A Study Of The Mechanism Of Icariin Inhibits Osteoclast Differentiation Of RAW264.7

1 Effects of Icariin on ROS and osteoclast formation of RANKL-induced RAW264.7 cellsObjective: The changes in ROS content and ROS-catalyzed enzymes NOX1 and NOX4 during the osteoclast differentiation induced by RANKL were determined to determine whether ROS,NOX1 and NOX4 were involved in the osteoclast differentiation process.Different concentrations of Icariin（ICA）were used to intervene in the osteoclast differentiation of RAW264.7 cells induced by RANKL.The expression levels of ROS,NOX1,and NOX4 and the expression changes of NFATc1 and TRAP,the characteristic markers of osteoclast formation,were determined to explore the regulatory mode of ICA in inhibiting osteoclast differentiation.Methods: 1.RAW264.7 cells were divided into 4 groups: Control group,RANKL group,RANKL+ICA 10^-8 M group,and RANKL+ICA 10^-6 M group.RANKL 50ng/m L was added.2.The effects of ICA and RANKL at different concentrations on RAW264.7 cell activity were determined by the CCK^-8 method.3.ROS generation in the Control group,RANKL group,RANKL+ICA 10^-8 M group,and RANKL+ICA 10^-6 M group were detected by DCFH-DA and DHE fluorescent probes.4.The expression of NOX1 and NOX4 in the Control group,RANKL group,RANKL+ICA 10^-8 M group,and RANKL+ICA 10^-6 M group at different time points were determined by qRT-PCR and Western Bolt.5.The expression of NFATc1 and TRAP,the characteristic markers of osteoclasts,in the Control group,RANKL group,RANKL+ICA 10^-8 M group,and RANKL+ICA 10^-6 M group at different time points were determined by qRT-PCR and Western Bolt.Results: 1.CCK8 detected the effect of ICA on the cell activity of RAW264.7 cells.It was found that ICA 10^-8 M group,ICA 10-7 M group,ICA 10^-6 M group,and ICA 10-5 M group were treated with RAW264.7 cells for 24 hours,48 hours,and 72 hours.The growth curve was drawn and ICA concentration did not affect RAW264.7 cell proliferation.The effect of the RANKL group,RANKL+ICA 10^-8 M group,and RANKL+ICA 10^-6 M group on cell activity was measured by the CCK8 method.There was no difference in cell activity between the Control group,RANKL group,RANKL+ICA 10^-8 M group,and RANKL+ICA 10^-6 M group after 24 h culture after CCK8 addition.The cell activity of the RANKL group was lower than that of the Control group at 48 and 72 h,and the difference was statistically significant.There was no difference in cell activity between the RANKL+ICA 10^-8 M group and the RANKL group.The activity of the RANKL+ICA 10^-6 M group was higher than that of the RANKL group,and the difference was statistically significant.2.The fluorescence intensity of DCF and DHE of RAW264.7 cells induced by RANKL was measured after 3 days of culture.The results showed that the fluorescence intensity of DCF and DHE in the RANKL group was about 3 times and 2 times higher than that in the Control group,respectively,and the difference was statistically significant.3.Compared with the RANKL+ICA 10^-6 M group,the fluorescence intensity of DCF and DHE decreased,and the difference was statistically significant.The fluorescence intensity of DCF and DHE in the RANKL+ICA 10^-8 M group was not significantly decreased compared with the RANKL group,and the difference was not statistically significant.4.The m RNA expressions of NOX1 and NOX4 in RAW264.7 cells were measured by qRT-PCR,and the m RNA expressions of NOX1 and NOX4 in the RANKL group were significantly higher than those in the Control group at 3,5,and 7 days,with statistically significant differences.WB was used to detect the expression levels of NOX1 and NOX4 proteins in the RANKL group at 3,5,and 7 days,which were significantly higher than those in the Control group,and the differences were statistically significant.The m RNA expression of NOX4 in the RANKL+ICA 10^-8 M group was lower than that in the RANKL group at 3,5,and 7 days,and the difference was statistically significant.The m RNA expression of NOX1 in the RANKL+ICA 10^-8 M group was lower than that in the RANKL group at 5 and 7 days,and the difference was statistically significant.However,the m RNA expression of NOX1 in the RANKL+ICA 10^-8 M group was lower than that in the RANKL group at 3 days,but the difference was not statistically significant.The relative expression of NOX1 and NOX4 proteins in the RANKL+ ICA 10^-8 M group showed no significant difference compared with the RANKL group at 3,5,and 7 days,and the difference was not statistically significant.The m RNA and protein relative expression levels of NOX1 and NOX4 in the RANKL+ICA 10^-6 M group were lower than those in the RANKL group at 3,5,and 7 days,with statistically significant differences.5.The m RNA and protein expressions of NFATc1 and TRAP in osteoclast markers were determined,and the m RNA and protein expressions of NFATc1 and TRAP in the RANKL group were significantly higher than those in the Control group at 3,5,and 7 days,with statistically significant differences.The m RNA expression of NFATc1 in the RANKL+ICA 10^-8 M group was lower than that in the RANKL group at 3,5,and 7 days,and the difference was statistically significant.The m RNA expression of TRAP in the RANKL+ICA 10^-8 M group was lower than that in the RANKL group at 5 and 7 days,and the difference was statistically significant.However,the m RNA expression of TRAP in the RANKL+ICA 10^-8 M group was lower than that in the RANKL group at 3 days,but the difference was not statistically significant.The relative expression levels of NFATc1 and TRAP protein in the RANKL+ICA 10^-8 M group were not significantly different from that in the RANKL group at 3,5,and 7 days,and the difference was not statistically significant.The m RNA expressions of NFATc1 and TRAP in the RANKL+ICA 10^-6 M group were significantly lower than those in the RANKL group at 3,5,and 7 days,and the differences were statistically significant.The protein expressions of NFATc1 and TRAP in the RANKL+ICA 10^-6 M group were also lower than those in the RANKL group,and the differences were statistically significant.Conclusion: Intracellular ROS increased significantly in RAW264.7 cells during osteoclast differentiation induced by RANKL,suggesting that ROS is involved in osteoclast differentiation.In addition,NOX1 and NOX4 were significantly increased at different time points during osteoclast differentiation,indicating that ROS was generated mainly through NOX1 and NOX4 catalysis during osteoclast differentiation.ICA can inhibit the expression of NOX1 and NOX4 induced by RANKL to inhibit ROS generation,thus inhibiting the differentiation process of osteoclasts.2 Relationship between icariin and estrogen receptor in Osteoclast differentiation of RAW264.7 cells induced by RANKLObjective: ICI 182780 was used to block the activation of estrogen receptor（ER）to explore whether Icariin（ICA）can inhibit the osteoclast differentiation of RAW264.7 induced by RANKL by binding with estrogen receptor ER.Methods: 1.RAW264.7 cells were divided into 6 groups: Control group,RANKL group,RANKL+ICA 10^-6 M group,RANKL+ICI 10^-6 M group,RANKL+ICI 10^-6 M+ICA 10^-8 M group,and RANKL+ICI 10^-6 M+ICA 10^-6 M group.RANKL was 50ng/ml.ICI refers to the concentration of ICI 182780 at 10^-6 M.2.The changes in ROS production in the Control group,RANKL group,RANKL+ICA 10^-6 M group,RANKL+ICI 10^-6 M group,and RANKL+ICI 10^-6 M+ICA 10^-6 M group were detected by DCFH-DA and DHE fluorescence probes.3.Control group,RANKL group,RANKL+ICI 10^-6 M group,RANKL+ICI 10^-6 M+ ICA 10^-8 M group,and RANKL+ICI 10^-6 M+ICA 10^-6 M group were determined the expression changes of NOX1 and NOX4 at different time points by qRT-PCR and Western Bolt.4.Control group,RANKL group,RANKL+ICI 10^-6 M group,RANKL+ICI 10^-6 M+ ICA 10^-8 M group,and RANKL+ICI 10^-6 M+ICA 10^-6 M group were determined the expression of NFATc1 and TRAP at different time points by qRT-PCR and Western Bolt.Results: 1.ROS content measured by DCFH-DA and DHE under different treatments showed that ROS in RANKL+ICI 10^-6 M group increased significantly compared with the RANKL group,and the difference was statistically significant.However,ROS in the RANKL+ICI 10^-6 M+ICA 10^-6 M group was significantly lower than that in the RANKL+ICI 10^-6 M group,and the difference was statistically significant.2.The m RNA and protein expressions of NOX1 and NOX4 in RAW264.7 cells were measured by qRT-PCR and WB,and the m RNA expressions of NOX1 and NOX4 in the RANKL+ICI 10^-6 M group were significantly higher than those in the RANKL group at 3,5,and 7 days,with statistically significant differences.WB was used to detect the expression levels of NOX1 and NOX4 protein at 3,5,and 7 days,which were significantly higher than those of the RANKL group,and the difference was statistically significant.Comparison between RANKL+ICI 10^-6 M+ICA 10^-6 M group and RANKL+ICI 10^-6 M group showed that: The m RNA and protein expression levels of NOX1 and NOX4 in RANKL+ICI 10^-6 M+ICA 10^-6 M groups were lower than those in RANKL+ICI 10^-6 M group at 3,5,and 7 days,and the differences were statistically significant.However,compared with the RANKL+ICI 10^-6 M+ICA 10^-8 M group,m RNA expression levels of NOX1 and NOX4 in the RANKL+ ICI 10^-6 M+ICA 10^-8 M group were higher at 3,5,and 7 days than those of RANKL+ICI 10^-6 M group.However,the relative expression levels of NOX1 and NOX4 protein in the RANKL+ICI 10^-6 M+ICA 10^-8 M group did not decrease at 3,5,and 7 days compared with RANKL+ICI 10^-6 M group,and the difference was not statistically significant.3.The expression of osteoclast marker NFATc1 and TRAP was measured by qRT-PCR and WB,and the m RNA and protein relative expression levels of NFATc1 and TRAP in the RANKL+ICI 10^-6 M group were significantly higher than those in the RANKL group at 3,5,and 7 days,with statistically significant differences.RANKL+ICI 10^-6 M+ICA 10^-6 M group compared with RANKL+ICI 10^-6 M group,The m RNA and protein relative expression levels of NFATc1 and TRAP in the RANKL+ICI 10^-6 M+ICA 10^-6 M group were lower than those in the RANKL+ICI 10^-6 M group at 3,5,and 7 days,and the differences were statistically significant.Compared with the RANKL+ICI 10^-6 M+ICA 10^-8 M group,The m RNA expressions of NFATc1 and TRAP in the RANKL+ICI 10^-6 M+ICA 10^-8 M group were lower than those in the RANKL+ICI 10^-6 M group at 3,5,and 7 days,and the differences were statistically significant.However,the relative expressions of NFATc1 and TRAP proteins in the RANKL+ICI 10^-6 M+ICA 10^-8 M group did not decrease at 3,5,and 7 days compared with the RANKL+ICI 10^-6 M group,and the difference was not statistically significant.Conclusion: ICI 182780 could not block the inhibitory effect of Icariin on osteoclast differentiation of RAW264.7 cells induced by RANKL,suggesting that the inhibitory function of ICA on osteoclast differentiation was not dependent on the binding and activation of estrogen receptor ER.ICI 182780 can promote ROS generation by increasing the expression of NOX1 and NOX4 in RANKL-induced RAW264.7 cells,thus promoting osteoclast differentiation.3 Effect of Icariin on Nrf2/KEAP1 pathway of osteoclast differentiation of RAW264.7 cellsObjective: To investigate the effect of Icariin（ICA）on the Nrf2/KEAP1 pathway of osteoclast differentiation of RAW264.7 cells induced by RANKL.Methods: 1.RAW264.7 cells were divided into 7 groups: Control group,RANKL group,RANKL+ICA 10^-8 M group,RANKL+ICA 10^-6 M group,RANKL+ICI 10^-6 M group,RANKL+ICI 10^-6 M+ICA 10^-8 M group,and RANKL+ICI 10^-6 M+ICA 10^-6 M group.2.After three days of culture in the same environment,cells in the seven groups were collected and Nrf2 protein and KEAP1 protein in cytoplasm and nucleus were extracted.The relative expression levels of each protein were determined.Results: 1.Nrf2/KEAP1 was involved in RANKL-induced osteoclast differentiation of RAW264.7 cells.The relative expression levels of cytoplasmic Nrf2 protein and nuclear Nrf2 protein in the RANKL group were lower than those in the Control group,and the differences were statistically significant.The relative expression of KEAP1 protein in the RANKL group was significantly increased compared with the Control group,and the difference was statistically significant.2.Icariin can regulate Nrf2 and KEAP1 expression of RAW264.7 cells during osteoclast differentiation induced by RANKL.Compared with the RANKL group,the relative expression level of nuclear Nrf2 protein in the RANKL+ICA 10^-6 M group was higher than that in the RANKL group,and the difference was statistically significant.The relative expression level of Nrf2 protein in the cytoplasm of the RANKL+ICA 10^-6 M group was lower than that of the RANKL group,and the difference was statistically significant.The relative expression levels of cytoplasmic Nrf2 protein and nuclear Nrf2 protein in the RANKL+ICA 10^-8 M group showed no significant change compared with the RANKL group,and the difference was not statistically significant.3.ICI 182780 can regulate Nrf2/KEAP1 expression.The relative expression level of Nrf2 protein in the cytoplasm of the RANKL+ICI 10^-6 M group was higher than that of the RANKL group,and the difference was statistically significant.The relative expression level of nuclear Nrf2 protein in the RANKL+ICI 10^-6 M group was lower than that in the RANKL group,and the difference was statistically significant.The relative expression level of KEAP1 protein in the cytoplasm of the RANKL+ICI 10^-6 M group was higher than that of the RANKL group,and the difference was statistically significant.4.After blocking the estrogen receptor,ICI 182780 could not inhibit ICA regulation of Nrf2/KEAP1.The relative expression level of Nrf2 protein in the cytoplasm of the RANKL+ICI 10^-6 M+ICA 10^-6 M group was significantly lower than that of the RANKL+ICI 10^-6 M group,and the difference was statistically significant.The relative expression level of Nrf2 protein in the RANKL+ICI 10^-6 M+ICA 10^-6 M group was significantly higher than that in the RANKL+ICI 10^-6 M group,and the difference was statistically significant.The relative expression level of KEAP1 protein in the cytoplasm of the RANKL+ICI 10^-6 M+ICA 10^-6 M group was significantly lower than that of the RANKL+ICI 10^-6 M group,and the difference was statistically significant.The relative expression level of Nrf2 protein in the cytoplasm of the RANKL+ICI 10^-6 M+ICA 10^-8 M group was not significantly different from that of the RANKL+ICI 10^-6 M group,and the difference was not statistically significant.The relative expression level of nuclear Nrf2 protein in the RANKL+ICI 10^-6 M+ICA 10^-8 M group was significantly higher than that in the RANKL+ICI 10^-6 M group,and the difference was statistically significant.The relative expression level of KEAP1 protein in the cytoplasm of the RANKL+ICI 10^-6 M+ICA 10^-8 M group was lower than that of the RANKL+ICI 10^-6 M group,and the difference was statistically significant.Conclusion: ICA can regulate and inhibit RANKL-induced RAW264.7 osteoclast differentiation through Nrf2/KEAP1 pathway.ICI 182780 could affect Nrf2/KEAP1 expression of RANKL-induced RAW264.7 osteoclast differentiation.ICI 182780 could not block the regulation of ICA on Nrf2/KEAP1.4 Effects of ICA on gene expression pattern and function of osteoclast differentiation in RAW264.7 cellsObjective: To investigate the effect of Icariin on the gene expression profile of osteoclast differentiation in RAW264.7 cells induced by RANKL.Methods: 1.RAW264.7 cells were divided into 3 groups: Control group,RANKL group,and RANKL+ICA 10^-6 M group.2.After the three groups of cells were cultured in the same environment for 7 days,the cells were collected and the total RNA of each group was extracted and purified to construct a c DNA library,which was sent to Lianchuan Biological Company using Illumina Novaseq^TM 6000 platform for transcriptome sequencing to screen the differentially expressed genes among the three groups.The differentially expressed genes were analyzed by functional analysis and KEGG pathway analysis.Results: 1.Comparison after sequencing showed that there were 228 differentially analyzed genes in the Control group and the RANKL group,including 56 up-regulated genes and 172 down-regulated genes;Compared with the RANKL+ICA 10^-6 M group,61 differentially expressed genes were found in the RANKL group,including 23 up-regulated genes and 38 down-regulated genes.There were 215 differentially expressed genes in the Control group compared with the RANKL+ICA 10^-6 M group,including 61 up-regulated genes and 154 down-regulated genes.2.Compared with the RANKL group,GO enrichment analysis of the RANKL+ICA 10^-6 M group showed that the first 10 GO annotations,GO_ID and differentially expressed genes on this ID of the biological process were as follows according to enrichment scores:（t RNA Wobble adenosine to Inosine Editing,GO:0002100,Gm49322）;（alanyl-t RNA aminoacylation,GO:0006419,Gm27029）;（T-helper 1 cell differentiation,GO:0045063,Hmgb1）;（positive regulation of cytosolic calcium ion concentration,GO:0007204,Cxcr5,Hmgb1）;（positive regulation of dendritic spine development,GO:0060999,Adam1a）;（positive regulation of JNK cascade,GO:0046330,Hmgb1）;（tumor necrosis factor secretion,GO:1990774,Hmgb1）;（negative regulation of CD4-positive,alpha-beta T cell differentiation,GO:0043371,Hmgb1）;（aspartate transmembrane transport,GO:0015810,Lrrc8d）The first 10 GO annotations of molecular function,GO_ID and differentially expressed genes on this ID are shown in the following order according to enrichment score:（Exodeoxyribonuclease III activity,GO:0008853,Trex1）;（RNA-DNA hybrid ribonuclease activity,GO:0004523,Fen1）;（5’-flap endonuclease activity,GO:0017108,Fen1）;（crossed form four-way junction DNA binding,GO:0000402,Hmgb1）;（open form four-way junction DNA binding,GO:0000401,Hmgb1）;（t RNA-specific adenosine-34 deaminase activity,GO:0052717,Gm49322）;（flap endonuclease activity,GO:0048256,Fen1）;（3’-5’-exodeoxyribonuclease activity,GO:0008296,Trex1）;（calcium-dependent protein serine/threonine kinase activity,GO:0009931,Adam1a）;（C-X-C chemokine receptor activity,GO:0016494,Cxcr5）.The first 10 GO annotations of cellular component,GO_ID and differentially expressed genes on this ID are as follows according to the enrichment score :（Recycling endosome membrane,GO:0055038,Scamp4）;（trans-Golgi network membrane,GO:0032588,Scamp4）;（cytoplasmic microtubule,GO:0005881,Dynlt1a）;（nuclear chromosome,telomeric region,GO:0000784,Fen1）;（early endosome,GO:0005769,Hmgb1）;（nuclear speck,GO:0016607,Cbll1）;（lysosome,GO:0005764,H2-Ob）;（septin cytoskeleton,GO:0032156,Adam1a）;（secretory vesicle,GO:0099503,Dynlt1a）;（MHC class II protein complex,GO:0042613,H2-Ob）.3.Using the KEGG database,we carried out Pathway analysis on the genes encoding different proteins.The top 10 KEGG annotations and KEGG_ID:（Systemic lupus erythematosus,ko05322）;（Base excision repair,ko03410）;（Necroptosis,ko04217）;（Alcoholism,ko05034）;（non-homologous end-joining,ko03450）;（Asthma,ko05310）;（DNA replication,ko03030）;（Intestinal immune network for Ig A production,ko04672）;（Allograft rejection,ko05330）;（Graft-versus-host disease,ko05332）.Conclusion: Through sequencing analysis of the frontal transcriptome of the RANKL+ICA 10^-6 M group and the RANKL group,a total of 61 differentially expressed genes were found,38 up-regulated and 23 down-regulated.After GO enrichment analysis,an osteoclast differentiation-related gene Hmgb1 was down-regulated,and ICA may inhibit osteoclast differentiation by regulating Hmgb1.After KEGG enrichment analysis,the Necroptosis pathway had the highest down-regulation trend in the RANKL+ICA 10^-6 M group.In addition,the Cell adhesion Molecules（CAMs）pathway and the Chemokine Signaling Pathway are related to ICA regulation of osteoclast differentiation,which is worthy of further study.