Research On The Effect And Mechanism Of Urolithin B Inhibits Osteoclastogenesis And Attenuates Ovariectomy-induced Bone Loss

Background: osteoporosis(OP)is a systematic bone disease caused by multiple reasons,characterized by decreased bone density and destruction of the microstructure of bone tissue,resulting in increased bone fragility and susceptibility to fracture.Osteoporotic fracture is a serious complication of OP and is a major cause of disability and death in older patients.OP has become a major public health problem in the world and is receiving high attention.Excessive absorption and differentiation of OCs are the main pathological causes of osteoporosis.Urolithin B(UB),as a decomposition product of ellagic acid,has higher stability and bioavailability.UB has potential anti-inflammatory,antioxidant and anti-atherosclerotic biological activities,and its development has attracted the attention of many researchers.Objectives:(1)Receptor activator of nuclear factor-κB ligand(RANKL)induced RAW264.7 cells to establish mature osteoclast model in vitro.To investigate the effects of UB on osteoclast formation,differentiation and molecular mechanisms.(2)To establish an animal model of osteoporosis in ovariectomized rats,and to detect the morphological effect of UB on the bone loss model in ovariectomized rats.Methods:(1)In vitro experiments: RANKL(40 ng/m L)independently induce differentiation of mononuclear macrophage RAW264.7 into mature multinuclear osteoclasts,and different concentrations of UB(0,10,30,50,100 μM)were added for intervention.The effects of different concentrations of UB on osteoclast differentiation and bone resorption were evaluated by tartrate-resistant acid phosphatase(TRAP)staining and cytoskeletal fibrous actin(F-actin)ring staining.The effects of UB on molecules related to NF-κB,MAPK and ROS signaling pathway during osteoclast differentiation and the expression of specific genes in osteoclast differentiation were detected by Western blotting and RT-PCR.DCFH-DA staining was used to detect the effect of UB at different concentrations on the expression of ROS during osteoclast differentiation.(2)In vivo experiments: In vivo verification was carried out through animal experiments.The animal model of osteoporosis was established using ovariectomized rats as the subject of the study.Thirty-two female SD rats were randomly divided into sham operation group,OVX+ same dose of saline group,OVX+ low-dose UB group(OVX+20 mg/kg UB)and OVX+ high-dose UB group(OVX+60 mg/kg UB).At the end of the experiment,morphological evaluation of bone parameters was performed by Micro-CT scanning of the femur of each group,and histological evaluation was performed by H&E staining of the distal femur.To investigate the effect of UB on osteoporosis in ovariectomized rats.Results:(1)The RANKL-induced mature multinuclear osteoclasts model was established.Morphologically,UB inhibited RANKL-induced osteoclast differentiation and actin ring formation in a dose-dependent manner,especially at 100μM concentration.Mechanically,UB suppresses the phosphorylation of key signaling molecules of NF-κB,MAPK and ROS in the downstream of osteoclast differentiation induced by RANKL and attenuates nuclear translocation of the NF-κB p65 subunit.Furthermore,UB inhibited the expression of osteoclast-specific genes,including TRAP,CTSK,OC-STAMP,MMP-9,c-Fos,and NFATc1.In addition,UB can inhibit ROS production in osteoclasts and has antioxidant properties.(2)To establish rat model of osteoporosis induced by ovariectomy.Compared with the ovariectomized group,the UB group was able to increase bone loss caused by ovariectomized,increase bone density,densely-arranged bone trabeculae in a relatively orderly way,and significantly improve the damaged structure.Conclusion: Urolithin B,the most biologically active intestinal metabolite of ellagitannin,has powerful anti-inflammatory and antioxidant effects.In vitro experiments,UB has shown potent anti-osteoporosis properties in both in vitro cell models and in vivo animal tests.UB can inhibit osteoclast differentiation and function by inhibiting RANKL-induced activation of NF-κB,MAPKs and ROS,reducing the expression of TRAP,CTSK,OC-STAMP,MMP-9,c-Fos and NFATc1.UB could attenuate nuclear translocation of the NF-κB p65 subunit,prevent ROS production,and ultimately inhibit osteoclast differentiation and maturation.In vivo experiments show that UB improved bone loss induced by ovary removal,increased bone density enhance the microstructure of trabecular bone.UB may be a novel therapeutic strategy for osteoporosis and osteolysis-related diseases.