The Effect Of Osteoclast Cell And Plasma TRACP5b On The Pathogenesis Of Osteoporosis In Type 2 Diabetic Rats

Objective: Type 2 diabetes is a commonly and frequently-occurring disease,it has become a worldwide health problem. Every year there are millions of patients died of its complications, which causing social ecomomical burden Diabetic osteoporosis is one of the complications. With the process of aging in our population, the incidence of diabetic osteoporosis is also increasing. Therefore,early diagnosis and treatment to prevent bone fracture and reduce disability has become a major concern in our social life.Studies have found the osteoclast plays an important role in the pathogenesis of bone loss in diabetes.The number and function of osteoclast are closely related with diabetic osteoprosis.TRACP5b is an enzyme specially secreted by osteoclast. the level of serum TRACP5b may reflect osteoclastic activity and bone resorption station.Its concentration can not be affected by diurnal variation,diet,liver and kidney disease.In this study,we established type 2 diabets osteoprosis rat modes. The alerations of osteoclast and plasma TRACP5b levels were measured at different time point in normal, osteoporosis,type 2 diabetes and type 2 diabetic osteoporosis groups.The effects of osteoclast in type 2 diabetic osteoporosis will be explored.Methods: 100 Wistar rats were randomly divided into four groups after 1 week feeding: normal contorl(NC,N=24) group, normal ovariectomy (NOVX,N=26) group,diabetes control(DC,N=24) group,diabetes and ovariectomy(DOVX,N=24)group.NC and NOVX groups were fed with normal feedstuff while DC and DOVX groups were fed with high-sugar-fat feedstuff(mixed with 20% sugar,2.5% cholesterin, 15% cooked lard) for 8 weeks. STZ(30mg/kg) were injectded at left lower quadrant after 12 hours fasting. At the end of the 9th week, rats with fasting blood glucose(FBG) equal or more than 7.8mmol/L were considered as T2DM. NOVX group and DOVX group were ovariectomied 1 week after T2DM model was set.On the day of surgery(week 0),four rats were chosen from four groups and were killed and served as baseline. Body weight ,fasting blood glucose and FINS were measured at 0,2,4,6,8,10,and 12 weeks after ovariectomy, femur bone marrow were collected from 4 groups at 4,8 and also 12 weeks after ovariectomy and curtured. They were cultured in the presence of macrophage colony stimulating factor(M-CSF),and receptor activator of nuclear factor kappa-B ligand(RANKL). Bone marrow cavity was flushed withα-MEM. The marrow cells were washed twice withα-MEM and counted 1.5×106cells/ml in 24-well-plate and were cultured inα-MEM which containing 10% FCS and 30ng/ml RANKL,10ng/ml M-CSF. Medium was changed every other day. At day 7 the cell were fixed and stained,TRAP positive and with three nuclei or more nulcea were considered as osteoclast cell.The number of osteoclast were measured every well under the inverted phase contrast microscope. Serum TRACP5b were measured by ELISA. The experimental data was presented as mean±standard deviation.Variance Analysis or nonparametric test were used to check differences between groups by SPSS 11.5, p<0.05 was considered statistically significant.Results:1 The comparison of body weight, blood glucose and insulin in each group at different time point1.1 body weightThe body weight in NOVX and NC group were increased, especially the NOVX group due to ovariectomy. The body weight in DOVX rats has no change. The body weight of DC group was gradually reduced because of high glucose.0 week:There are no difference found in body weight among NC group,DC group and DOVX group(p>0.05). There no difference found in body weight between NC group and NOVX group(p>0.05).There were statistical differences found in body weight among NOVX group,DC group and DOVX group(p<0.05).2 week:There were no statistical difference found in body weight among NC group,DC group and DOVX group(p>0.05). NOVX group had significantly higher body weight than the other groups(p<0.05). 4 week:It was found that DC group had significantly lower body weight than NC group and NOVX group(p<0.05),while NOVX group had significantly higher body weight than the other groups(p<0.05).6 and 8 weeks: There are significant differences found between any two groups (p<0.05). The body weight of DC group was the lowest while NOVX was the highest.10 week: Rats in DC group had significantly lower body weight than the other groups(p<0.05),while NOVX group had significantly higher than the other groups(p<0.05). 12 week: It was found that NOVX group animals had significantly higher than the other groups(p<0.05).There were significant differences between DC group and NC group(p<0.05).There were no significant differences among DOVX and DC,NC groups(p>0.05).1.2 The comparison of fasting blood glucose0 week: FBG of diabetic rats(DC,DOVX) had significantly higher FBG than non-diabetic rats(NC,NOVX)(p<0.05).2 week:The FBG of DC and DOVX groups were decreased.The FBG of DC group had significantly higher than NC and NOVX groups(p<0.05). There were significant differences found in FBG between DOVX and NOVX(p<0.05). The FBG of DC group had significantly higher than DOVX group(p<0.05).There were no differences found in FBG between NC and NOVX (p>0.05) . The FBG of DOVX was higher than NC,but there were no differences between them(p>0.05).4 week: The FBG of DC and DOVX groups tend to be decreased but there no differences found in all groups(p>0.05).6 week: The FBG of diabetic rats had significantly higher than non-diabetic rats(p<0.05).There were no differences found in FBG between NC and NOVX(P>0.05),DC and DOVX(p>0.05).8 week:The FBG of DC and DOVX groups had significantly higher than NC and NOVX groups(p<0.05). There were no differences found in FBG between NC and NOVX,(p>0.05),DC and DOVX(p>0.05)separately. 10 and 12 week:There were no differences found in FBG in all groups(p>0.05)1.3 The comparison of insulin 0 week:The diabetic rats(DC and DOVX) had significantly higher insulin than the non-diabetic rats(NC and NOVX)(p<0.05). 2 week:DC group had significantly higher insulin than NC,NOVX and DOVX group (p<0.05). NOVX group had significantly lower insulin than the other groups(p<0.05). There were no differences found in insulin between NC and DOVX(p>0.05).4 week: It was found that DC group had significantly higher than NC,NOVX and DOVX group(p<0.05). The other had no differences between every two group(p>0.05).6 week:There were no differences found in insulin in all groups(p>0.05).8 week: DC and DOVX group had significantly higher insulin than NOVX (p<0.05). The other had no differences between every two group(p>0.05).10 week and 12 week: There were no differences found in insulin in all groups(p>0.05).2 The number of osteoclast and serum TRACP5b concentration in each group at different times2.1 The number of osteoclast0 week: There were no differences found in the number of osteoclast in all groups(p>0.05).4 and 8 weeks: It was found that DOVX group had higher osteoclast numbers than NC and DC groups(p<0.05),but was similar to NOVX(p>0.05). NC group had significantly lower number of osteoclast than the other groups(p<0.05).12 week: DOVX group had significant higher osteoclast number than the other groups(p<0.05),while NC group had significant lower(p<0.05).There were no significant differences between DC and NOVX group(p>0.05).2.2 Serum TRACP5b concentration0 week: There were no differences found in the TRACP5b concentration in all groups(p>0.05).2 week:Compared with NC and DC,the serum TRACP5b concentration in NOVX and DOVX were higher(p<0.05),but there were no significant difference between NOVX and DOVX groups (p>0.05). NC group had significant lower serum TRACP5b concentration than the other groups(p<0.05).4 week: TRACP5b concentration in NOVX was significant higher than DC and DOVX groups,and NC had much lower than the other groups(p<0.05).There were no significant differences in TRACP5b concentration between DC and DOVX groups(p>0.05).6 week:NC group had significantly lower than the other groups(p<0.05).There were no significant differences among NOVX, DC, DOVX(p>0.05).8 week:There were no significant differences found in all groups(p>0.05).10 week:NC had significant lower TRACP5b concentration than the other groups(p<0.05).There were no significant differences among DC,NOVX and DOVX(p>0.05). 12 week:DOVX had significantly higher TRACP5b concentration than the other groups (p<0.05). There were no significant differences among NC,DC and NOVX(p>0.05).3 Results of correlate analysis3.1 Correlation between osteoclast number and serum TRACP5b concentrationThe number of osteoclast was positively corralted with serum TRACP5b concentration(r=0.789,p<0.000).3.2 The correlation osteoclast number,TRACP5b and blood glucose No correlation was found between the number of osteoclast and blood glucose(r=-0.290,p=0.275),TRACP5b and blood glucose(r=-0.306,p=0.249).Conclusions:1 The results of the study indicated that type 2 diabetes and the loss of ovary function increase the derivation of bone marrow cells to osteoclast.Increasing the osteoclast formation might be the cause for osteoporosis in type 2 diabetes rat.2 Compared with normal control group, serum TRACP5b concentration was significantly higher in osteoporosis in type 2 diabetes group, which provides early signs for osteoprosis early detection and prevention.3 Osteoclast number and serum TRACP5b concentration are relatively stable with the development of type 2 diabetes osteoporosis.4 The number of OLCs positively correlated with TRACP5b concentration.5 Osteoclast number,TRACP5b had no correlation with blood glucose.