| BackgroundOsteoporosis is a progressive systemic skeletal disease characterized by disruption of the bone resorption/bone formation balance resulting in abnormally active bone resorption,decreased bone mass,increased bone fragility and susceptibility to fracture.The key pathological basis for the development of OP is the abnormal bone remodeling function and decreased bone mass dominated by osteoblasts and osteoclasts,which ultimately result from the independent or combined effects of various factors such as aging,gender characteristics,hormonal changes,lifestyle habits,and medication history.The traditional Chinese medicine Red Yeast Rice has the effect of activating blood circulation and removing blood stasis,eliminating food and stomach.Studies have shown that Red Yeast Rice and its extracts have certain therapeutic effects on osteoporosis,but the mechanisms are explored from the perspective of regulating bone formation,and no relevant reports based on regulating bone resorption have been found.Monascin,a secondary metabolite and one of the main active ingredients of Red Yeast Rice,has been shown to have significant antiinflammatory and antioxidant effects,but its effect on osteoclast differentiation has not been reported.Based on the above background,this paper observed the effects of Monascin on osteoclast differentiation and function through in vitro experiments,and also preliminarily explored its molecular mechanism,in order to provide new perspectives and ideas for the prevention and treatment of bone metabolic diseases in traditional Chinese medicine.ObjectiveIn order to obtain a more complete and efficient culture system,we firstly observed the key factors affecting the differentiation of RAW264.7 cells into osteoclasts by using different induction methods and different cell densities,and established more stable induction conditions.To investigate the effects of Monascin on osteoclastogenesis and function in vitro,the second part of the paper observed the effects of Monascin on osteoclast differentiation and maturation,actin ring formation,and bone resorption function using different administration times and different administration concentrations,respectively.In order to explore the molecular mechanisms by which Monascin inhibits osteoclastogenesis and function in vitro,the third part of the paper observes the effects of Monascin acting on osteoclast signature genes,MAPKs pathway,and p65 nuclear translocation of NF-κB pathway.MethodsIn the first part of the paper,10th-15th generation RAW264.7 cells were applied with different induction methods(50ng/mL RANKL,100ng/mLRANKL,50ng/mLRANKL+50ng/mL M-CSF,100ng/mLRANKL+50ng/mL M-CSF)and different cell densities(5×103/well,1×104/well,2×104/well,4×104/well)were observed on the key factors affecting the differentiation of RAW264.7 cells to osteoblasts.In the second part of the paper,the CCK8 method was applied to detect the cytotoxic effects of different concentrations(0-20μM)of Monascin;TRAP staining,F-actin fluorescence staining and bone resorption trap assay were used to observe the effects of 0,5,10,15 and 20μM Monascin given on day 1 of induction and 20μM Monascin given on days 1,2,3 and 4 after RANKL addition on osteoclastogenesis,actin ring formation and bone resorption function,respectively.In the third part of the paper,RT-qPCR was used to observe the effects of administration of 0,10 and 20μM Monascin on day 1 of induction and 20μM Monascin on days 1,2,3 and 4 after the addition of RANKL on the key transcription factors c-Fos and NFATc1 in osteoblasts and their downstream regulatory genes TRAP,CTSK,MMP-9,ITGβ3,OSCAR and DC-STAMP expression,respectively.Western Blot assay was used to observe the effect of Monascin on the phosphorylated protein expression of p38,ERK and JNK in the MAPKs pathways in osteoblasts;cellular immunofluorescence staining was used to observe the effect of Monascin on the nuclear translocation of p65 in the NF-κB pathway.ResultsThe first part of the paper found that 50-100ng/mL RANKL effectively induced RAW264.7 cells into osteoclasts,while the addition of 50ng/mL M-CSF co-cultured with RANKL resulted in reduced generation of mature osteoclasts and lower induction efficiency.Cell density was closely related to osteoclast production,and in a 24-well plate with a bottom area of 2 cm2,for example,the induction efficiency was higher at a density of 1×104/well to 2×104/well,and either too high or too low could reduce the induction efficiency.In the second part of the paper,it was found that Monascin at concentrations≤20μM had no significant toxic effect on RAW264.7 cells.5-20μM Monascin inhibited osteoclastogenesis and actin ring formation,and the inhibitory effect was stronger at higher concentrations,mainly at the early stage of osteoclast differentiation.5-20μM Monascin effectively reduced the number and depth of RANKL-induced bone resorption traps in a concentration-dependent manner,and decreased the resorptive capacity of osteoclasts.5-20μM Monascin can effectively reduce the number and depth of bone resorption traps caused by RANKL in a concentration-dependent manner and decrease the resorptive capacity of osteoclasts.The third part of the paper found that Monascin significantly inhibited the expression of key osteoclast transcription factors c-Fos and NFATc1 at an early stage and suppressed the expression of osteoclast-associated genes TRAP,CTSK,MMP-9,ITGβ3,OSCAR,and DC-STAMP in a concentration-dependent manner.Monascin significantly reduced the phosphorylated protein expression of p38,ERK,and JNK in MAPKs pathway and effectively blocked the occurrence of p65 nuclear translocation in NF-κB classical pathway.ConclusionsMonascin effectively inhibited osteoclast formation and significantly reduced bone resorption function in vitro by downregulating the expression of osteoclast transcription factors c-Fos and NFATc1 and their downstream signature genes.Monascin may inhibit the activation of key transcription factors c-Fos and NFATc1 by suppressing the phosphorylation of p38,ERK,and JNK proteins in MAPKs pathway and blocking the nuclear translocation of p65 in NF-κB pathway.Our study suggests that Monascin may be a potential therapeutic strategy for the treatment of abnormal bone resorption/osteolytic diseases. |
