Study On Serum Osteocalcin Levels In Type 2 Diabetic Patients And Mechanism Of 17β-estradiol Affecting Osteoporosis In Ovariectomized Rats

Chapter 1.Serum carboxylated and non-carboxylated osteocalcin levels and genetic variation of osteocalcin gene in type 2 diabetic patientsStudy Background:In recent years,researchers have suggested that bone can be seen as an endocrine organ involved in the regulation of energy metabolism.Osteocalcin(OC)plays an important role as an "energy messenger" in the interaction between glucose metabolism and bone metabolism.Animal experiments have found that OC can promote islet cell secretion and improve insulin resistance.Some clinical trials have also reached similar conclusions,but there are also clinical studies suggesting that there is no significant correlation between OC levels and blood glucose levels and diabetes risk.At the same time,type 2 diabetes mellitus(T2DM)and osteoporosis(OP)are both multiplex genes related diseases.Studies on that whether the level of OC is related to OC gene polymorphism is less.Therefore,in order to further analyze the relationship between OC and glucose metabolism and the relationship between OC level and OC gene polymorphism,this research was conducted to try to find new targets for prevention and treatment of T2DM.Objective:To evaluate the association of serum osteocalcin and under carboxylated osteocalcin and carboxylated osteocalcin with glucose metabolism in Chinese patients with type 2 diabetes mellitus(T2DM)and to observe the relationships between single-nucleotide polymorphisms(SNPs)and haplotypes in and around the osteocalcin gene with osteocalcin.Methods:A retrospective study was conducted with 456 type 2 diabetic patients and 224 control individuals.Physical examination was performed,related index of biochemistry,bone metabolism and glucose metabolism were measured.Nine SNPs of the osteocalcin gene(rsl543294,rs1800247,rs759330,rs2842880,rs933489,rs2241106,rs2758605,rs2277872 and rs 12563631)were genotyped.Results:(1)The level of OC,under-carboxylated osteocalcin(ucOC),carboxylated osteocalcin(cOC)and ucOC/cOC was significantly lower than the normal control group(P<0.01).(2)There was a negative correlation between cOC and HbA1c in type 2 diabetic patients(P<0.01),and there was no statistical correlation between level of OC and related index of glucolipid metabolism(P>0.01).(3)On SNP site Rs933489,the level of cOC on site CC and CT genotypes was significantly different,CC genotype showed lower level of ucOC than TT genotype among T2DM patients.Conclusion:The level of OC,ucOC,cOC in T2DM patients were significantly lower than the normal control group,and there was a negative correlation between cOC and HbAlc in Chinese type 2 diabetic patients.Common genetic variants of osteocalcin gene may not contribute to level of OC.Chapter 2:The embedded study of 17β-estradiol attenuates osteoporosis in ovariectomized rats by suppressing Eph A2/ephrin A2 signaling pathwayStudy Background:Estrogen,a steroid hormone,is well-known to have important effects on the circulatory,reproductive,and cardiovascular systems,and it has a significant effect on bone homeostasis by inhibiting bone resorption and increasing bone formation.Estrogen has 3 types,including 17β-estradiol(E2),estron and estriol.E2,which is secreted in the ovarium of normally cycling adult women,has the highest estrogenic potent.Estrogen deficiency could cause both the early and late forms of osteoporosis in postmenopausal women.Current studies demonstrate that estrogen and progesterone replacement are effective in preventing bone loss in postmenopausal women.However,because this method has many side effects,the curative effect is limited.If we can further understand the mechanism of osteoblasts and osteoclasts by regulating several signal pathways of bone reconstruction,that can help us to treat osteoporosis clinically.In recent years,the Eph/Ephrin pathway has been explored in depth,and it has been found that it can regulate the differentiation of osteoclasts and osteoblasts,and has the function of bidirectional regulation.EphrinA2-EphA2 signaling regulates bone remodeling at the initiation phase.EphrinA2 and EphA2 in osteoclast precursors enhanced differentiation of multinucleated osteoclasts,EphA2 on osteoblasts generates anti-osteoblastogenic signals presumably by up-regulating RhoA activity.Whether 17p-estradiol inhibits EphA2/ephrin A2 signaling pathway has not been completely clarified,so this study has been designed to solve this problem.In this paper,we aimed to explore the potential role of 17β-estradiol on ovariectomy-induced osteoporosis.In the first part,we selected 21 cases of Sprague-Dawley rats(Twelve-week-old female)and divided them into 3 groups:Sham group,ovariectomy group(OVX)and ovariectomy+17β-estradiol group(OVX+E2).Then,body weight,serum Ca,urinary Ca,urinary Cr,b-ALP and TRAP-5b in rats of the 3 groups were determined.We determined the bone mineral density and bone histomorphology of the experimentally rats.In the second part,we used qRT-PCR to determine the mRNA levels of bone metabolism-related genes ALP,TRAP,Runx2,Osterix,Collal,OPG and NFκ-Bligand(RANKL).We used qRT-PCR and western blotting to determine the expression of EphA2 and ephrinA2 in Sham,OVX and OVX+E2 group.Then,we determined the effect of EphA2 and ephrinA2 knock down on osteoclastogenesis.Part 1:Effects of E2 on OVX-induced osteoporosisObjective:1.To explore the effect of E2 on OVX-induced body weight gain and bone turnover marker;2.To explore the effect of E2 on bone mineral density(BMD)and bone histomorphology.Methods:1.We selected 21 cases of Sprague-Dawley rats(Twelve-week-old female),and divided them into 3 group:Sham group(n=10),OVX group(n=10)and OVX+E2 group(n=10).2.After treatment for 2,4,6 and 8 weeks,we determined and compared the body weight in rats of the 3 groups.3.After treatment for 2,4,6 and 8 weeks,we used an automatic analyzer to determine serum calcium(Ca),urine Ca and creatinine(Cr),Serum bone-specific alkaline phosphatase(b-ALP)and bone resorption item tartrate-resistant acid phosphatase-5b(TRAP-5b)concentration.Use electrochemiluminescence analyze P1NP and β-CTX.4.Dual energy X-ray absorptiometry was used to determine BMD of the left tibias;5.HE staining was used histopathological analysis on left tibias,and light microscope(Leica,Germany)and image analyzer were used to determine static parameters,such as bone volume per tissue volume(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N),and trabecular separation(Tb.Sp).Results:1.Body weight gain was typically and consistently higher in the OVX rats compared with the sham-operated control rats.After E2 treatment,the OVX-induced body weight gain was significantly suppressed in a time-dependent manner.2.The serum Ca concentration in E2 group was significantly high than that in OVX group.After detecting the urinary Ca/Cr for 8 weeks,the obvious enhancement in OVX group was observed while it decreased immediately after E2 treatment.The b-ALP、TRAP-5b、P1NP、β-CTX concentrations in serum were also detected,E2 treatment effectively attenuated the enhancement of b-ALP and TRAP-5b concentrations induced by OVX in rats.3.After 4 and 8 weeks treatment.OVX notably decreased the tibias BMD compared with the sham group.And E2 treatments significantly increased the tibias BMD compared with the OVX group.4.OVX-induced rats had a reduced BV/TV,Tb.N and Tb.Th compared to the control group.Undoubtedly,E2-treated significantly prevented those reductions.E2-treated significantly prevented the induction of the Tb.Sp value as there were significant differences noted between OVX and the E2 treatment groups.Conclusions:This study indicated that OVX could induce body weight gain,and the increase of serum calcium(Ca),urine Ca and creatinine(Cr),Serum bone-specific alkaline phosphatase(b-ALP)、procollagen type I N-terminal propeptide(P1NP)and bone resorption item tartrate-resistant acid phosphatase-5b(TRAP-5b)、β-C-terminal cross-linked telopeptides of type I collagen(β-CTX)concentration.E2 treatment effectively attenuated the enhancement.The significantly low BV/TV,Tb.N and Tb.Th,along with the significantly high Tb.Sp in OVX group were clear evidences of bone loss,mainly due to trabecular perforation,trabecular thinning and loss of trabecular connectivity,and they were all attenuated by treatment of E2.Part 2:The study mechanism of 17β-estradiol affecting osteoporosis in ovariectomized ratsObjective:To explore the effect of E2 on expression levels of the bone metabolism-related genes.To explore whether E2 influence the formation and fuction of osteoclasts via EphA2/ephrinA2 pathway.Methods:We used qRT-PCR to determine the mRNA levels of ALP,TRAP,Runx2,osterix,collal,osteoprotegerin(OPG)and receptor activator of NFκ-B ligand(RANKL)in Sham,OVX group and OVX+E2 group.2.We used qRT-PCR and western blotting to determine the expression of EphA2 and ephrinA2 in Sham,OVX and OVX+E2 group.3.SiNRA transfection was used to make phA2 or ephrinA2 knockdown,and we determined the number of osteoclasts after treating with RANKL or M-CSF.Results:1.The expression of ALP,TRAP and RANKL in OVX group was significantly higher than Sham group,E2 treatment effectively attenuated the up-regulation of ALP and TRAP and RANKL.2.The expression of Runx2,osterix and Collal in OVX group was significantly lower than Sham group,E2 treatment effectively attenuated the down-regulation of ALP and TRAP and RANKL.3.The expression of OPG in the 3 groups had no significant difference.4.The qRT-PCR and immunoblots assay showed that E2 significantly decreased the OVX-stimulated expression of ephA2 and ephrinA2 protein.5.Osteoclast differentiation were induced by RANKL and M-CSF in vitro;EphA2 and ephrinA2 knockdown significantly suppressed osteoclastogenesis.Conclusions:This study incicated,E2 not only inhibited bone resorption function of osteoclasts by inhibiting the production of TRAP,RANKL,but also inducing bone formation function of osteoblasts by prompting the Runx2,Osterix and Collal expressions.EphA2 or ephrinA2 knockdown could significantly suppress osteoclastogenesis,indicating their stimulating role on osteoclast differentiation.