Effect Of Metformin Inhibition Of HMGB1 And RAGE On Osseointegration In Type 2 Diabetic Rats And Bone Protection In Non-diabetic Rats

Objective:To investigate the effects of metformin on the expression of HMGB1 and RAGE around implants in type 2 diabetic rats and the possible mechanism of its influence on osseointegration.To investigate the effect of metformin on osseointegration in non-diabetic rats.Methods:40 SPF male SD rats from 6 to 8 weeks were randomly divided into 4 groups:normal group（n=10）（T0 group）,normal group+metformin group（n=10）（T1 group）,diabetic group（n=10）（T2 group）,diabetes+metformin group（n=10）（T3 group）.After 3 weeks of high fat and high sugar diet in T2 and T3 groups,1%,35mg/kg streptozotocin was injected to create type 2 diabetes model.After the model was established,pure titanium implants were implanted in bilateral tibial epiphyseal of all rats.On the day of implantation,T1 and T3 groups were intragastric with the therapeutic dose of metformin 300mg·kg^-1·d^-1,and T0 and T1 groups were intragastric with the same amount of normal saline once a day.At 4 and 8 weeks,5 rats in each group were sacrificed randomly,and the morphology and microstructure of bone tissue were observed by H&E staining and Micro-CT.The expression of HMGB1 and RAGE were detected by immunohistochemistry.Serological ELISA was used to detect the contents of HMGB1,RAGE,OCN and TNF-αaround implants.Results:At the 4th and 8th week after metformin intervention,body weight of T3 group[（372.97±26.34）and（484.02±15.58）g]was significantly increased compared with T2group[（301.78±29.12）and（255.20±43.12）g]（P<0.01）.The random blood glucose levels[（7.60±9.25）and（6.09±0.82）mmol/L]were significantly decreased[（19.78±3.73）and（19.23±3.41）mmol/L]（P<0.01）.Micro-CT analysis,metformin intervention for 4and 8 weeks,T3 group bone volume fraction（BV/TV）[（0.50±0.04）and（0.56±0.04）%],bone trabecular number（Tb.N）[（1.63±0.05）and（1.81±0.04）/mm],bone trabecular thickness（Tb.Th）[（0.76±0.04）and（0.82±0.02）mm]were significantly higher than those in T2 group[（0.42±0.01）%,（0.39±0.01）%,（1.39±0.11）/mm,（1.55±0.05）/mm,（0.65±0.03）mm and（0.53±0.04）mm]（P<0.01）.Tb.Sp[（0.53±0.08）and（0.26±0.04）mm]were significantly lower than those in T2 group[（0.90±0.01）and（0.93±0.06）mm]（P<0.01）.At the 4th week,BV/TV[（0.57±0.01）%]and Tb.N[（1.70±0.06）/mm]in T1group were higher than those in T0 group[（0.52±0.03）%]and[（1.57±0.05）/mm]（P<0.05）.At the 8th week,Tb.N in T1 group[（2.32±0.34）/mm]was higher than that in T0 group[（1.88±0.06）/mm]（P<0.05）.At 4 and 8 weeks after HE staining,the trabecular structure of T2 group was disorganized and slender,with fewer neovascularization,less mature lamellar bone structure and more osteoclasts than that of T0 group.After 4 and 8weeks of immunohistochemistry,the positive expression of HMGB1 and RAGE decreased in T3 group.The average optical densities of HMGB1（61.92±13.60）,（62.80±16.80）and RAGE（28.58±14.78）,（33.71±23.97）were lower than those of T2group（339.82±58.31）,（368.05±61.58）,（170.23±73.02）and（157.80±49.70）（P<0.01）.At4 and 8 weeks,serum HMGB1 in T3 group[（0.64±0.30）and（0.47±0.23）μg/L]was detected by ELISA.The content of RAGE[（63.26±7.44）and（45.78±5.85）pg/ml],TNF-α[（93.15±6.93）and（76.56±9.09）ng/L]were significantly lower than T2 group[（4.15±0.47）μg/L,（3.22±0.42）μg/L,（89.66±10.29）pg/ml,（62.62±7.68）pg/ml,（110.85±7.39）and（83.62±2.34）ng/L]（P<0.01）.The content of OCN[（1.62±0.43）and（0.81±0.16）ng/L]was significantly higher than that in T2 group[（0.82±0.24）and（0.39±0.15）ng/L]（P<0.05）.At 8 weeks,the content of RAGE in T1 group[（35.49±2.77）ng/L]was significantly lower than that in T0 group[（44.92±7.99）ng/L],and the content of OCN in T1 group[（1.32±0.19）ng/L]was significantly higher than that in T0 group[（0.89±0.26）ng/L]（P<0.01）.Conclusions:1.Metformin can reduce the expression of HMGB1 and RAGE in type 2 diabetic rats,which may be one of the mechanisms promoting osseointegration.2.Metformin has bone protective effect on non-diabetic rats.