Plastrum Testudinis Extracts Via Controling Orientation Of Macrophage Polarization Ameliorates Senile Osteoporosis And Promote Fracture Healing

Objective1.To explore the method of constructing SOP model in C57 mice and research the role of plastrum testudinis extracts in the treatment of SOP and to explore the mechanism based on macrophage polarization pathway.2.Based on the classical C57BL/6 mouse bone defect model,we explored the role of different macrophage polarization subtypes in regulating fracture healing.And try to extract macrophages from human blood for forward culture and identification.3.To detect the serum TNF-α expression level of SOP patients and initially explore the osteoimmune mechanism of SOP.Methods1.To explore the effect of plastrum testudinis extracts in the treatment of SOP based on macrophage polarization pathway.Sixty 8-week-old male C57 mice were randomly divided into control group,SOP group and SOP+plastrum testudinis extracts intervention group,10 mice in each group.After 10 weeks,micro-CT was applied to examine the microstructure of L2 bone,HE to observe the morphology of hippocampal tissue in the brain,HE staining to observe the morphology of bone tissue in L4,HE staining was used to observe the microscopic morphology of the bone tissue,TRAP staining to observe the number of osteoclasts in L4,and Western Blot to detect the protein expression levels of INOS and ARG1 in the lumbar spine.2.In vitro targeted induction of macrophage polarization followed by local injection of different macrophage polarization subtypes to observe their effects on fracture healing.Forty-eight 15-week-old male C57 mice were selected and randomly divided into PBS,M1 and M2 groups,with 8 mice in each group and 6 groups in total.The tibial bone defects were molded in each group,and PBS,M1 macrophages and M2 macrophages were injected locally into the bone defects immediately and every 3 days after the molding.The mice were sampled at the 2nd and 4th weeks after surgery.After sampling,micro-CT was used to examine the structure of the tibial bone defect,HE staining was used to observe the microscopic morphology of the bone tissue,TRAP staining was used to observe the number of osteoclasts and the percentage of osteoclast circumference in the tibial bone defect,and the protein expression levels of INOS and ARG1 in the tibial bone defect were measured by the set spectral density method.3.Clinical study Eighty serum specimens were collected from each of the elderly and non-elderly groups,and the TNF-α levels were measured in both groups.Results1.To explore the effect of plastrum testudinis extracts in the treatment of SOP based on macrophage polarization pathway(1)Comparison of microstructural parameters of lumbar vertebrae: in male mice,compared with the control group,BV/TV and Tb.N were reduced and Tb.Sp was increased in the SOP group,and these were reversed after plastrum testudinis extracts intervention.All the above changes were statistically significant.(2)HE,Nissler staining to observe the morphology of hippocampal tissue in the brain: in male mice,hippocampal neuronal cells in the SOP group showed abnormal changes with reduced cell volume,irregular nuclei and scattered cell arrangement compared with the control group.Compared with the SOP group,hippocampal neuronal cells in the SOP+PTE group were restored.(3)HE staining of lumbar vertebral bone tissues: in male mice,compared with the control group,the number of bone trabeculae was reduced,the bone trabeculae were slender and broken,and the medullary cavity was enlarged in the SOP group.Compared with the SOP group,the number of trabeculae in the SOP+PTE group increased,the trabeculae were thickened and continuity was significantly improved,and the medullary cavity was relatively smaller.(4)TRAP staining of lumbar vertebral bone tissue: there was no difference in the number of osteoclasts(Oc.N/BS)(/mm)and the percentage of osteoclast perimeter(Oc.S/BS)(%)in the SOP group compared with the control group.Both of these were significantly reduced in the SOP+PTE group compared with the SOP group after the application of plastrum testudinis extracts’ intervention(p < 0.05).(5)Comparison of protein expression in lumbar spine: in male mice,i NOS protein expression decreased in SOP+PTE group compared with SOP group;ARG1 protein expression increased.2.Local injection of different macrophage polarization subtypes to observe their effects on fracture healing(1)Comparison of bone microstructural parameters of tibial bone defects: both at the early stage of healing(2 weeks)and at the late stage of healing(4 weeks),the bone cortex of tibial bone defects in the M2 group healed better,with higher healing rate and higher BV/TV compared with the PBS and M1 groups(p < 0.05).(2)HE at tibial bone defect,Trap staining: after 2 weeks of modeling,the healing rate of bone defects was higher in the M2 group compared with the PBS and M1 groups(p < 0.05);and after 4 weeks of modeling,the healing rate of bone defects was higher in the M2 group compared with PBS(p < 0.05).However,there was no difference in the number of osteoclasts between the 3 groups,whether after 2 or 4 weeks.(3)Protein expression levels of INOS and ARG1: At 2 weeks of modeling,i NOS protein expression was lower in the M2 group compared to the PBS and M1 groups(p < 0.05),but ARG1 expression was not statistically different.However,at 4 weeks of staging,ARG1 protein expression was lowest in the M2 group compared with the PBS and M1 groups(p <0.05).(4)Extraction,culture of human blood macrophages: it has been possible to extract and culture viable,differentiable human macrophages directly from different populations of human blood.3.Serum TNF-α expression levels were significantly higher in the older male group compared to the younger male group(P < 0.05).There was no statistical significance between the female groups.Conclusions1.The SOP model obtained by using detesticular surgery + galactose intervention was consistent with the changes of senile osteoporosis.And because of inhibiting the polarization of M1 macrophages and promoting the polarization of M2 macrophages,the plastrum testudinis extracts could ameliorate the bone loss and structural degeneration of hippocampal neurons caused by SOP by2.Directly changing the M1/M2 balance of macrophage polarization by local injection of M2 macrophages can promote bone regeneration.3.Clinical studies: Validation of the tilt of the macrophage polarization balance in the male internal environment toward the pro-M1 endpoint may be an important factor mediating the development of SOP.