Simvastatin Alleviates The Bone Loss Caused By Ketogenic Diet:an In Vivo Study In Mice

ObjectiveTo evaluate the therapeutic effect of simvastatin in alleviating bone loss caused by ovariectomy and ketogenic diet,respectively.Specifically to analyze distal femoral microstructure,mechanical properties and bone morphology in mice treated with simvastatin(ST)in the two animal models.The differences between the two animal models were compared by analyzing the changes of bone microstructure,mechanical properties and bone morphology of L4 vertebral body and femur.MethodsA total of 40 C57 mice were randomly and evenly divided into sham group,ovariectomized group(OVX),ovariectomized with simvastatin treatment group(OVX + ST),ketogenic diet group(KD)and the ketogenic diet with simvastatin treatment group(KD + ST)group,respectively.The mice in the OVX and OVX+ST groups underwent ovariectomy and fed with standard diet.The mice in the KD and KD+ST groups were fed with ketogenic diet whose ratio of carbohydrate and fat is 1:3.Simvastatin were administered to the mice with a dosage of 20mg/(kg·d)by gavage.Blood ketone,blood glucose,serum calcium and serum phosphorus were measured at the end of the study of 12 weeks.The trabecular microstructure,biomechanical properties and bone morphological changes of femur and L4 lumbar body were evaluated by MicroCT scan and/or three-point bending tests,finite element analysis and HE staining.ResultsThe blood ketone levels were 1.28mmol/L and 1.48mmol/L in the KD group and KD+ST group,respectively,and significantly higher than that in the sham group(0.60mmol/L).There are no significant differences in body weight,blood glucose,blood calcium or phosphorus between groups.Compared with the sham group,connectivity density(36 and 69mg HA/CCM),bone volume fraction(4%and 7%),trabecular connection density(16 and 39 mm-3)and trabecular number(2.06 and 3.38 mm'1)of the distal femoral cancellous bone in the OVX group decreased while trabecular separation increased(0.48 and 0.3 mm),respectively.However,the changes of cortical bone in the middle femur were not significant.The stiffness of femoral finite element compression(47 and 320 N/mm)and three-point bending stiffness(41 and 43 N/mm)decreased.The histology of distal femur also showed that the trabeculae of distal femur cancellous bone in the OVX group was significantly less than that in the sham group.Simvastatin treatment improved the cancellous bone,the femoral finite element compression stiffness and three point bending stiffness of the OVX + ST group.Such as distal femur cancellous bone volume fraction increased from 4%to 6%,the finite element compression stiffness increased from 47 N/mm to 233 N/mm,femur three point bending stiffness increased from 41 N/mm to 57 N/mm.Histological observation also showed that distal femur cancellous bone trabecular of the OVX + ST group improved significantly than that of the OVX group.Compared with the Sham group,bone volume fraction(0.10 and 0.16),number of bone trabeculae(3.0 and 3.6 mm-1)and bone trabeculae connectivity(37 a:nd 60 mm-3)of L4 vertebral in the OVX group were decreased.However,the trabecular separation increased(0.34 and 0.29 mm).The axial compression stiffness of L4 vertebral body also decreased(1591 and 1984 N/mm).Histology also showed that the trabecular bone in OVX group was significantly reduced.Compared with the sham group,connectivity density(39 HA/ccm),bone volume fraction(4%),trabecular connection density(11 Imm-3)and trabecular number(2.1 mm-1)of the distal femoral cancellous bone in the KD group decreased while trabecular separation increased(0.44 mm),respectively.However,the changes of cortical bone in the middle femur were not significant.The stiffness of three-point bending(35 mm-1)decreased.The histology of distal femur also showed that the trabeculae of distal femur cancellous bone in the KD group was significantly less than that in the sham group.Simvastatin treatment improved the cancellous bone,the femoral finite element compression stiffness and three point bending stiffness of the KD + ST group.Such as distal femur cancellous bone volume fraction increased to 6%,the finite element compression stiffness increased to 161 N/mm and femur three point bending stiffness increased to 60 N/mm.Histological observation also showed that distal femur cancellous bone trabecular of the OVX + ST group improved significantly than that of the OVX group.There were no significant differences between KD group and Sham group in L4 vertebral bone volume fraction(0.16),number of bone trabeculae(3.3 mm-1),bone trabeculae connectivity(53.4 mm-3),bone trabeculae separation degree(0.3 mm)and axial compression stiffness(1717 N/mm).No significant change of L4 vertebral body was observed by histological observation.The above indicators also failed to change after ST treatment.The K.D group and OVX group had similar changes in cancellous bone of distal femur and cortical bone of middle femur,while those indexes ofL4 vertebral body in the OVX group were lower than those in KD group or Sham group.ConclusionsBoth of the ketogenic diet and ovariectomy induced osteoporosis and lead to the bone loss in the long bones of the limbs,such as the femur,with most affected part is cancellous bone.However,for axial bones,such as the lumbar vertebra,ovariectomy can still result in bone loss in the lumbar vertebra,while ketogenic diet could not lead to corresponding bone loss.Simvastatin treatment alleviated the bone loss caused by OVX and ketogenic diet,improving the bone volume fraction of femoral cancellous bone,three-point bending stiffness,finite element compression stiffness and the number of bone trabeculae in mice.