Licochalcone B Inhibits Osteoclastogenesis Via Suppression Of RANKL-induced Signaling Pathway And Alleviates Hypoestrogen-induced Bone Loss In Ovariectomized Mice

In the aging population,especially postmenopausal women,bone mass and bone microarchitecture decrease rapidly,leading to the occurrence of osteoporosis and other osteolytic diseases.The essence of osteoporosis is the destruction of bone microstructures as a result of an imbalance between osteoblast-dominated bone formation and osteoclast-dominated bone resorption,primarily bone resorption.Therefore,the research on the formation and function of osteoclasts and the development of new therapeutic targets and drugs have become the focus of treatment for such osteolytic diseases.At present,drugs for osteoporosis such as bisphosphate and dinoseb monoclonal antibody have obvious therapeutic effects,but their side effects cannot be ignored.Licochalcone B(LCB),a flavonoid compound extracted from Radix et Rhizoma Glycyrrhizae,has been proved to have multiple biological effects,but it is rarely mentioned in bone metabolism.In previous research centers,we found that LCB remarkably inhibited the generation of osteoclasts,and the number of osteoclasts was negatively correlated with the LCB dose.In this study,we will further explore its deep mechanism and observe the effect of LCB in resisting estrogen deficiency-induced bone loss in mice.Objective: To investigate the mechanism of LCB intervention on osteoclastogenesis and osteoclast bone resorption in vitro,and to observe the effect of LCB on anti-estrogen deficiency-induced bone loss in mice in vivo.Methods: 1.C57BL/6 mice bone marrow mesenchymal stem cells(BMMSCS)were extracted and induced into bone marrow mononuclear macrophages(BMMS)by macrophage colony stimulating factor(M-CSF)and then induced by nuclear factor κB receptor activating factor ligand(RANKL)and intervene by LCB with different doses.Then the activity of LCB was observed by using the CCK-8 method,and the effect of LCB on osteoclastogenesis was observed by using the differentiation experiment,and the effect of LCB on bone resorption function of osteoclasts was observed by using the bone resorption experiment.2.Real-time fluorescent quantitative PCR(q RT-PCR)was used to determine the effect of LCB on the expression of specific genes of osteoclasts.Western Blot was used to verify the effects of the drugs on osteoclastogenesis and osteoclast functional signaling pathways.3.Based on the in vitro cell experiments,we constructed a mouse model of estrogen decline(OVX model)and evaluated the inhibitory effect of the drugs on osteoclasts and the therapeutic effect on osteoporosis caused by estrogen deficiency in vivo using immunohistochemistry and Micro-CT analysis.The metabolic toxicity of LCB in mice was analyzed by H&E staining on pathological sections of liver,kidney and small intestine.The mice were randomly divided into four groups(n = 6 per group): sham group,OVX/Vehicle group,low-dose group(Group L)and high-dose group(Group H).Results: 1.CCK-8 assay showed that LCB had no effect on the normal proliferative activity of BMMS cells.The differentiation experiments showed that different concentrations of LCB(2.5,5,7.5,and 10 μM)significantly inhibited the differentiation and formation of osteoclasts,and the number of osteoclasts was negatively correlated with LCB concentration(P < 0.001).The staining of Podsome Belt/ F-actin Ring in pseudopodosomes showed that different concentrations of LCB significantly inhibited the osteoclast nuclear fusion and the area of actin ring formation(P < 0.05).Bone resorption experiments showed that the intervention of LCB effectively inhibited the erosion and dissolution of bone fragments by osteoclasts(P < 0.001).The oxidative stress test(ROS)showed that LCB significantly inhibited the oxidative stress response during the formation and function of osteoclasts,and the difference in fluorescence flux data was statistically significant(P < 0.05).2.Real-time fluorescent quantitative PCR assay showed that LCB could effectively inhibit the specific expressions of CTSK,DC-Stamp,C-fos,TRAc P,NFATc1 and MMP-9 and other related genes in the process of osteoclastogenesis,and the differences were statistically significant(P < 0.001).At the same time,LCB also promoted the oxidative stress-related free radical scavenging factors(P < 0.05).Western Blot results showed that LCB inhibited the phosphorylation of JNK and ERK in the MAPK signaling pathway during osteoclast activation,and significantly inhibited the ubiquitination and P65 phosphorylation activation of IκB in the NFκB pathway(P < 0.05).At the same time,LCB significantly activated HO-1,Catalase and GSR in the ROS pathway(P < 0.05).3.In the in vivo experiment of mouse OVX model,Micro-CT analysis showed that LCB could reduce the bone loss of mouse femur caused by estrogen decline(P <0.05),namely,it increased the bone volume fraction(BV/TV),number of trabecular bones(Tb.N),connection density of trabecular bones(Conn.Dn)and thickness of trabecular bones(Tb.Th)of the mice after surgery.The same conclusion was reached by H&E staining of mouse femur tissue sections,and TRAc P staining of mouse femur showed that compared with the placebo group,there were significantly fewer osteoclasts in mice treated with LCB(P < 0.05).Pathological sections of the mouse liver,kidney,and small intestine did not reveal tissue abnormalities.Conclusions: 1.LCB at the experimental concentration has no effect on the proliferation activity of osteoclast precursor.LCB significantly inhibited osteoclastogenesis and bone resorption with a negative correlation.LCB inhibited the formation of Podsome Belt/ F-actin of osteoclasts and thus inhibited the dissolution and absorption function of bovine bone fragments by osteoclasts.2.LCB inhibits the differentiation and function of osteoclasts by inhibiting genes related to osteoclast formation and promoting the expression of oxidative stress response-related free radical scavengers such as HO-1,Catalase and GSR,thereby inhibiting the expression of ERK and JNK in the MAPK pathway as well as the ubiquitination and degradation of IκB in the NFκB pathway.3.LCB has a therapeutic effect on osteoporosis induced by estrogen deficiency in mice,and there is no metabolic toxicity.