| The world’s population has grown and life expectancy has been extended,leading China to enter an aging society.This has caused a significant rise in Osteoporosis(OP),a health issue that poses a danger to the middle-aged and elderly.OP can be divided into primary osteoporosis and secondary osteoporosis,among which postmenopausal osteoporosis and senile osteoporosis are the most common in primary osteoporosis.Often low-energy injuries can lead to spine,wrist,and hip fractures,causing great pain to patients and increasing the medical burden.Bone is a multifunctional and constantly changing organ that can support the activity of the motor system and maintain the metabolic balance of minerals such as calcium and phosphate.In order to ensure the homeostasis of the bone microenvironment and meet the needs of physical activities,bone tissue is continuously ablated and regenerated,which is called bone remodeling.Bone remodeling is mainly accomplished by Osteoclasts(OC),osteoblasts(OB),and osteocytes in the bone remodeling cavity.Imbalance of bone remodeling is a major factor in bone microstructural damage in osteoporosis patients,and osteoclasts develop from blood-derived mononuclear precursor cells,which are the only bone resorptive cells.They can produce enzymes related to acid dissolution of mineral salts and degradation of organic matter in bone tissue;They will adhere to the surface of bone matrix for bone resorption,and when osteoclasts are overexpressed in certain pathological conditions,bone remodeling is unbalanced and bone microstructure is damaged,leading to OP.Anti-osteoporosis drugs improve OP but also cause many side effects,including gastrointestinal intolerance,osteonecrosis,osteosarcoma,and cancer.Therefore,there is a great need for safe and more effective anti-osteoporosis drugs.The wide variety of materials and few side effects of natural plant extracts have made them increasingly attractive as a potential remedy for human diseases in recent years.Vitexin,a C-glycosylation derivative of apigenin and active flavonoid in many traditional Chinese medicines,is widely found in many fruits and vegetables in nature.Its biological activities include antioxidant,anti-cancer and neuroprotective properties.This study seeks to explore the molecular and effectual effects of isvitexin on the differentiation of osteoclast precursors into osteoclasts in vitro,as well as to identify potential drug targets for treating osteoclast-associated osteoporosis.Despite IVX’s purported ability to protect cells from inflammation by regulating the Nrf2/NF-κB signaling pathway,there is no evidence of osteoclastogenesis induced by NF-κB signaling pathway.Methods: 1.An osteoclast induction culture system was created by stimulating the receptor activator of NF-κB ligand(RANKL)in vitro,inducing RAW264.7 cells to differentiate into osteoclasts(OC).Subsequently,RAW264.7 cells were recovered,cultured,and passedaged in DMEM complete medium,and α-MEM medium containing40 ng/ml recombinant Ran KL was chosen to achieve directional differentiation.Using tartrate-resistant acid phosphatase staining(TRAP staining),osteoclasts’ differentiation activity was determined.2.In order to obtain its safe concentration,cell viability assay(cck-8)was used to detect and analyze the toxic effects of different concentrations of isvitexin on osteoclast precursors and osteoclast differentiation.3.Corning bone testing microplate was used to evaluate the inhibitory effect of 20μM,40μM,and 60μM of isvitexin on the bone resorption function of osteoclasts.4.Phalloidin staining was used to observe the effect of isvitexin on the formation of mature F-actin ring.5.Using PCR,the expression of osteoclast differentiation-related genes c-Fos and NFATc1 was determined after being exposed to 20μM,40μM,and 60μM of isvitexin.Western blotting was then employed to assess the influence of varying concentrations of isvitexin on the expression of c-Fos and NFATc1.6.Using SPSS26.0 software,one-way ANOVA test was used to analyze the statistical characteristics of the experimental targets.All experiments were repeated at least three times.Results: 1.The RAW264.7 cell line-induced osteoclast system was successfully established in vitro.2.By using different concentrations of isvitexin(20μM,40μM,60μM),the cytotoxicity test showed that at the experimental concentration,isvitexin was not toxic to the differentiation of osteoclasts,including osteoclasts and osteoclast precursor cells.In terms of osteoclast differentiation,after stimulation with different concentrations of isvitexin in the experiment,the number of osteoclasts was significantly reduced,and its differentiation was inhibited.In the set concentration range,with the increase of the concentration,the inhibitory effect was significantly enhanced.3.At the concentration of 20μM,40μM,and 60μM of isvitexin,the experimental group’s pit area was significantly reduced in comparison to the control group’s in the osteoclast resorption detected by bone detection microplate.This difference was statistically significant at60μM(P < 0.05)and the more isvitexin concentration,the smaller the area of bone pits,the more significant the inhibitory effect.The results of actin ring staining showed that with the increase of isvitexin concentration,the number of actin rings in OC was inhibited,and the most obvious was at 60 μM.4.(4)To analyze the m RNA expression levels of c-Fos and NFATc1 in the osteoclasts stimulated by isvitexin.Compared with the control group,the transcriptional activity of the experimental group was expressed in the range of 20μM,40μM and 60μM,and the inhibitory effect was gradually enhanced with the increase of IVX concentration,which was most obvious at 60μM.There were statistical differences in the comparison between groups with significant features.Western blot analysis showed that isvitexin at the range of 20μM,40μM,and 60μM could reduce RANKL-mediated c-Fos and NFATc1 protein expression,which further confirmed the inhibitory effect of isvitexin on osteoclasts at the protein and molecular levels.Conclusions: In vitro,isvitexin inhibited osteoclast differentiation and the production of transcription factors c-Fos and NFATc1 by regulating RANKL-mediated signaling pathway.Within the concentration range of 20-60μM,isvitexin inhibited the production of related proteins in a concentration-dependent manner.This study provides a feasible target for the development of drug treatment for osteoporosis related to pathological overexpression of osteoclasts. |
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