1.Mechanism Study Of The Effects Of Omentin-1on The Proliferation Of Human Osteoblasts2.Effect Of Ghrelin On The Arterial Calcification Of Osteoprotegerin Gene Knockout Mice

Part one:Mechanism study of the Effects of Omentin-1on the Proliferation of Human osteoblastsObjective:Omentin-1is a newly discovered adipokine which is mainly secreted by omental adipose tissue. Researches showed that Omentin-1participated in multiple physiological processes including insulin action, cardiovascular function, and inflammatory response and bone metabolism. Our previous study demonstrated that Omentin-1could alleviate bone loss in osteoprotegerin-deficient or ovariectomized mice by regulating the proliferation and differentiation of the mouse osteoblast. However, research concerning mechanism of the effects of Omentin-1on human osteoblast (hOB) proliferation remains relatively poor. The present study investigates the effect of omentin on hOB proliferation and the signaling pathway involved.Methods:Primary hOB was isolated from the human trabecular bone. HOB was incubated with25,50,100,200ng/ml Omentin-1for48h. The proliferation of hOB was determined by measuring [3H] thymidine incorporation. Activation of Akt (phosphatidylinositol-3kinase downstream effector) was assessed by Western blot. Akt small interfering RNA (Akt-siRNA), LY294002(a selective PI3K inhibitor) and HIMO (a selective Akt inhibitor) were used for investigating the effect of Omentin-1on hOB proliferation and signal pathway involved. Results:25-200ng/ml Omentin-1dose-dependently promoted the proliferation of hOB with the maximum effect at the concentration of200ng/ml (p<0.05); Omentin-1induced the activation of Akt and transfection of hOBs with Akt siRNA inhibited Akt protein expression. Furthermore, Akt-siRNA, LY294002and HIMO could all abolish the hOB proliferation induced by Omentin-1.Conclusion:Omentin-1induced hOB proliferation via the PI3K/Akt signaling pathway. Part Two:Effect of Ghrelin on the arterial calcification in osteoprotegerin gene knockout miceObjective:Ghrelin is a28amino acid acyl peptide that has multiple physiological function such as promoting release of growth hormone (GH), increasing of food intake, and regulating energy homeostasis. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays a key role in arterial calcification. Our previous study found that Ghrelin attenuated osteoblastic differentiation of mouse vascular smooth muscle cells via ERK and PI3K/Akt signal pathway. This study was to investigate the effect of Ghrelin on arterial calcification in osteoprotegerin gene knockout (OPG-/-) mice.Methods:OPG-/-mice were treated with Ghrelin adenovirus (Ad-Ghr) or β-galactosidase adenovirus (Ad-Gal) or Phosphate buffered saline (PBS) of the same volume as group of Ghrelin, group of negative control and group of blank control, respectively. Wide type(WT)mice was treated with PBS as group of WT. They were treated every2weeks and8weeks later, the mice were euthanized. The thoracic aortas were dissected, western blot was used to detect the expression of Ghrelin. Alizarin Red S staining and Vonkossa staining were used to detect calcification. Aortic segments from aorta arch to the iliac bifurcation were removed and the colorimetric quantification of calcium was achieved. The ALP activity of supernatant was measured by spectrophotometric measurement of P-nitrophenol release. Immunohistochemistry was used to detect the expression of OC, RANKL and RUNX2.Results:Expression of Ghrelin in group of Ghrelin was higher than other three groups, which suggested that Ghrelin adenovirus overexpressed Ghrelin successfully. Alizarin Red S staining and Vonkossa staining showed that no significant arterial calcification was detected in group of WT with slight arterial calcification in group of Ghrelin and obvious arterial calcification in group of negative control and blank control. Compared with WT mice, aortic calcium contents and ALP activity in OPG-/-mice were higher. However, they decreased apparently in group of Ghrelin than that in groups of negative control and blank control. Immunohistochemistry suggested that compared with WT mice, expression of OC, RANKL and RUNX2in OPG-/-mice were stronger. While in group of Ghrelin, the expression were weaker relative to groups of negative control and blank control.Conclusion:Ghrelin attenuated arterial calcification in OPG-/-mice and suggested that Ghrelin played a protective role against arterial calcification.