| Objective:1.This study intends to establish a primary osteoblast senescence model to verify the role of food-derived small molecule compounds ferulic acid in delaying the aging of primary osteoblasts and restoring the osteogenic function of senescent osteoblasts in vitro.2.The SD rat model of ovariectomized osteoporosis was established to discuss the effect of ferulic acid in delaying bone loss of postmenopausal osteoporosis and molecular mechanism in vivo.It provides theoretical basis and experimental basis for the development of functional food and provides more scientific dietary nursing guidance for clinical prevention of osteoporosis and delay of bone loss in patients with osteoporosis.Methods:1.Establishment of senescence model of primary osteoblasts in vitro In this experiment,the senescence model of primary osteoblasts in vitro was established by continuous passage.By observing the morphological changes of osteoblasts of different passages,detecting the viability and βgalactosidase content of osteoblasts of different passages,the representative passages were determined.Alkaline Phosphatase(ALP)staining,Alizarin red S(ARS)staining,Reactive oxygen species(ROS),apoptosis,mitochondrial membrane potential,Malondialdehyde(MDA),Superoxide Dismutase(SOD)and Glutathione reductase(GR)activities were performed to analyze the changes of osteogenic function and antioxidant capacity of osteoblasts of different passages.2.Protective effect of ferulic acid on aging osteoblastsBased on the aging model of primary osteoblasts in vitro,the effects of ferulic acid on the survival rate,differentiation and mineralization ability of representative generations of osteoblasts were studied,and the concentration and dosage time were determined.The effects of ferulic acid on osteogenic function,antioxidant capacity and mitochondrial function of aging osteoblasts were further studied.3.Protective effect of ferulic acid on bone loss in ovariectomized ratsThe ovariectomized rat model of osteoporosis was established by using 3-month-old female SD rats as the study object in vivo.During the study,48 rats were divided into Sham operation control group(Sham),ovariectomized group(OVX),Estradiol group(E2),low-,medium-and high-dose ferulic acid group(FA-L/M/H).The Sham group and the OVX group were administered with suspension medium by intragastric administration,the E2 group was given intragastric administered with the concentration at 50 μg/kg of estradiol,the FA-L/M/H groups were given intragastric administered respectively with the concentration of 20 mg/kg,50 mg/kg and 100 mg/kg of ferulic acid for a total of 3 months.After all rats were sacrificed,ELISA was performed on serum samples,three-point bending test was performed on femur of rats,and HE staining was performed on upper tibia and uterus.Results:1.Establishment of senescence model of primary osteoblasts in vitroAfter continuous passage to 10-14 passages,the cytoplasm and nucleus of osteoblasts increased significantly,and the viability,differentiation ability,mineralization ability,GR activity,and mitochondrial membrane potential of osteoblasts decreased significantly.The β-galactosidase activity,ROS level,apoptosis rate,MDA level and SOD activity of osteoblasts were significantly increased.2.Protective effect of ferulic acid on aging osteoblastsFerulic acid can significantly improve the viability,differentiation and mineralization ability of representative osteoblasts.Ferulic acid can significantly reduce the ROS level of aging osteoblasts,reduce the apoptosis rate,and restore the mitochondrial membrane potential.It can also increase the expression of osteogenic functional gene proteins,anti-apoptotic protein and mitochondrial quality control proteins.3.Protective effect of ferulic acid on bone loss in ovariectomized ratsIn animal experiments,the serum E2 level of OVX group was significantly lower than that of Sham group.The serum Testosterone(T),1,25dihydroxyvitamin D3(1,25(OH)2D3),Bone Alkaline Phosphatase(BALP),Tartrate Resistant Acid Phosphatase 5b(TRACP-5b),C-Terminal Telopeptides of Type Ⅰ Collagen(CTX-I),Interleukin-1(IL-1),Interleukin-6(IL-6)and Tumor necrosis factor-a(TNF-α)were significantly increased.After ferulic acid intervened in ovariectomized rats for 3 months,compared with the OVX group,the serum E2 level was significantly increased,the serum T,1,25(OH)2D3,BALP,TRACP-5b,CTX-I,IL-1,IL-6 and TNF-α were significantly decreased.Ferulic acid can significantly reduce the maximum external inner diameter of the femur,and tend to increase the maximum load,maximum stress and maximum strain of the femur.Ferulic acid significantly increased the percent trabecular area and trabecular thick in the upper tibia of rats,decreased the trabecular separation,and had no significant effect on uterine coefficient and and endometrial thick.Conclusion:1.The 10-14 primary osteoblasts derived from SD rat calvaria can be used for the screening of drugs for preventing osteoporosis.2.Senescence model of primary osteoblasts in vitro,it was verified that ferulic acid can improve the osteogenic function of senescent osteoblasts and delay the senescence of osteoblasts,which may be related to the fact that ferulic acid improves the antioxidant capacity of aging osteoblasts,reduces the rate of apoptosis,and maintain the steady state of mitochondrial fusion and fission.3.In ovariectomized rats with osteoporosis,it was confirmed that ferulic acid can reduce the rate of bone transformation,increases the secretion of sex hormones,improves the biomechanical properties of rat femurs,and alleviates the decrease in bone mass caused by estrogen deficiency,with less side effects than estrogen therapy.Ferulic acid may be a safer candidate for functional food to prevent postmenopausal osteoporosis. |
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