In order to examine the role of astacene on mice body development and the expression of energy metabolism related genes in mice, we treated mice (Kunming white) and primary culture of mouse muscle cells with astacene of higher and lower concentration. Then the total mRNA was extracted from the muscle tissue and cells respectively, and the mRNA levels of UCP3 and LXRalpha were detected by RT-PCR in all the samples. Compared with the control group, the body weight of mice in high concentrations of astacene group grown slowly, and the expressions of UCP3 genes decreased significantly in muscle tissue of the 10th day and the 30th day as well as the cells of treated for 24 h (P<0.05). The expression of LXRalpha gene increased significantly in all samples (P<0.05) and reached its peak at 72 h (P<0.01). With the treatment of lower concentration of astacene, the expressions of UCP3 and LXRalpha gene mRNA in muscle tissue did not alter much, but in muscle cells treated for 24 h, the mRNA level of UCP3 gene decreased significantly (P<0.05), and LXRalpha gene increased significantly (P<0.05). The results suggest that astacene has a role in regulating the energy use in mice muscle.
Animals ; Carotenoids ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Energy Metabolism ; drug effects ; genetics ; Ion Channels ; genetics ; metabolism ; Liver X Receptors ; Male ; Mice ; Mitochondrial Proteins ; genetics ; metabolism ; Muscle, Skeletal ; cytology ; metabolism ; Orphan Nuclear Receptors ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Uncoupling Protein 3

BACKGROUND:Previous studies have found that neohesperidin can delay bone loss in ovariectomized mice and has the potential to treat osteoporosis,but its specific mechanism of action remains to be explored. OBJECTIVE:To explore the key targets and possible mechanisms of neohesperidin in the treatment of osteoporosis based on bioinformatics and cell experiments in vitro. METHODS:The gene expression dataset related to osteoporosis was obtained from GEO database,and the differentially expressed genes were screened and analyzed in R language.The osteoporosis-related targets were screened from GeneCards and DisGeNET databases,and the neohesperidin-related targets were screened from ChEMBL and PubChem databases,and the common targets were obtained by intersection of the three.The String database was used to construct the PPI network of intersection genes,and the key targets were screened.The DAVID database was used for GO and KEGG enrichment analysis.The AutoDock software was used to verify the molecular docking between the neohesperidin and the target protein.The effect of neohesperidin on osteogenic differentiation of C57 mouse bone marrow mesenchymal stem cells was detected.Complete medium was used as blank control group;osteogenic induction medium was used as the control group;and osteogenic induction medium containing different concentrations of neohesperidin(25,50 μmol/L)was used as experimental group.The expression of alkaline phosphatase,the degree of mineralization,the expression of osteogenic-related genes and target genes during osteogenic differentiation of cells were measured at corresponding time points. RESULTS AND CONCLUSION:(1)9 253 differentially expressed genes,2 161 osteoporosis-related targets,and 326 neohesperidin-related targets were screened.There were 53 common targets among the three.All 53 genes were up-regulated in osteoporosis samples.The PPI network screened the target gene PRKACA of research significance.GO function and KEGG pathway enrichment analysis showed that neohesperidin's treatment of osteoporosis through PRKACA target mainly depended on biological processes such as protein phosphorylation and protein autophosphorylation,acting on endocrine resistance,proteoglycan in cancer,and estrogen signaling pathway to play a therapeutic role.Molecular docking results showed that neohesperidin had a certain binding ability to the protein corresponding to the target PRKACA.(2)The results of alkaline phosphatase staining showed that neohesperidin could promote the expression of alkaline phosphatase in the early stage of osteogenic differentiation of mesenchymal stem cells.Alizarin red staining showed that neohesperidin could promote the mineralization of osteogenic differentiation of mesenchymal stem cells.RT-qPCR results showed that neohesperidin could increase the mRNA expression of alkaline phosphatase,PRKACA,and osteocalcin.(3)These results indicate that neohesperidin may promote osteogenic differentiation through PRKACA target on the estrogen signaling pathway to prevent and treat osteoporosis.