A Study Of The Mechanisms Involved In The Inhibition Of Osteoclast Differentiation By Helenalin Through The RANKL-mediated Signalling Pathway

Background: Countries around the world are entering the ageing phase to varying degrees,and this has made osteoporosis a major disease in terms of human health.Osteoporosis,which is caused by various factors,is a type of metabolic disease that is characterized by a decrease in the quality and density of bone tissue,a certain degree of destruction of the microstructure,and a high risk of bone fragility and fracture damage.In terms of osteoporosis-related treatment,the cost of treatment in China has been increasing year by year in recent years,which has caused greater problems for the entire health care system.Therefore,research on its prevention and treatment are also current activities that must be carried out.Purpose:This experiment was conducted to analyze the inhibitory effect produced by helenalin during the induction of osteoclast precursor subdivision in vitro,and to understand its mechanism of action and analyze the specific effects,so as to provide a reliable theoretical basis for the use of helenalin in the treatment of corresponding diseases such as osteoporosis.Method: 1.In vitro culture: RAW264.7 cells were stimulated with RANKL(40 ng/ml)to induce differentiation into osteoclasts(OCs),and anti-tartrate acid phosphatase staining(TRAP staining)was used to verify whether the induced differentiated cells were the osteoclasts required for the experiment.2.Cytotoxicity assay: the experimental concentration of cardiolactone was clarified.The concentrations of cardiolactone were set at 0μM,2.5μM,5μM,10μM and 40μM.The CCK-8 cytotoxicity assay was used to analyze whether cardiolactone was toxic to osteoclasts at various concentrations and to determine the concentration gradient of cardiolactone in the experimental group.3.Actin ring staining:The number of actin rings was clarified by using the ghost pen ring peptide staining experiment,and then the effect of various concentrations of cardiolactone on the bone resorption function of osteoclasts was studied.4.RT-PCR: The effects of different concentrations of cardiolactone on the expression of c-Fos,nuclear factor of activated T cells 1(NFATc1),DH3 a,Ndg2 and ATP5 b m RNA were analyzed by RT-PCR.5.Protein immunoblotting assay: Western blot assay was performed to clarify the effects of different concentrations of cardiolactone on NF-κB,c-Fos,NFATc1,PGC-1β,CREB,MYC,ERRα,Nrf2 nerve expression in osteoclast differentiation.6.Reactive oxygen generation assay:The effect of different concentrations of cardiolactone on RANKL-induced reactive oxygen species(ROS)was determined by 2’ and 7’ dichlorodihydrofluorescein diacetate(DCFH-DA)staining.7.7.Statistical analysis: For the experimental results,statistical analysis was performed by SPSS 23.0 software,and one-way analysis of variance(ANOVA)and multiple comparisons(LSD)/Student-Newman-Keul test were used to determine their significance;p<0.05,then the differences were statistically significant.Result:An in vitro differentiation culture system was specifically established for the induction of RAW264.7 cells.1 From the results of the experiments,the experimental groups of helenalin at the concentrations of 2.5 μM,5 μM,and 10 μM did not show cytotoxicity compared to the control group.In terms of osteoclast differentiation,all concentrations of helenalin showed a greater inhibitory effect,and the higher the concentration,the more obvious this inhibitory effect was,and all gradient experimental groups showed statistically significant differences compared to the control group.The results of ghost pen cyclic peptide experiments showed that the cytoskeletal actin ring exhibited more significant inhibition during the differentiation and maturation of osteoclastic precursor cells at increasing concentrations(p < 0.05).2.It was indicated by RT-PCR experiments that for osteoclastic precursor cells,with the addition of the same10μmol/L of helenalin come,with increasing time,the experimental group compared with the control group in NFATc1,c-Fos,IDH3 a,Ndg2 and ATP5 b m RNA expression showed significant decreases at the measured time points 12 h,24h and 48h(all p < 0.05).3,The inhibition of ROS production by helenalin could be measured by DCFH-DA staining.4.The results of real protein immunoblotting(WB)also showed that the same concentration of helenalin in osteoclastic precursor cells reduced the RANKL-induced protein expression levels of NF-κB,c-Fos,NFATc1,PGC-1β,CREB,MYC,ERRα,Nrf2 at all time points in this cell.Conclusion: 1.Helenalin can show inhibitory effects on in vitro RANKL-induced differentiation and maturation of RAW264.7 cells and bone resorption.in the concentration range of 2.5-10 μM,the intensity of inhibition was positively correlated with both concentrations,and inhibited the formation of actin rings,osteoclastogenesis,osteoclast-specific gene expression and ROS.2.as shown by RT-PCR helenalin significantly inhibited the RANKL-induced expression of NF-κB,c-Fos and NFATc1.The decreased production of mitochondria-related genes Ndg2,IDH3 a and ATP5 b indicated diminished mitochondrial proliferation and activity,reduced energy supply,and inhibition of osteoclast differentiation and function.3.Protein blot analysis corroborated the effects of helenalin on the production of c-Fos,NFATc1,PGC-1β,CREB,MYC,ERRα by inhibition and NF-κB and Nrf2 expression was increased.This suggests that helenalin inhibits the expression of relevant cytokines in the nucleus of osteoclasts through the RANKL pathway,also has an inhibitory effect on the activation of the mitochondrial pathway,and reduces its differentiation of osteoclasts through an increase in ROS catabolism.The decrease in transferrin 1 and calcium-regulated neurophosphatase may be associated with a decrease in ROS toward In conclusion,our results confirm that helenalin,a polyphenolic compound,can be used as a potential treatment for osteoclast-associated skeletal diseases such as osteoporosis.