The Mechanism Of Type 1 Diabetes Affecting Bone Remodeling In Ovariectomized Rats

Background:Osteoporosis is a systemic bone disease which has always been paid close attention by many researchers and clinical professionals. The main manifestations of osteoporosis are osteopenia, increased osteopsathyrosis and bone fracture. Along with the arrival of the aged in our country and even the world range, primary osteoporosis including postmenopausal osteoporosis has been a profound medical and social problem. Moreover, some other factors such as systemic diseases which causes secondary osteoporosis are also serious threats to human health. As one kind of secondary osteoporosis, diabetic osteoporosis has been paid more and more attention by domestic and international researchers, just because it shows serious bone damage, high rate of bone fracture and so on. In recent years, the incidence of postmenopausal osteoporosis with diabetes is increased year by year. To provide theoretical basis and improve life quality of senile women, the mechanism and bone remodeling of postmenopausal osteoporosis with diabetes should be further elucidated.Purpose:In the present study, experimental animal models of postmenopausal osteoporosis, type 1 diabetic osteoporosis, and postmenopausal osteoporosis with type 1 diabetes were established. The mechanism of bone remodeling in ovariectomized rats with type 1 diabetes was evaluated systematically, so that the theoretical supports of clinical intervention and treatment of osteoporosis were provided eventually.Methods:Part 1:40 healthy 8 weeks old Wistar female rats were randomly divided into control group(BC, n=10), ovariectomy group (ED, n=10), type 1 diabetes group (DM, n=10) and ovariectomy with type 1 diabetes group (ED+DM, n=10). The bilateral ovaies of rats in ED group and ED+DM group were resected to establish postmenopausal osteoporosis models. After 4 weeks, the rats in DM group and ED+DM group were intraperitoneally injected with streptozotocin (1%STZ, pH4.5, 50mg/kg) to establish type 1 diabetes osteoporosis, and postmenopausal osteoporosis with type 1 diabetes. When the rats in every groups were 21weeks, five rats of each group were randomly selected and sacrificed by taking off necks. Then the bilateral tibias were separated and a series of steps were processed such as dipped in 4% paraformaldehyde solution, flushed with PBS buffer solution, clipped, and detected in Micro-CT at last. The scanning parameters were scanning voltage 80kVp, electric current 500μA, resolution ratio10μm, and exposure time 2500ms. After using Inveon Research Workplace 2.2 for 3D reconstruction, the regions of interesting (ROI) were selected from the area blowing tibia growth plate lmm. The spongy bone values of BV/TV、Tb.Th、Tb.N、Tb.Sp、Tb.Pf and BMD were detected.Part 2:Animal models and the groups were just the same with part1. The other five rats of each groups were narcotized by 10% chloral hydrate and flooded inside the blood circulation system through apex cardiac by 4% paraformaldehyde solution, and the bilateral hind femur and tibia of rats were separated. After series of steps with fixed, decalcified, dehydration, transparent, paraffin, embedding, et al,5 micrometer sections were got serially. To evaluate the tibia histomorphology, ALP, CK and TRAP expression, the sections were stained by haematoxylin-eosin solution, IHC and enzyme chemical dyeing. After selecting 10 sections in each specimens and 3 different views in each sections, the IPP software was used to count the number of osteoclasts and detect immune staining intensity.Part 3:Animal models and the groups were just the same with part1. The bilateral hind femurs of rats in part one were separated. Trizol method was used to extract total RNA of bone tissues. The mRNA relative expression of RANKL、OPG、 RANKL/OPG、TRAF6、NF-κB、LRP5、β-catenin、Runx2 were detected by real-time quantitative PCR.Results:Part 1:① Fasting glucoses levels:After STZ injection, the blood glucose levels of DM and ED+DM groups were positively correlated with time(P< 0.01)and the ED+DM group levels were slightly higher than DM group. There was no statistical difference in BC and ED groups. ②Weight changes:The body weight levels of ED group were positively correlated with time(P<0.01). On the contrary, the weight levels of DM and ED+DM groups were negatively correlated with time(P<0.01). There was no statistical difference in BC group. ③Parameters of trabecular bone structure:The values of BV/TV、Tb.Th、Tb.N and BMD in ED、 DM and ED+DM groups were all statistically lower than those in BC group(P< 0.05), and the statistically lowest indexes were found in ED+DM group. The values of BV/TV and Tb.N in ED group were statistically lower than those in DM group(P <0.05). The values of Tb.Th and BMD in ED group were statistically higher than those in DM group(P<0.05). The values of Tb.Sp were increasing in ED and ED+DM groups compared with BC group(P<0.05), and there was no statistical difference in DM group. The values of Tb.Sp in ED、DM and ED+DM groups were increased(P<0.05), the statistically highest indexes were found in ED+DM group. There were no statistical difference in DM and ED groups.Part 2: ① ALP staining:The highest levels of ALP express were found in ED group compared with the other three groups(P<0.05), there were more ALP positive osteoblasts on the bottom of epiphyseal growth plate and trabecular surfaces. The lowest levels of ALP expression were found in ED+DM group(P<0.05), there were only tiny ALP positive cells on the bottom of growth plate and almost invisible on trabecular surfaces. The levels of ALP expression in DM group were higher than ED+DM group and lower than BC and ED groups(P<0.05). ALP positive cells were dispersedly distributed on the bottom of growth plate, and a small amount ALP positive osteoblasts on trabecular surfaces were found. ②CK staining and TRAP positive osteoclast counting:The numbers of TRAP positive osteoclasts and the levels of CK expression were the highest in ED group(P<0.05). The osteoclasts were larger and the number of nucleus were even more. The numbers of TRAP positive osteoclasts and the levels of CK expression were lower in DM and ED+DM groups(P<0.05). The osteoclasts were smaller and the number of nucleus were less. There were no statistical differences of osteoclast numbers and CK expression levels in ED+DM group compared with DM group.Part 3:① mRNA expression of Wnt/β-catenin pathway:The expression levels of LRP5 were the highest in ED group(P<0.01) and lowest in ED+DM group(P< 0.01), there were no statistical differences in BC and DM groups. The expression levels of β-catenin were the highest in ED group(P<0.01) and lowest in ED+DM group(P<0.05). The expression levels of Runx2 were increased obviously in ED group(P<0.01), the levels were lower in DM and ED+DM groups(P<0.05), but there was no statistical difference in ED+DM group compared with DM group. ② mRNA expression of NF-κB pathway:The expression levels of RANKL were little increased in ED group(P<0.05), and increased obviously in ED+DM group(P< 0.01), and no statistical difference in DM group. Four groups of OPG expression had no statistical differences. The expression trends of RANKL/OPG ratio were similar with RANKL expression. The expression levels of TRAF6 were higher in ED、DM and ED+DM groups(P<0.05) and ED group had obviously changes(P< 0.01). But there were no significant differences in ED、DM and ED+DM groups. The expression levels of NF-κB were increased in ED group and decreased in DM and ED+DM groups(P<0.01), and there was no significant difference in ED+DM group compared with DM group.Conclusions:1. There had remarkable differences of bone trabecular forms in osteoporosis caused by ovariectomization and type 1 diabetes. The former mainly showed the number reduction and missing of bone trabecular, the latter showed that the trabecular becoming thinner and breakage. Ovariectomized rats twith type 1 diabetes showed that the numbers of trabecular were obviously decreased, bone cavity was increased obviously, sclerotin damage and missing were most serious.2. In the ovariectomized rats, the NF-κB and Wnt/β-catenin signal pathway were activated. Then the bone formation and resorption increased, which showed typical high turnover of bone remodeling.3. When osteoporosis only caused by type 1 diabetes, expression of NF-κB and Wnt/β-catenin were down regulated. The bone resorption and formation were restrained, which showed low turnover of bone remodeling.4. When postmenopausal osteoporosis with type 1 diabetes, high turnover of bone remodeling was reversed to low turnover, which increased the bone mass missing. The details of action mechanism remained to be further research.