Compound Kidney And Blood Activating Granules Regulate The Balance Between Osteogenic Differentiation And Adipogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells Through Trb3

Background:Osteoporosis-induced fractures are an important cause of death in the elderly,and the search for drugs that can be taken for a long time and are effective against osteoporosis is an important task now and in the future.The disorder of osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)can cause osteoporosis,which is a new direction for the prevention and treatment of osteoporosis.Compound Kidney-Invigorating Granule(CKG),which is based on the pathogenesis of "kidney deficiency and blood stasis",can significantly improve the symptoms related to postmenopausal osteoporosis,but the exact molecular mechanism is not clear.In our previous work,we found that CKG medicated serum significantly promoted the osteogenic differentiation of human BMSCs and mouse BMSCs,and further studies revealed that the formula-containing serum promoted the entry of Trb3,a key protein for osteogenic/lipogenic differentiation of BMSCs,into the cell nucleus.Based on the results of the previous study and literature reports,we propose the hypothesis that "compound kidney tonic and blood activating granules have a dual effect on anti-osteoporosis through Trb3 regulation of bone lipid homeostasis".In this study,based on the previous work,we will clarify the effect of this formula on osteogenesis/lipogenesis differentiation of BMSCs through in vitro cellular experiments to reveal the molecular mechanism of antiosteoporosis of compound kidney and blood particles,and to lay a solid foundation for further compounding studies.Objective:To observe the effect of CKG-medicated serum on osteogenic/adipogenic differentiation of BMSCs,and to identify Trb3 as a key "molecular switch" that mediates osteogenic differentiation and adipogenesis to determine the spectrum fate of hBMSCs.Methods:1.In vitro culture systems of hBMSCs from patients with OP were constructed,and morphological observation and flow identification of cell surface antigens were performed for clear characterization;CKG-medicated serum was prepared,and the optimal concentration interval of CKG-medicated serum and rat blank serum were used to intervene with hBMSCs from patients to screen the optimal medicated serum concentration interval,respectively.2.Osteogenic and adipogenic differentiation of hBMSCs by CKG-medicated serum was observed by ALP activity and staining,and oil red O staining.The relative quantitative analysis of transcript levels and translation levels of osteogenic markers Runx2,Osterix,ALP,OCN and adipogenic markers PPARy and FABP4 were observed by qPCR,Western blot assay.3.The effect of CKG-medicated serum on Trb3 was observed by qPCR and Western blot to clarify whether CKG-medicated serum had regulated Trb3 and then knocked down the expression of endogenous Trb3.After that,the key "molecular switch"of Trb3 on osteogenesis and lipogenesis was observed by ALP activity and staining,oil red O staining,and relative quantitative analysis of marker proteins of osteogenesis and lipogenesis at transcriptional and translational levels.4.Transfection of hBMSCs with Trb3 siRNA reduced Trb3 expression in cell endogenous,and the following assays were done after CKG-medicated serum culture:after cell fixation,Trb3,Smad4 and β-catenin primary antibodies with different wavelengths of fluorescence recognized the corresponding proteins,respectively,and the subcellular observation of Trb3 and Smad4,Trb3 and β-catenin co-localization.The total cell protein,cytoplasm and nucleus were extracted respectively,and the distribution changes of Trb3,Smad4 and β-catenin in the cytoplasm and nucleus were analyzed by Western blot.Results:1.hBMSCs from osteoporotic patients were successfully isolated and cultured,and flow cytometry identified high expression of CD73(97.6%),CD105(98.6%),CD90(97.6%)and low expression of CD14(0.02%),CD34(1.21%),CD45(0.974%)of cell surface antigens;CKG-medicated serum concentrations were in the range of 1%～5%range,promoting the growth of hBMSCs in a dose-and time-dependent manner.2.CKG-medicated serum enhanced ALP activity in hBMSCs,upregulated Runx2,Osterix,ALP,OCN gene and protein expression and inhibited PPARy,FABP4 gene and protein expression and lipid accumulation.3.CKG-medicated serum promoted Trb3 expression and reversed the effect of compound kidney tonic blood granule-containing serum to promote osteogenic differentiation and inhibit lipid differentiation when endogenous Trb3 was knocked down.4.The expression of Trb3,β-catenin and Smad4 was increased in the nucleus of cells in the CKG-medicated serum-treated group;immunofluorescence confocal localization showed that CKG medicated serum promoted increased Trb3/Smad4 or Trb3/β-catenin subcellular co-localization,and mainly localized to the nucleus.Conclusion:This study reveals that CKG enhances osteoblast differentiation and limits adipocyte differentiation in hBMSCs via Trb3.CKG-medicated serum in combination with Trb3 siRNA reversed the effect of CKG-medicated serum in promoting osteoblast differentiation and inhibiting adipocyte differentiation.CKG promotes Trb3 entry into the nucleus and synergistically activates BMP/Smad4 and Wnt/β catenin signaling.