Fig 1: R13 and anti-Rank-L antibody treatments display the similar effect on blocking trabecular bone loss induced by ovariectomy in WT female mice.Femoral bone structures were assessed by in vitro µCT in wild-type mice which were obtained ovariectomy at 12 weeks old, and some of which were treated with R13 (21.8 mg/kg, by oral gavage) or anti-RANK-L antibody. a Representive images of the femoral indices of trabecular bone structure measured by in vitro µCT scan. b µCT scanning measurements of BV/TV, Conn.D., SMI, Tb.N, Tb.Sp, Tb.Th. Data are shown as mean ± SEM, n = 8 mice per group, one-way ANOVA. c µCT scanning measurements of cortical bone Ct.Ar, Ct.Th and Ct.Ar/Tt.Ar. Data are shown as mean ± SEM, n = 8 mice per group, one-way ANOVA. d Serum levels of osteocalcin, CTX, RANK-L, OPG and RANK-L/OPG ratio. Data are shown as mean ± SEM, n = 6 mice per group, one-way ANOVA.
Fig 2: AEP knockout improves trabecular bone density in ovariectomy female mice.Femoral bone structures were assessed by in vitro µCT in AEP wild-type, AEP knockout (AEP KO) mice with or without ovariectomy. a Representative images of the femoral indices of trabecular bone structure measured by in vitro µCT scan. b µCT scanning measurements of trabecular bone volume fraction (BV/TV), connectivity density (Conn.D), structure model index (SMI), trabecular number (Tb.N), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th). Data are shown as mean ± SEM, n = 5 mice per group for AEP WT sham group, n = 6 mice per group for AEP KO group and n = 7 mice per group for AEP WT and AEP KO OVX group, one-way ANOVA. c µCT scanning measurements of cortical bone cortical area (Ct.Ar), average cortical thickness (Ct.Th) and relative cortical bone area to tissue area (Ct.Ar/Tt.Ar). Data are shown as mean ± SEM, n = 5 mice per group for AEP WT sham goup, n = 6 mice per group for AEP KO group and n = 7 mice per group for AEP WT and AEP KO OVX group, one-way ANOVA. d Serum levels of osteocalcin (a marker of bone formation), C-terminal telopeptide (CTX) (a marker of bone resorption), RANK-L, OPG, RANK-L/OPG ratio and serum BDNF level in wild-type and AEP knock out mice with and without ovariectomy. Data are shown as mean ± SEM, left to right: n = 5, 5, 7, 7 mice per group for osteocalcein and CTX measurement, n = 5 mice per group for BDNF, RANK-L, and OPG measurement, one-way ANOVA.
Fig 3: R13 treatment increases serum OPG level and blocks trabecular bone loss induced by ovariectomy in both WT and BDNF+/- female mice.Femoral bone structures were assessed by in vitro µCT in wild-type and BDNF+/- mice which were with (OVX) or without (sham) ovariectomy at 12 weeks old, and some of which administrated R13 (21.8 mg/kg) or vehicle for 8 weeks (6 days per week) by oral gavage. a Representative images of the femoral indices of trabecular bone structure measured by in vitro µCT scan in WT sham, BDNF+/- sham, WT OVX, BDNF+/- OVX, and WT OVX + R13, BDNF+/- OVX + R13 group. b µCT scanning measurements of BV/TV, Conn.D., SMI, Tb.N, Tb.Sp, Tb.Th. Data are shown as mean ± SEM, n = 8 mice per group (n = 7 mice for BDNF+/- sham group), one-way ANOVA. c µCT scanning measurements of cortical bone Ct.Ar, Ct.Th and Ct.Ar/Tt.Ar. Data are shown as mean ± SEM, n = 8 mice per group (n = 7 mice per group for BDNF+/- sham group), one-way ANOVA. d Serum levels of osteocalcin, CTX, RANK-L, OPG, RANK-L/OPG ratio, and serum BDNF level. Data are shown as mean ± SEM, n = 5 mice per group for BDNF measurement, n = 6 mice per group for RANK-L and OPG measurement, n = 7 mice per group for osteocalcin and CTX measurement, one-way ANOVA.
Fig 4: 7,8-DHF positively regulates OPG expression via activating CREB.a MC3T3 cells cultured in OIM were treated with 7,8-DHF in different time points. Western blotting showed that 7,8-DHF inhibited C/EBPß, increased Akt (S473), MAPK (p38), C-Jun, CREB phosphorylation. b Relative protein level of C/EBPß, p- C/EBPß, AEP, phosphorylated C-Jun, CREB, Akt, MAPK, and TrkB in MC3T3 cells treated with 7,8-DHF in different time points. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA. c Western blotting showed that knockdown of CREB blunted 7,8-DHF-induced OPG expression. d Relative protein levels of RANKL, OPG, and RANKL/OPG ratio. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA. e qPCR results showed that knockdown of CREB inhibited OPG mRNA expression induced by 7,8-DHF. Data are shown as mean ± SEM of three independent experiments, one-way ANOVA.
Fig 5: R13 and anti-RANK-L antibody treatments block the changes in bone turnover induced by ovariectomy in female mice.a Representative images of hematoxylin and eosin (H&E) staining of the distal femur bone in WT mice with sham, OVX, OVX + IgG, OVX + R13, and OVX + anti-RANK-L antibody group (n = 5 mice per group). (Scale bar, 500 µm). b Representative images of tartrate resistant acid phosphatase-stained (TRAP-stained) sections of the distal femur bone were shown at low magnification (upper panel) and higher magnification (lower panel) (n = 5 mice per group). (Scale bar, 500 µm (upper panels), 20 µm (lower panels)). d Representive calcein double-fluorescence labeling images of the trabecular bone (n = 6 mice per group) (Scale bar, 30 µm). d Histomorphometric indices of distal femur. N.Oc/BS and Oc.S/BS are indices of bone resorption. N.Ob/BS, Ob.S/BS, MAR, BFR/BS, and MS/BS are indices of bone formation. Data are shown as mean ± SEM, n = 6 mice per group, one-way ANOVA. e The schematic diagram of R13 treatment on osteoporosis via elevating OPG and inhibiting AEP via activating BDNF/TrkB signaling.
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