1. The Role Of RANKL In Mechanism Of Ghrelin Inhibiting Osteoblastic Differentiation Of Vascular Smooth Muscle Cells2. Mechanism Of Inhibitory Effects Of Ghrelin On Apoptosis Of Osteoblastic MC3T3-E1Cells

Part one The role of RANKL in mechanism of Ghrelin inhibiting osteoblastic differentiation of vascular smooth muscle cellsChapter one Ghrelin inhibits RANKL expression in mouse vascular smooth muscle cellsObjective:To investigate the expression of receptor activator for nuclear factor-K B ligand (RANKL) in the process of osteoblastic differentiation of mouse vascular smooth muscle cells (VSMCs), and the effect of Ghrelin on RANKL expression.Methods:Mouse VSMCs were isolated and then smooth muscle a-actin (SMa-actin) was detected by immunocytochemistry and Western blot assay to identify the phenotype of VSMCs. lOmM β-glycerophosphate (β-GP) was adoptted to induce osteoblastic differentiation of mouse VSMCs. The mRNA expressions of Runt-related transcription factor2(Runx2), bone morphogenetic protein-2(BMP-2), and RANKL were determined by real-time PCR. Alkaline phosphatase (ALP) activity was detected by ALP assay kit. The extent of mineralized matrix was determined by Alizarin Red S staining. Protein levels of RANKL were determined in culture media supernatants of cells with commercially available enzyme linked immunosorbent assay (ELISAs). Results:(1) Mouse VSMCs were successfully isolated and the cells were identified by positive staining with SMa-actin.(2) Data showed that (3-GP significantly stimulated the expression of Runx2and BMP-2, increased ALP activity, and promoted the formation of mineralization nodule. The expression of RANKL was significantly increased in the process of osteoblastic differentiation of mouse VSMCs.(3)10-6～10-8M Ghrelin significantly inhibited ALP activity and RANKL expression in dose-and time-dependent manner.Conclusion:The cell model of mouse VSMCs differentiating into osteoblast-like cells was successfully constructed by β-GP. Ghrelin inhibited the osteoblastic differentiation of mouse VSMCs, and blocked the expression of RANKL in the process. Chapter two RANKL mediates inhibitory effects of Ghrelin on osteoblastic differentiation of mouse vascular smooth muscle cellsObjective:To investigate the role of RANKL for Ghrelin inhibiting the osteoblastic differentiation of vascular smooth muscle cells.Methods:RANKL overexpression plasmids were constructed. RANKL overexpression plasmids were adoptted to transfect into mouse VSMCs.10-6M Ghrelin recombinant protein was used to incubate mouse VSMCs transfected with RANKL overexpression plasmids. RANKL and Runx2mRNA expressions were detected by real-time PCR and ALP activity was determined by ALP assay kit.Results:(1) The transfection of RANKL overexpression plasmids significantly increased the expression of RANKL.(2) The overexpression of RANKL blocked inhibitory effects of Ghrelin on ALP activity and Runx2expression, suggesting that could abolish the inhibitory effects of Ghrelin on osteoblastic differentiation of mouse VSMCs.Conclusion:RANKL mediated inhibitory effects of Ghrelin on osteoblastic differentiation of mouse vascular smooth muscle cells. Chapter three Mechanisms of inhibitory effect of Ghrelin on osteoblastic differentiation of mouse vascular smooth muscle cellsObjective:To investigate mechanisms of inhibitory effect of Ghrelin on osteoblastic differentiation of mouse vascular smooth muscle cells.Methods:10"6M Ghrelin recombinant protein was used to incubate mouse VSMCs for5～60min. Western blot assay was adoptted to detect the phosphorylated proteins of MAPK and PI3K/Akt. The specific ERK inhibitor PD98059and PI3K inhibitor LY294002were used to determine the mechanisms involved. The expressions of RANKL and Runx2were determined by real-time PCR. The ALP activity was determined by ALP kit.Results:(1) Ghrelin stimulated the expressions of p-ERK and p-Akt, but did not activate the phosphorylation of p38and INK.(2) ERK inhibitor PD98059and PI3K inhibitor LY294002could respectively abolish inhibitory effects of Ghrelin on RANKL expression, Runx2expression and ALP activity.Conclusion:Ghrelin attenuated osteoblastic differentiation of mouse vascular smooth muscle cells via ERK and PI3K/Akt signal pathway by down-regulating RANKL expression. Part two Mechanism of inhibitory effects of Ghrelin on apoptosis of osteoblastic MC3T3-E1cellsChaper one Ghrelin inhibits serum deprivation-induced apoptosis of mouse osteoblastic MC3T3-E1cellsObjective:To investigate the effects of10-9～10-11M Ghrelin on serum deprivation-induced apoptosis of mouse osteoblastic MC3T3-E1cells.Methods:10-9～10-11M Ghrelin was used to incubate MC3T3-E1cells. TUNEL and ELISA assay were adoptted to determine the apoptosis of MC3T3-E1cells. The protein expressions of Bcl-2and Bax, as well as caspase-3secretion were determined by Western blot assay.Results:(1)10-9～10-11M Ghrelin significantly inhibited serum deprivation-induced MC3T3-E1cell apoptosis in a dose-dependent manner.(2)10-9～10-11M Ghrelin could promote Bcl-2expression, and inhibit Bax expression and caspase-3secretion in a dose-dependent manner.Conclusion:Ghrelin inhibited serum deprivation-induced apoptosis of mouse osteoblastic MC3T3-E1cells. Chapter two Ghrelin inhibits mouse osteoblastic MC3T3-E1apoptosis through GHSR/ERK and GHSR/Akt signaling pathwayObjective:To explore mechanism of inhibitory effects of Ghrelin on apoptosis of mouse osteoblastic MC3T3-E1.Methods:Expressions of GHSR were determined by RT-PCR and Western blot assay. RNA interference was used to knockdown the expression of GHSR.10-9M Ghrelin was adoptted to incubate MC3T3-E1cells. Expressions of p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-Akt and Akt were determined by Western blot assay. The specific ERK inhibitor PD98059and PI3K inhibitor LY294002were used to determine the mechanisms involved. Cell apoptosis was detected by ELISA kit.Results:(1) GHSR was expressed in mouse osteoblastic MC3T3-E1cells. The expression of GHSR could be significantly knocked down by GHSR-siRNA.(2) Ghrelin stimulated the expression of p-ERK and p-Akt, but did not activate the phosphorylation of p38and JNK. ERK inhibitor PD98059, PI3K inhibitor LY294002, or GHSR-siRNA could respectively abolish the effects of Ghrelin on ERK and Akt activation.(3) The blockade of ERK, Akt signal pathway, or the knockdown of GHSR could abolish the inhibitory effects of Ghrelin on MC3T3-E1cell apoptosis. Conclusion:Ghrelin inhibited mouse osteoblastic MC3T3-E1apoptosis through GHSR/ERK and GHSR/Akt signaling pathway...