The Protective Effects Of Collagen Peptides From Walleye Pollock Skin(CPWPS) Against Dexamethasone Induced Rat Osteoporosis

Aim: The aim of this study was to establish dexamethasone induced rat osteoporosis model and osteoblast cytotoxicity model as well as to investigate the preventative and therapeutic effects of collagen peptides from walleye pollock skin(CPWPS) against osteoporosis. Additionally, High-performance liquid chromatography(HPLC) methods for simultaneous detection of Pro-Hyp and Gly-Pro-Hyp were established and the biological activities of these small peptides were assessed. Methods: In vivo study: 100 male wistar rats(8 week old) were randomly assigned into five groups: control group, dexamethasone model group, CPWPS prevention group, CPWPS treatment high and low dose groups. Since the beginning of study, rats in CPWPS prevention received CPWPS 1 g/kg/d via gavage throughout the whole study. Meanwhile, other animals received equivalent amount of saline via gavage. After four weeks, all animals except for those in control group received dexamethasone sodium phosphate 1 mg/kg(count as dexamethasone) via intramuscular injection into glutaeus muscle twice a week for a total of six weeks. After the dexamethasone treatment, rats in CPWPS treatment groups started to receive CPWPS 0.5 g/kg/d(low dose group) or 1 g/kg/d(high dose group) for a total of 13 weeks. X-ray photograph of rat femur was taken after 10, 16 and 23 weeks treatment. Blood samples were collected at each time point from abdominal aorta. ELISA was utilized to detect the concentration of BALP, PINP, CTX-1 and TRAP. Hematoxylin and eosin(HE) staining was used to measure the ratio of knee joint bone trabecula area to total bone area and the thickness of bone cortex. Immunohistochemistry was used to assess the expression level of TGF-β on knee joint bones. HPLC conditions were determined as follows: chromatographic column: venusic ASB-C18(250 mm x 4.6 mm, 5 um); column temperature: 40 ℃; mobile phase: gradident acetonitrile and water; flow velocity: 1.0 ml/min; detection wavelength: 220 nm. In vitro study: primary rat osteoblast from cranium was established and identified, on which the dexamethasone induced cytotoxicity model was established. Cells were assigned into control group, dexamethasone group, CPWPS treatment group(100, 200 and 400 ug/ml), Gly-Pro-Hyp treatment group(200 ug/ml) and positive control group(dexamethasone antagonist, RU486). Cell viability was determined with CCK-8 method. Additionally, OCN protein expression level was assessed with western blotting. Results: In vivo experiments: the dexamethasone induced rat osteoporosis model was successfully established. Grey scale analysis of X-ray photos revealed significant decrease in photos from dexamethasone model group(64.49±2.71) relative to control group(109.23±1.48)(P<0.01) at all time points tested. CPWPS prevention group rats(administered for ten weeks) exhibited higher grey scale value relative to contemporary model rats, and the value continued to rise as administration duration increased. Higher grey scale values were observed in rats of both CPWPS treatment groups(low and high doses) relative to model group rats(76 and 84, respectively, P<0.01), in which high dose CPWPS treatment exhibited better therapeutic effects than low dose treatment. HE staining of knee joint bone revealed decreased bone trabeculaquantity in model group rats, the ratio of bone trabecula area to total bone area was decreased by 50%, along with larger gaps and enlarged cavities. Partial bone cortex rupture and thinning were also present. On the other hand, 16 week preventive administration of CPWPS remarkably improved bone morphology. Ratio of bone trabecula area to total bone area was increased by 35% as compared to those of model group(47% vs 56%, P<0.01). The improvement continued overtime. Both CPWPS treatment groups also improved bone morphology comparing to model group time-dependently; high dose group exhibited better effects than low dose group. Immunohistochemistry on knee joint bone indicated significantly lower expression of TGF-βin samples from model group rats, comparing to those from control group. CPWPS prevention for ten weeks resulted in remarkably elevated TGF-βexpression time-dependently. Both CPWPS treatment groups had elevated TGF-βexpression levels starting after six weeks treatment, which also followed time-dependent manner(P<0.05). In model group serum samples, BALP and PINP levels were significantly lower relative to those of control groups as measured with ELISA(P<0.01), while CPWPS groups all had remarkably higher levels comparing to model group time-dependently(P<0.01), among the groups. Meanwhile, elevated CTX-1 and TRAP levels were detected in model group serum samples, while significantly lower levels of these were observed in all CPWPS groups time-dependently(P<0.05). The HPLC detection range of serum Pro-Hyp concentration was between 0.005 mg/ml and 0.5 mg/ml(R2=0.9999), and the range of Gly-Pro-Hyp was between 0.01 mg/ml and 1 mg/ml(R2=0.9998). Average recovery rate for Pro-Hyp(low middle and high concentration) were 99.26%, 98.77% and 99.07%, respectively. Relative standard deviation(RSD) were 1.70, 1.20 and 0.84, respectively, indicating good recovery rate. Serum concentration of Pro-Hyp and Gly-Pro-Hyp in control group rats were 0.096 and 0.079 mg/ml, respectively, which significantly decreased in those of model groups(0.029 and 0.044 mg/ml, respectively). Meanwhile, CPWPS prevention significantly increased these small peptide serum peptides(0.148 and 0.107 mg/ml, respectively)(P<0.01). In vitro experiments: 10-5 m M dexamethasone exposure for 48 h effectively established osteoblast cytotoxicity model. All the three doses of CPWPS significantly improved osteoblast viability by 70%, however no statistical difference among doses were observed(P>0.05). The Gly-Pro-Hyp improved osteoblast viability by as high as 139%(P<0.01). OCN protein expression level were significantly elevated by both CPWPS and Gly-Pro-Hyp treatment, as indicated by western blotting. Gly-Pro-Hyp had the most prominent effects. Conclusions: At doses tested, CPWPS time-dependently prevented and treated dexamethasone induced osteoporosis in rats. The serum concentration of Gly-Pro-Hyp and Pro-Hyp may be negatively associated with osteoporosis. The biological active olegopeptides were associated with the anti-osteoporosis effects of CPWPS. Furthermore, both CPWPS and Gly-Pro-Hyp could directly promote the proliferation and differentiation of primary rat osteoblast from cranium, in which Gly-Pro-Hyp exhibited the best effects.