The Experimental Study Of Polygonatum Sibiricum Polysaccharide On Anti-osteoporosis Effect In Vivo And Promotion Of Osteogenic Differention Of Bone Mesenchymal Stem Cells In Vitro

Background and Objective: Osteoporosis(OP)is a chronic metabolic bone disease which characterised by low bone mineral density and microarchitectural deterioration of bone tissues,leading to reduced bone strength and a consequent increase in bone fragility and vulnerability to fractures.At present,most of the anti-osteoporosis drugs are mostly bone resorption inhibitors.The drugs are expensive,the long-term efficacy is unclear,and the adverse reactions are obvious.A natural,effective and side-effect phytochemical drug has become a hot topic of research.In our previous study,we found that Polygonatum Sibiricum Polysaccharide(PSP)effectively promoted the osteogenic differentiation of mouse BMSCs.The possible mechanism is that PSP promotes osteogenic differentiation of BMSCs by promoting the expression and into the nucleus of downstream effector molecules ?-catenin in the Wnt/?-catenin pathway,but not through the upstream LRP5 molecule activation pathway.Therefore,we speculate that there may be other pathways or signal pathways involved in the PSP promote osteogenic differentiation of BMSCs.In our study,we explored the anti-osteoporotic effects of PSP on ovariectomized(OVX)rats in vivo.Then we further explored the effect of PSP on the osteogenic differentiation of BMSCs and possible molecular mechanisms in vitro.To provides experimental evidence for the clinical application of PSP in the treatment osteoporosis.Methods: 1.Construction of the ovariectomized(OVX)rat model.(1)Rats were matched with their body weights and randomly divided into 5 groups according to the random number table,including high-and medium-dose PSP group,zoledronate(ZOL)group,model group and sham-operated group(SHAM);(2)Rats in each group were given intragastric administration every other day,and body weight was measured every week to assess changes;(3)Collecting rats uterus after intervention for 12 weeks;(4)Micro-CT was used to perform a metrological analysis of bone mineral density(BMD)and bone morphology,including the number of bone ridges(Tb.N),bone volume fraction(BV\TV),trabecular thickness(Tb.Th),trabecular separation(Tb.Sp);(5)HE staining for histopathological observation of bone;(6)Real-time PCR analysis for mRNA expression of key genes in rat bone.2.Construction of the BMSCs.(1)Inverted microscope was used to observate cell growth;(2)Cell proliferation was detected by CCK-8 assay.(3)Alkaline phosphatase(ALP)and Alizarin red S staining were performed after 7 and 28 days by PSP osteoblast-inducing culture.The mRNA expressions of ALP,Runx2 and OCN were determined by Real-time PCR;(4)The relative expression of Hippo pathway related genes and proteins(MST1,p-MST1,TAZ,p-TAZ)were analyzed by Real-time PCR or Western-bloting.Results: There was no significant difference in body weight between the groups at 0 week.At 12 th week,the body weight of the model group was significantly higher than that of the SHAM group.The weight of the rats in the ZOL and PSP groups was significantly lower than that in the model group.(2)At the 12 th week,the uterine morphology of the SHAM group was normal,and OVX rats had different degrees of atrophy.Compared with the uterine wet weight of the model group,there was no significant difference in the PSP groups',but the increased significantly in ZOL group's.(3)Compared with the SHAM group,the BMD,BV/TV,and Tb.N of the model group were significantly lower,but the Tb.Sp was significantly increased;Compared with the model group,BMD,BV/TV,and Tb.N were significantly increased in ZOL and PSP groups,but the Tb.Sp was significantly reduced,and the Tb.Th was no statistical difference between the groups.(4)With HE staining,the SHAM group showed normal,compact and uniform trabeculae with intertrabecular spaces,and filled numerous cells;the model,OVX and M-PSP groups were filled numerous fat cells and showed significant expansion of the trabecular space caused by sparse and disorderly trabecular bone;(5)Real-time PCR analysis of bone tissue showed that compared with the SHAM group,the differentiation-related gene expression of osteoblasts(ALP,Col1a1,Runx2,OCN)and osteoclasts(ACP5,CTSK)was significantly enhanced in model group;Compared with the model group,high-dose PSP can significantly promote osteogenic differentiation-related gene expression,inhibit osteoclast differentiation-related gene expression;Compared to the model group,ZOL group can significantly inhibit osteoclast differentiation-related gene expression,but had no effect on the osteogenic differentiation-related genes;(6)BMSCs P6 were long shuttle-shaped,with long synaptic cells,colony growth,and vortex-like growth.After osteogenesis induced,the cells became short shuttle-type and the cytoplasmic black granules increased.(7)CCK-8 experiments showed that PSP concentration(0-50mg/L)did not affect cell proliferation at 24 th hour,48 th hour,and 72 th hour.(8)Osteogenesis induced by 7 days and 28 days respectively,ALP staining and Alizarin red S staining showed that all concentration PSP treatment groups promoted osteogenic differentiation and mineralization of BMSCs,and the effect of 25 mg/L PSP group was most obvious;25 mg/L PSP significantly promoted the expression of osteogenic differentiation-related genes.(9)BMSCs were induced by 25 mg/L PSP for 24 h,48 h,and 72 h,qPCR analysis showed that PSP promoted the expression of TAZ gene at 48 th hour,but had no effect on MST1 gene expression.Western-blot result showed that PSP significantly inhibited TAZ phosphorylation at 48 th and 72 th hour,but had no effect on MST1 protein expression.Conclusion:(1)After 12 weeks of ovariectomy,rats weight gained,uterine atrophy,bone density and bone mass decreased,and bone microstructure was destroyed.(2)PSP can exert the prevention and treatment of osteoporosis in vivo,and can prevent bone density,bone loss and bone microstructural destruction.(3)PSP had no effect on cell proliferation in BMSCs.(4)PSP promoted osteogenic differentiation and mineralization of BMSCs,and promoted the differentiation-related gene expression of osteoblasts(ALP? Col1a1? Runx2? OCN).(5)The effect of PSP on skeleton formation is associated with regulation of the Hippo pathway in vitro.