| BackgroundSteroid-induced avascular necrosis of femoral head is a common type of refractory hip orthopedic diseases, which has been gradually to the first place of traumatic avascular necrosis, but its exact etiology and pathogenesis is unclear. Femoral head microcirculation dysfunction caused by the injury of bone microvascular endothelial cell induced by Hormones and their metabolites may be the important reasons in occurrence and development of avascular necrosis of the femoral head. Long-term and high-dose application of hormone can affect the protein and gene expression of bone microvascular endothelial cells, which cause microvascular endothelial cell apoptosis, new blood vessels form, vasomotor dysfunction and abnormal function of blood coagulation and fibrinolysis, resulting in blood circulation dysfunction within the femoral head, finally give rise to bone tissue necrotic. Previous studies suggested that icariin can promote angiogenesis, regulating vasomotor and protecting vascular endothelial cells from oxidative stress damage and other pharmacological effects, and our laboratory pilot study cellular level also suggested that icariin can protect the injury of bone microvascular endothelial cell induced by glucocorticosteroid, but specific molecular mechanism remains to be further research.ObjectiveThis topic research includes two parts, the first one attempt to explore the effect of icariin on the proteomic expression profile, apoptosis and migration ability of bone microvascular endothelial cells of human femoral head against steroids-induced lesion using protein chip system in vitro. The second one, the model of BMECs injury induced by glucocorticoid and icariin preconditioning was established, and then explore the effect of icariin on mi-RNAs expression of bone microvascular endothelial cells of human femoral head against steroids-induced lesion in vitro. Look for possible role of mi-RNAs molecular targets after icariin preconditioning; determine the function of the significantly differentially mi-RNAs expression.MethodThe first part of the research content, bone micro vascular endothelial cells in cancellous bone of femoral head were isolated and harvested in vitro. The model of BMECs injury induced by glucocorticoid and icariin preconditioning was established. The apoptosis of BMECs were detected with TUNEL methods. The migrations of BMECs were detected with scratch assay. The proteomic expression profile of each group BMECs were detected by protein chip system.The second part of the research content, bone microvascular endothelial cells in cancellous bone of femoral head were isolated and harvested in vitro. The model of BMECs injury induced by glucocorticoid and icariin preconditioning was established. Their differential expression profiles and transcriptomes were tested by micro-RNAs microarrays. The most differentially expressed mi-RNAs in microarray analysis were further confirmed by real-time quantitative PCR; meanwhile detect the effect of icariin on the expression of the significantly differentially mi-RNAs expression. Finally the possible target genes and signaling pathways were predicted using bioinformatics software.ResultThe first part of the research content, The apoptosis rate of BMECs in hydrocortisone groups (46.93±5.8%,78.03±8.5%) significantly higher than normal group (4.80±1.3%), meanwhile the apoptosis rate of BMECs in icariin and hydrocortisone groups (15.17±7.7%,44.57±5.3%) significantly decreased compared with hydrocortisone group (P< 0.001). The migration distance of BMECs in hydrocortisone groups (13.24±0.32) significantly higher than normal group(5.28±0.31), meanwhile the migration distance of BMECs in icariin and hydrocortisone groups (6.12±0.15) significantly decreased compared with hydrocortisone group (P<0.05). The results of protein chip showed that the expression of G-CSF in the hydrocortisone group decreased compared with the control group, meanwhile the expression of IL-4 increased. The expression of G-CSF in the icariin and hydrocortisone groups increased compared with the hydrocortisone group, meanwhile the expression of IL-4 decreased. No appreciable change was found in the expression of apoptotic factors.The second part of the research content, According to the microarray analysis, one mi-RNA (miR-339) were up-regulated, four mi-RNAs (miR-23、miR-100、miR-22 and miR-933) were down-regulated in glucocorticoid group(Fold> 2, P< 0.05), Results of RT-qPCR revealed that miR-23b was down-regulated and miR-339 was up-regulated in glucocorticoid group, which were in agreement with the microarray analysis (P< 0.05). Icariin pretreatment can effectively prevent the imbalances of miR-23b expression induced by glucocorticoid (P< 0.01). The prediction of the possible target genes and signaling pathways shows that there were 23 possible target genes, including ADAMTSL4, STAT5A, TBX15, PRICKLE2, NRCAM, PPAR-y, STON1, TCEAL4, GPHN, CISH, CLCN2, MAN1C1, SMIM3, ST3GAL6, SMPDL3A, FAM117B, Sprouty2, Sema6A, Sema6D, Apaf-1, TNFAIP1, TNFAIP2 TNFAIP3 and 9 possible signaling pathways, including JAK-Stat5 signaling pathways, Notch signaling pathways, Wnt/JNK signaling pathways, RPTPβ signaling pathways, PPARγ signaling pathways, PI3K/Akt/mTOR signaling pathways, ERK1/2 signaling pathways, RTK signaling pathways, MAPK signaling pathways. The function of the possible target genes and signaling pathways is regulation of cell proliferation, cell differentiation cell apoptosis, and formation of blood vessels, participation in immune regulation and Mediation to inflammatory response.Conclusion1. Icariin can significantly inhibit the apoptosis of bone microvascular endothelial cell induced by glucocorticoid, keep migration ability of bone microvascular endothelial cell induced by glucocorticoid, and improve the expression of pro-angiogenic factor G-CSF, meanwhile suppress the expression of inflammatory cytokines IL-4. The therapy effect of icariin to steroid-induced necrosis of femoral head might be related with the protective action to BMECs and the moderating effect on the proteins expression of pro-angiogenesis factor and inflammatory factor.2. Steroids-induced significant changes in transcripts of bone microvascular endothelial cells such as miR-100, miR-222, miR-933, miR-339 and miR-23, however icariin can effectively prevent hormone-induced bone microvascular endothelial cell injury caused by the imbalances of miR-23b expression. Icariin may participate in the pathological process of steroid-induced femoral head necrosis through regulating the expression of miR-23b. Thus we speculate that icariin may regulate the expression of miR-23b to suppress the apoptosis and angiogenesis related target mRNA gene expression, which can influence the expression the apoptosis or angiogenesis related proteins, finally participate in the pathological process of steroid-induced femoral head necrosis. The therapeutic effect of miR-23b mediated may be the new sight to the treatment of steroid-induced avascular necrosis of femoral head, which deserves the further investigation. |
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