Normalisation of High Bone Remodelling due to Oestrogen Deficiency by Traditional Chinese Formulation Kang Shuai Lao Pian in Ovariectomised Rats

Abstract

Postmenopausal osteoporosis transpires due to excessive osteoclastic bone resorption and insufficient osteoblastic bone formation in the presence of oestrogen insufficiency. Kang Shuai Lao Pian (KSLP) is a red ginseng-based traditional Chinese medicine known for its anti-ageing properties. However, studies on its effect on bone loss are lacking. Thus, the current study examined the skeletal protective effects of KSLP in an ovariectomised rodent bone loss model. Three-month-old female Sprague Dawley rats (n=42) were randomised into baseline, sham and ovariectomised (OVX) groups. The OVX rats were supplemented with low- (KSLP-L; 0.15 g/kg), medium- (KSLP-M; 0.30 g/kg), high-dose KSLP (KSLP-H; 0.45 g/kg) or calcium carbonate (1% w/v). The daily supplementation of KSLP was performed via oral gavage for eight weeks. Gavage stress was stimulated in the ovariectomised control with distilled water. The rats were euthanised at the end of the study. Whole-body and femoral bone mineral content and density scans were performed at baseline and every four weeks. Blood samples were obtained for the determination of bone remodelling markers. Histomorphometry and biomechanical strength testing were performed on femurs and tibias. High bone remodelling typically due to oestrogen deficiency, indicated by the elevated bone formation and resorption markers, osteoclast surface, single-labelled surface and mineralising surface/bone surface ratio, was observed in the untreated OVX rats. Whole-body BMD adjusted to body weight and Young's modulus decreased significantly in the untreated OVX rats. High-dose KSLP supplementation counteracted these degenerative changes. In conclusion, KSLP improves bone health by normalising bone remodelling, thereby preventing bone loss and decreased bone strength caused by oestrogen deficiency. Its anti-osteoporosis effects should be validated in patients with postmenopausal osteoporosis.

Keywords: Traditional Chinese medicine; postmenopausal osteoporosis; skeletal health.

Figure 1

Body weight (A), fat mass (B), fat percentage (C), bone mineral content (D), bone mineral density (E) and whole-body bone mineral density/body weight (F) of each study group (n=6/group) during the experiment. The data are expressed as mean ± standard error mean. Mixed-design ANOVA is used to analyse the time and group differences. ^ap < 0.05 vs sham, ^bp < 0.05 vs OVX control, ^cp < 0.05 vs CaCO₃, ^ep < 0.05 vs KSLP-M within the same month; ^*p < 0.05 vs M0, ^#p < 0.05 vs M1 within the same group. Abbreviations: CaCO₃, calcium carbonate; KSLP-L, KSLP-low dose; KSLP-M, KSLP-medium dose; KSLP-H, KSLP-high dose; OVX, ovariectomised or negative; M0, month 0; M1, month 1; M2, month 2; %, percentage; BMD, bone mineral density.

Figure 2

Osteocalcin (A) and CTX-1 (B) concentration of each study group (n=6/group) before and after the experiment. The data are expressed as mean ± standard error mean. Mixed-design ANOVA is used to analyse the time and group differences. ^ap < 0.05 vs sham; ^bp < 0.05 vs OVX control; ^cp < 0.05 vs CaCO₃; ^dp < 0.05 vs KSLP-L; ^ep < 0.05 vs KSLP-M with the same time; ^*p < 0.05 vs before treatment in same group. Abbreviations: CaCO₃, calcium carbonate; KSLP-L, KSLP-low dose; KSLP-M, KSLP-medium dose; KSLP-H, KSLP-high dose; OT/BGP, Osteocalcin/Bone Gla protein; OVX, ovariectomised or negative; CTX-1, Type I collagen cross-linked c-telopeptide.

Figure 3

Micrograph of femur sections (100 × magnification) stained using the von Kossa method/silver nitrate (A-G) for each group (n=6/group). The structural histomorphometric indices of the femur evaluated are BV/TV (H), Tb.Th (I), Tb.N (J), and Tb.Sp (K). The quantitative data are expressed as mean ± standard error mean. The differences between groups are evaluated by one-way ANOVA with Tukey's post hoc test. Abbreviations: CaCO₃, calcium carbonate; KSLP-L, KSLP-low dose; KSLP-M, KSLP-medium dose; KSLP-H, KSLP-high dose; OVX, ovariectomised or negative; BV/TV, bone volume/total volume; Tb.Th., trabecular bone thickness; Tb.N, trabecular bone number; Tb.Sp., trabecular bone separation

Figure 4

Micrograph of H & E- (A-G) (100 × magnification) and TRAP-stained (H-N) (100 × magnification) femur sections for each study group (n=6/group). Static histomorphometric indices of femur bone evaluated are Ob.S/BS (O), Oc.S/BS (P), ES/BS (Q), OS/BS (R) and OV/BV (S). The data are expressed as mean ± standard error mean. The differences between groups are evaluated by one-way ANOVA with Tukey's post hoc test. ^ap < 0.05 vs baseline; ^bp < 0.05 vs sham; ^cp < 0.05 vs OVX control. Abbreviations: CaCO₃, calcium carbonate; KSLP-L, KSLP-low dose; KSLP-M, KSLP-medium dose; KSLP-H, KSLP-high dose; Ob.S/BS, osteoblast surface; Oc.S/BS, osteoclast surface; OVX, ovariectomised or negative; ES/BS, eroded surface; OS/BS, osteoid surface; OV/BS, osteoid volume; Ob, osteoclast; Oc, osteoclast; ES, eroded surface; OS, osteoid

Figure 5

Micrograph of calcein-labelled femur sections (A-G) (200 × magnification) for each study group (n=6/group); Dynamic histomorphometric indices of femur bone evaluated are sLS/BS (H), dLS/BS (I), MS/BS (J), MAR (K) and BFR (L). The data are expressed as mean ± standard error mean. One-way ANOVA with Tukey's post hoc test is used to compare the difference between groups. ^ap < 0.05 vs baseline; ^bp < 0.05 vs sham; ^cp < 0.05 vs OVX control. Abbreviations: CaCO₃, calcium carbonate; KSLP-L, KSLP-low dose; KSLP-M, KSLP-medium dose; KSLP-H, KSLP-high dose; OVX, ovariectomised or negative; sLS/BS, single-labelled surface; dLS/BS, doubled-labelled surface; MS/BS, mineralizing surface; MAR, mineral apposition rate; BFR, bone formation rate

Figure 6

Bone mechanical strength indices of each group (n=6/rats) evaluated based on three-point-bending test. The parameters generated are load (A), displacement (B), stiffness (C), stress (D), strain (E) and Young's modulus (F). The data are expressed as mean ± standard error mean. One-way ANOVA with Tukey's post hoc test is used to compare the difference between groups. ^bp < 0.05 vs sham. Abbreviations: CaCO₃, calcium carbonate; KSLP-L, KSLP-low dose; KSLP-M, KSLP-medium dose; KSLP-H, KSLP-high dose; OVX, ovariectomised or negative.