| Objective: This study has aimed to assess the effects of naringenin on osteoporotic fracture(OPF) healing in rats, and to observe the impact of naringenin on bone mineral density(BMD), biomechanics, imaging, histology in OPF rats caused by ovariectomy surgery. Further research the effects of naringenin on OPG/RANKL/RANK system in OPF rats to explore its mechanism.Methods:1 Animal Grouping and administration SD rats, 60, were randomly divided into control group(Control), model group(Model), the low dose naringenin group(Nar-L): 35 mg/kg group, the medium dose naringenin group(Nar-M):70mg/kg group and the high dose naringenin group(Nar-H):140 mg/kg group.Each group of 12 animals, with 10 ml/kg volume, were continuous lavaged for eight weeks. Control group and model group were given an equal volume of0.5% CMC-Na solution.2 Preparation of model Ovariectomized rat model of osteoporosis preparation:Rats were anesthetized with sodium pentobarbital, with axillary midline costal1 cm under waist as the center, successively performed ovariectomy surgery after iodine, alcohol disinfection. Conventional breeding ovariectomized rats12 weeks after surgery, resulting in osteoporosis rats. Anesthetized rat model of osteoporosis, exposure right femur, femoral cut with a hacksaw, causing transverse fracture, after anterograde implantation 1.5mm diameter Kirschner,and rat osteoporotic fracture model was prepared.3 Observation indexes and test methods3.1 Bone mineral density BMD test: Rats were anesthetized to measure the right femoral bone mineral density BMD in rats by dual-energy X-ray absorptiometry.3.2 Observation of Radiology:To observe the fracture line rat growth and fracture callus line situation by dual-energy X-ray.3.3 Blood biochemical parameters:Anesthetized animals were sacrificed. The abdominal aortic blood centrifuged.Calcium Ca, phosphorus P, alkaline phosphatase, ALP, acid phosphatase ACP contents in serum calcium were measured by biochemical analyzer.3.4 Bone biomechanical testing:Using the universal testing machine operated side femur three-point bending test, measure biomechanical parameters including the maximum bending strength, flexural strength, fracture deflection,elastomeric block end.3.5 Histological HE dyeing:Histological observation of the fracture callus under a microscope by HE staining.3.6 OPG, RANKL and RANK m RNA expression: The mothod of real-time fluorescent quantitative reverse transcription PCR were taken.3.7 OPG, RANKL and RANK protein expression: The mothod of western blot were taken.Results:1 Blood biochemistry analysis in each group Compared with the control group, serum Ca, P level obviously decreased(P<0.01), the ALP level increased obviously(P<0.01) in model group. Compared with model group,Ca level were significantly increased in the medium, high dose naringenin group(P<0.01). P level were significantly increased in the low, medium and high dose naringenin group(P<0.01). ALP level were decreased significantly in low, medium and high dose naringenin group(P<0.01), and ACP level in each group had no obvious change.2 BMD analysis in each group Compared with the control group, BMD level obviously increased in model group(P<0.01). Compared with model group, BMD level were significantly increased in the medium, high dose naringenin group(P <0.01).3 Biomechanical parameters analysis in each group Compared with the control group, the maximum bending strength, flexural strength, fracturedeflection, elastomeric block end obviously decreased in model group(P<0.01). Compared with model group, the maximum bending strength were significantly increased in the low, medium, high dose naringenin group(P<0.01). Flexural strength were increased in the high dose naringenin group(P<0.01). Fracture deflection, elastomeric block end were increased in the medium, high dose naringenin group(P<0.01).4 Dual energy X-ray radiography results analysis X-ray abserved that in the model group, a significant fracture line, obvious callus growth around were showed. Compared with the model group, fracture gap decreased in naringenin group. High dose naringenin reduced callus volume small, promote the most obvious fracture healing.5 Histological HE dyeing Optical microscope observed that in the control group there is a lot of mature trabecular bone formation, bone trabecular coarser and arrange compact and orderly, the number is more, line up with the direction of principal stress direction. In model group, visible cartilaginous callus, a few mature trabecular bone cells, bone trabecular thickness uneven and disordered arrangement, with the principal stress direction, staggered into mesh each other. Naringenin improved the pathological changes of the trabecular bone coarser, increase in the number,arrangement relatively orderly.6 OPG, RANKL, RANK m RNA expression changes in femur of the rats Compared with the control group, OPG m RNA expression decreased significantly(P<0.01), m RNA expression of RANKL, RANK increased of femur in the model group(P<0.01). Compared with the model group, OPG m RNA expression was significantly increased in naringenin group of low,medium and high dose group(P<0.01). RANKL and RANK m RNA expression of low, medium, high dose naringenin group decreased significantly(P <0.01).7 OPG, RANKL, RANK protein expression changes in femur of the rats Compared with the control group, OPG protein expression decreased significantly, RANKL, RANK protein expression increased of femur in themodel group(P<0.01). Compared with the model group, OPG protein expression was significantly increased in naringenin group of low, medium and high dose group(P<0.01). RANKL protein expression of high dose naringenin group decreased significantly(P<0.05). RANKL protein expression of medium, high dose naringenin group decreased significantly(P<0.01).Conclusion: Naringenin has the effect of promoting fracture healing for ovarian rats osteoporosis fracture, related to regulate OPG/RANKL/RANK system. |
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