| ObjectiveOsteonecrosis of Femoral Head is the main disease leading to loss of hip joint function and even disability in young and middle-aged patients.The causes include hip joint trauma,alcohol abuse and high-dose glucocorticoid use.Trauma Induced Osteonecrosis of Femoral Head(TIONFH)is a serious complication secondary to Femur Neck Fracture(FNF).The smooth progress of bone repair in TIONFH determines the prognosis of the disease.The method of Huoxue Tongluo is an important method to promote bone repair in femoral neck fracture and osteonecrosis of Femoral Heads.A previous study found abnormal fucosylation modification in peripheral blood of steroid-induced osteonecrosis of Femoral Head.In order to exclude the interference of glucocorticoids and underlying diseases,and focus on bone repair and fucosylation modification,this study selected TIONFH with a relatively simple etiology and pathogenesis as the research object.This study intends to analyze the potential mechanism of fucosylation modification in TIONFH bone repair and the intervention effect of tanshinone IIA,a traditional Chinese medicine for Huoxue Tongluo method,through clinical and in vitro studies.MethodsIn the first part of the clinical study,the target gene X and the corresponding glycosylation pathway that may be involved in the TIONFH repair reaction were analyzed by lectin chip and whole transcriptome sequencing derived from network data,and verified in clinical samples.Specifically:(1)Firstly,the reliability and accuracy of Magnetic resonance imaging(MRI)in the diagnosis of early TIONFH at different time periods after FNF were analyzed through the study of inter-observer and intra-observer consistency.Based on this,the target cases were included for follow-up lectin chip detection.(2)The differentially expressed lectins in the peripheral blood of early TIONFH patients were identified by the lectin chip,combined with the bioinformatics results of the whole transcriptome data of TIONFH lesions,it was speculated that the target gene X and the corresponding glycosylation may be involved in the early bone repair response of TIONFH modification pathway.(3)In the femoral head specimens of TIONFH,the correlation between the expression of target gene X and osteogenesis-related genes in the "repair zone" and "necrosis zone" was detected by quantitative real-time PCR(RT-qPCR).In the second part of the in vitro experiments,it is planned to explore the characteristics and roles of the target gene X and the corresponding glycosylation pathway in the phenotypes of mouse preosteoblast cell line MC3T3-E1 cell proliferation,migration and osteogenic differentiation.The next step the intervention effect of tanshinone IIA was analyzed.(1)The osteogenic differentiation model of MC3T3-E1 cells was simulated in vitro to activate and inhibit,and the changes of target gene X and the corresponding glycosylation pathway in the phenotypes of MC3T3-E1 cells such as proliferation,migration and osteogenic differentiation were analyzed.(2)Through the construction of shRNA silencing lentiviral vector,the role of target gene X in activating or inhibiting the proliferation,migration and osteogenic differentiation of MC3T3-E1 cells was analyzed.(3)To analyze the effect of Tanshinone IIA on the proliferation,migration and osteogenic differentiation of MC3T3-E1 cells,as well as the effect on the target gene X and the corresponding glycosylation modification in the process.Specifically,the cell proliferation ability was determined by CCK-8,the osteogenic differentiation ability of cells was observed by ALP alkaline phosphatase staining,the cell migration ability was observed by scratch test,the corresponding glycosylation modification level was detected by lectin immunofluorescence staining,quantitative real-time PCR(RT-qPCR)was used to analyze mRNA expression.ResultsThe first part of the clinical study:(1)We found that MRI diagnosis of TIONFH in "46 months" and "7-12 months" after FNF had good inter-observer and intra-observer agreement.Therefore,we included 5 patients with early TIONFH diagnosed by MRI between 4 and 7 months as the observation group to draw peripheral blood for lectin chip analysis.(2)Compared with 3 healthy people as controls,the results of lectin chip indicated that there were 29 differential lectins in TIONFH.Among them,the expression of RS-FUC,a lectin that specifically binds to fucose,was significantly increased.According to whole transcriptome sequencing,3347 differentially expressed mRNAs were screened in TIONFH lesion tissue samples;among them,the expression of core fucosylation-modified transferase FUT8 in TIONFH was significantly decreased.(3)RT-qPCR results showed that the expression of FUT8 in TIONFH repair area was significantly higher than that in necrosis area.β-catenin,LRP5,BMP2,Osx and other tissues in the repair area have significantly higher expression levels of genes that promote osteogenic differentiation than those in the necrotic area.Based on the above results,it is speculated that there are differences in the expression of lectins related to fucosylation modification in the peripheral blood of TIONFH,which may be related to the activation of FUT8 during the repair of TIONFH.The differential expression of FUT8 in TIONFH may be related to the osteogenic regulation of Wnt/β-catenin signaling pathway.The second part of basic research:(1)In the CHIR-99021-mediated osteogenesispromoting environment,the proliferation,migration,osteogenic differentiation and osteogenesis-related gene expression(Osx,Runx2,Opn and ALP)of MC3T3-E1 cells were significantly enhanced.In the context of DKK1-mediated osteogenesis inhibition,the proliferation,migration,osteogenic differentiation and osteogenesis-related gene expression(Runx2,ALP,Opn and Osx)of MC3T3-E1 cells were significantly attenuated.LCA immunofluorescence staining showed that CHIR-99021 and Dkkl promoted and inhibited the level of fucose modification in MC3T3-E1 cells,respectively.After CHIR-99021 and DKK1 intervened in MC3T3-E1 cells,they promoted and inhibited the expression of FUT8,respectively.CHIR-99021 was significantly up-regulated.TCF1 expression was inhibited;DKK1 significantly inhibited the expression of TCF/LEF-1.(2)Inhibition of FUT8 attenuated the proliferation,migration,osteogenic differentiation and osteogenesis-related gene expression(Runx2,ALP,Opn and Osx)of MC3T3-E1 cells,and significantly interfered with the effect of CHIR-99021 on MC3T3-E1 cell proliferation,migration,and osteogenesis.Osteogenic differentiation and promotion of osteogenesisrelated gene expression(Runx2,ALP,Opn and Osx).At the same time,the expression of βcatenin,a key factor in the Wnt/β-catenin pathway,and the target gene C-myc were significantly reduced after FUT8 inhibition.(3)10-9M and 10-8M tanshinone ⅡA can promote cell proliferation.10-8M Tanshinone ⅡA promoted MC3T3-E1 cell migration,osteogenic differentiation and related gene expression(Runx2,ALP,Opn and Osx).Tanshinone ⅡA significantly promoted the fucosylation modification of MC3T3-E1 cells,and at the same time promoted the expression of FUT8 and important transcription factors C-myc,β-catenin and TCF/LEF-1 in Wnt/β-catenin pathways.ConclusionThere are differences in the expression of lectins related to fucosylation modification in the peripheral blood of TIONFH,which may be related to the activation of FUT8 during the repair of TIONFH.FUT8 can act as a positive regulator to synergize with the Wnt/p-catenin signaling pathway to promote osteoblast proliferation,migration and osteogenic differentiation.In addition,tanshinone ⅡA,an extract from Salvia miltiorrhiza,a traditional Chinese medicine for Huoxue Tongluo method,can promote the proliferation,migration and osteogenic differentiation of MC3T3-E1 cells,and significantly enhance the expression of FUT8 and FUT8-mediated core glycosylation in the process. |
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