Effects of salvianolate on bone metabolism in glucocorticoid-treated lupus-prone B6.MRL-Fas^lpr/J mice

Figures & data

Table 1 Bone biomechanical properties and serum biochemical analyses of lupus mice undergoing different treatments

Group	WT group	Lupus group	GC group	Sal group	GC + Sal group
ULT (N)	20.68±2.25	14.32±1.96A	14.31±0.93A	16.87±2.03A,b,c	15.74±2.03A
YLD (N)	17.32±1.80	8.77±1.72A	8.49±0.58A	11.93+0.96A,b,c	10.61±2.32A
STIFF (N/mm)	138.29±14.6	50.5±5.33A	44.48±8.47A	68.82±10.01A,b,c	61.01±2.28A,b,c
TRACP 5b (U/L)	0.72±0.04	1.01±0.04A	1.05±0.06A	0.92±0.03A,B,C	0.96±0.02A,b,C
OPG (pg/mL)	209.31±27.04	77.85±12.77A	106.88±33.4A	208.8±34.8B,C	175.8±29.5B,C

Notes:

A P<0.01 vs WT;

b P<0.05 vs lupus;

B P<0.01 vs lupus;

c P<0.05 vs GC;

C P<0.01 vs GC. Values are presented as mean ± SD.

Abbreviations: GC, glucocorticoid; Sal, salvianolate; SD, standard deviation; STIFF, stiffness; TRACP 5b, tartrate-resistant acid phosphatase 5b; ULT, ultimate load; WT, wild-type; YLD, yield load; OPG, osteoprotegerin.

Figure 1 Representative micro-CT photographs of PTM in lupus mice undergoing different treatments.

Notes: The micro-CT photos were selected according to the mean BV/TV in different groups. Color images represent the thickness distribution of bone inside or outside the tibial metaphysis. Color changes from green to red indicate a gradual increase in bone thickness. The analysis area was located between 0 and 2 mm distal to the growth plate–epiphyseal junction. WT group, wild type mice; lupus group, lupus mice (B6.MRL-Fas^lpr/J); GC group, lupus mice with prednisone treatment; Sal group, lupus mice with salvianolate treatment; GC + Sal group, lupus mice with prednisone plus salvianolate treatment.
Abbreviations: BV, bone volume; GC, glucocorticoid; micro-CT, micro-computed tomography; PTM, proximal tibial metaphysis; Sal, salvianolate; TV, tissue volume.

Figure 2 Micro-CT analysis of proximal tibial metaphysis (PTM) and monitored body weight data of lupus mice undergoing different treatments.

Notes: (A) BV/TV, (B) Conn.D, (C) Tb.N, (D) SMI, (E) Tb.Th, (F) Tb.Sp, (G) BS/BV, (H) Body weight data for lupus mice within different treatment groups. WT group, wild type mice; lupus group, lupus mice (B6.MRL-Fas^lpr/J); GC group, lupus mice with prednisone treatment; Sal group, lupus mice with salvianolate treatment; GC + Sal group, lupus mice with prednisone plus salvianolate treatment. Values are presented as mean ± SD, ^aP<0.05 vs WT; ^AP<0.01 vs WT; ^bP<0.05 vs lupus; ^BP<0.01 vs lupus; ^cP<0.05 vs GC; ^CP<0.01 vs GC.
Abbreviations: BS/BV, bone surface to bone volume; BV/TV, bone volume to tissue volume; Conn.D, connectivity density; GC, glucocorticoid; micro-CT, micro-computed tomography; Sal, salvianolate; SD, standard deviation; SMI, structure model index; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.

Figure 3 Representative bone fluorescence micrographs of PTM (calcein fluorescence, magnification: 40×), osteoblast morphology (undecalcified sectioning with toluidine blue staining, magnification: 40×), and bone histomorphometry analysis in lupus mice undergoing different treatments.

Notes: The regions of interest for analyses were located between 1 and 3 mm distal to the growth plate–epiphyseal junction. (A) Representative bone fluorescence micrographs and histomorphometry analysis. (B) Representative toluidine blue staining images of PTM and histomorphometry analysis of osteoblast and osteoclast. WT group, wild-type mice; lupus group, lupus mice (B6.MRL-Fas^lpr/J); GC group, lupus mice with prednisone treatment; Sal group, lupus mice with salvianolate treatment; GC + Sal group, lupus mice with prednisone plus salvianolate treatment. Values are presented as mean ± SD. ^aP<0.05 vs WT; ^AP<0.01 vs WT; ^bP<0.05 vs lupus; ^BP<0.01 vs lupus; ^cP<0.05 vs GC; ^CP<0.01 vs GC; ^DP<0.01 vs Sal. Red arrows point to osteoblasts.
Abbreviations: BFR/BS, bone formation rate to bone surface; BV/TV, bone volume to tissue volume; GC, glucocorticoid; LBGR, longitudinal bone growth rate; MAR, mineral apposition rate; MS/BS, the ratio of mineralizing surface to bone surface; Ob.S/BS, osteoblast surface per unit of bone surface; Oc.S/BS, osteoclast surface per unit of bone surface; PTM, proximal tibial metaphysis; Sal, salvianolate; SD, standard deviation.

Figure 4 Immunoblotting analysis of RANKL and IL-6 expression in lumbar vertebrae and immunohistochemistry staining for IL-6 expression in the distal femur.

Notes: (A) Representative immunoblotting images of RANKL and IL-6. (B) RANKL expression. (C) IL-6 expression. (D) Immunohistochemistry staining for IL-6 expression, arrows indicate positive expression of IL-6. (scale 100 μm, magnification: 20×) (E) IL-6 expression in the distal femur. WT group, wild type mice; lupus group, lupus mice (B6.MRL-Fas^lpr/J); GC group, lupus mice with prednisone treatment; Sal group, lupus mice with salvianolate treatment; GC + Sal group, lupus mice with prednisone plus salvianolate treatment. Values are presented as mean ± SD. ^aP<0.05 vs WT; ^AP<0.01 vs WT; ^BP<0.01 vs lupus; GC; ^CP<0.01 vs GC; ^dP<0.05 vs Sal; ^DP<0.01 vs Sal.
Abbreviations: GC, glucocorticoid; IL, interleukin; RANKL, receptor activator of nuclear factor kappa-B ligand; Sal, salvianolate; SD, standard deviation.

Figure 5 Immunoblotting analysis of ROS, PPARγ, and RUNX2 in the lumbar vertebrae of lupus mice undergoing different treatments.

Notes: (A) Representative immunoblotting analysis images of ROS, PPARγ, and RUNX2; (B) RUNX2; (C) PPARγ; (D) ROS; WT group, wild type mice; lupus group, lupus mice (B6.MRL-Fas^lpr/J); GC group, lupus mice with prednisone treatment; Sal group, lupus mice with salvianolate treatment; GC + Sal group, lupus mice with prednisone plus salvianolate treatment. Values are presented as mean ± SD. ^AP<0.01 vs WT; ^BP<0.01 vs lupus; ^CP<0.01 vs GC; ^DP<0.01 vs Sal.
Abbreviations: GC, glucocorticoid; PPARγ, peroxisome proliferator activated receptor gamma; ROS, reactive oxygen species; RUNX2, runt-related transcription factor 2; Sal; salvianolate; SD, standard deviation.

Figure 6 Renal pathological changes in lupus mice undergoing different treatments (magnification: 10×, H&E stained).

Notes: (A) Quantification of the abnormal glomeruli ratio in each treatment group. (B) The region of interest of the renal cortex, in half of the renal section. (1×) Gray box/arrow shows the renal pathological analysis located in the region of renal cortex, in half of the renal section (C). White box/arrow shows the renal interstitial space (D). (C) Glomeruli in the renal cortex in half of the renal section. (D) Inflammatory cell invasion in the renal interstitial space. WT, wild-type mice; lupus, lupus mice (B6.MRL-Fas^lpr/J); GC group, lupus mice with prednisone treatment; Sal group, lupus mice with salvianolate treatment; GC + Sal group, lupus mice with prednisone plus salvianolate treatment. Values are presented as mean ± SD. ^BP<0.01 vs lupus.
Abbreviations: GC, glucocorticoid; H&E, hematoxylin and eosin; Sal, salvianolate; SD, standard deviation.