| Diabetes mellitus(DM)is a group of chronic metabolic diseases characterized by hyperglycemia.With the improvement of people's living standards and the aggravation of population ageing,the prevalence of diabetes mellitus has been increasing year by year.It is estimated that there will be nearly 550 million people worldwide with diabetes by 2030.China's latest epidemiological survey show that the prevalence of diabetes mellitus is as high as 11.2%.Type 2 diabetes mellitus(T2DM)accounted for more than90.0%.The main characteristic of type 2 diabetes mellitus is insulin resistance.Long-term derangements of blood glucose,lipid and protein metabolism can lead to multi-system damagement,which not only leads to chronic progressive diseases of the eyes,kidneys,nerves,heart and blood vessels,but also has the influence on the bone metabolism.Back in 1948,Albright reported that chronic poor blood glucose control in diabetes mellitus can lead to osteoporosis.Osteoporosis is a kind of systemic metabolic bone disease which is prone to fracture due to the reduction of bone mass,the destruction of bone microstructure and the increase of bone fragility.Although bone mineral density may be reduced,normal,or increased in type 2 diabetes mellitus,the risk of osteoporosis and fracture risk is increased.It is estimated that between20% and 60% of people with type 2 diabetes mellitus develop osteoporosis.The study showed a 69.0% increased risk of fracture in patients with type 2diabetes mellitus compared with healthy controls.In addition,type 2 diabetes mellitus affects the effectiveness of osteoporotic treatment.Increasing bone fragility is now considered to be one of the chronic complications of type 2diabetes mellitus.Type 2 diabetes mellitus may lead to bone loss,destruction of bone microstructure and increase of bone fragility,which may lead to osteoporosis or osteoporotic fracture,but the mechanism is not completely clear.Recent studies have found that antidiabetic drugs also have some effects on bone.Metformin is a biguanide that has been used clinically for more than60 years.As a first line treatment for type 2 diabetes mellitus,metformin is currently one of the most widely used oral anti-diabetic agents in the world,with a definite hypoglycemic effect,metformin alone or combination can effectively reduce fasting blood glucose and postprandial blood glucose in patients with type 2 diabetes mellitus.Its main pharmacological effects are through inhibiting liver gluconeogenesis,reducing liver glucose output,improving muscle glycogen synthesis,reducing free fatty acid,improving peripheral tissue sensitivity to insulin and insulin resistence,increasing glucose uptake and utilization,inhibiting glucose absorption by intestinal wall cells.In addition to lowering blood glucose,metformin can also improve blood lipids,increase fibrinolytic activity,inhibit platelet aggregation,improve endothelium function and so on.In recent years,more and more attention has been paid to the role of metformin except its hypoglycemic effect.There have been conflicting reports about the effects of metformin on bone metabolism.Studies have shown that metformin can improve bone loss from ovariectomy and ketogenic diet induced bone loss,and reduce the risk of osteoporosis and fractures.But some studies suggest that metformin has no significant effect on bone metabolism.In Vitro studies have shown that metformin not only promotes proliferation,differentiation and mineralization of the osteoblast cell line MC3T3E1,it can also promote the differentiation of bone marrow mesenchymal stem cells(BMSCs)and adipose stem cells(ASCs)into osteoblasts(OB),but the mechanism has not been fully elucidated.The present study established the rats model with type 2 diabetes and type 2 diabetic osteoporosis to observe the changes of bone mineral density,bone strength and serum bone metabolic indexs in type 2 diabetes and type 2diabetic osteoporosis rats treated with metformin and investigate the effects and the underlying mechanisms of metformin on the differentiation of BMSCs into osteoblasts in type 2 diabetes and type 2 diabetic osteoporosis rats.Finally,the effects of different doses of metformin on serum bone metabolic markers in elderly men with type 2 diabetes mellitus were observed.The present study was divided into three parts: the first part is the effects of metformin on bone mineral density,biomechanics,and serum bone metabolic markers in type 2 diabetic osteoporosis rats.The second part is about the effect of metformin on the differentiation of BMSCs into osteoblasts in type 2 diabetic rats with osteoporosis and the underlying mechanisms.The third part is the effects of different doses of metformin on serum bone metabolic markers in elderly men with type 2 diabetes mellitus.Part one The effect of metformin on bone mineral density,biomechanics and serum bone metabolic markers in type 2 diabetic rats with osteoporosisObjective: To investigate the effects of metformin on bone mineral density,bone strength and serum bone metabolic markers in type 2 diabetic rats with osteoporosis.Methods:1.60 female Wistar rats aged 2.5-3 months,weighing 180-200 g,were randomly divided into two groups: routine diet group(N=20)and high sugar and high fat diet group(N=40).The rats in the routine diet group were given normal diet,and the rats in the high-sugar and high-fat Diet group were given the high-sugar and high-fat diet,for 8 weeks.After 12 hours of fasting,rats in the high-sugar and high-fat diet group were injected with a small dose of streptozocin(STZ 0.1 mmol/l,sodium citrate preparation,PH4.2)30 mg/kg in the left lower abdominal cavity,and rats in the routine diet group were injected with the same amount of normal saline in the left lower abdominal cavity.After 1 week of STZ injection,rats were fed with high-sugar and high-fat diet,fasting blood glucose(FBG)?7.8 mmol/l and insulin resistance were measured as T2 DM animal models.2.One week after the establishment of T2 DM animal model,the rats were randomly divided into normal control group(NC group,N = 10)and ovariectomized group(NOVX group N = 10),the rats in the high-sugar and high-fat diet group were randomly divided into four groups: type 2 diabetic control group(T2DM group,N = 10),type 2 diabetic plus metformin treatment group(T2DM + M group,N = 10),type 2 diabetic ovariectomized group(DOVX group,N = 10)and type 2 diabetic ovariectomized plus metformin treatment group(DOVX + M group,N = 10).Ovariectomy was performed in NOVX group,DOVX group and DOVX + M group.Sham operation was performed in DM group and DM + M group.3.4 weeks after ovariectomy,rats in DM + M group and DOVX + M group were given metformin 100 mg/kg for 8 weeks,the body weight,fasting blood glucose(FBG),fasting insulin(FINS),bone alkaline phosphatase(B-ALP),tartrate-resistant acid phosphatase 5b(TRAP-5b),bone mineral density(BMD)and L3-load were measured.Calculate the HOMA-IR index based on FBG and FINS.Results:1.After 8 weeks of meteformin treatment,compared with the NC group,the body weight,FBG,FINS,HOMA-IR index,serum TRAP-5b significantly increased,whereas serum B-ALP,BMD and L3-Load significantly decreased in the DM group(P < 0.05).FBG,FINS,HOMA-IR index,serum TRAP-5b and B-ALP of the NOVX group were significantly increased,whereas BMD and L3-Load significantly decreased(P < 0.05).Serum levels of B-ALP and TRAP-5b in the NOVX group were significantly higher than those in the DM Group(P < 0.05),but there was no significant differences in BMD and L3-Load between the two groups(P > 0.05).Compared with the NOVX group,the serum levels of B-ALP,TRAP-5b,BMD and L3-Load were significantly decreased in the DOVX group(P < 0.05).Compared with the DM group,the body weight,FBG,FINS,HOMA-IR index,serum TRAP-5b significantly decreased,serum B-ALP,BMD and L3-Load significantly increased in the DM + M group(P < 0.05).Compared with the DOVX group,the body weight,FBG,FINS,HOMA-IR index,serum TRAP-5b significantly decreased,serum B-ALP,BMD and L3-Load significantly increased in the DOVX + M group(P < 0.05).2.Further Pearson Correlation Analysis showed that serum B-ALP was negatively correlated with body weight,FBG,FINS,HOMA-IR index,and positively correlated with BMD(P < 0.05),but not significantly correlated with L3-Load(P >0.05).Serum TRAP-5b was positively correlated with body weight,FBG,FINS,HOMA-IR index,and negatively correlated with BMD and L3-Load(P < 0.05).Conclusions:1.Type 2 diabetes and type 2 diabetic osteoporosis rats showed decreased bone formation and increased bone resorption.2.Metformin may improves bone mineral density,bone strength and serum bone metabolism markers through body weight,FBG and IR in rats with type 2 diabetes mellitus and type 2 diabetic osteoporosis.Part two The effect of metformin on the differentiation of bone marrow mesenchymal stem cells into osteoblasts in type 2 diabetic rats with osteoporosis and the underlying mechanismsObjective: To investigate the effect of metformin on the differentiation of bone marrow mesenchymal stem cell(BMSCs)into osteoblasts in type 2diabetic rats with osteoporosis after 8 weeks treatment.Methods:1.BMSCs of rats in NC group,DM group,DM + M group,NOVX group,DOVX group,and DOVX + M group of the first part of the present study,were were isolated from the femurs by whole bone marrow adherent method and passaged to the 3rd generation.Osteoblast differentiation was induced to the 7th day and the alkaline phosphatase(ALP)activity was detected by ALP staining,the total RNA and protein were extracted,and the RNA and protein levels of Runx2 were detected by Real-time RT-PCR and Western Blot respectively.2.At the 14 th day,the RNA and protein levels of OCN were detected by Real-time RT-PCR and Western blot respectively.Calcium deposits were detected by AR staining.3.Western blot was used to detect the expression of phosphorylated AKT and AKT on the 3rd day after osteoblastic differentiation.Results:1.Compared with the NC group,ALP activity of osteoblasts in the DM,DM + M,NOVX,DOVX and DOVX + M groups decreased significantly(P <0.05).Compared with the NOVX group,ALP activity of osteoblasts in the DOVX group was significantly decreased(P < 0.05).Compared with the DM group,ALP activity of osteoblasts in the DM + M group was significantly increased(P < 0.05).Compared with the DOVX group,ALP activity of osteoblasts in the DOVX + M group was significantly increased(P < 0.05).2.Compared with the NC group,the RNA levels of RUNX2 and OCN in osteoblasts of the DM,DM + M,NOVX,DOVX and DOVX + M groups were significantly decreased(P < 0.05).Compared with the NOVX group,the RNA levels of RUNX2 and OCN of osteoblasts in the DOVX group significantly decreased(P < 0.05).Compared with the DM group,the RNA levels of RUNX2 and OCN of osteoblasts in the DM + M group significantly increased(P < 0.05).Compared with the DOVX group,the RNA levels of RUNX2 and OCN of osteoblasts in the DOVX + M group significantly increased(P < 0.05).3.Western blot further confirmed the results,compared with the NC group,the protein levels of RUNX2 and OCN were significantly decreased in the DM,DM + M,NOVX,DOVX and DOVX + M groups(P < 0.05).Compared with the NOVX group,the protein levels of RUNX2 and OCN were significantly decreased in the DOVX group(P < 0.05).Compared with the DM group,the protein levels of RUNX2 and OCN in the DM + M group significantly increased(P < 0.05).Compared with the DOVX group,the protein levels of RUNX2 and OCN of osteoblasts in the DOVX + M group significantly increased(P < 0.05).4.Semi-quantitative results of Alizarin Red(AR)showed that,compared with the NC group,the calcium deposition in the DM,DM + M,NOVX,DOVX and DOVX + M groups significantly decreased(P < 0.05).Compared with the NOVX group,the calcium deposition in the DOVX group significantly decreased(P < 0.05).Compared with the DM group,the calcium deposition in the DM + M group significantly increased(P < 0.05).And compared with the DOVX group,the calcium deposition in the DOVX + M group significantly increased(P < 0.05).5.The expression of p AKT and AKT was detected by Western blot at the3 rd day of culture of osteoblasts.The results showed that the p AKT/AKT ratios of osteoblasts in the DM,DM + M,NOVX,DOVX and DOVX + M groups significantly decreased compared with the NC group(P < 0.05).Compared with the NOVX group,p AKT/AKT ratio significantly decreased in the DOVX group(P < 0.05).Compared with the DM group,p AKT/AKT ratio significantly increased in the DM + M group(P < 0.05).Compared with the DOVX group,p AKT/AKT ratio significantly increased in the DOVX + M group(P < 0.05).Conclusions:1.The differentiation of BMSCs into osteoblasts was inhibited,the activity of alkaline phosphatase and the calcium deposition were decreased in type 2 diabetes and type 2 diabetic osteoporosis rats.2.Metformin may improve BMSCs differentiation into osteoblasts,enhance alkaline phosphatase activity,and increase calcium deposition in type2 diabetic and type 2 diabetic osteoporosis rats through AKT phosphorylation.Part three Effects of different doses of metformin on serum bone metabolic markers in elderly male patients with type 2 diabetes mellitusObjective: To investigate the effects of different doses of metformin on serum bone metabolic markers in elderly male patients with type 2 diabetes mellitus.Methods:1.From July 2018 to June 2019,120 elderly male patients with type 2diabetes were enrolled in the endocrinology department of Cangzhou Central Hospital in Hebei province.The patients were randomly divided into experimental Group(N = 60)and control Group(N = 60).2.Patients in the experimental group were given 0.5g metformin 4times per day,while those in the control group were given 0.5g metformin 2times per day,for 12 weeks.Serum P1 NP,N-MID,?-CTX and 25(OH)D were measured before and after treatment.Results:1.There was no significant differences of therapeutic effect on blood glucose between experimental group and control group after the treatment(P>0.05).2.There was no significant differences in serum bone metabolism markers between the two groups before treatment(P>0.05).After treatment,the bone metabolism markers were improved in both groups.The serum25(OH)D levels in the experimental group were significantly higher than those in the control group(P < 0.05).The serum N-MID and ?-CTX levels in the experimental group were significantly lower than those in the control group(P < 0.05).There was no significant difference in serum PINP levels between the experimental group and control group.Conclusions:1.Both high and low doses of metformin are effective in controlling blood glucose in elderly men with type 2 diabetes mellitus.2.Both high and low doses of metformin contribute to the improvement of serum markers of bone metabolism in elderly male with type 2 diabetes mellitus,and depending on doses. |
PDF Full Text Request