Arg, avidin-peroxidase, and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-mouse IGF-I antibody,biotinylated anti- mouse IGF-I antibody, and recombinant mouse IGF-I were purchased from R＆D Systems (Minneapolis, MN, USA). Phosphorylated (p)STAT5 antibody was purchased from Invitrogen Corp. (Camarillo, CA, USA). STAT5 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Male ICR mice (4 weeks old) and diets were purchased from Dae-Han Experimental Animal Center (Eumsung, Republic of Korea), acclimated for 7 days, and then randomly assigned for 2 weeks to adequate protein (CON, 20% protein) or low protein diet (PEM, 4% protein) (Reeves et al., 1993). The protein source used was casein. Except for the protein content, the two diets were identical and isocaloric (Table 1). After 2 weeks, mice were divided into five groups, CON (adequate protein diet + distilled water (DW)-administered group); PEM (low protein diet + DW-administered group); KBS (low protein diet + KBSadministered group); Arg (low protein diet + Arg-administered group); Glu (low protein diet + Glu-administered group). The mice were fed indicated diet, administered each material three times a week for 12 weeks, housed four to six per cage in a laminar air-flow room, and maintained at a temperature of 22 ± 1℃, a relative humidity of 55 ± 1% throughout the study. The research was conducted in accordance with internationally accepted principles for laboratory animal use and care as found in US guidelines (NIH publication #85-23, revised in 1985).

KBS obtained from the Kyungheebaroker Clinic of Korea. Traditional Medicine (Seoul, Republic of Korea) consisted of 15 Korean medicinal drugs (Table 2). Among 15 drugs, Hominis Placenta extract is the most abundant component in KBS. UNICENTA^®(UNIMED PHARM, INC., Seoul, Republic of Korea) was substitubed for Hominis Placenta extract, because UNICENTA^® is a manufactured medicine using Hominis Placenta collected from hospital. UNICENTA^® contains Glu and Arg (manufacturer’s specifications). Dilutions were made in distilled water then filtered through a 0.45-μm syringe filter.

To measure the level of IGF-I in serum, a modified sandwich ELISA method was used (Moon and Kim, 2011; 2012). In brief, 100 μl aliquots of anti-mouse IGF-I monoclonal antibody in PBS were coated in 96-well microplate. The plate was incubated overnight at 4℃, washed with PBS containing 0.05 % tween-20 (Sigma, St. Louis, MO, USA), and blocked for 1 h with PBS containing 1% BSA, 5% sucrose and 0.05% NaN₃. After washing the plates again, avidin-peroxidase was added and the plate was incubated for 30 min at 37℃. The well was again washed and TMB substrate (Pharmingen) was added. Color developmentwasmeasured at 405 nm using an automated microplate ELISA reader. A standard curve was run on the plate using recombinant mouse IGF-I in serial dilutions.

Using an easy-BLUE^TM RNA extraction kit (iNtRON Biotech, Republic of Korea), total RNA was isolated from liver tissues according to the manufacturer’s specifications, as previously described (Han et al., 2011; Moon et al., 2012). The concentrations of total RNA in the final elutes were determined by a spectrophotometer. Total RNA (1 μg) was heated at 65℃ for 10 min and then chilled on ice. Each sample was reversetranscribed to cDNA for 90 min at 37℃ using a cDNA synthesis kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The PCR was performed with the following primer for mouse IGF-I (5’ CCG GAC CAG AGA CCC TTT G3’; 5’ CCT GTG GGC TTG TTG AAG TAA AA3’); GAPDH (5’GGC AAA TTC AAC GGC ACA3’; 5’ GTT AGT GGG GTC TCG CTC CTG3’). Quantitative real-time PCR was performed using a SYBR Green master mix and the detection of mRNA was analyzed using an ABI StepOne real-time PCR System (Applied Biosystems, Foster City, CA, USA). The level of the target mRNA was normalized to the level of the GAPDH and compared with the control. All data were analyzed using the ΔΔCT method.

The supernatants of homogenized liver tissues were prepared in a sample buffer containing sodium dodecyl sulfate (SDS). The samples were heated at 95℃ for 5 min and briefly cooled on ice. Following the centrifugation at 15,000 × g for 5 min, the proteins in the lysates were separated by 10% SDSpolyacrylamide gel electrophoresis and transferred to nitrocellulose paper. The nitrocellulose paper was blocked with 5% skim milk in PBS-tween-20 for 1 h at room temperature and then incubated with primary and secondary antibodies. Finally, the protein bands were visualized by an enhanced chemiluminesence solution purchased from Amersham Co. (Newark, NJ, USA) following the manufacturer’s instructions.

Tissue samples were immediately fixed with 4% formaldehyde and embedded in paraffin. After dewaxing and dehydration, sections were blocked with bovine serum albumin followed by 60 min of incubation with anti–mouse pSTAT5 (Santacruz, CA, USA) at a concentration of 1 μg/ml. The secondary antibody, TRITC-conjugated anti-rabbit IgG (invitrogen), was added to the sections for 30 min. All specimens were examined with a confocal laser-scanning microscope. Sections were coded and randomly analyzed by three blinded observers.

High-resolution microcomputed tomography (μCT) was used to provide the 2D and 3D information on bone geometry. The mice were sacrificed; femora and tibiae were dissected, cleaned of soft tissue, and fixed in 4% formaldehyde before storage. The source of the open tube type and the minimum focal spot size was 8 μm. Reconstruction was carried out using a modified Feldkamp algorithm using the Sky Scan Nrecon software (Sky Scan, Ltd., Kartuizersweg, Kontich, Belgium). The x-ray source was set at 75 kV and 100 μA. Four hundred projections were acquired over an angle of 180°. The image slices were reconstructed using cone-beam reconstruction software based on the Feldkamp algorithm (Dataviewer; Skyscan, Belgium). The trabecular bone was extracted by drawing ellipsoid contours with the CT analyzer software. Trabecular bone volume (BV/TV; percentage) and trabecular number (Tb.N) of femur epiphysis and proximal tibial metaphysis were calculated by the mean intercept length method. Trabecular thickness (Tb.Th; mm) was calculated according to the method of Hildebrand and Ruegsegger (Ngueguim et al., 2012). 3D parameters were based on analysis of a Marching cubes-type model with a rendered surface. To analyze 3D parameters intibias, whole bone was scanned, and 600 slices of 8 μm in thickness were placed through the former area. Bone volume/ tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), connection density (Conn.D), and total porosity were recorded. The thickness of excised bone growth plate was determined using the Sky Scan 1076 as described in a previously published protocol (Sharan et al., 2011).

Statistical analysis was performed using SPSS software (version 14.0, SPSS Inc, Chicago, IL, USA). All results are expressed as the mean ± SEM. The statistical evaluation of the results was performed by an independent t-test. The results were deemed significant at p < 0.05.

The proximal tibia growth plate length was measured to examine the longitudinal bone growth using a μCT. The lengths of proximal tibia growth plate in the CON and PEM groups were 112.82 ± 4.18 and 86.43 ± 1.47, respectively. The growth plate lengths in the KBS, Arg, and Glu groups were 119.05 ± 6.48, 118.75 ± 4.81, and 87.82 ± 6.38, respectively. KBS and Arg significantly enhanced the longitudinal bone growth, whereas Glu did not (p < 0.05, Fig. 1). Thus, we performed subsequent studies with KBS and Arg except for Glu.

We performed μCT measurements to evaluate whether KBS or Arg could affect developmental bone growth. The KBS or Argadministered mice showed an increase in the bone mineral density (BMD) (Fig. 2A). The 3D μCT reconstruction of trabecular bone images, converted into parameters representing trabecular connectivity, revealed an increase in the BV/TV, Tb.Th, Tb.N, and Conn.D, and a fall in the total porosity at tibia (Fig. 2B-F).

To examine whether KBS or Arg can elevate IGF-I level in the serum, we measured the level of IGF-I by means of sandwich ELISA method. The serum IGF-I level was significantly upregulated by KBS or Arg (p <0.05, Fig. 3A). Then we analyzed the IGF-I mRNA expression in the liver, because majority of plasma IGF-I is biosynthesized in the liver. As expected, hepatic IGF-I mRNA level was found to be significantly elevated in KBS or Arg-administered group (p < 0.05, Fig. 3B).

To determine whether the longitudinal bone growth by KBS or Arg is mediated by the phosphorylation of STAT5, we performed Western blot analysis and immunohistochemistry. As shown in Fig. 4A, the phosphorylation of STAT5 in the liver decreased by malnutrition. However, the decreased STAT5 phosphorylation was up-regulated by KBS or Arg. Furthermore, STAT5 phosphorylation increased by KBS or Arg in the tibia (Fig. 4B).