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. 2022 Mar 9:13:803880.
doi: 10.3389/fphar.2022.803880. eCollection 2022.

Mogrol Attenuates Osteoclast Formation and Bone Resorption by Inhibiting the TRAF6/MAPK/NF-κB Signaling Pathway In vitro and Protects Against Osteoporosis in Postmenopausal Mice

Yongjie Chen  1   2 , Linlin Zhang  1 , Zongguang Li  1 , Zuoxing Wu  3 , Xixi Lin  3 , Na Li  2   3 , Rong Shen  2 , Guojun Wei  1 , Naichun Yu  1 , Fengqing Gong  1 , Gang Rui  4 , Ren Xu  2   3   5 , Guangrong Ji  1   2
Affiliations

Mogrol Attenuates Osteoclast Formation and Bone Resorption by Inhibiting the TRAF6/MAPK/NF-κB Signaling Pathway In vitro and Protects Against Osteoporosis in Postmenopausal Mice

Yongjie Chen et al. Front Pharmacol. .

Abstract

Osteoporosis is a serious public health problem that results in fragility fractures, especially in postmenopausal women. Because the current therapeutic strategy for osteoporosis has various side effects, a safer and more effective treatment is worth exploring. It is important to examine natural plant extracts during new drug design due to low toxicity. Mogrol is an aglycon of mogroside, which is the active component of Siraitia grosvenorii (Swingle) and exhibits anti-inflammatory, anticancer and neuroprotective effects. Here, we demonstrated that mogrol dose-dependently inhibited osteoclast formation and function. To confirm the mechanism, RNA sequencing (RNA-seq), real-time PCR (RT-PCR), immunofluorescence and Western blotting were performed. The RNA-seq data revealed that mogrol had an effect on genes involved in osteoclastogenesis. Furthermore, RT-PCR indicated that mogrol suppressed osteoclastogenesis-related gene expression, including CTSK, ACP5, MMP9 and DC-STAMP, in RANKL-induced bone marrow macrophages Western blotting demonstrated that mogrol suppressed osteoclast formation by blocking TNF receptor-associated factor 6 (TRAF6)-dependent activation of the mitogen-activated protein kinase nuclear factor-B (NF-κB) signaling pathway, which decreased two vital downstream transcription factors, the nuclear factor of activated T cells calcineurin-dependent 1 (NFATc1) and c-Fos proteins expression. Furthermore, mogrol dramatically reduced bone mass loss in postmenopausal mice. In conclusion, these data showed that mogrol may be a promising procedure for osteoporosis prevention or therapy.

Keywords: MAPK; NF-κB; RANKL; TRAF6; mogrol; osteoclast; osteoporosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Mogrol has no toxic effect on BMMs. (A) Mogrol’s chemical structure. (B) After culturing for 48 or 96 h, a CCK-8 assay was conducted to study the impact of mogrol on BMM proliferation (n = 6). (C) Flow cytometry was utilized to analyze apoptosis of BMMs treated with 20 μM mogrol for 48 h. The histograms show the percentages of apoptotic and dead cells.
FIGURE 2
FIGURE 2
Mogrol attenuated RANKL-activated osteoclastogenesis by time- and dose-dependently. (A) BMMs cultured with 0, 5, 10, or 20 μM mogrol with the stimulation of 30 ng/ml RANKL (or not). After 5 days, these cells were fixed and stained with TRAP (n = 3). (B) TRAP-positive multinucleated (there or more) cells were regarded as mature osteoclasts. Quantity, area and size of mature osteoclasts were quantified and analyzed. (C) Images of TRAP staining of BMMs stimulated without (negative control group) or with RANKL and treated without (positive control group) or with 20 μM for 1, 3, and 5 days (n = 3). (D) Osteoclast number, osteoclast area, and osteoclast size were all quantified.
FIGURE 3
FIGURE 3
Mogrol inhibited the formation of the podosomal belt and bone resorption. (A) BMMs were cultivated with (or without) 20 μM mogrol with stimulation of 30 ng/ml RANKL. After 5 days, rhodamine-conjugated phalloidin was utilized to stain the podosomal belts of osteoclasts, and DAPI were used to label their nuclei (n = 3), scale (B) Images of the bone resorptive area captured by scanning electron microscopy. (n = 3). (C) ImageJ software was utilized to measure the resorption pit area and F-actin belt length. bar = 100 μm.
FIGURE 4
FIGURE 4
RNA-seq analysis and osteoclast marker gene expression examined by RT–PCR. Identification of genes that are regulated by mogrol. RNA was extracted from BMMs cultured with or without mogrol treatment for 5 days under RANKL stimulation. (A) Volcano plots of RNA-seq data in mogrol-treated BMMs compared to control BMMs. (B) The log2-fold change and p value of some osteoclastogenesis-related genes. (C,D) Gene Ontology. (C) GO terms are displayed by gene p values. (D) KEGG pathways in the differentially expressed genes are illustrated by gene numbers. (E) Mogrol inhibits osteoclastogenesis marker gene expression. The expression levels of ACP5, DC-STAMP, MMP9, CTSK and ATP6V0d2 were measured using RT–PCR.
FIGURE 5
FIGURE 5
Mogrol inhibited the RANKL-dependent TRAF6/NF-κB/MAPK signaling pathways. (A) The impact of mogrol on RANKL-induced activation of ERK, JNK, and p38. BMMs were starved for 4 h with or without 20 μM mogrol following activation with 50 ng/ml RANKL at various time points (0, 5, 10, 20, 30, or 60 min). Specific antibodies were employed to identify the total and phosphorylated forms of ERK, JNK, and P38. (B) Phosphorylated ERK, JNK, and P38 Gy levels were quantified and normalized to total proteins. (C) RANKL-induced NF-κB p65 phosphorylation and IκBα degradation after mogrol treatment. (D) Using ImageJ, the gray levels of p65 or IκBα were measured and normalized to GAPDH (n = 3). (E) BMMs were cultivated with or without mogrol for 0, 1, 3, and 5 days with the stimulation of RANKL. The proteins were identified with antibodies against NFATc1, c-Fos, TRAF6, Siglec-15, or GAPDH. (F,H) The relative expression of NFATc1, c-Fos, TRAF6 and Siglec-15 normalized to GAPDH was quantified by the gray level using ImageJ. (G) Nuclear translocation of P65 was determined by immunofluorescence staining using a p65 antibody and secondary antibody (red) and observed using a confocal fluorescence microscope. Scale bar = 50 μm.
FIGURE 6
FIGURE 6
Mogrol inhibited bone mass loss in OVX mice. (A) 3D reconstructions of images from micro-CT scans for femurs from sham, vehicle, and mogrol groups. (B) Bone parameter quantitative analysis, comprising BV/TV, BS/BV, Tb. Th, Tb. N, Tb. Sp, and Cs. th. (n = 6).
FIGURE 7
FIGURE 7
Mogrol relieved bone mass loss in OVX mice by suppressing osteoclast differentiation or activity. (A) Images of Von Kossa staining of the femurs. (B) Images of TRAP staining. (C) Quantitative analysis of BV/TV in Von Vossa, Oc. S/BS, and N. Oc/B in TRAP staining (n = 6).
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