis. RANKL induced phosphorylation of p38, ERK, and JNK in BMMs (Fig. 4A) and RAW264.7 cells (Fig. 4B). Acteoside prevented these RANKL-induced increases in p-p38, p-ERK, and p-JNK. This result was supported by immunometric analysis, which that pretreatment with 10 mM acteoside significantly inhibited the levels of phosphorylated MAPKs in these macro- phages (Fig. 4C). RANKL treatment increased the DNA-binding of NF-kB, whereas acteoside inhibited RANKL-induced activation of NF-kB-DNA binding (Fig. 5A). This inhibition was more prominent in BMMs than in RAW264.7 cells. Acteoside also diminished RANKL-stimulated p65 and IkBa phosphorylation in BMMs and RAW264.7 cells (Figs. 5B and C). Adding 10 mM acteoside almost completely inhibited both the degradation and activation of IkBa in BMMs (Fig. 5B). To further confirm that NF- kB activation is involved in the action of acteoside, kB promoter-luciferase constructs were transiently transfected into RAW264.7 cells. The cells incubated with 100 ng/ml RANKL had 3-fold higher kB promoter activity, which was significantly attenuated by 10 mM acteoside (Fig. 5D).
Acteoside Suppresses the Production of Inflammatory Cytokines and the Expression of TNF-a, c-Fos, and NFATc1 in RANKL-Stimulated Macrophages TNF-a, IL-1b, and IL-6 are important in osteoclast formation and function, which is mediated by NF-kB signaling in RANKL-stimulated macrophages. RANKL stimulated the production of these cytokines, and this production was markedly reduced by 10 mM acteoside pretreatment in BMMs (Fig. 6A). Similarly, acteoside attenuated the RANKL-induced production of cytokines, except IL-6, in RAW264.7 macrophages (Fig. S3). To understand the molecular mechanisms of acteoside action in osteoclastogenesis, we further examined the effect of acteoside on TNF-a, c-Fos, and NFATc1 expression. RANKL up-regulated the mRNA expression of these factors in BMMs and RAW264.7 cells (Figs. 6B and C). Pretreatment with 10 mM acteoside significantly inhibited the RANKL-induced expression of these factors in both
BMMs and RAW264.7 cells. Acteoside pretreatment also strongly reduced the protein levels of c-Fos and NFATc1 in RANKL- stimulated BMMs (Fig. 6D). These results suggest that acteoside down-regulates RANKL inducing mediators of osteoclast formation at the gene and protein levels.
CISTANCHE EXTRACT: ACTEOSIDE
Acteoside Diminishes Intracellular ROS Generation in BMMs in a Dose-Dependent Manner
Since it is known that intracellular ROS production is correlated with RANKL-stimulated osteoclastogenesis, we investigated whether acteoside inhibits ROS production during RANKL-mediated osteoclast differentiation using a cell-permeable, oxidation-sensitive dye, DCFH-DA. Flow cytometry analysis showed that the mean fluorescence signal specific to DCF in BMMs was apparently right-shifted after stimulation with RANKL, compared to the untreated control cells (Fig. 7A). This shift was similar to the case that RAW264.7 macrophages were observed after RANKL stimulation (data not shown). Treating BMMs with acteoside reduced the signal intensity of the DCF in a dose-dependent manner. Pretreatment with 10 mM acteoside almost completely reduced the levels of intracellular ROS produced during osteoclast differentiation to the un-treated control levels (Fig. 7B).
Oral Acteoside Administration Inhibits Alteration of Osteoporotic Biochemical Markers and Bone Loss in Ovariectomized Mice
To explore the effect of acteoside on bone loss, we prepared an osteoporotic animal model by ovariectomy. There was no significant difference in body weight between OVX and Sham mice during the experimental period (data not shown). The OVX group had significantly higher serum levels of IL-1b and IL-6 than the Sham group (Fig. 8). Ovariectomy-induced increases in these inflammatory cytokines were attenuated by oral acteoside administration (AC group). The serum levels of bone turnover markers such as ALP, calcium, TRAP5b, and OC were significantly increased in OVX group. Of these osteoporotic markers, the increased levels of calcium, TRAP5b, and OC in OVX mice were apparently inhibited by acteoside treatment, whereas serum level of ALP was not changed by the treatment. The average maximum fracture load to the middle of the right femoral shaft was significantly lower in the OVX group than in the Sham group (Fig. 9A). Acteoside treatment raised the maximum fracture back up to that of the Sham group. When the cortical bone of the femur was dissected and observed by optic microscopy, the osteoporotic features shown in OVX group had almost completely disappeared in AC mice (Fig. 9B). To verify the effect of acteoside on OVX- induced osteoporosis model, BMD and bone morphologic parameters in trabecular of the light proximal femur were analyzed by micro-CT. As shown in Fig. 9C, an alteration of the femoral trabecular architecture was found in OVX mice, whereas this change was diminished by treatment with acteoside. The results from the micro-CT analysis revealed that BMD, a measure of bone strength, was dramatically reduced in OVX mice (Fig. 9D). Compared to the Sham group, OVX mice also showed significant changes in BV/TV, Tb. Sp, and Tb. N, but not in Tb.Th. Oral acteoside treatment of OVX mice significantly prevented the alteration in BMD as well as BV/TV and Tb.N.
Acteoside does not Affect Osteoblastogenesis in Bone Marrow Cells
The role of acteoside on osteoblastic differentiation was further investigated using bone marrow cells. As shown in Fig. 10A, DAG treatment increased the number of alizarin red-stained cells and this was not changed by 10 mM acteoside pretreatment. The amount of dye present indicated that the combined acteoside and DAG treatment did not change mineralization (Fig. 10B). Similarly, DAG-induced increases in the intracellular calcium content (Fig. 10C) and mRNA levels (Fig. 10D) of bone-specific markers, such as Runx2, osterix, BSP, and OC, were not affected by pretreatment with acteoside.
effect of cistanche acteoside
Discussion
Bone remodeling is tightly regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. Prolonged and excessive bone resorption causes an imbalance of skeletal turnover, resulting in bone-resorptive diseases. To explore the effects of acteoside on osteoclastogenesis, we used two macrophages, primary cultured BMMs and RAW264.7 cells.
These cells were stimulated with RANKL to differentiate into osteoclasts in the presence and absence of acteoside. We showed for the first time that acteoside inhibits osteoclast differentiation and formation. Acteoside itself at the concentrations examined did not cause a decrease of the viability of the primary cultured macrophages in both conditions of growth and differentiation.
Acteoside treatment also decreased the resorption activity of mature osteoclasts. These results suggest that acteoside suppresses osteoclastic formation from macrophages and osteoclast resorption activity. The results from our culture system, which did not include osteoblasts or stromal cells, also suggest that acteoside prevents osteoclast formation by directly acting on osteoclast precursors.
RANKL activates MAPKs including p38, ERK, and JNK. These three kinases are involved in early osteoclastic differentiation and thus their inhibition pharmacologically or with a dominant-negative JNK transfection suppresses RANKL-induced osteoclas- togenesis [29]. Our results revealed that acteoside pretreatment inhibited all these kinases, indicating a non-specific down-regulation of MAPKs. This result differed in part from the previous report that EGCG, the major anti-inflammatory compound in green tee, specifically attenuated JNK activation without affecting ERK or p38 activation in RANKL-stimulated BMMs [7]. Paeonol, an anti-inflammatory compound derived from a Chinese herb, has also been reported to inhibit ERK and p38, but not JNK, phosphorylation in RANKL-stimulated RAW264.7 cells [30]. In contrast, silibinin, a novel inhibitor in bone, attenuated RANKL-induced activation of p38, ERK, and JNK [31]. These findings suggest that the effects of anti-resorptive compounds on MAPK activation by RANKL depend on the compound, though all three MAPKs are involved in early osteoclastogenesis.
Considering the observation that acteoside attenuated p-JNK
levels in RANKL-stimulated BMMs, even at 1 mM, the blockage ofJNK rather than of p38 MAPK or ERK appeared to be more specific event in acteoside-mediated anti-osteoclastogenesis in the cells. Although acteoside at the same concentration did not reduce the number of osteoclasts in BMMs, there was a significant decrease in pit formation by acteoside treatment. We also found that pretreatment with SP600125, a pharmacological inhibitor specific to JNK, dramatically prevented the formation of osteoclasts (data not shown). Collectively, these findings suggest that JNK-mediated signaling is closely related to the acteoside- mediated suppression of osteoclastogenesis stimulated by RANKL.
NF-kB signaling regulates cellular events, including apoptosis, cell-cycle progression, cell adhesion, cytokine production, and survival in macrophages [32]. NF-kB signaling is also required for osteoclast development, which has been demonstrated by the appearance of osteopetrosis in NF-kB-knockout mice [33,34]. Therefore, inhibiting NF-kB is proposed to be an effective target for anti-resorptive agents to down-regulate osteoclast activity and treat osteoporosis. Post-translational modification of NF-kB subfamily proteins is crucial in modulating NF-kB activity. Especially, phosphorylation of the p65 subunit and IkB kinase is critical for NF-kB to induce osteoclastogenesis [7]. Our present findings showed that RANKL stimulation increased DNA binding activity of NF-kB and phosphorylation of the p65 subunit and IkBa in both BMMs and RAW264.7 cells. Pretreatment with acteoside inhibited these RANKL-induced increases, resulting in down-regulated NF-kB activity. Consequently, these results suggest that, in addition to MAPKs, NF-kB signaling is the main target of acteoside in inhibiting osteoclast differentiation and formation from RANKL-stimulated macrophages.
In addition to NF-kB signaling, the c-Fos/c-Jun/NFATc1 pathway plays key roles in osteoclast development, thus the lack of any of these proteins can arrest osteoclastogenesis [35,36]. In this study, we found that acteoside prevented the RANKL-induced c- Fos and NFATc1 expression at the mRNA and protein levels. JNK is an upstream kinase of c-Jun, which is required for NFATc1 expression and osteoclastogenesis in response to RANKL [29].
Blocking the the theJNK/c-Jun pathway with a junk inhibitor diminished RANKL-induced osteoclast formation and c-Fos and NFATc1 expression [7]. Our results and previous findings suggest that inhibiting JNK-mediated signaling by acteoside is closely associated with preventing RANKL-mediated c-Fos and NFATc1 expression, which suppresses osteoclast differentiation in macro- phages. Differences in the effects of acteoside on BMMs and RAW264.7 cells are at least in part due to differences in the sensitivity to JNK inhibition.
TNF-a can induce osteoclastogenesis independent of RANKL- RANK signaling [37]. IL-1 is a potent mediator of pathological bone destruction induced by estrogen deficiency or inflammation [7]. Disrupting the type I IL-1 receptor or IL-1 signaling can reverse bone loss induced by ovariectomy [38] or rheumatoid arthritis [39]. This study demonstrated the ability of acteoside to reduce the production of inflammatory cytokines such as TNF-a, IL- 1b, and IL-6 in macrophages. Acteoside is thought to inhibit inflammatory cytokine production by suppressing p38 kinase and ERK signaling, because activating ERK1/2, p38 MAPK, or both is required for lipopolysaccharide-induced production of these cytokines in macrophages [40,41]. Luteolin, an inflammatory compound, had also been reported to suppress the production of inflammatory mediators by inhibiting p38 MAPK activation [42]. In this study, we also found that acteoside attenuated bone loss in ovariectomized mice, as evidenced by the restored maximum fracture force at the midshaft of the right femur and the disappearance of osteoporotic cortical bone. Oral acteoside administration down-regulated the ovariectomized-induced in-creases in serum IL-1b and IL-6 levels, but not ALP. The increased serum levels of calcium, TRAP and OC in OVX were also inhibited by oral treatment with acteoside, suggesting that acteoside attenuates alteration of biomarkers specific for bone formation as well as resorption. As osteoporosis is characterized by a reduced mass density and deteriorated trabecular bone microarchitecture, OVX-induced trabecular bone loss and morphometric parameter alteration were significantly inhibited by oral acteoside administration. These findings suggest that acteoside may be used as an anti-resorptive agent to treat osteoporosis by reversing imbalanced osteoclast activation. However, osteoblasts are the primary factor responsible for new bone formation. Thus an agent able to increase osteoblast proliferation or differentiation is needed to enhance bone formation [30]. In contrast, we found that acteoside did not affect osteoblast differentiation or mineralization in DAG-treated bone marrow cells. Taken together, our results suggest that acteoside has an anti-resorption effect but does not directly affect bone formation. More detailed experiments analyzing bone-specific parameters in vivo and in vitro are needed to clarify whether or not acteoside benefits osteoblastogenesis.
The present study highlights the inhibitory effect of acteoside on osteoclast differentiation and bone resorption by suppressing MAPKs and several transcriptional factors such as NF-kB, c-Fos, and NFATc1. The data suggest two possible mechanisms by which acteoside has these benefits.
CISTANCHE ECHINACOSIDE AND ACTEOSIDE CAN Treat osteoporosis
References
1. RhoJ, Takami M, Choi Y (2004) Osteoimmunology: interactions of the immune and skeletal systems. Molecules and Cells 17: 1–9.
2. Del Fattore A, Capannolo M, Rucci N (2010) Bone and bone marrow: the same organ. Archives of Biochemistry Biophysics 503: 28–34.
3. Rachner TD, Khosla S, Hofbauer LC (2011) Osteoporosis: now and the future. Lancet 377: 1276–1287.
4. Sturge J, Caley MP, Waxman J (2011) Bone metastasis in prostate cancer: emerging therapeutic strategies. Nature Reviews Clinical Oncology 8: 357–368.
5. Goltzman D (2002) Discoveries, drugs, and skeletal disorders. Nature Reviews Drug Discovery 1: 784–796.
6. Rodan GA, Martin TJ (2002) Therapeutic approaches to bone diseases. Science 289: 1508–1514.
7. Lee JH, Jin H, Shim HE, Kim HN, Ha H, et al. (2010) Epigallocatechin-3- gallate inhibits osteoclastogenesis by down-regulating c-Fos expression and suppressing the nuclear factor-kappaB signal. Molecular Pharmacology 77: 17– 25.
8. Kim HN, Lee JH, Jin WJ, Ko S, Jung K, et al. (2012) MS-275, a benzamide histone deacetylase inhibitor, prevents osteoclastogenesis by down-regulating c-Fos expression and suppresses bone loss in mice. European Journal of Pharmacology 691: 69–76.
9. Kim T, Ha H, Shim KS, Cho WK, MaJY (2013) The anti-osteoporotic effect of Yijung-tang in an ovariectomized rat model mediated by inhibition of osteoclast differentiation. Journal of Ethnopharmacology 146: 83–89.
10. Nakanishi A, Litsuka N, Tsukamoto J (2013) Fish oil suppresses bone resorption by inhibiting osteoclastogenesis through decreased expression of M-CSF, PU.1, MITF and RANK in ovariectomized rats. Molecular Medicine Reports 7: 1896–1903.
11. Bar-Shavit Z (2007) The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell. Journal of Cellular Biochemistry 102: 1130– 1139.
12. Takahashi N, Maeda K, Ishihara A, Uehara S, Kobayashi Y (2011) Regulatory mechanism of osteoclastogenesis by RANKL and Wnt signals. Frontiers in Bioscience 16: 21–30.
