Uric Acid Promotes Osteogenic Differentiation Of Human Bone Mesenchymal Stem Cells By Upregulating PL Cβ3

Objective We plan to validate the role of higher concentrations of uric acid(UA)in promoting osteogenic differentiation of human bone marrow mesenchymal stem cells(h BMSCs),screen for differentially expressed proteins that may be up-or down-regulated by UA in the osteogenesis process by mass spectrometry sequencing and bioinformatics analysis,and explore the possible mechanisms in UA contributing to the osteogenesis.Methods ⑴hBMSCs from patients who underwent lumbar spine surgery without other underlying diseases was isolated,purified and cultured.The third-generation h BMSCs were selected for further experiments;⑵The h BMSCs were divided into six groups: control group(with complete culture medium)and osteogenesis induction group(with osteogenesis induction medium),and the latter group was divided into five sub-group by UA concentration gradients(0 mmol/L,0.1 mmol/L,0.2 mmol/L and 0.4 mmol/L).The growth state and morphology of the cells were observed using inverted microscopy;⑶14 days after induction,osteogenic differentiation of h BMSCs was identified using alizarin red staining and alkaline phosphatase(ALP)activity;⑷Cell samples induced by 0.1 mmol/L and 0.4 mmol/L UA were prepared.Mass spectrometry sequencing was performed using 4D label-free quantification(4D-LFQ)technology;⑸ The differentially expressed proteins were further analyzed by bioinformatics,using Gene Ontology(GO)functional annotation and enrichment analysis to describe the biological processes,molecular functions and cellular components of the candidate differential proteins.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway annotation and enrichment analysis were used to describe the signaling pathways of the candidate differential proteins.In addition,heat map,protein interaction network construction,hub gene analysis and database search were used to further screen for significant differential proteins;⑹Western blotting was used to detect differential protein expression.Results ⑴hBMSCs were successfully isolated and cultured by Ficoll density gradient centrifugation,and h BMSCs were induced towards osteoblasts;⑵The number of calcium nodules formed in osteogenesis increased with increasing UA concentration,in which the difference was more significant when UA>0.2 mmol/L.The number of calcium nodules in the 0.2 mmol/L and 0.4 mmol/L groups were 19.33±1.53 and 27.67±1.53(P<0.001).In addition,the intracellular ALP activity was also increased,and the ALP activity in the 0 mmol/L,0.1 mmol/L,0.2 mmol/L and 0.4 mmol/L groups were 0.23±0.02 gold unit/g protein,0.33±0.01 gold unit/g protein,0.41±0.01 gold unit/g protein,0.75±0.01 gold unit/g protein,0.75± 0.01 gold unit/g protein(P<0.001);⑶After mass spectrometry analysis andidentification,86 significantly differentially expressed proteins were finally obtained.By bioinformatics analysis,these possible differentially expressed proteins were possibly involved in biological processes such as organelle transport and axon development,involved structural molecules,transcriptional regulators and other functions,presented in various intra-and extra-cellular components,related to vascular generation,thermogenesis,autophagy and other signaling pathways.Heat map screening identified RABEP2,AGTRAP,MAGED2,SRSF5 genes encoding proteins.Protein mutual network construction and hub gene analysis identified MRPL12,MRPL43,MRPL48,COG7,NDUFB3 as possible key genes.And protein PLCβ3,Col16α1 and KIF11 were selected for further database searches.Finally,PLCβ3 was screened as differentially expressed proteins,which showed correlation with bone metabolism in previous studies and could be continued to be validated at the antibody level;⑷By Western blotting assay,the UA promoted osteogenic differentiation of h BMSCs,the expression of PLCβ3 was increased.Conclusion This experiment verified the promoting effect of UA on osteogenic differentiation of h BMSCs,while using mass spectrometry sequencing,bioinformatics analysis and antibody validation,we found that PLCβ3 was highly expressed in the process of UA promoting osteogenic differentiation and was a possible target for this process.