Study On The Mechanism Of Puerarin Inhibiting Postmenopausal Osteoporosis Through Wnt4/β-catenin Signaling Pathway

Objective: Osteoporosis has become a global public health problem.The efficacy of puerarin in the treatment of osteoporosis has been confirmed,but the mechanism remains unclear.Wnt/β-catenin signaling pathway is essential for bone development and bone homeostasis.Osteoclasts are the only cells with bone resorption function in bone tissue.This study:(1)The osteoporosis mouse model was established by dorsally bilateral ovariectomy.Puerarin was used to intervene the osteoporosis mouse model to observe the improvement effect of puerarin on osteoporosis mice,and to explore the mechanism of Wnt/β-catenin signaling pathway in osteoporosis mice.(2)Mouse bone marrow mononuclear macrophages were cultured and induced to form osteoclasts,and puerarin intervention was used to investigate the regulatory effect of puerarin on the Wnt4/β-catenin signaling pathway in osteoclasts.Through animal and cell experiments,the molecular mechanism of puerarin inhibiting osteoporosis through Wnt4/β-catenin signaling pathway was revealed,in order to provide theoretical basis for clinical application of puerarin.Methods:(1)Ten female C57BL/6 mice without specific pathogen(SPF)were randomly divided into two groups,one group was ovariectomized(OVX)group and the other group was Sham operation group(Sham),Serum biochemistry was used to detect the contents of calcium,phosphorus,magnesium and ALP in serum,three-dimensional reconstruction of Micro CT scan,HE staining of pathological tissue,TRAP/ALP staining and immunofluorescence staining were used to detect TRAP protein expression.(2)Eighteen pathogen-free(SPF)grade female C57BL/6 mice were randomly divided into Sham operation group(Sham-NS),ovariectomized normal group with saline(OVX-NS),and ovariectomized group with puerarin(OVX-Pu),with 6 mice in each group.C57BL/6 mice model of osteoporosis was established by dorsal bilateral ovariectomy.After successful identification of the model,the ovaries were not removed in Sham-NS group,and normal saline was injected intraperitoneally.Bilateral ovaries were removed in OVX-NS group,and normal saline was injected intraperitoneally.OVX-Pu group was intraperitoneally injected with 2.5mg/100μL/kg puerarin solution after ovary removal for 12 weeks/day,and the behavioral changes of mice were observed every day.The changes of calcium,phosphorus,magnesium,alkaline phosphatase(ALP)and three biochemical indexes of bone metabolism(parathyroid hormone,calcitonin and osteocalcin)were detected by serum biochemical assay.Histopathological staining and imaging were used to detect the pathological changes of femur tissue in mice;Real-time PCR was used to detect m RNA relative expression levels of Wnt4,β-catenin,C-myc,Cyclin D1,s FRP2,TCF1 and other related indexes in bone tissues before and after drug intervention.The protein expressions of Wnt4,β-catenin,Cyclin D1,HNF and s FRP2 in femur tissues were determined by immunohistochemical staining.(3)Bone marrow mononuclear macrophages of mice were extracted and cultured to form osteoclasts.After intervention with puerarin,the formation and proliferation activity of osteoclasts were observed by TRAP(Tartrate resistant acid phosphatase,TRAP)staining,and the expression of TRAP and Wnt4 protein was determined by immunofluorescence technique.Results:(1)A mouse model of osteoporosis could be established 8 weeks after dorsal bilateral ovariectomy.(2)Mice in the OVX-Pu group and Sham-NS group had normal activities,good diet and shiny hair,while mice in the OVX-NS group were prone to hair loss,dark hair color,decreased activity,prolonged reflex,increased excretion,and less diet and water.(3)Serum biochemical tests showed that the content of ALP in OVX-Pu group was higher than that in OVX-NS group,the difference was statistically significant.The changes of calcium,phosphorus,magnesium and bone metabolism were all improved,but the difference was not obvious.(4)Micro CT scanning of femoral tissue showed that the number and thickness of bone trabecula and the thickness of bone plate decreased after ovary removal,it showed a trend of improvement after puerarin intervention.(5)After the intervention of puerarin,fat vacuoles in the medullary cavity decreased,the medullary cavity became smaller,and vascular hyperplasia was obvious.(6)Real time PCR results showed that after 12 weeks of puerarin intervention,the m RNA relative expression levels of Wnt4,β-catenin,TCF1,Cyclin D1 and C-myc in bone tissue of OVX-Pu group were higher than those of OVX-NS group,and the expression level of s FRP2 in OVX-NS group was more than that of OVX-Pu group,the difference was statistically significant.(7)After puerarin intervention,the expression of Wnt4,β-catenin,Cyclin D1 and HNF positive particles increased,but the difference was not significant.(8)Bone marrow primary mononuclear macrophages were induced and cultured to form osteoclasts by Macrophage colony-stimulating factor(M-CSF)and nuclear factor-kappa B receptor activating factor(RANKL).TRAP staining and immunofluorescence detection after puerarin intervention showed that TRAP expression was significantly decreased in the 50μg/ml puerarin concentration group,while Wnt4 expression was increased(compared with that in the M-CSF+RANKL group),the difference was statistically significant.Conclusion:(1)The mouse model of postmenopausal osteoporosis can be successfully established by bilateral dorsal ovariectomy,the comprehensive evaluation method could objectively reflect the success or failure of the mouse model of osteoporosis.(2)Puerarin can inhibit osteoporosis through Wnt4/β-catenin signaling pathway,and it may promote the expression of Wnt4/β-catenin signaling pathway factors Wnt4,β-catenin,TCF1,Cyclin D1 and C-myc in osteoporosis bone tissue by inhibiting the expression of s FRP2,thus delaying the occurrence and development of osteoporosis.(3)Puerarin can inhibit the proliferation of osteoclasts by promoting the expression of Wnt4.