| Research background and purpose:Postmenopausal osteoporosis(PMOP)is a common metabolic bone disease in postmenopausal women,and brittle fracture is the most common clinical complication,which seriously affects the quality of life of patients and even increases the risk of death.It is bound to cause a certain burden to the family,medical treatment and society.Therefore,timely and effective prevention and treatment of PMOP is very important and has become the focus of attention.The clinical treatment methods of Chinese and western medicine vary,mainly western medicine,mainly focused on promoting bone formation and inhibiting bone damage,there are many shortcomings such as long treatment cycle,many side effects.Electroacupuncture(EA)treatment as a kind of external therapy in traditional medicine in our country,many clinical studies have confirmed its acupuncture and cure effect to affirm PMOP and is a useful intervention means to prevent the development of PMOP.Studies on the prevention and treatment mechanism of EA mainly focus on the regulation of endocrine and body fluid circulation,the expression of bone metabolity-related proteins,and the expression of bone cell signal transduction pathways.In recent years,it has been suggested that there is an association between osteogenesis and angiogenesis,especially the close association between H-type blood vessels and bone formation in bone tissue.Therefore,the theory of "angiogenesis-osteogenesis coupling" provides a new breakthrough for the prevention and treatment of PMOP,while the effect of EA on "angiogenesis-osteogenesis coupling" has not been confirmed by relevant studies.Therefore,in this study,the changes of H-type blood vessels in bone tissue of PMOP were taken as the entry point,and Ovariectomy(OVX)rats were taken as the research object.Through EA intervention,the serum levels of biochemical indicators of bone metabolism,bone tissue morphological changes,HIF-1α/VEGF expression and H-type blood vessel expression in OVX rats were observed.Validation EA the regulation of bone metabolism imbalance in OVX rats and the effect and influence on osteogenesis and into the blood vessels,confirm the EA to promote H angiogenic potential pathways mechanism,for the EA through regulating "into the blood vessels-osteogenesis decoupling union" effect of prevention and treatment of PMOP provide reliable experimental evidence,to provide new insights for EA prevention and treatment of PMOP,has important clinical value and research significance.Methods and materials:Chapter 1 Establishment of OVX rat modelObjective: The vaginal smear method was used to evaluate the success of bilateral ovarian tissue removal in rats through the lower abdominal surgical incision to provide a reliable OVX rat model for the subsequent experiments in this study.Methods: Twelve Specific pathogen free(SPF)female Sprague Dawley(SD)rats were fed adaptively for one week and divided into sham operation group and model group using random number table method with 6 rats in each group.The rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium(40mg/kg),and the abdomen was opened through a median incision in the lower abdomen.The rats in the model group had bilateral ovarian tissue removed,and the rats in the sham operation group only had equal amount of adipose tissue around the ovaries removed.Building after 1 week,for 5consecutive days of each group rats vaginal exfoliated cells smear,then HE staining,observe whether regular estrus rats,to determine whether a castrated rats successfully.Results: Regular estrus cycle was observed in vaginal exfoliated cell smears of the sham operation group,but not in the model group.Conclusion: The modeling method used in this study can provide a reliable experimental animal model for subsequent experiments in this study.Chapter 2 Effect of EA on osteogenesis and bone metabolism in OVX ratsObjective: The effects of EA intervention on bone formation and resorption in OVX rats were observed.Methods: Thirty SPF female SD rats aged 12 weeks were fed adaptively for 1 week and divided into sham operation group,model group and electroacupuncture group by random number table method,with 10 rats in each group.OVX rats in the model group and the electroacupuncture group were constructed according to the modeling method used in the first chapter of this study.In the sham operation group,only the same amount of adipose tissue around the ovary was removed,and intervention was performed 2 weeks after modeling.EA intervention was performed after fixation in electroacupuncture group,while fixation in sham operation group and model group was performed only,5 times a week.After 10 weeks of intervention,the rats were sacrificed,and Bone formation and bone resorption in each group were evaluated by the detection of biochemical parameters of bone metabolism,Bone mineral density(BMD)and bone histomorphology.Results: After 10 weeks of intervention in each group,the results of biochemical indexes of serological bone metabolism showed as follows: Compared with the rats in the sham operation group,the Calcium,Ca,Phosphorus,and Alkaline phosphatase(ALP)in the model group were significantly decreased(P<0.05).Significantly increased Tartrateresistant acid phosphatase(TRAP)(P<0.05);Compared with model group,ALP and Bone Gla-protein(BGP)in electroacupuncture group were significantly increased(P<0.05),Ca and P had an increasing trend(P>0.05),and TRAP was significantly decreased(P<0.05).BMD detection of femur tissue and morphological changes of bone tissue showed:Compared with the sham group,Trabecular separation(Tb.sp)was significantly increased in the model group(P<0.05),and Trabecular thickness(Tb.Th)and BMD were significantly decreased(P<0.05).Compared with model group,Percent bone volume(BV/TV),Tb.Th,Trabecular number(Tb.N)and BMD in electroacupuncture group were significantly increased(P<0.05),while Tb.Sp was significantly decreased(P<0.05).Microcomputed tomography(Micro-CT)femur scan images and bone histopathology results show that compared with the sham operation group,the number of bone trabeculae in the electroacupuncture group and the model group is significantly reduced,and the arrangement of bone trabeculae is sparse and irregular,and the reduction is more obvious in the model group.Conclusion: EA intervention can promote the synthesis of Ca and P and the expression of ALP and BGP in OVX rats,inhibit TRAP expression,promote bone formation and inhibit bone resorption,and effectively improve the imbalance of bone metabolism in OVX rats.EA intervention can increase BV/TV,Tb.Th,Tb.N and BMD in OVX rats,while decrease Tb.Sp,indicating that EA intervention can increase BMD in OVX rats and improve the trabecular and bone status in OVX rats.Chapter 3 Effect and mechanism of EA on type H vessel formation in OVX ratsObjective: To verify whether EA intervention affects the formation of H-type blood vessels in OVX rats through HIF-1α/VEGF signaling pathway,and then affects the osteogenesis and bone metabolism of OVX rats,to preliminarize the mechanism of EA affecting H-type blood vessels,and to provide new insights for EA prevention and treatment of PMOP.Methods: Immunohistochemistry and fluorescence staining were used to detect the expressions of HIF-1α,VEGF and H-type blood vessels in femoral tissues of rats in each group after the intervention of EA in Chapter 2 of this study.Results: The results of immunohistochemistry of femur tissue of rats in each group showed that compared with model group,the percentage expression of HIF-1α and VEGF positive cells in electroacupuncture group was increased(P<0.05).The results of immunofluorescence staining showed that the number of H-type vessels decreased in both the model group and the electroacupuncture group compared with the sham operation group.The number of H-type vessels in electroacupuncture group was higher than that in model group.Conclusions:(1)EA intervention may promote H-type angiogenesis in OVX rats by regulating HIF-1α/VEGF pathway;(2)EA intervention promotes H-type angiogenesis in OVX rats,and can improve the imbalance of bone metabolism in OVX rats by regulating the "angiogenesis-osteogenesis coupling",thus preventing and treating PMOP. |
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