| Experiment 1: Experimental study on the effects of stilbene glycosides on osteoporotic fractures in postmenopausal rats and the selection of the best effective concentration.Purpose: To investigate the effect of stilbene glycoside(TSG)on fracture healing in rats after ovariectomy,and find the best effective concentration.Material and method: 1.Forty-three-month-old SD female rats were randomly divided into 6 groups according to the random number table method,each group of 8 rats:sham operation group(Sham),model group(OVX),and stilbene glycosides.Dose group(H-TSG),stilbene glycoside medium dose group(M-TSG),stilbene glycoside high dose group,raloxifene group.A postmenopausal osteoporotic rat model was established,and bilateral castration(ovary removal)was adopted.After successful operation,the animals were reared for 12 weeks,and the dual-energy X-ray bone densitometer was used to evaluate the effect of modeling.2.After castration successfully established a rat model of postmenopausal osteoporosis,all rats established an open fracture model in the right femur,and the right middle femoral transverse fracture was fixed with the Kirschner wire in the femur.Intragastric administration was started 3 days after the operation,and continued for 7 weeks.3.After 7 weeks of gavage,rats in each group were anesthetized intraperitoneally,blood was collected from the abdominal aorta,and the supernatant was collected after centrifugation.The serum total type I collagen amino-terminal elongation peptide(P? NP),bone alkaline phosphatase(BALP),automatic biochemical analyzer to analyze serum calcium and phosphorus content.Dual-energy X-rays were used to determine the bone density of the left femur.Pathological tissue sections were examined for morphology of epiphysis using HE staining.Universal material tester was used to measure biomechanical parameters of epiphysis.Small animal CT was used to observe fracture healing.Results:1.After castration,the weight of Sham group was significantly lower than that of OVX group,and the difference was statistically significant(P <0.05).After 7 weeks of intervention,the body weight of the high,medium,and low dose TSG and RALOXIFENE groups was significantly lower than that of the OVX group,and the difference was statistically significant(P <0.05).The uterine weight of the OVX group was lower than that of the Sham group,and the difference was statistically significant(P <0.05).There was a statistically significant difference in TSG and RALOXIFENE group weight compared with OVX uterine weight(P<0.05).The uterine coefficient of the OVX group was smaller than that of the Sham group,and the difference was statistically significant(P <0.05).The TSG medium dose and the RALOXIFENE group had a larger uterine coefficient than the OVX,and the difference was statistically significant(P <0.05).2.Compared with the Sham group,the trabecular bone in the OVX group was significantly sparse,the number of trabecular bone was small,the continuity was poor,and the total area of??the trabecular bone was small.There are fewer newly formed bone cells in the epiphysis,and a larger proportion of immature bone cells and osteoblasts.Compared with the OVX group,the TSG medium-dose group has dense trabeculae,a large number of trabeculae,good continuity,a large total area of trabeculae,abundant new bone cells,and mature osteoblasts and bone cells Large,the degree of mineralization is also better than the OVX group.Compared with the OVX group,the trabecular structure of the TSG high and low dose groups and the RALOXIFENE group was also improved,and osteoblasts were also richer and the degree of mineralization was better than that of the OVX group.3.The serum calcium and serum phosphorus contents in the OVX group were lower than those in the Sham group,but the differences were not statistically significant(P> 0.05).Compared with the OVX group,serum calcium and phosphorus in each group were slightly higher,but the difference was not statistically significant(P> 0.05).The comparison of calcium and phosphorus products between the groups was not statistically significant(P>0.05).4.Compared with Sham group,serum PINP content in OVX group was lower,and the difference was statistically significant(P <0.05).Compared with the OVX group,the high andmedium dose TSG and RALOXIFENE groups had higher levels of PINP,and the difference was statistically significant(P <0.05).Compared with the OVX group,the PSG content in the low-dose TSG group was higher,but the difference was not statistically significant(P> 0.05).Compared with the Sham group,the serum BALP content in the OVX group was lower,and the difference was statistically significant(P <0.05).Compared with the OVX group,the BALP content in the TSG high-,medium-dose and RALOXIFENE groups was higher,and the difference was statistically significant(P <0.05).Compared with the OVX group,the BASF content in the low-dose TSG group was higher,but the difference was not statistically significant(P> 0.05).5.Compared with the Sham group,the bone mineral density of the OVX group was significantly reduced,and the difference was statistically significant(P <0.05).Compared with the OVX group,the bone density of the TSG high and middle dose groups and the RALOXIFENE group were significantly increased,and the difference was statistically significant.Compared with the OVX group,the bone density of the TSG low-dose group was higher,but the difference was not statistically significant(P> 0.05).6.Compared with the Sham group,the elastic modulus of the OVX group was lower,but the difference was not statistically significant(P> 0.05).Except for the high-dose TSG group,the elastic modulus of the other groups was higher than that of the OVX group,but the difference was not statistically significant(P> 0.05).Compared with the Sham group,the maximum force,breaking force and flexural strength of the OVX group were significantly reduced,and the differences were statistically significant(P <0.05).Compared with the OVX group,except for the TSG low-dose group,the maximum force and flexural strength of the other groups were increased,and the differences were statistically significant(P <0.05).The breaking force of the other groups was higher than that of the OVX group,but there was no significant difference in the other groups except the TSG medium dose group(P> 0.05).7.Compared with the Sham group,the OVX group has sparse epiphyses,severe fractures,discontinuous fracture lines,smaller epiphyseal ratios,slower epiphyseal healing,and lower mineralization.Compared with the OVX group,the epiphysis of each group was significantlyimproved,especially in the TSG medium-dose group,the fracture line was continuous,there were more hard epiphyses,and epiphyseal shaping had begun in many places.Conclusion:1.Postmenopausal osteoporosis rats gain weight and uterine atrophy,TSG can significantly improve the weight gain and uterine atrophy caused by postmenopausal.2.TSG intervention can promote fracture healing and epiphyseal mineralization in rats,and improve trabecular structure.3.The effect of TSG intervention on serum calcium and phosphorus in rats is small.4.TSG intervention can improve the serum PINP and BALP in rats.5.TSG intervention can improve bone mineral density in postmenopausal osteoporotic rats.6.TSG intervention can improve the load and load of bone tissue in rats,and improve biomechanical performance.7.TSG intervention can promote osteoporotic fracture healing,accelerate epiphyseal mineralization,and reduce healing time?Experiment 2: To investigate the role of stilbene glycoside(TSG)in promoting osteoblast proliferation by regulating the Wnt / ?-catenin signaling pathway and the mechanism of its osteogenic differentiation ability.Purpose:1.To explore the effect and mechanism of stilbene glycoside(TSG)on osteogenic differentiation of osteoblast precursor cells MC3T3-E1,and to provide theoretical support for in vivo experiments.2.To study the effect of stilbene glycoside(TSG)on the proliferation of mouse osteoblast precursor cells MC3T3-E1 and the expression of related signaling molecules in the Wnt /?-catenin signaling pathway,and to explore the related mechanisms.Material and method:1.The osteoblast precursor cells MC3T3-E1 were evenly seeded in a twelve-well plate.Whenthe cells evenly covered the field of vision up to 90%,the osteoblast induction medium was replaced and the culture was continued.The experimental grouping was as follows:(1)Normal group: complete culture Base processing group.(2)Blank control group: osteogenic differentiation medium treatment group.(3)TSG group A: 10-2 mg / m L TSG + osteogenic differentiation medium treatment group(4)TSG group B: 10-3 mg / m L TSG + osteogenic differentiation medium treatment group(5)TSG group C: 10-4 mg / m L TSG + osteogenic differentiation medium treatment group.After 7d,14 d,and 21 d induction,the degree of mineralization of osteoblast precursor cells MC3T3-E1 was evaluated by alizarin red staining analysis.ALP staining and ALP detection kits were used for qualitative and quantitative analysis to evaluate the secretion and expression of osteoblast MC3T3-E1 differentiation activity marker ALP.2.Using mouse osteoblast precursor cell MC3T3-E1 cell line,add TSG medium at a concentration of 10-2,10-3,10-4,and 10-5 mg / m L to a 96-well plate,respectively.After 24,48,and 72 hours,the cell survival was measured by the CCK-8 method,the drug toxicity was evaluated,and the optimal effective concentration was selected.2.Using western blot technology to evaluate the expression levels of ?-catenin and cyclin-D1 at the protein level,and preliminary explain the related mechanism of TSG on the proliferation of osteoblast precursor cells MC3T3-E1.?Results:1.After 7 days of osteogenic induction differentiation medium intervention,no extracellular matrix calcium ion complex was deposited in each group.At 14 days,the extracellular matrix of osteogenic precursors gradually showed the deposition of calcium complexes,and calcium nodules reflected the degree of mineralization of osteoblasts.Compared with the normal medium,calcium ion nodules of different sizes appeared in each group added with osteogenic induction differentiation medium.Compared with the control group,the area of ??calcium nodule deposits formed after adding TSG in each group was larger,and this performance was more obvious at 21 days.2.After 7 days of osteogenic differentiation differentiation medium intervention,the amountof blue formation of ALP in each group of osteogenic differentiation inducer was significantly higher than that of the complete medium group,and it was clearly seen that the ratio of ALP blue formation in each group of TSG administration There was a significant increase without TSG intervention.At 14 days,the above situation was more prominent,and blue staining appeared in the cytoplasm of a large number of cells in each group treated by TSG.3.Compared with the normal group,the AKP activity of the blank control group and the TSG group with different concentrations was significantly increased,and the difference was statistically significant(P <0.05).Compared with the blank control group,the activity value of AKP in A cells of the TSG group was significantly higher than that of the blank control group,and the difference was statistically significant(P <0.05).In contrast,although the AKP activity values ??of T and B groups were higher than those of the blank control group,there was no significant difference(P> 0.05).4.At 24 hours,there was no significant difference in OD between the groups(P> 0.05).At 48 hours,except for the TSG of 10-5 mg / ml,the other groups had higher OD values ??than the blank control group.The difference was statistically significant.At 72 hours,compared with the blank control group,except for the 10-5 mg / ml group,the OD values ??of the other groups were increased,and the difference was statistically significant(P> 0.05).5.After TSG treatment,the protein ?-catenin expression increased significantly,which was significantly higher than that in the untreated group,and the difference was statistically significant(P <0.05).However,after different concentrations of TSG intervention,there were differences between the groups,but they were not statistically significant(P> 0.05).The downstream cyclin-D1 protein,as one of the important proteins affecting osteoblast differentiation,showed the same results as the core protein ?-catenin.The expression level of cyclin-D1 protein in the TSG treatment group was significantly higher than that in the untreated group.(P <0.05).However,the differences between the TSG groups were not statistically significant(P> 0.05).Conclusion:1.TSG intervention can promote the differentiation of osteoblast precursor cells and mineralization of extracellular matrix,resulting in greater deposition of calcium nodules.2.TSG intervention can promote the differentiation ability of osteoblast precursor cells and produce a large amount of active ALP / AKP in the cell.3.TSG intervention can promote the proliferation of osteoblast precursor cells without significant cytotoxicity.4.TSG can promote the proliferation and differentiation of osteoblasts by promoting the protein expression of Wnt / ?-catenin signaling pathway core protein ?-catenin and downstream cyclin-D1. |
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