| Part 1 To investigate the clinical significance of Apelin-13 in Nontraumatic Osteonecrosis Of Femoral HeadObjectiveThe purpose Of this study was to investigate the role Of Apelin-13 in the diagnosis Of Nontraumatic Osteonecrosis Of Femoral Head(NONFH)by analyzing the serum levels Of Apelin-13 in healthy individuals and in patients with NONFH,in order to provide evidence for the discovery Of new serum markers for NONFH.MethodsNinety-two patients with Nontraumatic Osteonecrosis Of Femoral Head(NONFH group)and 84 healthy subjects(Health group)matched with sex and age were randomly selected.Statistics of the gender,age,body mass index,general information,using Enzyme Linked Immunosorbent Assay to detect all samples,serum levels of Apelin-13 contrast analysis of NONFH and the level of serum Apelin-13 healthy subjects,and further analyze the cases of NONFH of different pathogenic factors,side,ARCO stage,Hariis score between the levels of Apelin-13.Correlation analysis was conducted to explore the correlation between serum Apelin-13 level and ARCO stage and Harris score.The value of serum Apelin-13 level in the diagnosis of NONFH was evaluated by Receiver Operating Characteristic curve(ROC).ResultsThere was no statistical difference in gender,age and body mass index between the NONFH group and the Health group,and they were comparable.Compared with the Health group,the level of serum Apelin-13 in the NONFH group was lower(892.48±224.28 pg/mL vs 1316.79±237.14 pg/mL),and the difference was statistically significant(P<0.01).There was no significant difference in serum Apelin-13 levels among steroid-induced,alcohol-induced and idiopathic femoral head necrosis(P>0.05).The level of serum Apelin13 in unilateral femoral head necrosis was higher than that in bilateral femoral head necrosis(957.56±233.19 pg/mL vs 852.53±210.84 pg/mL),and the difference was statistically significant(P<0.05).The difference of serum Apelin-13 level among different ARCO stages was statistically significant,and with the increase of ARCO stage,the serum Apelin-13 level gradually decreased.Spearman correlation analysis showed that serum Apelin-13 level was negatively correlated with ARCO stage(r=-0.473,P=0.000<0.001),and Pearson correlation analysis showed that serum Apelin13 level was positively correlated with Harris score(r=0.520,P=0.000,P<0.001).ROC curve analysis of serum Apelin-13 level in the NONFH group and the Health group showed that the optimal cut-off point was 1106.00pg/mL,the sensitivity was 83.33%,the specificity was 80.43%,the accuracy was 81.82%,and the area under the curve(AUC)was 0.894.ConclusionSerum Apelin-13 level has certain significance in the diagnosis of NONFH,and is a potential biomarker for the diagnosis of NONFH.The role of Apelin-13 in the pathogenesis of femoral head necrosis deserves further study.Part 2 To study the effect of Apelin-13 on bone metabolism of Rat Bone Marrow Mesenchymal Stem CellsObjectiveThe imbalance of bone marrow mesenchymal stem cells to osteoadipoblast differentiation plays an important role in the pathological changes of femoral head necrosis.The antiapoptotic effect of Apelin-13 on osteogenic precursor cells has been demonstrated.In this study,we investigated the effects of exogenous Apelin-13 on osteogenic differentiation in isolated Rat Mesenchymal Stem Cell-bone marrow(rBMSCs),and explored the mechanism of Apelin-13’s involvement in bone metabolism regulation through Wnt/βcatenin signaling pathway.MethodsrBMSCS was cultured,and the role of Apelin-13 in osteogenic differentiation of rBMSCs was studied by adding different concentrations of exogenous Apelin-13 into rBMSCs medium.The effect of Apelin-13 on rBMSCs proliferation was evaluated by CCK-8 assay at 4 h,24 h,72 h and 120 h.The morphology of rBMSCs cells after 14 days of osteogenic differentiation induced by the addition of exogenous Apelin-13 was observed under light microscope,and the intracellular calcium nodules were observed by Alizarin post-staining.Exogenous Apelin-13 culture was added,and RT-PCR assay was performed to detect the expression of COL1A1 and RUNX2 osteogenic genes of rBMSCs.Exogenous Apelin-13 was added to culture,and the expression of COL1A1,Runx2,total β-catenin(tβcat)and active β-catenin(aβ-cat),key proteins in Wnt/β-catenin signaling pathway,were detected by Western Blot assay.And expression of aβ-cat,COL1A1 and Runx2 after addition of Wnt/β-catenin signaling pathway inhibitor DKK1.ResultsCCK-8 results showed that Apelin-13(0.1,1,10,100,1000nM)had no significant effect on the proliferation of rBMSCs cells at 4 h,24 h,72 h,and 120 h compared with the control group(P>0.05).RT-PCR results showed that compared with the control group,the mRNA relative expressions of COL1A1 and RUNX2 in Apelin-13(0.1,1,10,100 and 1000nM)groups were significantly increased(P<0.05).Alizarin red staining showed that after 14 days of osteogenic differentiation,calcium nodules deposition gradually increased with the increase of Apelin-13 concentration.Western Blot results showed that compared with the control group,the relative expression of COL1A1 and RUNX2 proteins in Apelin-13(1,10 and 100nM)concentration groups were significantly increased(P<0.05).Compared with the control group,the relative expression levels of aβ-cat and tβ-cat in Apelin-13(1,10,100nM)concentration groups were significantly increased(P<0.05).Compared with the control group,the protein expressions of a β-cat,COL1A1 and Runx2 in Apelin-13(100nM)group were significantly increased(P<0.05),while the protein expressions of aβ-cat,COL1A1 and Runx2 in DKK1 group and DKK1+Apelin-13 group were significantly decreased(P<0.05).ConclusionApelin-13 can up-regulate COL1A1 and Runx2 expressions of rBMSCs,and may promote osteogenic differentiation of rBMSCs through Wnt/β-catenin signaling pathway.Part 3 To study the effects of cordycepin and Apelin13 on bone metabol ism of rBMSCsObjectiveCordycepin is one of the main active components of Chinese traditional medicine Cordycepin.Its anti-tumor,anti-virus,anti-oxidation and antiinflammatory properties have been reported.Oxidative stress can inhibit the osteogenesis of human bone marrow mesenchymal stem cells,and Cordycepin can protect the osteogenic inhibition induced by oxidative stress.Therefore,we speculated that Cordycepin may be a traditional Chinese medicine monomeric applied in the prevention and treatment of Osteonecrosis Of Femoral Head.The effect of Cordycepin on osteogenic differentiation of rBMSCs in vitro was studied.By detecting the expression of Cordycepin on osteogenic specific factors COL1A1,RUNX2 and Wnt/β-catenin signaling pathway inhibitor DKK1,the effect of Cordycepin and Apel in-13 on osteogenic differentiation was explored,so as to discover the potential value of cordycepin in prevention and treatment of ONFH.MethodsThe effect of Cordycepin and Apelin-13 on osteogenic differentiation and the interaction of Cordycepin and Apelin-13 on osteogenic differentiation were studied by co-culture of Cordycepin and Apelin-13 in rBMSCs medium.The effect of Cordycepin on rBMSCs proliferation was evaluated by CCK-8 assay at 4 h,24 h,72 h and 120 h.The morphology of rBMSCs cells after 9 days of osteogenic differentiation induced by Cordycepin co-culture with Apelin-13 was observed under light microscope,and the intracellular calcium nodules were observed by Alizarin poststaining.Cordycepin was co-cultured with Apelin-13(100nM)in rBMSCs medium,and the expression of osteogenic genes COL1A1 and RUNX2 of rBMSCs was detected by RT-PCR assay,and the expression of early osteogenic specific proteins COL1A1 and RUNX2 of rBMSCs,as well as protein DKK1 were detected by Western Blot assay.ResultsCCK-8 results showed that compared with the control group,Cordycepin(0.01,0.1,1,10,20 μg/mL)concentration groups at the 4 h,24 h,72 h,120 h had no significant difference on the proliferation activity of rBMSCs cells(P>0.05).RT-PCR results showed that compared with the control group and Apelin-13 group,the mRNA relative expressions of COL1A1 and RUNX2 in Cordycepin(0.1,1,10 μg/mL)+Apelin-13 co-culture group were significantly increased(P<0.05).Western Blot results showed that compared with the control group and Apelin-13 group,the relative expression of DKK1 protein in Cordycepin(1,10 μg/mL)+Apelin-13 co-culture groups was significantly decreased(P<0.05).Compared with the control group and Apelin-13 group,the relative expression of RUNX2 protein in Cordycepin(0.1,1,10 μg/mL)+Apelin-13 co-culture groups was significantly increased(P<0.05).Compared with the control group and Apelin-13 group,the relative expression level of COLIA1 protein in Cordycepin(1,10 μg/mL)+Apelin-13 co-culture groups was significantly increased(P<0.05).ConclusionCordycepin may play a bone-promoting role by inhibiting the expression of RUNX2 DKK1 and acting synergically with Apelin-13 through the Wnt/βcatenin pathway. |
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