Protective Effect Of Metformin On Bone And Its Relationship With The Expression Of TLR4/NF-κB

Objective: 1. To investigate the changes of BMD(bone mineral density) and bone turnover markers in high sucrose-fat diets with STZ- induced diabetic rats by metformin. 2. Observe the effect of metformin on the expression of osteoblast functional protein and bone turnover markers in both normal glucose and high glucose. 3. Analysis the relationship between the effect on bone and osteoblast functional protein by metformin and the expression of the TLR4/ NF-κB.Methods: 1. Diabetes rat model: 60 male sprague-dawlry(SD) rats were randomly divided into normal control group(NC group, n=15) and model group(n=45). Normal control group was fed with standard chow, while model group was fed with high sucrose-fat diets(HSFDs) for 8 weeks, the model group were injected intraperitoneally with streptozotocin(STZ, 30mg/kg) while the ones in group NC were injected intraperitoneally with the similar dose of normal saline. Subjects with FBG higher than 16.7mmol/L in three consecutive days were successfully modeled. 2. High glucose injury model: The MC3T3-E1 subclone 14 was set as an object of research. Cells were exposed to 25 mmol/L of glucose for 24 hours to mimic conditions of the high glucose injury model. Cells was divided into 4 groups, NC(normal glucose control team), HG(high glucose team), NC+MET(normal glucose metformin team) and HG+MET(high glucose metformin team). 3. Pharmaceutical intervention: 30 modeled rats were grouped into Type 2 diabetes mellitus group(T2DM group, n=15) and metformin treated group(MF group, 100mg/kg·d, n=15). After 2 weeks, the thervention by metformin. Group NC and group T2 DM were gavaged equal physiological saline. Group NC were fed with the standard chow, while the remaining two groups were fed with HSFDs. After20 weeks detecting the blood glucose and weight of each group rats. The MC3T3-E1 subclone 14 of both normal glucose and high glucose team cultured with 100umol/L metformin for 24 h. 4. The detection of BMD and bone turnover markers: DEXA was used to measure each part of whole body bone mineral density(BMD). The serum OCN and TRACP5 b activity were detected by ELISA; AMP-buffer tested serum ALP level. 5.The detection of protein: western detected the protein expression of TLR4、NF-κB、BMP-2 and Caspase 3 of the femur and osteoblast cells.Results: 1. Compared with group NC, FBG increased and body weight decreased significantly in group T2DM(P<0.05), FBG increased in group MF(P<0.05), body weight has no significant difference(P>0.05). Compared with group T2 DM, FBG in group MF decreased significantly(P<0.05), while body weight increased obviously(P>0.05). 2. Compared with group NC, BMD of lower limbs, upper limbs, spine and whole body in group T2 DM decreased(P<0.05); Compared with group T2 DM, BMD of limbs, upper limbs, spine and whole body increased in group MF(P<0.05). 3. Compared with group NC, the expression of serum ALP and OCN decreased(P<0.05), TRACP5 b increased(P<0.05) in group T2 DM, the expression of TRACP5 b increased(P<0.05) in group MF. Compared with group T2 DM, the expression of serum ALP and OCN increased(P<0.05), TRACP5 b decreased(P<0.05) in group MF. 4. Compared with group NC,The protein expression of TLR4、NF-κB in rat femur were significantly increased(P<0.05), the protein expression of BMP-2 was decreased(P<0.05) both in group T2 DM and group MF; Compared with group T2 DM, The protein expression of TLR4、NF-κB in rat femur were significantly decreased(P<0.05), the protein expression of BMP-2 was increased(P<0.05) in group MF. 5. Compared with group NC, the expression of serum ALP and OCN decreased(P<0.05) in group HG and HG+MET, while increased(P<0.05) in group NC+MET.Compaired with group HG, the expression of serum ALP and OCN increased(P<0.05) in group HG+MET. 6. Compared with group NC,The protein expression of TLR4、NF-κB and Caspase 3 were significantly increased(P<0.05), the protein expression of BMP-2 was decreased(P<0.05) both in group HG and group HG+MET, while protein expression of TLR4 was decreased(P<0.05) and BMP-2(P<0.05) was increased in group NC+MET. Compared with group HG, The protein expression of TLR4、NF-KB and Caspase 3 were significantly decreased(P<0.05), the protein expression of BMP-2 was increased(P<0.05) in group HG+MET.Conclusions: 1. Metformin could promote bone formation, inhibit bone resorption and increase BMD in diabetic rats(limbs, upper limbs, spine and whole body); 2. However normal glucose or high glucose, metformin hydrochloride can also enhance the expression of osteoblast functional protein; 3. Metformin may by reducing the expression of TLR4/NF-κB, to promote the proliferation of osteoblasts and expression of osteoblast functional protein, and play the role of bone protection of the diabetic rats...