Effect of gnetol on osteoporotic bone defect mice based on RANKL/RANK/TRAF6 and gut microbiota

Objective To explore the potential mechanism of gnetol in the treatment of osteoporotic bone defects and its effect on the gut microbiota.

Methods A total of 30 C57BL/6J mice were randomly divided into sham group, model group, alendronate sodium group, low-dose gnetol group, and high-dose gnetol group. An osteoporosis model was constructed by bilateral ovarian enucleation, and then a skull defect model was constructed using lipopolysaccharide (LPS) on this model. Micro-CT and hematoxylin-eosin (HE) staining were used to observe the bone mineral density and bone microstructure of the skull of mice, the expression of tumor necrosis factor- α (TNF-α) and C-terminal telopeptide of type Ⅰ collagen (CTX-I) in the serum was detected by enzyme-linked immunosorbent assay (ELISA), the expression of cathepsin K (CTSK) was detected by reverse transcriptionquantitative polymerase chain reaction (RT-qPCR), and the expression of NFATc1, c-fos, TNF-α, TRAP, CTSK, RANK, TRAF6, RANKL, p-p65 and p-IκBα was detected by western blotting. The abundance of gut microbiota in the sham, model and high-dose groups was detected by 16sRNA sequencing.

Results Compared with the sham group, the BMD and bone microstructure of the skull in the model group were significantly damaged. The protein expression levels of TRAP, CTSK, CTX-I, TNF-α, c-Fos, NFATc1, as well as those related to the osteoclast differentiation pathway (RANK, RANKL, TRAF6), inflammatory pathway (p-P65 and p-IκBα) were increased, and the expression of bone resorption marker CTSK gene was also increased. Moreover, the gut microbiota richness was increased, while the structure of the normal gut microbiota community was disrupted. Gnetol ameliorated impaired bone microarchitecture and bone loss caused by lipopolysaccharide (LPS) in osteoporotic states, inhibited the protein expression of TNF-α, c-Fos and NFATc1 which promote osteoclast differentiation, as well as the protein expression of the osteoclast differentiation pathways (RANK, RANKL, TRAF6) and inflammatory pathways (p-P65, p-IκBα), and reduced the gene expression of CTSK. It reduced the abundance of harmful bacteria such as Lachnospiraceae and Alistipes that have a negative impact on bone tissue, and increased the abundance of beneficial bacteria such as Erysipelothrichaceae, and Bifidobacteriaceae (including Bifidobacterium).

Conclusion Gnetol effectively ameliorates bone microstructural disruption and excessive bone resorption in the body due to LPS in osteoporotic states. It down-regulates the expression of the RANKL/RANK/TRAF6 signaling pathway, inhibits the activation of the NF-κB signaling pathway, and regulates the abundance of both beneficial and harmful bacteria.