Experimental And Clinical Study Of Qigu Capsule In Treating Postmenopausal Osteoporosis Based On Osteogenesis And Angiogenesis Coupling

Objective:To analyze the correlation between angiogenic factors and bone turnover markers and bone mineral density in postmenopausal osteoporosis(liver and kidney deficiency syndrome).And to observe the TCM syndrome scores,changes in the expression of bone turnover markers,bone mineral density,and angiogenic factors,and to explore the mechanism of the treatment of postmenopausal osteoporosis with Qigu Capsule.Method:(1)Using a cross-sectional study method,84 patients with postmenopausal osteoporosis and the bone mineral density and bone turnover markers were measured by general investigation and recording of TCM syndrome scores.ELISA was used to Slit3,VEGF,and IGF-1,Endothelin 1.The t-test and Spearman correlation analysis were used to analyze the correlation between angiogenic factors and bone turnover markers and bone mineral density in postmenopausal osteoporosis.(2)Randomized controlled trials were performed.78 patients who met the inclusion criteria were randomly divided into the experimental group and the control group.The control group was treated with calcium supplementation with oral Calcium D.The experimental group received oral Qigu capsules on the basis of basic treatment.The Qigu capsules were taken orally three times a day.(3)In vitro experiment : The femur of 1 month old rats was taken,and the primary bone marrow mesenchymal stem cells were isolated and passaged to the third generation.The cells were randomly divided into 5 groups: control group,low dose Qigu capsule,high dose Qigu capsule,icariin group,naringin group.The cells were induced by osteoblasts and endothelial cells in the corresponding drug-containing serum,and the cell proliferation rate was detected at 24 h,48h and 72 h respectively.After 10 days of culture,ALP activity was detected by p NPP method,and Western Blot and RT-PCR were used to detect the expression of proteins and genes after osteogenic differentiation and endothelial differentiation.(4)In vivo experiment: 50 SD rats of 6 months old,bilateral ovaries were removed.The rats were divided into 4 groups:Qigu low group,Qigu high group,model group and sham group.All experimental animals were excuted after intragastric administration for 3 months.Micro-CT,Microfil detector,HE staining and Immunohistochemistry stianing,were used to evaluate bone formation conditions.Western Blotting method and RT-PCR method were used to observe the expression of the related proteins and genes.Result:(1)Spearman correlation analysis found that there was a high correlation between Slit3,VEGF,IGF-1,and ET-1 in patients with osteoporosis,meanwhile Slit3 and P1NP(P=0.001,r=0.420),OC(P=0.002,r =0.408)also have a high correlation.CTX(P=0.009,r=0.344);VEGF(P=0.012,r=0.332),IGF-1(P=0.0030,r=0.287)ET-1(P=0.011,r=0.261)has a low correlation with OC.(2)Qigu Capsule has good effect on relieving low back pain,weak waist and knee weakness,and lower limb cramps in patients with osteoporosis.Compared with the control group,it can reduce TCM syndrome scores and relieve osteoporotic symptoms(p<0.05).P1 NP was significantly improved after 3 months of treatment(p<0.05).The OC and ?-CTX were significantly decreased in the treatment group,and IGF-1?Slit3 was significantly increased after treatment(p<0.05).(3)The high and low dose groups of Qigu capsule,icariin and naringin can promote the proliferation and differentiation of BMSCs,promote the expression of ALP after osteogenic induction,and up-regulate Runx2 and Osterix,and up-regulated the expression of CD31 gene and protein during endothelial differentiation.(4)Qigu Capsule can increase the bone mass of rats and improve the microstructural changes;increase the number and density of blood vessels in the distal femur of rats.HE staining showed that the Qigu capsule reduced the number of fat vacuoles and increased the number of bone cells.Immunohistochemical staining showed that the Qigu capsules could increase the expression of H-type vessels and Osterix protein.The Qigu capsule may activate the Wnt-? catenin pathway via the Slit3 protein.Conclusion:(1)Slit3 is closely related to the metabolic state of bone tissue in postmenopausal osteoporosis patients.VEGF,IGF-1,ET-1 have a certain correlation with bone tissue metabolism.The circulating levels of angiogenic factors Slit3,VEGF,IGF-1,and ET-1 in postmenopausal osteoporosis are associated with bone formation.(2)Qigu Capsule can significantly reduce TCM syndrome scores in postmenopausal osteoporosis patients,relieve clinical symptoms such as low back pain,weak waist and knee weakness,lower extremity cramps,and difficult walking.After treatment,it can increase P1 NP level and reduce OC and ?-CTX levels.It suggests that it has the ability to promote bone formation and inhibit bone resorption.It can also increase angiogenic factors IGF-1 and Slit3.(3)Qigu capsules and effective components can promote the differentiation of rat bone marrow mesenchymal stem cells into osteoblasts and endothelial cells.(4)Qigu capsules may activate the Wnt-? catenin pathway through Slit3,increase intravascular bone perfusion,increase H blood vessel formation,and prevent bone loss in OVX rats.