Inhibitory Effect Of Myricetin On Osteoclasts And Its Mechanism

In recent years,the incidence of osteoporosis has increased year by year in the world,and has become an important chronic disease that seriously threatens human health.Osteoporosis is a systemic bone disease in which bone mineral deposition is reduced and bone microstructural destruction is caused by an imbalance in activity between bone formation and bone resorption,which ultimately leads to increased bone fragility and increased risk of fracture.Osteoclasts are terminally differentiated multinucleated giant cells.Their precursor cells are derived from hematopoietic stem cells and can be differentiated into mature osteoclasts under the induction of various cytokines and hormones.Abnormalities in the number and activity of osteoclasts in bone are associated with a variety of bone metabolic diseases such as osteoporosis,osteopetrosis and rheumatoid arthritis.Therefore,inhibiting the formation and activity of osteoclasts is of great significance for the development of new drugs for the treatment of osteoporosis.Myricetin,a pentahydroxyflavone-3-rhamnoside,is a polyphenolic hydroxyflavonoid glycoside compound,which is abundantly present in the fruit,bark,leaves and other natural plants of bayberry,and has improved microcirculation and resistance.A variety of pharmacological activities such as inflammation,anti-oxidation,and inhibition of apoptosis.This study established the RAW264.7 cell culture system and determined the optimal conditions for inducing RAW264.7 cells to differentiate into mature osteoclasts.The CHEN-8 cytotoxicity test,TRAP staining experiment and RT-PCR experiment confirmed the myricetin on inhibition of osteoclast differentiation and maturation.OBJECTIVE:The RAW264.7 cell line was established and differentiated mature osteoclasts were induced.To determine the effect of myricetin on the differentiation of RAW264.7 cells into osteoclasts.METHODS:The experiment is divided into two parts:1.Establishment of RAW264.7 cell line and induction of osteoclasts:The proliferation curve of RAW264.7 cells was drawn,and the passage time was determined according to the proliferation curve.After digestion with trypsin,the cell digestion state was observed under different time points to determine the trypsin digestion time;the cells were calculated according to the cell density at the time of passage.The density was frozen and verified.The optimal induction conditions of osteoclasts were determined by different cell seeding densities and different RANKL concentrations.After TRAP staining,the microscopic counts were used for statistical analysis.2.Effect of myricetin on the differentiation of RAW264.7 cells into osteoclasts:The effects of different concentrations(1.25mg/L,2.5mg/L,5mg/L,10mg/L,20mg/L)of myricetin on the survival of RAW264.7 cells were determined by CCK-8 method.RAW264.7 cells were induced with 50 ?g/L RANKL,50 ?g/L RANKL + 1.25 mg/L,2.5 mg/L,5 mg/L,10 mg/L of myricetin for 5 days,respectively.The formed osteoclasts were subjected to TRAP staining and counting,and TRAP staining was positive and the number and number of cells > 3 were considered mature osteoclasts.50?g/L RANKL,50?g/L RANKL+1.25mg/L,10mg/L myricetin cultured RAW264.7 cells for 24 hours,using fluorescent real-time quantitative PCR to detect the expression of the osteoclast differentiation-related genes C-Fos,NFATc1,Ctsk and TRAP.RESULTS:Part 1:(1)The passage time of RAW264.7 was 5-6 days.The digestion method was trypsin digestion for 1.5 minutes,then gently blew several times with medium,and the cryopreservation density of the cells was ?50×10 4 /m L.(2)The optimal induction conditions for osteoclasts were: cell seeding density of 3×104cells/m L,RANKL concentration of 50 ?g/L,and medium replacement with ?-MEM medium.Part 2:(1)When 1.25mg/L,2.5mg/L,5mg/L,10mg/L concentration of myricetin was applied to RAW264.7 cells,there was no obvious cytotoxicity,which could be used as the concentration of the subsequent experiments.(2)When RAW264.7 cells were treated with 1.25 mg/L,2.5 mg/L,5 mg/L,and 10 mg/L of myricetin,TRAP staining showed that jasmine inhibited RANKL-induced RAW264.7 cells to mature osteoclasts.Cell differentiation was dose dependent.(3)When 1.25mg/L and 10mg/L concentrations of myricetin were applied to RAW264.7 cells,RT-PCR results showed that myricetin can inhibit the expression of C-Fos,NFATc1,Ctsk and TRAP m RNAs related to osteoclast differentiation.CONCLUSION:The RAW264.7 cell line was successfully established,and the optimal conditions for inducing osteoclasts were determined.Myricetin is not toxic to RAW264.7 cells,and inhibits RANKL-induced osteoclast formation by down-regulating the expression of related genes.Bayberry can be used as a potential drug for the treatment of osteoporosis.