Effects of icariin on the fracture healing in young and old rats and its mechanism

Figures & data

Figure 1. Icariin accelerated fracture healing in the rat model of fractures. Digital radiography (DR) was used to observe fracture healing on the 1st day, 10th day and 20th day.

Table 1. Lane–Sandhu’s scores of DR in each group.

Groups	Time (days)	n	Model	L-Icariin	M-Icariin	H-Icariin
Young	1	6	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	10	6	2.33 ± 0.52	3.33 ± 0.52^#	5.16 ± 0.41*^,#	5.50 ± 0.55*^,#
	20	3	4.67 ± 0.58	5.33 ± 0.58^#	8.33 ± 0.58*^,#	9.33 ± 0.58*^,#
Old	1	6	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	10	6	2.17 ± 0.41	2.33 ± 0.52	4.17 ± 0.41*	4.83 ± 0.41*
	20	3	4.00 ± 1.00	4.33 ± 0.58	5.00 ± 0.00*	5.67 ± 0.58*

* p< 0.05 vs. corresponding time’s model group.

# p< 0.05 vs. corresponding time’s old group.

Figure 2. HE staining images of the fracture site in the rat model of fractures. Magnification ×40 and ×400. Yellow arrow: trabecular bone. Red arrow: chondrocytes. Green arrow: osteoblasts.

Figure 3. Icariin activates the BMP-2/Smad5/Runx2 pathway in the rat model of fractures. (A) RT-qPCR was performed to detect the mRNA expression levels of Runx2, Osterix, BMP-2 and Smad5. GAPDH was used as the control. (B) The expression levels of Runx2, Osterix, BMP-2, p-Smad5 and t-Smad5 proteins were detected by Western blotting. (1) Model group, (2) L-icariin group, (3) M-icariin group and (4) H-icariin group. (C) The relative expression levels of Runx2, Osterix, BMP-2, p-Smad5 and t-Smad5 were calculated by normalizing to the levels of GAPDH. Data are represented as the means ± SD (n = 3). *p< 0.05, **p< 0.01 and ***p< 0.001 vs. the model group. ^#p< 0.05 vs. the corresponding L-icariin group.

Figure 4. Icariin induces the expression of osteocalcin in the rat model of fractures. (A) Immunohistochemistry to detect the expression of osteocalcin protein. (B) The MOD of osteocalcin expression was calculated using Image-Pro Plus 6.0 software. Data are represented as the means ± SD (n = 3). *p< 0.05, **p< 0.01 and ***p< 0.001 vs. the model group. ^#p< 0.05 vs. the corresponding young group.

Table 2. The concentrations of OC, BAP, NTX-1 and CTX-1 in our rat model of fractures on the 10th day (n = 3).

Groups	Markers	Model	L-Icariin	M-Icariin	H-Icariin
Young	OC (ng/mL)	1.78 ± 0.065	1.79 ± 0.067	1.83 ± 0.057	2.27 ± 0.064*^,#
	BAP (U/mg prot)	36.3 ± 1.57	41.05 ± 1.81	48.86 ± 1.10*	60.85 ± 2.03*^,#
	NTX-1 (ng/mL)	1.05 ± 0.057	1.15 ± 0.062	1.21 ± 0.062*	1.59 ± 0.071*^,#
	CTX-1 (ng/mL)	0.50 ± 0.041	0.55 ± 0.048	0.67 ± 0.052*	1.18 ± 0.067*^,#
Old	OC (ng/mL)	0.94 ± 0.058	1.42 ± 0.061*	1.51 ± 0.063*	1.86 ± 0.064*^,#
	BAP (U/mg prot)	22.45 ± 1.64	25.34 ± 2.15	32.69 ± 1.50*	49.65 ± 2.41*^,#
	NTX-1 (ng/mL)	0.71 ± 0.052	0.85 ± 0.049	0.95 ± 0.058*	1.20 ± 0.063*^,#
	CTX-1 (ng/mL)	0.28 ± 0.058	0.41 ± 0.053*	0.51 ± 0.055*	0.96 ± 0.053*^,#

Data are represented as the means ± SD (n = 3).

* p< 0.05 vs. model group.

# p< 0.05 vs. L-Icariin group.

Figure 5. Immunofluorescence identification of the rBMSCs isolated from young and old rats. (A) Immunofluorescence was used to identify the purity of rBMSCs using a fluorescently labelled antibody. (B) The percentage of CD29-positive, CD44-positive and CD45-negative rBMSCs from young and old rats. Magnification ×400. Data are represented as the means ± SD (n = 5).

Figure 6. The rBMSC viability (A, B) and ALP activity (C) assay of rBMSCs from young and old rats after icariin treatment. Data are represented as the means ± SD (n = 6). *p< 0.05; **p< 0.01 and ^#p< 0.05 vs. its control group.

Figure 7. Icariin activated the BMP-2/Smad5/Runx2 pathway in rBMSCs from young and old rats. (A) RT-qPCR was performed to detect the mRNA expression levels of Runx2, Osterix, BMP-2 and Smad5. GAPDH was used as the control. (B) The expression levels of Runx2, Osterix, BMP-2, p-Smad5 and t-Smad5 proteins were detected by Western blotting. (1) 5% icariin-control group, (2) 5% icariin-containing group, (3) 20% icariin-control group and (4) 20% icariin-containing group. (C) The relative expression levels of Runx2, Osterix, BMP-2, p-Smad5 and t-Smad5 were calculated by normalizing to the levels of GAPDH. Data are represented as the means ± SD (n = 4). *p< 0.05 vs. its icariin-control group. ^#p< 0.05 vs. the 5% icariin-containing group.