Vaspin Improves Postmenopausal Osteoporosis In Rats By Modulating Wnt/β-Catenin Signaling And OPG/RANKL Ratio

Objective :To investigate whether visceral adipose tissue-derived serine protease inhibitor(Vaspin)ameliorates postmenopausal osteoporosis(PMOP)in rats by modulating Wnt/β-catenin signaling and the OPG/RANKL ratio.Methods :Forty six-month-old SPF-grade female SD rats were selected and randomly divided into two groups: sham-operated group(Sham group,n=10)and ovariectomized group(OVX group,n=30),and the PMOP rat model was established by the dorsal bilateral ovariectomy method.At eight weeks ovariectomy,the success of the modeling was verified by measuring bone conversion markers and serum estradiol(E2)levels,bone mineral density,bone trabecular morphological structure,and bone morphometric parameters.After successful model identification,the rats in the successfully modeled OVX group were randomly divided into model control group(OVX group,n=6),Vaspin intervention group(OVX+Vaspin group,n=6),Vaspin+s FRP1(Wnt/β-catenin pathway inhibitor)intervention group(OVX+Vaspin+s FRP1 group,n=6),OVX+ Vaspin group was injected intraperitoneally with Vaspin solution(1000 ng/kg),OVX+Vaspin+s FRP1 group was injected intraperitoneally with Vaspin solution(1000 ng/kg)and s FRP1 solution(1000ng/kg),Sham and OVX groups were injected intraperitoneally with equal doses of saline.Rats in all groups were injected once daily for eight weeks of intervention.The changes of serum bone conversion markers: alkaline phosphatase(ALP)and c-terminal peptide of type1 collagen(CTX)levels in rats were detected using ELISA assay after the end of the intervention.Changes in bone mineral density of the rat femur were examined using small animal dual-energy x-ray.Morphological and structural changes in the trabecular bone of the rat femur were observed by pathological histological sections of the femur using HE staining and analyzed for changes in bone morphometric parameters: number of trabeculae(Tb.N),percent trabecular area(%Tb.Ar),trabecular separation(Tb.Sp).Changes in the number of osteoblasts and osteoclasts in the rat femur were analyzed using ALP staining and tartrate resistant acid phosphatase(TRAP)staining.Osteogenic and osteoclast-specific transcription factors: Runx2 and NFATc1 protein expression were detected using immunohistochemical assays.The expression of related proteins in the Wnt/β-catenin and OPG/RANK/RANKL pathways in rat tibia: β-catenin,Runx2,BSP,NFATc1,MMP9,OPG,RANKL were detected by western blot assay.Results :1.Eight weeks after ovariectomy,the results of ELISA showed that the serum E2 and ALP levels were significantly lower(both P<0.05)and CTX levels were significantly higher(P<0.05)in the OVX group compared with the Sham group.The results of bone mineral density assay showed that the bone mineral density was significantly lower(P<0.05)in the OVX group compared with the Sham group.The HE staining results showed that compared with the Sham group,the femoral trabeculae in the OVX group were disordered and sparse in structure,and Tb.N and %Tb.Ar were significantly lower(both P<0.05)and Tb.Sp was significantly increased(P<0.05),which proved that the PMOP model was successfully constructed.2.After the end of the Vaspin intervention,ELISA results showed that Vaspin administration increased serum ALP levels(P<0.05)and decreased serum CTX levels(P<0.05)in ovariectomized rats compared to the OVX group,suggesting that Vaspin promotes bone formation and inhibits bone resorption and enhances bone turnover.In contrast,the above effects of Vaspin were inhibited by the addition of the inhibitor s FRP1 compared with the OVX+Vaspin group(both P<0.05).3.At the end of the Vaspin intervention,bone mineral density assay results showed that Vaspin administration promoted bone mineral density recovery in rats with ovaries removed compared to the OVX group(P<0.05).In contrast,the improving effect of Vaspin on bone mineral density was inhibited by the addition of the inhibitor s FRP1 compared with the OVX+Vaspin group(P<0.05).4.At the end of the Vaspin intervention,HE staining showed that Vaspin administration promoted the morphological structure recovery of femoral trabeculae in rats with ovaries removed compared to the OVX group,with Tb.N and %Tb.Ar significantly increased(both P<0.05)and Tb.Sp significantly decreased(P<0.05),suggesting that Vaspin could improve bone microarchitecture to alleviate bone loss.In contrast,the above effects of Vaspin were inhibited by the addition of the inhibitor s FRP1 compared with the OVX+Vaspin group(all P<0.05).5.At the end of the Vaspin intervention,ALP staining and TRAP staining results showed an increase in ALP staining positive osteoblasts and a significant increase in Ob.S/BS(P<0.05),and a decrease in TRAP staining positive osteoclasts and a significant decrease in Oc.S/BS(P<0.05)in the femur of ovariectomized rats after Vaspin intervention treatment compared with the OVX group,suggesting that Vaspin could promote bone formation and inhibit bone resorption to mitigate bone loss.In contrast,the above effects of Vaspin were inhibited by the addition of the inhibitor s FRP1 compared with the OVX+Vaspin group(both P<0.05).6.At the end of the Vaspin intervention,immunohistochemical results showed that the expression of Runx2 protein in the femur of ovariectomized rats was significantly increased(P<0.05)and NFATc1 protein expression was significantly decreased(P<0.05)after Vaspin intervention treatment compared with the OVX group,suggesting that Vaspin could promote bone formation and inhibit bone resorption to alleviate bone loss.However,the above effects of Vaspin were inhibited by the addition of the inhibitor s FRP1 compared with the OVX+Vaspin group(both P<0.05).7.After the end of the Vaspin intervention,western blot results showed that compared with the OVX group,the expression of β-catenin,Runx2,BSP,and OPG protein was significantly increased(all P<0.05),NFATc1,MMP9,and RANKL protein expression was significantly decreased(all P< 0.05),and OPG/RANKL ratio was significantly increased(P<0.05),suggesting that Vaspin may alleviate bone loss by activating Wnt/β-catenin signaling and upregulating OPG/RANKL ratio to promote bone formation and inhibit bone resorption.However,these effects of Vaspin were inhibited by the addition of the inhibitor s FRP1 compared with the OVX+Vaspin group(all P<0.05).Conclusion :Vaspin may ameliorate PMOP in rats by activating Wnt/β-catenin signaling and upregulating the OPG/RANKL ratio to promote bone formation and inhibit bone resorption,providing a new theoretical basis for the prevention and treatment of PMOP.