Study Of The Mechanism Of Geraniin On Osteogenic Differentiation Mediated By Promoting β-catenin Expression In Gsk3β Gene Silenced Rat BMSCS

Objective:Using gene silencing technology to construct a system with Gsk3β gene silenced osteoporotic rat’s bone mesenchymal stem cells(BMSCs),to research β-catenin and key signaling molecule Gsk3β in classic Wnt pathway,as well as to investigate the effects of geraniin on expression of β-catenin/Gsk3β and finally to verify the molecular mechanism of geraniin against osteoporosisMethods:1.40 SD rats were divided into sham operation group and model group.After ovariectomy,the rat osteoporotic model was duplicated and BMSCs were isolated and cultured.The sham operation group was labeled as S-BMSCs group and the model group as M-BMSCs group,the M-BMSC intervened with geraniin(10-9mol/L)was labeled as M-BMSC+geraniin group.2.ALP activity was used to detect the effect of geraniin on osteogenic differentiation of BMSCs,and compared with sham operation group and model group,respectively.3.RNAi silencing technology was used to design and construct the interference sequence,and package the lentivirus,so as to use the lentivirus by Gsk3β gene as the vector to transfect the above BMSCs,construct the stable BMSCs cell line,and use RTQ-PCR to verify the stable system.The groups were as follows:①S-NC group②S-Gsk3β group③M-NC group④M-Gsk3β group⑤S-NC+geraniin group⑥S-Gsk3β+geraniin group⑦M-NC+geraniin group⑧M-Gsk3β+geraniin group(S:sham operation group;M:model group;NC:negative control;Gsk3β:Gsk3βgene silencing)4.After 14 days of intervention with geraniin,Alizarin red staining was used to observe the osteogenic differentiation.CCK8 was used to detect the proliferation of BMSCs,and quantitative PCR and western blot were used to detect the effect of geraniin on the expression of Gsk3β and β-catenin,respectively.Results1.ALP method showed that 10-9mol/L geraniin could increase ALP enzyme activity,which further verified that geraniin could promote osteogenic differentiation of BMSCs.2.The three interference sequences designed by gene silencing technology were used to verify that the sequence of the plasmid constructed for the target gene was completely correct,and the construction of Gsk3β interference lentivirus was successful.BMSCs were successfully transfected with Gsk3β interference lentivirus,and then Gsk3β gene silenced stable cell line was constructed.3.Compared with the group of S-NC,the group of S-Gsk3βsignificantly increased the cell survival rate due to the silencing of the Gsk3β gene(aP<0.05);compared with the group of M-NC,the group of M-Gsk3β significantly increased the cell survival rate due to the silencing of the Gsk3β gene(bp<0.05);compared with the group of S-Gsk3β,the group of M-Gsk3β decreased the cell survival rate(CP<0.05),the group of S-Gsk3β+geraniin increased the cell survival rate(cP<0.05);compared with the group of M-Gsk3β,the group of M-Gsk3 β+geraniin significantly increased the cell survival rate(dP<0.05).4.Compared with S-NC group,the number of calcium nodules in S-Gsk3β group increased significantly(aP<0.05);compared with M-NC group,the number of calcium nodules in M-Gsk3β group increased significantly(bP<0.05),which indicated that the osteogenic differentiation of BMSCs in vitro could be increased by silencing Gsk3β cells;compared with S-NC +geraniin group,the number of calcium nodules in S-Gsk3β+geraniin increased significantly(cP<0.05);compared with the group of M-NC +geraniin,the number of calcium nodules in M-Gsk3β+geraniin significantly increased(dP<0.05);compared with the S-Gsk3β group,the number of calcium nodules in M-Gsk3β group was significantly decreased(eP<0.05),and significantly increased in the S-Gsk3β+geraniin group(eP<0.05),while the number of calcium nodules in the S-Gsk3 β+geraniin was not significantly difference;compared with M-Gsk3β group,the number of calcium nodules in M-Gsk3β+geraniin was significantly increased(fP<0.05).5.Compared with the S-NC group,the expression of Gsk3 β protein and its mRNA in the S-Gsk3βgroup decreased significantly,while the expression of β-catenin protein and its mRNA increased significantly(aP<0.05);compared with the M-NC group,the expression of Gsk3β protein and its mRNA in the M-Gsk3β group decreased significantly,and the expression of β-catenin protein and its mRNA increased significantly(bP<0.05);compared with S-NC+geraniin group,the expression of Gsk3β protein and its mRNA in S-Gsk3β+geraniin group decreased significantly,while the expression of β-catenin protein and its mRNA increased significantly(cP<0.05);compared with M-NC+geraniin group,the expression of Gsk3β protein and its mRNA in M-Gsk3β+geraniin group decreased significantly,the expression of β-catenin protein and mRNA increased significantly(cP<0.05).Compared with S-Gsk3β group,the expression of Gsk3βprotein and its mRNA in M-Gsk3β group was significantly increased,and the expression of β-catenin protein and its mRNA was significantly decreased(eP<0.05);compared with M-Gsk3β group,the expression of Gsk3β protein and its mRNA in M-Gsk3β+geraniin group was significantly decreased,and the expression of β-catenin protein and its mRNA was significantly increased(fP<0.05).Conclusions1.Geraniin can obviously promote the osteogenic differentiation of BMSCs,especially the osteogenic differentiation ability of BMSCs from osteoporotic rats in vitro.2.Gsk3β gene silencing can up-regulate the expression of protein and mRNA of β-catenin,and promote the osteogenic differentiation of BMSCs in vitro.3.Geraniin can promote the osteogenic differentiation of BMSCs by reducing the expression of Gsk3β gene and increasing the expression of β-catenin in Gsk3β-silenced BMSCs.