The Preventive Effect Of Apple Polyphenols On Bone Loss In Obese Mice And Its Mechanism

Background and Objective:Osteoporosis is a disease characterized by reduced bone mass and deterioration of bone microstructure,which is one of the main health problems in the elderly population worldwide.Meanwhile,obesity has a certain impact on the body’s bone metabolism balance,and obese people are potential high-risk groups for bone loss.However,related correlation studies on the Chinese population are scarce.At present,drug therapy and lifestyle intervention are the main methods to prevent and treat bone loss.However,considering the side effects of drug therapy and poor compliance with lifestyle intervention,nutritional supplementation has become an important strategy for delaying and preventing bone loss.Therefore,developing and finding nutrients that can prevent obesity-related bone loss has significant implications for preventing bone loss in obese elderly people and improving their quality of life.Apple is a common fruit that is rich in phytochemicals such as polyphenols,which have certain effects on human health and disease prevention.Apple polyphenols have a significant effect on reducing body weight and body mass in obese individuals,but their impact on bone metabolism balance in obese individuals is not yet fully understood.Evaluating the impact of apple polyphenols on bone metabolism balance in obese individuals and exploring their potential mechanisms will provide a new approach to developing intervention methods for osteoporosis prevention and treatment.Methods:Part One:A study on the correlation between physique,body fat and the bone health of elderly people was conducted using a cluster random sampling method in 11 natural villages in Licha Town,Jiaozhou City,Qingdao,targeting elderly people over 60 years old.The study collected the general demographic characteristics,physical information,and disease or health status of the study subjects through face-to-face questionnaire surveys,standardized physical examinations,and laboratory tests.The bone density values of the calcaneus of the study subjects were measured using an ultrasound bone density meter.Cochran-Armitage trend test was used to analyze the trend of bone mineral density（BMD）with changes in the obesity index.Linear regression analysis was used to explore the correlation between different levels of obesity indexes and bone density.Binary logistic regression model was used to analyze the correlation between different levels of obesity indexes and osteoporosis.Part Two:The preventive effect and mechanism of apple polyphenols on obesity-induced bone loss.40 male 8-week-old C57BL/6J mice were used and obesity models were established by feeding them with high-fat diet.At the same time,apple polyphenols intervention experiments were conducted through gavage.The specific groups were as follows（n=10 per group）:control group（CON）,obesity model group（HFD）,resveratrol group（positive control,RES）,apple polyphenol group（APP）.The CON group and the HFD group were gavaged with normal saline.The RES group was administered with100mg/kg/day of resveratrol normal saline suspension.The APP group was gavaged with800mg/kg/day of apple polyphenol normal saline suspension.The intervention time was 8weeks,during which weight and food intake were recorded.After sacrificing the mice,epididymal fat,femur,and blood were collected and serum was separated.The success of the obesity model was determined by calculating weight,body fat,and detecting serum lipid metabolism indicators.Micro-CT was used to observe and analyze the structure and microstructural parameters of trabecular bone and cortical bone of the femur.Three-point bending test and finite element method were used to test the stiffness and strength of the mouse femur and evaluate changes in its biomechanical properties.Enzyme-linked immunosorbent assay,Western Blot,immunohistochemical staining,and high-temperature burning in the Muffle furnace were used to detect the levels of bone absorption,bone formation,and bone mineralization-related markers.Results:（1）This cross-sectional study included a total of 782 study subjects,including 352males and 430 females.Linear correlation analysis showed that BMD values were significantly negatively correlated with body mass index（BMI,r=-0.091,P=0.011）,body fat percentage（BFP,r=-0.250,P<0.001）,visceral fat area（VFA,r=-0.104,P=0.004）,and waist-height ratio（WHt R,r=-0.164,P<0.001）.Multivariate linear regression analysis showed that BFP（b=-0.003,t=-4.301,P<0.001）and WHt R（b=-0.578,t=-3.605,P<0.001）were negative predictive factors for BMD values.Trend analysis showed a reverse U-shaped relationship between BMI and BMD values in the overall population,with significant decreases in BMD values as BFP(P_trend<0.001),VFA(P_trend<0.001),and WHt R(P_trend<0.001)increased.In the male population,there were significant decreases in BMD values with increasing BMI(P_trend=0.014),BFP(P_trend=0.004),VFA(P_trend=0.013),waist circumference(WC,P_trend=0.007),and WHt R(P_trend=0.044).In the female population,BMI and BFP showed a reverse U-shaped relationship with BMD values,while there was no significant linear change in BMD values with increasing VFA,WC,WHt R,and Waist hip rate（WHR）.Binary logistic regression analysis showed that,after controlling for some confounding factors,the highest quartile of BFP in the male population had a 3.827-fold higher risk of osteoporosis than the lowest quartile（P<0.05）.（2）After 8 weeks of high-fat feeding,the body weight,weight gain rate,and body fat content of the HFD group mice were significantly higher than those of the CON group（P<0.05）;the serum triglycerides（TG）,total cholesterol（TC）,and low-density lipoprotein cholesterol（LDL-C）levels were significantly higher than those of the CON group（P<0.05）;HOMA-IR was significantly higher than that of the CON group（P<0.01）.These results indicated that the obesity model was successfully established.The body weight,weight gain rate,and body fat content of mice in the APP and RES groups were significantly lower than those in the HFD group（P<0.05）;the serum TG,TC,and LDL-C levels of mice in the APP and RES groups were significantly lower than those in the HFD group（P<0.05）,and HOMA-IR was significantly lower than that in the HFD group（P<0.001）.Micro-CT analysis showed that the three-dimensional structure of the femoral trabecular bone in mice of the HFD group was damaged,and the trabecular bone density and bone volume fraction were significantly lower than those in the CON group（P<0.001）;the femoral elastic modulus in the HFD group was significantly lower than that in the CON group（P<0.001）.The three-dimensional structure of femoral trabecular bone in mice of the APP and RES groups was relatively intact,and the trabecular bone density,bone volume fraction,and elastic modulus were significantly higher than those in the HFD group（P<0.05 and P<0.01,respectively）.The serum levels of tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）,tartrate-resistant alkaline phosphatase（TRACP）,and parathyroid hormone（PTH）in the APP and RES group mice were significantly lower than those in the HFD group（P<0.05）;the TRACP-positive cell count in the femur of the RES group mice was significantly lower than that in the HFD group（P<0.05）,but there was no statistical difference between the APP and HFD groups.The levels of the bone-specific transcription factor（Runt-related transcription factor 2,Runx2）in the femur and serum of the APP group mice were significantly higher than those of the HFD group（P<0.05）;the protein level of osteoprotegerin（OPG）in the femur was significantly higher than that in the HFD group（P<0.01）.The protein level of receptor activator for nuclear factor-κB ligand（RANKL）in the femur of the RES group was significantly lower than that in the HFD group（P<0.01）.The ratio of OPG/RANKL in the APP group mice was significantly higher than that in the HFD group（P<0.01）,but there was no statistical difference between the RES and HFD groups.The collagen area fraction in the femur of the APP group mice was significantly higher than that in the HFD group（P<0.01）;the ash content in the femur was significantly higher than that in the HFD group（P<0.05）;and the serum level of procollagen I N-terminal propeptide（P1NP）was significantly higher than that in the HFD group（P<0.001）.Conclusion:In the elderly population of Jiaozhou city in Qingdao,obesity index is negatively correlated with bone density.Animal model experiments show that high-fat diet-induced obesity in mice can cause serious bone loss.Supplementation of apple polyphenols can increase bone density and bone microstructure parameters,enhance femur biomechanical performance,inhibit the level of serum bone resorption markers,significantly increase the level of bone formation markers,and improve bone loss in obese mice.