Osteoking Inhibits Apoptosis Of BMSCs In Osteoporotic Rats Via PI3K/AKT Signaling Pathway

ObjectiveTo explore whether Osteoking inhibits the apoptosis of BMSCs in osteoporotic rats through PI3K/AKT signaling pathway and to investigate it thoroughly,in order to provide a theoretical basis for clinical Osteoking treatment of osteoporosis.Methods1 Establishment of osteoporosis rat model: Bilateral ovariectomy(OVX)was used to prepare a rat osteoporosis model.Equal amount of adipose tissue around the bilateral ovaries was removed in the sham-operated group.The rats were molded 16 weeks after bilateral ovariectomy.The model was evaluated by bone densitometry and HE staining to observe bone microstructure.2 The osteoporotic rats were divided into four groups(Sham,OVX,Osteoking and positive drug control)of 10 rats each.Osteoking gavage treatment(0.23 ml/100 g.2d)was given according to the body surface area conversion method.Teriparatide(0.23ug/100 g.d)was given to the positive drug control group.Bone density was measured after 10 weeks of treatment.The left tibia was taken for bone biomechanics.The left femur for RNA-seq.The right tibia for histomorphological staining and Masson staining.Peripheral serum was collected to determine the levels of E2,PINP,BALP,CTX,TRACP-5b and IL-1β.The remaining bone tissues were used to validate the RNA-seq results.The efficacy of Osteoking in the treatment of osteoporosis was determined comprehensively by comparative analysis and a preliminary determination of the mechanism was made.3 For the extraction and identification of primary BMSCs,this experiment used the bone marrow apposition method to culture primary BMSCs.the P3 generation cells were identified.The morphology was observed under an inverted microscope.Cell surface markers were analyzed by flow cytometry.The differentiation potential was analyzed and identified using the directed induction culture method.4 Determine the proliferation and osteogenic-lipogenic differentiation ability of extracted BMSCs from OVX and Sham groups of rats.Osteogenic and lipogenic induction media were prepared using L-DMEM with 10% fetal bovine serum,respectively.The expression of core regulators Runx2 and PPAR-γ was determined by protein extraction and the cells were stained for osteogenic and lipogenic induction of differentiation.5 Screening of intervention conditions for Osteoking and LY294002 by CCK8 method.Different concentrations of Osteoking-containing serum and LY294002 were set up to screen the appropriate intervention concentrations for drug administration.6 The effect of Osteoking-containing serum intervention on lipogenesis induction in BMSCs of OVX rats was observed.The expression of PI3K/AKT/m TOR,osteogenesis-related regulatory factors and apoptosis-related regulatory factors were detected after treatment of BMSCs of OVX rats with Sham serum,OVX serum,OVX+LY294002 serum and Osteoking+LY294002 serum,respectively.Results1 Osteoking significantly improved the whole-body BMD and bone biomechanical index in OVX rats.It also significantly increased the expression levels of BALP,E2 and PINP in the serum of OVX rats(P<0.05).Decreased the levels of TRACP-5b,CTX and IL-1β(P<0.05).and significantly improved bone microarchitecture and promoted the formation of new bone.2 RNA-seq detected 23538 genes.Padj<0.05,|log2foldchange|>1 were used as the criteria for significance of differences.A total of 146 differential genes were derived.They were analyzed for GO and KEGG enrichment.The final selection of validation pathways were enriched in PI3K/AKT signaling pathway,PPAR-γ and inflammatory signaling pathway.3 Osteoking increased the expression of Runx2 in OVX rats,decreased the expression of lipogenic marker PPAR-γ,and inhibited inflammatory signaling pathways in vivo(P<0.05).Phosphorylated proteins of PI3K/AKT signaling pathway were found to be significantly increased and the expression of apoptosis-related proteins was inhibited by WB assay(P<0.05).4 The extracted primary BMSCs were identified.Cells were grown apposed to the wall and osteogenic lipogenesis was successfully induced in P3-generation cells.The surface markers of BMSCs were identified by flow cytometry.CD45 showed negative low expression.CD29 and CD90 showed positive high expression.5 Comparison of the proliferative capacity of BMSCs in the Sham and OVX groups revealed that the proliferation capacity of BMSCs in the OVX group was significantly lower compared to that in the Sham group.Subsequently,OVX and Sham rat sera were used to treat BMSCs in the OVX group separately,and it was observed that the OVX group rat sera significantly inhibited the growth of BMSCs compared with the Sham group rat sera.The treatment of Osteoking drug-containing serum with different concentration gradients significantly promoted the proliferation of BMSCs in the OVX group.The fastest proliferation was observed when the cells were cultured with 100% drug-containing serum.The proliferation of BMSCs from OVX rats treated with different concentration gradients of LY294002 was found to be significantly reduced at a concentration of 10 u M.6 The expression level of PPAR-γ was significantly increased in the OVX group relative to the Sham group of BMSCs after lipogenesis induction.Lipid droplet formation was also significantly increased.Subsequently,the lipogenesis induction medium of the OVX group was cultured with Sham serum,OVX serum and Osteoking serum.The lipogenic differentiation of BMSCs was significantly reduced by the Osteoking-containing serum.7 Osteoking-containing serum significantly improved the reduced osteogenic differentiation of BMSCs caused by LY294002,increased the expression of cellular alkaline phosphatase(P<0.05).8 PI3K/AKT signaling pathway-related proteins were detected after treatment of BMSCs from OVX rats with Sham serum,OVX serum,OVX+LY294002 serum and Osteoking+LY294002 serum.The results showed that OVX serum could attenuate PI3K/AKT/m TOR signaling pathway by decreasing the phosphorylation of proteins in PI3K/AKT/m TOR signaling pathway(P<0.05).LY294002 treatment significantly increased the expression of Bax and Caspase3 and inhibited the expression of Bcl2(P<0.05).Treatment by Osteoking-containing serum increased the expression of Bcl2 and decreased the expression of Caspase3 and Bax(P<0.05).Conclusions1 Osteoking significantly increased bone density and bone biomechanics in OVX rats and improved bone microarchitecture in rats.RNA-seq revealed that its treatment may exert efficacy through multiple mechanisms.It involves PI3K/AKT signaling pathway,inflammation and Wnt-related signaling pathway etc.2 Osteoking significantly prevents apoptosis in the bone microenvironment.3 Osteoking-containing serum can significantly enhance the proliferation of BMSCs in OVX rats.It also reversed the imbalance in the spectrum of BMSCs with enhanced lipogenic differentiation and diminished osteogenic differentiation in OVX rats.4 Osteoking-containing serum prevented apoptosis of BMSCs in OVX rats via PI3K/AKT signaling pathway.