Experimental Study Of Genistein Promoting Bone Defect Repair In Obese Mice

Objective:Obesity is an increasingly common metabolic disease,which can lead to a significant decrease in bone density and accelerate bone resorption in the bone defect area.Genistein(GEN),a plant-derived flavonoid,has bone-protective effects and can treat osteopenia and osteoporosis caused by estrogen deficiency.Compared with estrogen,GEN has less adverse reactions and can reduce the occurrence of endometrial cancer and breast cancer.This project aims to clarify the role of GEN in the proliferation and differentiation of osteoblasts through in vitro experiments.In addition,the obese C57 mice were used to establish a skull extreme defect model,and the effect of GEN on the repair process of bone defects was observed in the obese state,which provided a new theoretical basis for the treatment of local bone defects in obese patients.Methods:1.In the in vitro experiments,we used mouse embryo osteoblast precursor cells(MC3T3-E1)as the experimental object,and the CCK8 method was used to determine the effect of GEN(0,0.1,1,10,50 μmol/L)on cell proliferation within 7 days.2.Alkaline phosphatase(ALP)staining and quantitative detection of ALP activity were used to clarify the changes of ALP activity in cells after drug addition;Real-time quantitative PCR-related gene detection method was used to detect ALP,osteopontin(OPN)and osteocalcin(OCN)RNA expression levels in the process of osteogenic differentiation;Western blotting was used to explore the change trend of ALP,OPN and OCN protein expression in each group of cells under the action of GEN;Alizarin red staining was used to clarify the effect of GEN on the number of calcium nodules in MC3T3-E1.3.Scanning electron microscopy was used to observe the changes of ultrastructure of platelet-rich fibrin(PRF)drug-loaded and PRF-unloaded drugs to verify the reliability of PRF drug loading.4.In the in vivo experiment,C57 was used as the experimental object,and it was randomly divided into two groups,the normal diet group(6 animals)and the high-fat diet group(24 animals).The rats were fed with conventional rat chow and high-fat rat chow containing 60%calorie for 11 consecutive weeks,and their body weights were measured once a week.A glucose tolerance test(GTT)was performed at week 12 to confirm modeling success.5.A skull defect model(3mm in diameter)was established on the basis of obese mice.The mice in the normal diet group were not placed with any biological materials and drugs after operation.Obese mice were randomly divided into four groups:HFD group(no biomaterials and drugs were placed in the bone defect area)、HFD+PRF group(PRF without any drug was placed in the bone defect area)、HFD+PRF+0.1 μmol/L GEN group(PRF with a loading concentration of 0.1 μmol/L GEN was placed in the defect area)、HFD+PRF+0.1 μmol/L GEN group(PRF with a loading concentration of 1.0 μmol/L GEN was placed in the defect area).Mice were sacrificed at the 5th week after surgery,and Micro CT scanning was performed to clarify the effect of GEN on the repair process of calvarial defects in obese state.Results:1.The results of CCK8 showed that 0.1μmol/L and 1 μmol/L GEN within 7 days could promote cell proliferation(p<0.05);10 μmol/L GEN had no significant effect on cell proliferation;From the 2nd day,the growth activity of the cells was significantly inhibited,and it was cytotoxic(p<0.05).Therefore,we selected 0.1 μmol/L,1 μmol/L and 10μmol/L of GEN for follow-up research.2.During the osteogenic differentiation of MC3T3-E1,the osteogenic effect of 0.1 μmol/LGEN was not significant;both 1 μmol/L and 10 μmol/L GEN significantly promoted the osteogenic differentiation of MC3T3-E1 because they enhanced ALP activity(p<0.05),up-regulated the RNA and protein expression levels of osteogenesis-related genes ALP,OPN and OCN(p<0.05),and promoted the formation of calcium nodules in MC3T3-E1(p<0.05).3.Scanning electron microscope shows that the interior of PRF presents a three-dimensional network structure,which provides space for loading drug molecules for local sustained release.4.The weight of the mice in the high-fat diet group(average weight of about 40.1g)was greater than 27.7%of the weight of the normal diet group(average weight of about 31.4g)(p<0.05);At each detection time point,the fat diet mice had higher blood glucose values than the normal diet mice,and had abnormal glucose tolerance,which proved that the obesity model was successfully established(p<0.05).Using micro-CT to scan the femur of mice,it showed that compared with the normal diet group,the number of trabecular bone in the femur of obese mice was decreased(P<0.05),the spacing of trabecular bone was widened,and the bone density was decreased(P<0.05).5.Micro CT scanning of obese mouse calvaria showed that loading GEN(0.1 μmol/L and 1.0μmol/L)with PRF on the bone defect area could increase the bone volume fraction(P<0.05)of the mouse calvaria and repair it to a certain extent bone defect.And the 0.1 μmol/L GEN loaded with PRF has the most ideal repair effect on the calvarial defect of obese mice.Conclusion:GEN can significantly promote the osteogenic differentiation of MC3T3-E1,and can effectively repair the calvarial defect in obese mice after loading with PRF.The results of this study provide a new theoretical basis for repairing local bone defects and reducing alveolar bone resorption in obese patients.