| Diabetes mellitus affects bone metabolism and leads to osteopenia and osteoporosis, but its pathogenic mechanism remains unknown. Insulin has positive effects on bone growth and differentiation, but the underlying mechanism is not clear. To address these problems, here we investigate the relation between insulin deficiency and osteoporosis in vivo animal model, and the effect of insulin on osteoblast proliferation, differentiation and maturation in vitro cell culture system.Nonpharmacologic management of osteoporosis includes nutrition guidelines, dietary supplementation and so on and is very important to improve bone heath for a patient at risk of osteoporosis. Screening anti-osteoporosis drugs from Chinese herbal medicine hold a very broad appliance future. Here, we investigate the biological mechanism of anti-osteoporosis of antler blood in ovariectomized rats. The thesis consists of eight chapters.First, we present a brief introduction to the mechanism of osteoporosis involved in the regulator, signaling pathway as well as transcription factor which effect on osteoblast growth and differentiation.Second, we revealed for the first time that loss of calcium (Ca), phosphorus (P), zinc (Zn) and strontium (Sr) element contents accounted for the bone mineral density (BMD) reduction in diabetic animal models. With synchrotron radiation X-ray fluorescence (SRXRF) microprobe analysis technique, we found that relative mineral content of Ca, P and Zn in diabetic femurs decreased significantly compared to controls. And Sr in diabetics reduced 11%(P= 0.09). Relative content of sulfur (S) in average was statistically higher (P< 0.01) in diabetics than that in controls. But no obvious difference was observed in relative content of chromium (Cr), iron (Fe), copper (Cu), and lead (Pb) between the two groups. Statistical analysis revealed that Ca correlated positively with P (R= 0.85, P< 0.001), with Sr (R= 0.38, P< 0.05) and with Zn (R= 0.37, P< 0.05). Whereas Zn correlated negatively with S (R=-0.40, P< 0.05).Third, we demonstrated for the first time that insulin treatment could restore BMD by increasing the Ca, P and magnesium (Mg) element contents which was determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) in type1 diabetic rat models. The results showed that the femoral BMD in diabetic group was significantly lower than that in normal group (P< 0.01) but reserved by insulin treatment (P< 0.05). ICP-AES analysis revealed that the element content of Ca, P, Mg, Sr, potassium (K) in diabetic group were remarkably lower than those in normal group (P<0.05) but only Ca, P and Mg content were significantly increased compared with diabetic group (P< 0.05) after insulin treatment. However, no significant differences were observed in element zinc (Zn) content among three groups.Fourth, we also showed that the decrease in serum levels of testosterone, insulin-like growth factor-1 (IGF-1) and osteocacin(OC) caused by insulin deficiency accounted for the bone mineral density (BMD) reduction in diabetic animal models. However, insulin treatment increased serum levels of testosterone, IGF-1 and OC and is capable of preserving bone loss. Results of BMD and histomorphometry revealed serious.bone loss in diabetes. Circling testosterone was significantly reduced in diabetic rats (P< 0.01). osteoprotegerin(OPG), IGF-1 and OC in diabetics also decreased noticeably (P< 0.01). A marked increase was observed in serum concentration of total alkaline phosphatase(ALP), bone ALP of diabetics (P< 0.01) compared to the controls. However insulin treatment induced a marked increase in the levels of testosterone, OPG, IGF-1, OC; and a decrease of total ALP, bone ALP. No significant differences were observed in serum Ca and P among three groups. Our results suggest that bone metabolism is seriously affected by diabetes. Insulin deficiency combining with testosterone depression are associated with alterations in bone turnover, resulting in the development of osteoporosis. And insulin treatment is capable of preserving bone loss and testosterone depression.Fifth, we reported the mechanism of the action of insulin on osteoblast growth and differentiation. Human osteoblastic cell line-MG63 was used and stimulated by insulin in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD98059, PI3-K inhibitor LY294002 or inhibitor PD98059+LY294002. Insulin receptor expression was determined by Confocal laser scanning microscopy (CLSM). Cell proliferation was detected by MTT assay. Cell apoptosis was determined by FACS. ALP activity was determined by ALP kit. Collagen 1 synthesis was determined by ELISA. The mineralized nodules were stained by Alizarin red stain. The mRNA expression of OC, Osx, Runx2, IGF-1, BMP-2 were quantified by real-time RT-PCR. Our results showed that insulin positively regulated the expression of its receptor. Insulin could promote cell proliferation, and blockage of each of the two pathways could decrease the proliferation and cause remarkable cell apoptosis. ALP activity and collagenl synthesis, OC expression, and mineralized nodule formation were increased in insulin treated group, whereas these indicators were decreased in both blockage groups. Down-regulation of Runx2 expression was reversed significantly in PI3K blocked group, but up-regulation of Osx expression was decreased significantly in MAPK blocked group, and so were IGF-1 and BMP-2 after insulin treatment. Therefore, insulin promoted osteoblast differentiation through MAPK signaling pathway to up-regulate Osx expression and PI3K signaling pathway to down-regulate Runx2 expression.In addition, it's the first time that we reported the antiosteoporotic effects of antler blood. The femoral BMD was analyzed by DXA and the element relative content was determined by SRXRF microprobe in ovariectomized rats. The results showed that the femoral BMD was significantly lower than that of sham-operated rats (p< 0.05) but reversed by antler blood (Cervus nippon Temminck) treatment (p< 0.05). A further study demonstrated that the relative contents of P, Ca, Zn and Sr were obviously lower in ovariectomized rats compared to sham-operated rats but only the relative contents of P, Ca and Zn were normalized by antler blood treatment (p< 0.05). Our experiments revealed that loss of element Ca, P, Zn and Sr was closely related to the BMD reduction in ovariectomized rats and the anti-osteoporotic effect of antler blood was mediated by increasing the contents of P, Ca and Zn.Forthermore, the antiosteoporotic effects of antler blood were evaluated in ovariectomized Wistar rats. Compared with SHAM group, serum 17β-estradiol (E2) level decreased significantly (p<0.05) and OC level increased significantly (p<0.05) in OVX group, indicating successful model of osteoporosis. However, serum levels of IGF-1 and testosterone (T) were lower obviously in OVX group than those in SHAM group (p<0.05) but reserved by antler blood treatment (p<0.05) and the BMD of the lumbar spine and left femur in OVX group decreased remarkably (p<0.05) compared to SHAM group but normalized by treated with the antler blood (p<0.05). No obvious changes in serum levels of Ca, P, total ALP and OPG were observed among three groups. These results showed that bone growth promoting effects of antler blood were mediated by high serum concentration of T and IGF-1.Finally, we summarize the contents of seven chapters above. |
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