| Objective(1)To investigate the expression of fucosyltransferase 8(Fut8)in steroid-induced osteonecrosis of the femoral head(SONFH)based on bioinformatics analysis techniques,and to verify the expression of Fut8 and osteogenic factor in SONFH in clinical specimens by immunohistochemistry and RT-qPCR.(2)To observe the effect of different doses of Compound Shengmai Chenggu Capsule on bone repair in SONFH rats,and the expression of Fut8,osteogenic gene and Wnt/β-catenin signaling pathway proteins in bone tissue by Compound Shengmai Chenggu Capsule.(3)To observe the expression of Fut8,osteogenic gene and Wnt/β-catenin signaling pathway in human bone marrow mesenchymal stem cells(hBMSCs)with different concentrations of compound Shengmai Chenggu capsule,and to explore the mechanism of compound Shengmai Chenggu capsule promoting osteogenic differentiation of hBMSCs.Methods(1)Through GEO(gene expression omnibus)database,search the relevant gene chip data of SONFH,screen differentially expressed genes,and perform gene ontology(GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes)signaling pathway analysis to construct PPI network,screen key genes,explore the expression of Fut8 in SONFH,and explore the relationship between Fut8 and SONFH by lectin chip detection;in addition,based on SONFH patient femoral head specimens,verify the expression of Fut8 and osteogenic factor in SONFH by immunohistochemistry and RT-qPCR.(2)Sprague Dawley(SD)rats were used in this study.SONFH was modeled by imiquimod and methylprednisolone.SONFH rats were intragastrically administered with equal volume of distilled water and low,medium and high doses of traditional Chinese medicine(Compound Shengmai Chenggu Capsule)(groups:blank group,model group,low dose group,medium dose group and high dose group).The number of bones(Tb)was analyzed by micro-CT scan.N),Bonevolume/Totalvolume(BV/TV),Trabecular thickness(Tb.Th)and osteopetrosis dispersion(Trabecular separation,Tb.Sp),etc.;hematoxylin-eosin staining(HE staining)was used to observe the empty bone defect rate in each group;in addition,the effect of Compound Shengmai Chenggu Capsule on the biomechanics of the femoral head in SONFH rats was observed by femoral head compression test;furthermore,the expression of Fut8,osteogenesis-related genes and Wnt/β-catenin signaling pathway in bone tissues was detected by RT-qPCR,Western blot and immunohistochemistry.(3)hBMSCs were used as research vectors to design different concentrations of(0,10-8,10-7,10-6,10-5 Mol)dexamethasone(Dex),and the effects of different concentrations of hormones on osteogenic differentiation of hBMSCs were observed by alkaline phosphatase staining(ALP staining)and alizarin red staining,and Fut8,Runx2,ALP,Ocn,Oxs and BMP2 expression were detected by RT-qPCR and Western blot;in addition,hBMSCs were infected with NC lentivirus,Fut8 overexpressing lentivirus,NC-shRNA lentivirus,Fut8 shRNA silencing lentivirus,BMP8 overexpressing lentivirus 10-5 M Dex)by RT-qPCR and alizarin red staining,and the effects of overexpression and silencing of Oc8 on osteogenic differentiation of hBMSCs were observed by RT-qPCR and Western blot.In addition,different concentrations of compound Shengmai Chenggu capsule medicated serum were prepared to design low,medium,and high concentrations of medicated serum on osteogenic differentiation of hBMSCs treated with high concentrations of Dex(grouping:model group,high concentration of medicated serum+silencing Fut8 group,low concentration of medicated serum group,medium concentration of medicated serum group,and high concentration of medicated serum group),and the effects of low,medium,and high concentrations of medicated serum on Fut8,osteogenesis-related genes,and Wnt/β-catenin signaling pathway of hBMSCs were observed by RT-qPCR,Western blot,and immunofluorescence techniques.Results(1)A gene chip data that met the requirements was obtained by GEO database search:GSE123568,including 30 serum samples from SONFH patients and 10 healthy controls,and 345 differentially expressed genes(up-regulated genes:82,down-regulated genes:263)were obtained after screening under set conditions,of which Fut8,Runx2 and DVL2 genes were lowly expressed in SONFH patients;GO and KEGG pathway enrichment analysis results showed that the GO functions of differentially expressed genes mainly involved myeloid cell differentiation,neutrophil degranulation,neutrophil activation involved in immune response,homeostasis of cell number,and myeloid cell homeostasis.The results of KEGG pathway enrichment analysis showed that the pathways of potential target genes mainly involved:cancer transcriptional dysregulation in cancer,animal mitophagy-animal,mTOR signaling pathway,protein processing in endoplasmic reticulum,interaction between viral proteins and cytokines and cytokine receptors,and NF-κB signaling pathway.The lectin microarray sample assay selected 19 differentially expressed mRNAs,all of which were up-regulated differential genes(including ORYSATA).In addition,compared with the normal area of femoral head specimens,immunohistochemistry and RT-qPCR verified that Fut8 and Runx2 were lowly expressed in the necrotic area(P<0.05).(2)Micro-CT scan revealed BV/TV and Tb in the low,medium,and high dose groups of traditional Chinese medicine.N and Tb.Th was significantly higher than the model group(P<0.05 for all)and increased in a concentration-dependent manner,while Tb.Sp was significantly lower than that in the model group(P<0.05).The femoral head compression test revealed that compared with the model group,the use of Compound Shengmai Chenggu Capsule could effectively increase the maximum load and elastic modulus of the femoral head in SONFH rats,and the maximum load and elastic modulus of the femoral head in the low,medium,and high dose groups of traditional Chinese medicine were greater than those in the model group(P<0.05 for all),and increased in a concentration-dependent manner.RT-qPCR results showed that the mRNA expressions of Fut8,Runx2,ALP,Ocn,Osx and BMP2 in the femoral head bone tissue of the model group were significantly lower than those of the blank group(P<0.05);compared with the model group,the mRNA expressions of the above genes were significantly increased after intervention with Compound Shengmai Chenggu Capsule(P<0.05),and the mRNA expression levels of Fut8,Runx2,ALP,Ocn,Osx and BMP2 were increased in a concentration-dependent manner by Compound Shengmai Chenggu Capsule.Western blot results showed that the protein expression of Fut8,Runx2,ALP,Ocn,Osx and BMP2 in femoral head bone tissue was significantly increased in the low,medium and high dose groups of TCM compared with the model group(P<0.05).The immunohistochemical results showed that the expressions of Fut8,Runx2,and BMP2 after intervention with low,medium,and high doses of Compound Shengmai Chenggu Capsules were also significantly higher than those in the model group(P<0.05).In terms of Wnt/β-catenin signaling pathway,RT-qPCR and Western blot results showed that the expression of Wnt2,LRP5 andβ-catenin in femoral head bone tissue was significantly increased(P<0.05)and SCK-3βexpression was significantly decreased(P<0.05)in the low,medium and high dose groups of TCM compared with the model group.(3)ALP and alizarin red staining revealed that high concentrations(10-5 M,10-6 M)of Dex significantly inhibited osteogenic differentiation of hBMSCs cells.RT-qPCR results showed that high concentrations of DEX significantly inhibited ALP,Ocn,Osx and BMP2 mRNA expression in hBMSCs cells compared with osteogenic induction alone(P<0.05 for all).Western blot results showed that high concentrations of Dex significantly decreased the protein expression of Fut8,Runx2,Osx and BMP2(P<0.05 for all),while low concentrations(10-7 M)of Dex significantly increased the protein expression of Fut8,Osx and BMP2(P<0.05 for all)compared with osteogenic induction alone.In addition,ALP and alizarin red staining revealed that overexpression of Fut8 enhanced osteogenic differentiation of hBMSCs cells,reversed the inhibitory effect of high concentrations of Dex on osteogenic differentiation of hBMSCs cells,and silenced Fut8 osteogenic differentiation weakened.RT-qPCR and Western blot results showed that the expression of osteogenesis-related genes such as Runx2,Ocn and BMP2 in the overexpression Fut8 lentivirus group was significantly greater than that in the NC lentivirus group(P<0.05 for all),while Runx2,ALP,Osx and BMP2 expression in the Fut8 shRNA-silenced lentivirus group was significantly lower than that in the NC-shRNA-silenced lentivirus group(P<0.05 for all).RT-qPCR and Western blot results showed that the Fut8-overexpressing lentivirus group could significantly promote gene expression such as Wnt2,LRP5 andβ-catenin compared with the NC lentivirus group(P<0.05 for all),while the Fut8-overexpressing lentivirus significantly inhibited GSK-3β expression(P<0.05).Compared with the NC-shRNA-silenced lentivirus group,the Fut8 shRNA-silenced lentivirus group significantly inhibited the expression of genes such as Wnt2,LRP5 andβ-catenin(P<0.05),while the Fut8 shRNA-silenced lentivirus significantly promoted the expression of GSK-3β(P<0.05).In addition,low,medium,and high concentrations of medicated serum could enhance alizarin red and ALP staining of hBMSCs;RT-qPCR and Western blot results showed that low,medium,and high concentrations of compound Shengmai Chenggu capsule medicated serum significantly increased Fut8 expression after intervention of hBMSCs compared with the model group(P<0.05 for all).In terms of osteogenesis-related genes,Ocn and ALP expression in the low,medium,and high concentration medicated serum groups was significantly higher than that in the model group(P<0.05 for all),and increased in a concentration-dependent manner;Ocn and ALP expression in the high concentration medicated serum+silenced Fut8 group was significantly lower than that in the high concentration medicated serum group(P<0.05 for all).For the Wnt/β-catenin signaling pathway,compared with the low,medium,and high concentration medicated serum groups,the expression of LRP5 and β-catenin in the model group was significantly decreased(P<0.05 for all),and the expression of LRP5 andβ-catenin in the high concentration medicated serum group was significantly higher than that in the high concentration medicated serum+silenced Fut8 group(P<0.05 for all).Immunofluorescence results showed that low,medium and high concentrations of medicated serum could significantly increase the fluorescence expression intensity of Fut8,Runx2 and β-catenin compared with the model group(P<0.05).The fluorescence expression intensities of Fut8,Runx2,Wnt2 and β-catenin in the high concentration medicated serum+silenced Fut8 group were significantly lower than those in the high concentration medicated serum group(P<0.05 for all).ConclusionMultiple molecules and pathways are involved in the pathogenesis of SONFH,and Fut8 expression is low in necrotic areas of femoral head specimens from patients with SONFH.Overexpression of Fut8 can activate Wnt/β-catenin signaling pathway to promote osteogenesis,and the mechanism of Compound Shengmai Chenggu Capsule in the treatment of SONFH may be through up-regulation of Fut8 expression,which in turn activates Wnt/β-catenin signaling pathway and promotes bone formation. |
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