| Objective Investigating the effects of alendronate on mi R-133a/Runx2 and femoral microstructures in Wistar rats’ formal tissue with diabetic osteoporosis to explore the mechanism that alendronate regulated mi R-133 a expression for anti-osteoporosis in diabetic osteoporosis(DOP),so as to provide theoretical evidence for the prevention and treatment of DOP.MethodsWe utilized 40 male Wistar rats(8 weeks of age,body weight: 190g-210g)from Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences.10 rats were randomly selected as control group after 4 weeks of adaptive feeding.Wistar rat model of diabetic mellitus induced with a single intraperitoneal injection of streptozotocin(STZ;30 mg/kg;Sigma).72 h following the injection of STZ,tail blood samples were obtained for measuring blood glucose concentration by using glucometer.Rat with random blood glucose ≥16.7 mmol/L was considered diabetic mellitus.Bone mineral density(BMD)was measured in all rats per 4 weeks after successfully establishing the diabetic models.There was a significant difference in BMD between the normal control group and diabetic groups,which demonstrated that DOP model was successfully established.Rats in the normal control group(NC control group,n=10)were treated with vehicle(normal saline,NS,gavage);rats in the diabetic group treated with vehicle(normal saline,NS,gavage)were utilized as the DOP control group(DM control group,n=10);rats in the diabetic group treated with mi R-133 a inhibitor solution(30μg/4days,tail vein injection)were utilized as DOP with MI control group(MI group,n=10);rats in the diabetic group treated with alendronate solution(5mg/kg/week,gavage)were utilized as DOP with alendronate control group(AD group,n=10)for 6 weeks.Femoral tissue from each rat was divided into two parts: one was immediately frozen in liquid nitrogen for Western blotting and RT-PCR,and the other was fixed with 4% paraformaldehyde for Harris hematoxylin and Shandon Instant eosin(H&E)and Masson-trichrome staining.Results General conditions: The diabetic rats had more to drink,polyuria,polyphagia,weight loss ofthe typical "more than three a little" symptom,yellow fur,unresponsiveness and moved slowly.72 hours after STZ injection,body weights of diabetic and intervention groups decreased,while body weights in the NC control group kept on growing(P<0.05).There were no significant differences in body weights among the three diabetic groups after STZ injection(P>0.05).Serum glucose suddenly exceeded the normal level in the diabetic groups after STZ injection and continued to be more than 16.7mmol/L.There were no significant differences in serum glucose among the three diabetic groups after STZ injection(P<0.05).RT-PCR detection: Compared with the NC control group,mi R-133 a was significantly enhanced in the DM group(P<0.05),while the expression of Runx2 and Osterix m RNAs were significantly decreased in the DM group(P<0.05).After the intervention of mi R-133 a inhibitor for 4 weeks,the expression of mi R-133 a was significantly decreased in the MI group compared with the DM control group(P<0.05),while the expression of Runx2 and Osterix m RNAs were significantly increased in the MI group(P<0.05).Meanwhile,after the treatment of alendronate for 6 weeks,the expression of mi R-133 a was significantly decreased in the AD group compared with the DM control group(P<0.05),while the expression of Runx2 and Osterix m RNAs were significantly increased in the AD group(P<0.05).Western Blotting detection: Compared with the NC control group,the expression of Runx2 and Osterix protein was significantly decreased in the DM group(P<0.05),while their expression was increased in MI group and AD group compared with the DM group(P<0.05).Bone metabolic markers detection: The serum ALP concentration was significantly decreased in the DM group,which was positively impacted by the intervention with mi R-133 a inhibitor and alendronate(P<0.05).The concentrations of serum PINP and PICP were significantly decreased in the DM group,which were promoted by the intervention with mi R-133 a inhibitor and alendronate(P<0.05).Micro-CT and histopathological detection: The femoral trabecular bone microarchitectures in the DM group had significant differences compared with that in the NC and AD groups.There were no significant effects between the NC and DM groups on femoral trabecular bone microarchitectures.The femoral BMD in the DM group had significant reduction compared with that in the NC,AD groups(P< 0.05).However,there were no significant difference between the NC and AD groups.BV/TV,Tb.Th,and Tb.N,decreased with diabetes mellitus,was significantly increased in AD groups(P< 0.05).In addition,increased Tb.Sp and SMI in the DM group compared with that in the NC group were decreased by intervention with alendronate(P< 0.05).the histological cross sections of the femur showed that the bony meshwork appeared in the metaphsis of each group.Compared with NC control group,there were disordered trabecular,enlargement of marrow cavity,and apparent reduction in trabecular number,trabecula thickness and collagen fibres in DM group.In contrast,the cancellous bones appeared to be added in the metaphyseal area of diabetic rats treated with alendronate.Conclusions: 1.In the femur of diabetic osteoporosis rats,the expression of mi R-133 a that regulated the osteoblasts differentiation increased,while the expression of Runx2/Osterix m RNA and protein decreased,and bone formation decreased.2.Alendronate could ameliorate DOP by inhibiting mi R-133 a expression and promoting expression of Runx2/Osterix m RNA and protein,which was one of the mechanisms to protect against DOP,and that mi R-133 a may be one of the therapeutic targets for alendronate in DOP. |
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