A Mixture of Humulus japonicus Increases Longitudinal Bone Growth Rate in Sprague Dawley Rats

Abstract

The aim of this study was to investigate the effects of administration of a mixture of Humulus japonicus (MH) on longitudinal bone growth in normal Sprague Dawley (SD) rats. We measured the femur and tibia length, growth plate area, proliferation of chondrocytes, and expression of insulin-like growth factor-1 (IGF-I) and IGF binding protein-3 (IGFBP-3), and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) phosphorylation after dietary administration of MH in SD rats for four weeks. The nose-tail length gain and length of femur and tibia increased significantly in the group that received MH for a period of four weeks. We performed H&E staining and Bromodeoxyuridine/5-Bromo-2'-Deoxyuridine (BrdU) staining to examine the effect of dietary administration of MH on the growth plate and the proliferation of chondrocytes and found that MH stimulated the proliferation of chondrocytes and contributed to increased growth plate height during the process of longitudinal bone growth. In addition, serum levels of IGF-1 and IGFBP-3 and expression of IGF-1 and IGFBP-3 mRNAs in the liver and bone were increased, and phosphorylation of JAK2/STAT5 in the liver was increased in the MH groups. Based on these results, we suggest that the effect of MH on longitudinal bone growth is mediated by increased JAK2/STAT5-induced IGF-1 production.

Keywords: IGF-1; IGFBP-3; growth plate; longitudinal bone growth.

Figure 1

The effect of dietary administration of a mixture of H. japonicus (MH) on femur and tibia lengths in SD rats. ND (normal diet control): AIN93M diet; growth hormone (GH): AIN93M diet + intraperitoneal injection of 0.37 mg/kg b.w./day recombinant growth hormone; MH50: AIN 93G diet supplemented with MH 50 mg/kg b.w.; MH150: AIN 93G diet supplemented with MH 150 mg/kg b.w.; MH300: AIN 93G diet supplemented with MH 300 mg/kg b.w. Values are presented as means ± SD. Different letters indicate a significant difference with p < 0.05 as determined by Duncan’s multiple range test.

Figure 2

The effect of dietary administration of a mixture of H. japonicus (MH) on architectural changes of tibia trabecular and cortical in SD rats. ND (normal diet control): AIN93M diet; GH: AIN93M diet + intraperitoneal injection of 0.37 mg/kg b.w./day recombinant growth hormone; MH50: AIN 93G diet supplemented with MH 50 mg/kg b.w.; MH150: AIN 93G diet supplemented with MH 150 mg/kg b.w.; MH300: AIN 93G diet supplemented with MH 300 mg/kg b.w.

Figure 3

The effect of dietary administration of a mixture of H. japonicus (MH) on the number of chondrocytes in SD rats. ND (normal diet control): AIN93M diet; GH: AIN93M diet + intraperitoneal injection of 0.37 mg/kg b.w./day recombinant growth hormone; MH50: AIN 93G diet supplemented with MH 50 mg/kg b.w.; MH150: AIN 93G diet supplemented with MH 150 mg/kg b.w.; MH300: AIN 93G diet supplemented with MH 300 mg/kg b.w. Values are presented as means ± SD. Different letters indicate a significant difference with p < 0.05, as determined by Duncan’s multiple range test.

Figure 4

The effect of dietary administration of a mixture of H. japonicus (MH) on growth plate in SD rats. Representative example of the proximal end of the tibia stained with H&E (A), growth plate (B) resting zone (C), proliferating zone (D), and hypertrophic zones (E). ND (normal diet control): AIN93M diet; GH: AIN93M diet + intraperitoneal injection of 0.37 mg/kg b.w./day recombinant growth hormone; MH50: AIN 93G diet supplemented with MH 50 mg/kg b.w.; MH150: AIN 93G diet supplemented with MH 150 mg/kg b.w.; MH300: AIN 93G diet supplemented with MH 300 mg/kg b.w. Values are presented as means ± SD. Different letters indicate a significant difference with p < 0.05, as determined by Duncan’s multiple range test.

Figure 5

The effect of dietary administration of a mixture of H. japonicus (MH) on serum insulin-like growth factor-1 (IGF-1) (A), serum IGF binding protein-3 (IGBBP-3) (B), IGF-1 mRNA in the liver (C), IGFBP-3 mRNA in the liver (D),protein expression of IGF-1, IGFBP-3, JAK2, p-JAK2, STAT5, and p-STAT5 in the liver (E), and IGF-1, IGFBP-3, and bone morphogenetic protein 2 (BMP-2) expression in bone (F). ND (normal diet control): AIN93M diet; GH: AIN93M diet + intraperitoneal injection of 0.37 mg/kg b.w./day recombinant growth hormone; MH50: AIN 93G diet supplemented with MH 50 mg/kg b.w.; MH150: AIN 93G diet supplemented with MH 150 mg/kg b.w.; MH300: AIN 93G diet supplemented with MH 300 mg/kg b.w. Values are presented as means ± SD. Different letters indicate a significant difference with p < 0.05, as determined by Duncan’s multiple range test.