Study On The Mechanism Of Bushen Qiangdu Recipe Intervening In Human Bone Marrow Mesenchymal Stem Cells Through LncRNA H19/miR-22/Wnt Pathway

Objective:To explore the effect of Bushen Qiangdu recipe on lncRNA H19/miR-22/Wnt pathway in the osteogenic process of human bone marrow mesenchymal stem cells(hBMSCs),and to explore the mechanism of Bushen Qiangdu recipe on osteogenic differentiation of hBMSCs.Methods:The serum containing Bushen Qiangdu decoction and western medicine(celecoxib)was prepared by Wister male rats.hBMSCs cells were divided into blank control groups,the western medicine control group,low,middle,and high dose groups.After the induction of osteogenic differentiation,the bone components were detected by alizarin red staining,the expression of osteogenesis-related markers and lncRNA H19 and miR-22 were detected by fluorescence quantitative PCR,and the expression of DKK-1 and β-catenin protein,the key factors of Wnt pathway,were detected by Western blot.LncRNA H19 overexpression lentivirus was used to construct lncRNA H19 overexpression hBMSCs.Fourteen days after induction of osteogenic differentiation,the osteogenesis was observed by alizarin red staining,the osteogenic markers were measured by fluorescence quantitative PCR,and the expression of miR-22,Western blot was detected to detect the expression of DKK-1 and β-catenin protein,which are the key factors of Wnt pathway.MiR-22 overexpression lentivirus was used to construct miR-22 overexpression.Fourteen days after induction of osteogenic differentiation,the osteogenesis was observed by alizarin red staining,the osteogenesis-related markers and the expression of lncRNA H19 were determined by fluorescence quantitative PCR,and the expression of DKK-1 and β-catenin protein,the key factors of the Wnt pathway,were detected by Western blot.In the process of osteogenic differentiation induced by hBMSCs overexpressed by lncRNA H19,high-dose serum-containing Bushen Qiangdu decoction was added to intervene.Fourteen days after induction of osteogenic differentiation,osteogenesis was observed by alizarin red staining.The osteogenic markers and the expression of lncRNA H19 and miR-22 were detected by fluorescence quantitative PCR.The expression of DKK-1 and β-catenin protein in the Wnt pathway was detected by Western blot.Results:After hBMSCs induced osteogenic differentiation,all groups of Bushen Qiangdu decoction and celecoxib control serum group could inhibit the formation of mineralized nodules caused by osteogenic differentiation of hBMSCs.The results of fluorescence quantitative PCR showed that compared with the blank control group,the Bushen Qiangdu recipe could inhibit the expression of osteogenic markers such as ALP,BMP2,RUNX2,and COL1A1 mRNA(P<0.05).The expression of β-catenin,the key protein of the Wnt pathway,decreased(P<0.05).and the expression of DKK-1 protein increased(P<0.05).Besides,compared with the blank control group,the expression of H19 in the high dose group of Bushen Qiangdu decoction decreased(P<0.05),and the expression of miR-22 increased(P<0.01).The hBMSCs cell line overexpressing lncRNA H19 was successfully constructed.After osteogenic differentiation,compared with the Vector group,the mineralized nodules in the lncRNA H19 overexpression group were significantly increased,the expression of osteogenesis-related markers ALP,BMP2,RUNX2,and COL1A1 were significantly increased(P<0.05).The expression of DKK1 protein,the key protein of);Wnt pathway and the level ofβ-catenin increased significantly(P<0.05).The hBMSCs cell line overexpressing miR-22 was successffully constructed.Compared with the Vector group,the number of mineralized nodules and the expression of osteogenesis-related markers ALP,BMP2,and RUNX2 in the miR-22 overexpression group decreased significantly after osteogenic differentiation,but there was no significant difference in the expression of lncRNA H19(P>0.05).The DKK-1 level of Wnt pathway key protein was significantly increased and the level of β-catenin was significantly decreased(P<0.05)In the process of inducing osteogenic differentiation of hBMSCs cell lines overexpressed by lncRNA H19,the drug-containing serum of Bushen Qiangdu Formula was added to intervene.Compared with lncRNA H19 overexpression group,lncRNA H19 overexpression plus Bushen Qiangdu recipe group significantly decreased mineralized nodules,osteogenesis-related proteins ALP.BMP2,RUNX2,and COL1A1 expression(P<0.05).The expression of β-catenin,the key protein of Wnt pathway,and DKK-1 expression decreased significantly,H19 expression decreased and miR-22 expression increasedConclusion:Bushen Qiangdu recipe can inhibit the osteogenic differentiation of hBMSCs in a dose-dependent manner.down-regulate the level of lncRNA H19 and up-regulate the level of miR-22,and regulate the expression of DKK-1 and β-catenin,the key proteins of the Wnt pathway.Both lncRNA H19 and miR-22 are closely involved in the osteogenic differentiation of hBMSCs.The overexpression of lncRNA H19 can promote the osteogenic differentiation of hBMSCs.Overexpression of miR-22 can inhibit the osteogenic differentiation of hBMSCs.Both of them can interfere with the expression of DKK-1 and β-catenin,the key proteins of the Wnt pathway.The medicated serum of the Bushen Qiangdu recipe can partially save the osteogenic differentiation of hBMSCs induced by overexpression of lncRNA H19,and up-regulate the level of miR-22.It is suggested that the Bushen Qiangdu method may interfere with osteogenic differentiation of hBMSCs through the lncRNA H19/miR-22/Wnt pathway,and this study plays a hint role in further exploring the mechanism of the Bushen Qiangdu recipe interfering with pathological osteogenesis of ankylosing spondylitis.