Mechanism Of Echinatin Inhibiting Osteoclast Differentiation And Preventing Bone Loss

Background and objectives: Osteoporosis is a degenerative systemic bone disease characterized by low bone mass and weak trabecular microstructure throughout the body,making the affected patients vulnerable to brittle fractures.Current anti-osteoporosis drugs can significantly reduce fractures,but some patients have serious side effects(osteonecrosis of the jaw,atypical femoral fractures)that have limited their long-term use due to safety concerns.Natural products play an important role in developing new anti-osteoporosis drugs with high safety,and have been considered as potential candidates for new therapies.Echinatin(Ecn),as the chemically active extract of common Chinese medicinal Radix et Rhizoma Glycyrrhizae,has the pharmacological effects of anti-oxidation,cardiovascular protection,anti-inflammation and anti-cancer.However,the effects of Ecn on osteoclast formation and function as well as on bone destruction have not been reported.Therefore,this study aimed to explore the effects of Ecn on RANKL-induced osteoclast differentiation and bone resorption,as well as its related molecular mechanisms,and whether it prevents bone loss in ovariectomized mice in vivo.The new effect and mechanism of Ecn on osteoclasts were then explored,which would provide a research basis for the development of new natural drugs to inhibit osteoclasts.Methods: 1.In vitro cell experiment: To verify the effects of Ecn on RANKL-induced osteoclastogenesis,bone resorption and related pathways.We used the CCK-8 test kit to verify the effects of different concentrations of Ecn on the proliferative activity of bone marrow mononuclear macrophages within 48 h to rule out the toxic effect of Ecn on osteoclast precursor cells.Next,RANKL and M-CSF were used to stimulate the BMMs to differentiate into osteoclasts,and different concentrations of Ecn were used for intervention.The formation of osteoclasts(the number of TRAP-positive cells with ≥3 nuclei)and the morphology of F-actin ring were observed by tartrate-resistant acidic phosphatase staining(TRAP staining)and rhodamine-phalloidin staining to verify the effect of Ecn on the formation of mature osteoclasts induced by RANKL.Moreover,in order to verify the effect of Ecn on the bone resorption of osteoclasts,we cultured osteoclasts on the bone slices and intervened with different concentrations of Ecn.The absorption pits on the surface of the bone slices were observed by scanning electron microscopy(SEM)and the area ratio of the absorption pits was counted to evaluate the effect of different concentrations of drugs on the bone resorption of osteoclasts.In order to further verify the molecular mechanism of Ecn affecting the formation and absorption of osteoclasts,we used different concentrations of Ecn to intervene RANKL-induced osteoclast differentiation,and determined osteoclast-related genes expression level by real-time quantitative PCR technology.Western-blot experiment was used to detect the effect of Ecn on RANKL-induced osteoclast differentiation-related signaling pathways(such as MAPK signaling pathway,NF-κB signaling pathway and AKT/GSK3β-c-Fos-NFATc1 signaling pathway).2.In vivo experiment: In order to verify the effect of Ecn on bone loss in OVX mice and its metabolic toxicity.Twenty-eight 10-week-old female mice were randomly divided into four groups: sham + vehicle group,OVX + vehicle group,OVX + Ecn(5 mg/kg)group,and OVX + Ecn(10 mg/kg)group.After six weeks of intervention treatment with Ecn,Micro-CT scanning and histopathological section were performed on the femur of mice to analyzed the related bone tissue parameters.Heart,liver,spleen and other major organs were analyzed by HE pathological section.Results: 1.In vitro: Ecn has no toxic effect on the proliferation of osteoclast precursor cells and can significantly inhibit osteoclast generation and bone resorption induced by RANKL.2.Ecn can decrease phosphorylation of MAPK signaling pathway ERK induced by RANKL,activation of AKT/GSK3β-c-Fos-NTATc1 pathway and expression of osteoclast differentiation related genes.3.In vivo: Ecn can prevent bone loss,bone density decrease and trabecular destruction in ovariectomized mice by inhibiting the number of osteoclasts in trabecular bone,and has no metabolic toxicity.Conclusion: Ecn inhibits osteoclast formation and bone resorption by down-regulating RANKL-induced ERK phosphorylation of MAPK signal pathway and activation of AKT/GSK3β-c-Fos-NTATc1 pathway,and prevent bone loss in ovariectomized mice.Ecn can be used as a new type of natural osteoclast inhibitor in the treatment of osteoclast-related osteolytic diseases.