Safranal inhibits estrogen-deficiency osteoporosis by targeting Sirt1 to interfere with NF-κB acetylation

AUTHORS

Sun-Ren Sheng, Yu-Hao Wu, Zi-Han Dai, Chen Jin, Gao-Lu He, Shu-Qing Jin, Bi-Yao Zhao, Xin Zhou, Cheng-Long Xie, Gang Zheng, Nai-Feng Tian

ABSTRACT

Background

Osteoporosis is a prevalent bone metabolic disease in menopause, and long-term medication is accompanied by serious side effects. Estrogen deficiency-mediated hyperactivated osteoclasts is the initiating factor for bone loss, which is regulated by nuclear factor-κB (NF-κB) signaling. Safranal (Saf) is a monoterpene aldehyde produced from Saffron (Crocus sativus L.) and possesses multiple biological properties, particularly the anti-inflammatory property. However, Saf's role in osteoporosis remains unknown.

Purpose

This study aims to validate the role of Saf in osteoporosis and explore the potential mechanism.

Study Design

The RANKL-exposed mouse BMM (bone marrow monocytes) and the castration-mediated osteoporosis model were applied to explore the effect and mechanism of Saf in vitro and in vivo.

Method

The effect of Saf on osteoclast formation and function were assessed by TRAcP staining, bone-resorptive experiment, qPCR, immunoblotting and immunofluorescence, etc. Micro-CT, HE, TRAcP and immunohistochemical staining were performed to estimate the effects of Saf administration on OVX-mediated osteoporosis in mice at imaging and histological levels.

Results

Saf concentration-dependently inhibited RANKL-mediated osteoclast differentiation without affecting cellular viability. Meanwhile, Saf-mediated anti-osteolytic capacity and Sirt1 upregulation were also found in ovariectomized mice. Mechanistically, Saf interfered with NF-κB signaling by activating Sirt1 to increase p65 deacetylation and inactivating IKK to decrease IκBα degradation.

Conclusion

Our results support the potential application of Saf as a therapeutic agent for osteoporosis.

MACF1 overexpression in BMSCs alleviates senile osteoporosis in mice through TCF4/miR-335–5p signaling pathway

AUTHORS

Kewen Zhang, Wuxia Qiu, Hui Li, Jun Li, Pai Wang, Zhihao Chen, Xiao Lin, Airong Qian

ABSTRACT

Background

The decreased osteogenic differentiation ability of mesenchymal stem cells (MSCs) is one of the important reasons for SOP. Inhibition of Wnt signaling in MSCs is closely related to SOP. Microtubule actin crosslinking factor 1 (MACF1) is an important regulator in Wnt/β-catenin signal transduction. However, whether the specific expression of MACF1 in MSC regulates SOP and its mechanism remains unclear.

Methods

We established MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-KI) mice, naturally aged male mice, and ovariectomized female mice models. Micro-CT, H&E staining, double calcein labeling, and the three-point bending test were used to explore the effects of MACF1 on bone formation and bone microstructure in the SOP mice model. Bioinformatics analysis, ChIP-PCR, qPCR, and ALP staining were used to explore the effects and mechanisms of MACF1 on MSCs’ osteogenic differentiation.

Results

Microarray analysis revealed that the expression of MACF1 and positive regulators of the Wnt pathway (such as TCF4, β-catenin, Dvl) was decreased in human MSCs (hMSCs) isolated from aged osteoporotic than non-osteoporotic patients. The ALP activity and osteogenesis marker genes (Alp, Runx2, and Bglap) expression in mouse MSCs was downregulated during aging. Furthermore, Micro-CT analysis of the femur from 2-month-old MSC-specific Prrx1 (Prx1) promoter-driven MACF1 conditional knock-in (MACF-cKI) mice showed no significant trabecular bone changes compared to wild-type littermate controls, whereas 18- and 21-month-old MACF1 c-KI animals displayed increased bone mineral densities (BMD), improved bone microstructure, and increased maximum compression stress. In addition, the ovariectomy (OVX)-induced osteoporosis model of MACF1 c-KI mice had significantly higher trabecular volume and number, and increased bone formation rate than that in control mice. Mechanistically, ChIP-PCR showed that TCF4 could bind to the promoter region of the host gene miR-335–5p. Moreover, MACF1 could regulate the expression of miR-335–5p by TCF4 during the osteogenic differentiation of MSCs.

Conclusion

These data indicate that MACF1 positively regulates MSCs osteogenesis and bone formation through the TCF4/miR-335-5p signaling pathway in SOP, suggesting that targeting MACF1 may be a novel therapeutic approach against SOP.

The translational potential of this article

MACF1, an important switch in the Wnt signaling pathway, can alleviate SOP through the TCF4/miR-335-5p signaling pathway in mice model. It might act as a therapeutic target for the treatment of SOP to improve bone function.

Abaloparatide has the same catabolic effects on bones of mice when infused as PTH (1-34)

AUTHORS

Carole Le Henaff Ph.D., Brandon Finnie B.S., Maria Pacheco M.S., Zhiming He M.S., Joshua Johnson M.S., Nicola C. Partridge Ph.D.

ABSTRACT

Abaloparatide is a peptide analog of parathyroid hormone-related protein (PTHrP 1-34) and was approved in 2017 as the second osteoanabolic peptide for treating osteoporosis. We have previously shown that intermittent abaloparatide is equally as effective as PTH (1-34). This study was designed to compare the catabolic effects of PTH (1-34) and abaloparatide on bone in young female wild-type mice. Two-month-old C57Bl/6J female mice were continuously infused with human PTH (1-34) or abaloparatide at 80  μg/kg BW/day or vehicle for 2 weeks. At euthanasia, DEXA-PIXImus was performed to assess bone mineral density (BMD) in the whole body, femurs, tibiae and vertebrae. Bone turnover marker levels were measured in sera, femurs were harvested for μCT analyses and histomorphometry, and tibiae were separated into cortical and trabecular fractions for gene expression analyses. Our results demonstrated that infusion of abaloparatide resulted in a similar decrease in BMD as infused PTH (1-34) at all sites. MicroCT and histomorphometry analyses showed similar decreases in cortical bone thickness and BMD associated with an increase in bone turnover from the increased bone formation rate found by in vivo double labeling and serum P1NP, and the increased bone resorption as shown by osteoclast numbers and serum CTX. Trabecular bone did not show major changes with either treatment. Osteoblastic gene expression analyses of trabecular and cortical bone revealed that infusion of PTH (1-34) or abaloparatide led to similar and different actions in genes of osteoblast differentiation and activity. As with intermittent and in vitro treatment, both infused PTH (1-34) and abaloparatide similarly regulated downstream genes of the PTHR1/SIK/HDAC4 pathway such as Sost and Mmp13, but differed for those of the PTHR1/SIK/CRTC pathway. Taken together, at the same dose, infused abaloparatide causes the same high bone turnover as infused PTH (1-34) with a net resorption in female wild-type mice.

A Novel RANKL-Targeted Furoquinoline Alkaloid Ameliorates Bone Loss in Ovariectomized Osteoporosis through Inhibiting the NF-κB Signal Pathway and Reducing Reactive Oxygen Species

AUTHORS

Puiian Wong, Zheng Lv, Jinglan Li, Qiushi Wei, LiangLiang Xu, Bin Fang, Yiwen Luo, and Mincong He

ABSTRACT

Dysregulation of osteoclast-osteoblast balance, resulting in abnormal bone remodeling, is responsible for postmenopausal osteoporosis (PMOP) or other secondary forms of osteoporosis. We demonstrated that dictamnine (DIC), a novel RANKL-targeted furoquinoline alkaloid, inhibits osteoclastogenesis by facilitating the activities of reactive oxygen species (ROS), NF-κB, and NFATc1 in vitro and prevents the development of OVX-induced osteoporosis mouse models in vivo. Methods. The docking mechanism of DIC and RANKL was initially identified by protein–ligand molecular docking. RNA sequencing was performed and analyzed to reveal the potential mechanism and signaling pathway of the antiosteoporosis effects of DIC. To verify the sequencing results, we examined the impact of DIC on RANKL-induced osteoclast differentiation, bone resorption, F-actin ring production, ROS generation, and NF-κB activation in osteoclasts in vitro. Moreover, a luciferase assay was performed to determine the binding and transcriptional activity of Nrf2 and NF-κB. The in vivo efficacy of DIC was assessed with an ovariectomy- (OVX-) induced osteoporosis model, which was analyzed using micro-CT and bone histomorphometry. Results. The molecular docking results indicated that DIC could bind particularly to RANKL. RNA-seq confirmed that DIC could regulate the osteoclast-related pathway. DIC suppressed osteoclastogenesis, bone resorption, F-actin belt formation, osteoclast-specific gene expression, and ROS activity by preventing NFATc1 expression and affecting NF-κB signaling pathways in vitro. The luciferase assay showed that DIC not only suppressed the activity of Nrf2 but also contributed to the combination of Nrf2 and NF-κB. Our in vivo study indicated that DIC protects against OVX-induced osteoporosis and preserves bone volume by inhibiting osteoclast activity and function. Conclusions. DIC can ameliorate osteoclast formation and OVX-induced osteoporosis and therefore is a potential therapeutic treatment for osteoporosis.

Bone-targeting delivery of platelet lysate exosomes ameliorates glucocorticoid-induced osteoporosis by enhancing bone-vessel coupling

AUTHORS

Gang Zheng, Hai-Wei Ma, Guang-Heng Xiang, Gao-Lu He, Han-Chen Cai, Zi-Han Dai, Yan-Lin Chen, Yan Lin, Hua-Zi Xu, Wen-Fei Ni, Cong Xu, Hai-Xiao Liu & Xiang-Yang Wang

ABSTRACT

Background

Glucocorticoids (GCs) overuse is associated with decreased bone mass and osseous vasculature destruction, leading to severe osteoporosis. Platelet lysates (PL) as a pool of growth factors (GFs) were widely used in local bone repair by its potent pro-regeneration and pro-angiogenesis. However, it is still seldom applied for treating systemic osteopathia due to the lack of a suitable delivery strategy. The non-targeted distribution of GFs might cause tumorigenesis in other organs.

Results

In this study, PL-derived exosomes (PL-exo) were isolated to enrich the platelet-derived GFs, followed by conjugating with alendronate (ALN) grafted PEGylated phospholipid (DSPE-PEG-ALN) to establish a bone-targeting PL-exo (PL-exo-ALN). The in vitro hydroxyapatite binding affinity and in vivo bone targeting aggregation of PL-exo were significantly enhanced after ALN modification. Besides directly modulating the osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs), respectively, PL-exo-ALN also facilitate their coupling under GCs’ stimulation. Additionally, intravenous injection of PL-exo-ALN could successfully rescue GCs induced osteoporosis (GIOP) in vivo.

Conclusions

PL-exo-ALN may be utilized as a novel nanoplatform for precise infusion of GFs to bone sites and exerts promising therapeutic potential for GIOP.

Sex-specific differences in Gsα-mediated signaling downstream of PTH1R activation by abaloparatide in bone

AUTHORS

Srilatha Swami, Joshua Johnson, Lawrence Vecchi, Matthew Kim, Beate Lanske, Rachelle Johnson, Joy Wu

ABSTRACT

Teriparatide, recombinant parathyroid hormone (PTH[1-34]), and abaloparatide, an analogue of PTH related-peptide (PTHrP[1-34]), are both anabolic medications for osteoporosis that target the PTH receptor PTH1R. PTH1R is a G protein-coupled receptor, and the stimulatory Gs protein is an important mediator of the anabolic actions of PTH1R activation in bone. We have published that mice lacking the α subunit of Gs in osteoprogenitors do not increase bone mass in response to PTH[1-34]. Unexpectedly, however, PTH[1-34] still increases osteoblast numbers and bone formation rate in male mice, suggesting that PTH1R may have both Gs-dependent and independent actions in bone. Here we examine the role of Gs signaling in the anabolic actions of abaloparatide. We find that abaloparatide increases bone formation in male mice with postnatal deletion of Gsα in Osx-expressing osteoprogenitors (P-GsαOsxKO mice), but not in female P-GsαOsxKO mice. Therefore, abaloparatide has anabolic effects on bone in male but not female mice that appear to be independent of Gs-mediated signaling.