Morroniside Inhibits Inflammatory Bone Loss through the TRAF6-Mediated NF-κB/MAPK Signalling Pathway

Abstract

Osteoporosis is a chronic inflammatory disease that severely affects quality of life. Cornus officinalis is a Chinese herbal medicine with various bioactive ingredients, among which morroniside is its signature ingredient. Although anti-bone resorption drugs are the main treatment for bone loss, promoting bone anabolism is more suitable for increasing bone mass. Therefore, identifying changes in bone formation induced by morroniside may be conducive to developing effective intervention methods. In this study, morroniside was found to promote the osteogenic differentiation of bone marrow stem cells (BMSCs) and inhibit inflammation-induced bone loss in an in vivo mouse model of inflammatory bone loss. Morroniside enhanced bone density and bone microstructure, and inhibited the expression of IL6, IL1β, and ALP in serum (p < 0.05). Furthermore, in in vitro experiments, BMSCs exposed to 0-256 μM morroniside did not show cytotoxicity. Morroniside inhibited the expression of IL6 and IL1β and promoted the expression of the osteogenic transcription factors Runx2 and OCN. Furthermore, morroniside promoted osteocalcin and Runx2 expression and inhibited TRAF6-mediated NF-κB and MAPK signaling, as well as osteoblast growth and NF-κB nuclear transposition. Thus, morroniside promoted osteogenic differentiation of BMSCs, slowed the occurrence of the inflammatory response, and inhibited bone loss in mice with inflammatory bone loss.

Keywords: TRAF6-mediated MAPK/NF-κB signaling pathway; inflammation; morroniside; osteogenic differentiation; osteoporosis.

Figure 1

(A) Representative micro-CT images of left femurs. (B) Representative HE staining images. Scale bar = 100 μm. (C−F) Three-dimensional BMD, BV/TV, Tb.Th, and Tb.Sp. (G−I) Biomechanical test results for stiffness, maximum deflection, and maximum load. (J−M) IL-1β, TNF-α, ALP, and TRAP levels in mice sera. Data are represented as the mean ± SD of three experiments. ** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.001 versus LPS group. Abbreviations: ALN, sodium alendronate.

Figure 2

Morroniside inhibits the osteogenic differentiation of BMSCs by inhibiting the TRAF6-mediated NF-κB/MAPK signaling pathway in vivo. (A) TRAF6 expression was detected using immunohistochemistry. (B) Positive-expression area of TRAF6 in femur tissue. (C−E) TRAF6, OCN, and Runx2 mRNA expression levels in tibia tissue. (F) The expression of TRAF6-mediated expression of NF-κB/MAPK signaling-pathway-related proteins (p-p65, TRAF6, p-ERK, and p-p38) were detected using Western blotting assays. (G−I) The levels of TRAF6, p-p65, and p-p38 were quantified using Image J and normalized to GAPDH. (J) The expression of the osteogenic differentiation proteins Runx2 and OCN was detected using Western blotting assays. (K−M) The levels of p-Erk, Runx2, and OCN were quantified using Image J and normalized to GAPDH. Data are presented as the mean ± SD of three experiments. ** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.001 versus LPS group. Abbreviations: ALN, sodium alendronate.

Figure 2

Morroniside inhibits the osteogenic differentiation of BMSCs by inhibiting the TRAF6-mediated NF-κB/MAPK signaling pathway in vivo. (A) TRAF6 expression was detected using immunohistochemistry. (B) Positive-expression area of TRAF6 in femur tissue. (C−E) TRAF6, OCN, and Runx2 mRNA expression levels in tibia tissue. (F) The expression of TRAF6-mediated expression of NF-κB/MAPK signaling-pathway-related proteins (p-p65, TRAF6, p-ERK, and p-p38) were detected using Western blotting assays. (G−I) The levels of TRAF6, p-p65, and p-p38 were quantified using Image J and normalized to GAPDH. (J) The expression of the osteogenic differentiation proteins Runx2 and OCN was detected using Western blotting assays. (K−M) The levels of p-Erk, Runx2, and OCN were quantified using Image J and normalized to GAPDH. Data are presented as the mean ± SD of three experiments. ** p < 0.01 versus control group; ^# p < 0.05, ^## p < 0.001 versus LPS group. Abbreviations: ALN, sodium alendronate.

Figure 3

Morroniside promotes the proliferation and osteogenic differentiation of BMSCs and delays the occurrence of inflammation. (A) Chemical structure of morroniside. (B) The proliferation of BMSCs was determined at 48 h using CCK8 assays. (C–F) Identification of BMSCs with CD90, CD29, CD34, and CD45 using flow cytometry (FCM) (G–J) Cell morphology was observed under an inverted microscope. (K,L)The levels of IL-1β and TNF-α in the supernatant of BMSCs were determined using ELISA. (M) Osteogenesis was determined using ALP staining. (N) Alizarin red staining results. Data are presented as mean ± SD of three experiments. ** p < 0.01 versus control group; ^## p < 0.01 versus LPS group.

Figure 4

Morroniside inhibits the osteogenic differentiation of BMSCs by inhibiting the TRAF6-mediated NF-κB/MAPK signaling pathway in vitro. (A) The expression of osteogenic differentiation proteins Runx2 and OCN was detected using Western blotting. (B,C) The expression of Runx2 and OCN was quantified using Image J (https://imagej.nih.gov/ij/) and normalized to GAPDH. (D) The expression of TRAF-mediated NF-κB/MAPK signaling-pathway-related proteins (p-p65, p65, TRAF6, p-Erk, Erk, p-p38, and p38) was detected using Western blotting assays. (E−H) TRAF6, p-p65/p65, p-ERK/ERK, and p-p38/p38 were quantified using Image J and normalized to GAPDH. (I) immunofluorescence staining of NF-κB p65 in BMSCs, with counterstaining using DAPI, as indicated (100 μm). Data are presented as mean ± SD of three experiments. ** p < 0.01 versus control group; ^## p < 0.01 versus LPS group.