Effect And Mechanism Of Panax Notoginseng Saponins On Proliferation And Differentiation Of Rat BMSCs Into Osteoblasts

Background:Osteoporosis(OP)has become one of the chronic diseases that seriously affect the health of middle-aged and old people with the aging of our population.OP is a systemic metabolic bone disease characterized by reduced bone mass,degraded bone microstructure,increased bone fragility,bone pain,and easy fracture,which seriously affects the normal life of patients.A large number of studies showed that traditional Chinese medicine and its active ingredients can induce bone Bone Marrow Stromal Cells(BMSCs)to differentiate into Osteoblast(OB).As the main functional cells of bone formation,OB can regulate and influence the formation and reconstruction of bones.Bone mass can be increased by stimulating the growth of OB,thus preventing and treating osteoporosis.Objectives:This study explored the effects and mechanisms of Panax Notoginseng Saponins(PNS)on the proliferation and differentiation of rat BMSCs to OB in vitro to explore the best conditions for clinical application of PNS,to provide a basis for the prevention and treatment of OP.Methods:1.Studies on the subculture of BMSCs in vitro and differentiation ability of osteogenic:Primary BMSCs(PO)of resuscitated and frozen rats were subcultured by whole bone marrow adherent method,and the third-generation cells(P3)were used for experiments and the morphological characteristics of BMSCs were observed under an inverted microscope.BMSCs were cultured with osteogenic inducers after 21 days,the formation of intracellular calcium nodules was detected by alizarin staining method,and the ability of rat BMSCs to differentiate into bone was identified.2.Studies on the effects of PNS inducers on the proliferation and differentiation of BMSCs to OB:The BMSCs of the third generation of rats were cultured with different culture mediums respectively,the control group was the DEME culture medium group,and the first observation group was the osteogenesis induction group.The BMSCs of the third generation of rats were cultured with different culture mediums respectively,the control group was the DEME culture medium group,the first observation group was the osteogenesis induction group,and the second to sixth observation groups were the PNS culture medium group,of which the concentrations of PNS were 10 mg/L,50 mg/L,100 mg/L respectively.The tests of each group were performed on 3d,7d,and 9d.The formation of intracellular calcium nodules was detected by alizarin staining.MTT method was used to detect the proliferation of BMSCs,alizarin staining method was used to detect the formation of intracellular calcium nodules.And on 14d RT-PCR methods were used to detect intracellular Aalkaline phosphatase(ALP),Core-binding factor 1(Cbfal),and Osteocalcin(OCN)of the mRNA expression.3.Studies on the mechanism of PNS inducers on the proliferation and differentiation of BMSCs to OB:The BMSCs of the third generation of rats were cultured with different culture mediums respectively,the control group was the Osteoinduction group.The optimal concentration of the PNS medium at 100mg/L was added to the osteogenic induction culture medium of the first observation group.Based on the first observation group,the ERK1/2 signaling pathway inhibitor U0126 10umol/L added in the culture mediums of the second observation group.The tests of each group were performed on 21 d,the formation of intracellular calcium nodules was detected by alizarin staining.And on 14d the mRNA expression of ALP,Cbfal and OCN in the cell were detected by RT-PCR.Results:1.After the primary BMSCs of rats were seeded,a few cells were adherent to the wall and gathered in clusters in 6h.The cell colony was enlarged and the cell fusion reached more than 80%on the 5th to 7th day,namely,the passage was carried out.After passage,the cell growth gradually became uniform and passed to the third passage.BMSCs were observed to grow adherent to the wall under the microscope with the same morphology and showing a spindle shape.The third generation BMSCs were successfully induced to osteogenesis by osteogenic inducer culture,and positive calcium nodule staining was observed after 21 days with erythrosin staining.2.At different concentrations of PNS osteoinductive medium,PNS promoted the proliferation and calcium nodule formation of BMSCs in a concentration-dependent manner,and the effect was most significant at a concentration of 100mg/L(P<0.05).Results of RT-PCR:The mRNA expression of ALP,Cbfal,and OCN in the cell were the highest in the osteoinduction group with an PNS concentration of 100mg/L(P<0.05).3.In different cultures medium,the amount of alizarin staining-positive calcium nodules in the osteoinductive solution with a PNS concentration of 100mg/L were better than those in the osteoinductive solution group and the U0126 inhibitor group(P<0.05).The osteoinductive fluid group with a PNS concentration of 100 mg/L was significantly different from the other two groups(P<0.05),while the U0126 inhibitor group was lower than the 100PNS bone induction group(P<0.05).Detection results of RT-PCR:The mRNA expression of ALP,Cbfal,and OCN in the cell were the highest in the osteoinduction group with an PNS concentration of 100mg/L(P<0.05),while the mRNA expression of ALP,Cbfal and OCN in the U0126 inhibitor group was lower than the 100PNS bone induction group(P<0.05).Conclusion:1.PNS promoted the proliferation and osteogenic differentiation of BMSCs in a concentration-dependent manner,and the effect was most significant when the PNS concentration was 100 mg/L.2.The mechanism of PNS to promotes the directed differentiation of BMSCs into osteoblasts may be through the ERKI/2 signaling pathway.