Mechanism Research In Polygonatum Sibiricumpolysaccharide Promoting Osteogenic Differentiation Through The GSK-3?/?-catenin Signaling Pathway

Background and Objective: OP(osteoporosis)is a kind of systemic metabolic disease,characterized by the bone loss and bone microstructure degradation,lead to increased risk of fragility fractures,easlily increasing the fragility fracture.International Osteoporosis Foundation(IOF)issued a set of data showed that with global aging society coming,World Health Organization(WHO)reported that osteoporosis has been an important health issue secondary to coronary heart disease.Early prevention and treatment of osteoporosis may help in developing a better method for preventing osteoporotic fractures.At present,the drugs used in the treatment of osteoporosis are bone resorption inhibitors and bone formation drugs.It is urgent to develop new anti-osteoporosis drugs,which have the advantages of economic and less side effects.Natural plant compound Polygonatum sibiricum polysaccharide is the extract of rhizoma polygonati,which is a kind of medicinal and edible plant.Past research found that polygonatum sibiricum polysaccharide had the enhancement immunity,anti-tumor,anti-aging and so on.Our studies have found that the polygonnatum sibiricum polysaccharide promoted osteogenetic differentiation in bone marrow mesenchymal stem cells by classical Wnt signaling.But its specific molecular mechanism remains unknown.Our research was investigated that the mechanism of in Polygonatum sibiricum polysaccharide promoting osteogenic differentiation by the glycogen synthase kinase-3?(GSK-3?)/ ?-catenin signaling pathway.Methods:(1)The bone marrow mesenchymal stem cells were isolated and cultured in vitro;(2)sh RNA-LRP5 virus,whose MOI=20,40,60,was transfected into BMSCs.The expression of green fluorescent protein in BMSCs accounted for more than 90% of the total cells under microscope.(3)The proliferation of BMSCs,infected with sh RNA-LRP5 virus,was evaluated by CCK-8method.(4)After recombinant lentiviral sh RNA-LRP5 transfection of BMSCs with phosphonomycin screening more than a week,the expression levels of LRP5 genes were further confirmed by quantitative real-time PCR and the expression of LRP5 protein was detected by western blot to detect efficiency of transfection.(5)BMSCs of the sh RNA-LRP5 group,Con group and NC group were cultured and induced in osteoblast medium.The gene expressions of osteogenesis-related genes(ALP,Runx2 and OCN)were detected by q RT-PCR.ELISA dected the expressions of osteogenesis-related proteins.(6)BMSCs of each group were cultured in osteoblast medium with 25 mg/L PSP,PBS buffer and Wnt3 a.After treatment for 21 days,PNP methods to detect the activity of alkaline phosphatase(ALP).After treatment for 28 days,alizarin red staining relative quantitative detection was measured.(7)TOPflash/FOPflash dual-luciferase report gene(dual luciferase report system testing,TOPflash and negative control FOPflash)detection test the nucleus in ?-catenin as the activity of transcription factors.(8)The expressions level of p-GSK-3? and GSK-3? in total extract protein were detected by Western blot,as well as nuclear protein?-catenin.Results:(1)Bone marrow stromal cells(BMSCs)were adherent,and the cells grew well and had strong proliferative ability,which could be induced to differentiate into osteoblasts after osteogenic induction.(2)After transfected with Virus for 96 hours,BMSCs were in good condition,and the expression efficiency of green fluorescent protein(GFP)was more than 90% under inverted fluorescence microscope.(3)CCK 8 method to detect the proliferation BMSCs Transfected with Virus(p > 0.05).(4)Compared with Con group,efficiency of transfection of BMSCs with sh RNA-LRP5 virus was above 70% examined by Real-time RCR and Western blot(p < 0.05).There was no statistically significant difference between Con group and NC group.(5)Compared with Con group,the osteogenesis-related genes were evaluated by q RT-PCR and the protein expressions were detected by ELISA in sh RNA-LRP5 group(p < 0.05).(6)The staining and relative quantitative test of ALP and AR result: in the Con group and NC group,PSP and Wnt3 a promoted BMSCs osteogenic differentiation.There was not statistically significant difference between PSP and Wnt3 a treatment in the Con group and NC group,p > 0.05.There was statistically significant difference between PSP treatment and NC group,p <0.05.In Sh RNA-LRP5 group,the treatment of PSP promoted BMSCs osteogenic differentiation.Compared with treatment of Wnt3 a and PBS buffer,there was not statistically significant difference between the treatment of Wnt3 a and PBS buffer,p > 0.05).(7)With PSP treatment,TOPFlash reporter gene luciferase activity was significantly enhanced in sh RNA-LRP5,Con,NC three groups;TOPFlash report gene luciferase activity was significantly enhanced in Wnt3a group,Con group and NC group.while the TOPFlash report sh RNA-group LRP5 gene luciferase activity was not significantly enhanced.PBS buffer solution,sh RNA-LRP5,Con,NC three groups of TOPFlash gene luciferase activity was not significantly enhanced.(8)In Con group and sh RNA-LRP5 group,the treatment of 25mg/L PSP compared with the treatment of PBS buffer increased the protein expression of nuclear ?-catenin and the difference was statistically significant,p<0.05.In Con group and sh RNA-LRP5 group,the treatment of 25mg/L PSP compared with the treatment of PBS buffer increased the total protein expression of p-GSK-3? and the difference was statistically significant,P<0.05.But there was not statistically difference between the treatment of 25mg/L PSP and PBS buffer about the total protein expression of p-GSK-3?,P >0.05.Conclusions:(1)Lentivirus-mediated LRP5 gene silencing in BMSCs knockdown the gene and protein in mouse bone marrow mesenchymal stem cells and silenced LRP5 gene to prevent BMSCs osteoblast differentiation.(2)PSP promoted osteoblast differentiation independ of LRP5 gene.The level of phosphorylation glycogen synthase kinase 3?(p-GSK-3?)was increased with PSP treatment,which increased the expression of ?-catenin and promoted its nuclear translocation,and enhanced TCF /LEF transcriptional activity.Together,our data indicated that PSP promoted osteoblastogenesis and bone formation by GSK-3?/?-catenin signaling.