Hormone-induced Femoral Head BMEC Injury, Exosomes, MicroRNA Expression, And The Intervention Effects Of Icariin And Shock Waves

Part ?:Effect of icariin on exosomes secreted by microvascular endothelial cells of the femoral headObjective:The use of glucocorticoid can directly damage bone microvascular endothelial cells(BMECs),thereby promoting the development of non-traumatic osteonecrosis of the femoral head and iatrogenic osteoporosis.Exosomes,as carriers of intercellular communication,play an important role in normal physiological functions of the body and the development of various diseases through the microRNAs and proteins they carry.This study was designed to evaluate the effect of icariin on glucocorticoid-induced damage of BMECs from the femoral head and to explore its effects on secreted exosomes.Methods:Patients receiving total hip arthroplasty due to femoral neck fracture,primary osteoarthritis of the hip or osteoarthritis secondary to development dysplasia of the hip were enrolled.BMECs were isolated from cancellous bone by enzyme digestion and magnetic activated cell sorting,and identified by immunofluorescent staining.Glucocorticoid(0.3 mg/mL)and different concentrations of icariin(0 M,1×10-6M,1×10-5M,5×10-5M and 1×10-4 M)were administrated and CCK-8 method was used to detect cell viability.Subsequently,the cells were divided into the icariin intervention group and the non-intervention group,After intervention,exosomes from different groups were isolated.The diameter and quantity of exosomes were evaluated by nanoparticle tracking analysis technique(NTA),and protein content was detected by BCA method.Expression of protein in exosomes was appraised by Western blot and differentially-expressed miRNAs by high-throughput sequencing and qPCR.Results:Isolated cells showed a high positive rate of CD31 and vWF,and manifested fusiform or polygonal shape and cobblestone-like appearance under inverted phase contrast microscope,indicating that isolated cells were indeed bone microvascular endothelial cells.Icariin can prevent the inhibitory effect of glucocorticoid on viability of BMECs.Cell viabilities in groups with a icariin concentration of being 0 M,1×10-6M,1×10-5M,5×10-5M and 1×10-4M were 103.96%±2.15%,107.16%±1.28%,110.15%±1.01%,129.72%±0.86%and 131.87%±2.13%,respectively;there were significant differences among them(F=207.467,p<0.001).The average diameters of exosomes in the non-intervention group and the intervention group were(86.68±1.97)nm and(85.35±1.65)nm,respectively,and the protein concentrations were(132.63±3.33)?g/mL and(138.56±9.58)?g/mL,respectively;there was no significant difference between them(p>0.05).The exosome concentrations in the two groups were(9.85±0.47)×106/mL and(1.54±0.08)×107/mL,respectively;the difference was statistically significant(t=-10.1915,p=0.001).The ratios of VEGFA/CD9 and TGF-?1/CD9 in exosomes of the intervention group were significantly higher than those of the non-intervention group,and the differences were statistically significant(p<0.01).At the same time,icariin intervention could up-regulate or down-regulate expression levels of various microRNAs(such as miR-1469,miR-133a-5p and miR-10b-5p)in exosomes.Conclusion:Icariin is able to alleviate glucocorticoid-induced endothelial cell damage in a dose-dependent and dose-saturated manner.Although icariin cannot change the morphology and size of exosomes secreted by bone microvascular endothelial cells,it can significantly increase the concentration of exosomes.At the same time,icariin cannot change the total protein content in exosomes,but can change its protein composition and increase the content of VEGFA and TGF-?1.Icariin intervention can alter expression of a variety of miRNAs associated with cell proliferation,migration,and apoptosis in exosomes.Part 2:Effects of endothelial cell-derived exosomes on glucocorticoid-induced injury and autophagy in microvascular endothelial cell of the femoral headObjective:To investigate the effects of endothelial cell-derived exosomes on glucocorticoid-induced damage and autophagy in bone microvascular endothelial cells of the femoral head,and whether pre-treatment of icariin can affect the effects of exosomes.Methods:Microvascular endothelial cells were isolated from femoral heads excised after total hip arthroplasty and divided into two groups:the blank control group and the icariin intervention group.Then exosomes were extracted from two groups,respectively and co-cultured with bone microvascular endothelial cells,which were set as the experimental group and the control group;another group of BMECs without intervention of exosomes was set as the blank control group.Cell viability was determined by CCK-8 assay,apoptosis was detected by flow cytometry,cell migration was detected by scratch assay,and neovascularization was detected by tube formation assay.Finally,Western blot was used to detect expression of proteins related to cellul autophagy,apoptosis and proliferation.Results:The apoptotic rates of BMECs in the blank control group,the control group and the experimental group were 35.67±1.99%,16.47±1.06%and 10.70±0.7%,respectively;the difference between three groups was statistically significant(F=512.61,p<0.001).After 1,2,3,and 4 days of treatment with hydrocortisone,the viabilities of BMECs co-cultured with exosomes was significantly higher than those of the blank control group,'and the cell viabilities in the experimental group was higher than that of the control group;the differences were statistically significant(p<0.01).At the same time,exosome intervention can increase the expression levels of pERK,eNOS and COX-2,and reduce the expression levels of cleaved caspase-3 and CDKN2C.In the blank control group,the control group and the experimental group,the expression of p62 protein increased,and the expression of LC3B-II protein showed a decreasing trend.The difference between any two of three groups was statistically significant(p<0.05).At 12h and 24h after hydrocortisone treatment,cell migration abilities of the control group and the experimental group were significantly higher than that of the blank control group,and the experimental group was significantly higher than the control group;the difference of the scratch closure rate of any two of three groups was statistically significant(p<0.05).When treated with hydrocortisone for 4 and 8 h,the number of lumens,number of sprouting vessels and length of tubule branches in the experimental group and the control group were significantly higher than those in the blank control group(p<0.05);length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group(p<0.05).Conclusion:Exosomes secreted by microvascular endothelial cells of the femoral head can improve apoptosis of BMECs induced by high concentration of glucocorticoid,reduce the autophagy of BMECs induced by low concentration of glucocorticoid,and promote endothelial cell migration and angiogenesis.Icariin pretreatment can enhance these effects of exosomes and act as a promising option for the treatment of glucocorticoid-induced bone disease.Part 3:Effects of extracorporeal shockwave on microRNA expression in microvascular endothelial cells from patients with glucocorticoid-induced osteonecrosis of the femoral headObjective:Injury and apoptosis of bone microvascular endothelial cells(BMECs)participates in the pathogenesis of glucocorticoid-induced osteonecrosis of the femoral head(ONFH),with extracorporeal shockwave(SW)being a non-invasive technique capable of altering this process.The aim of this study is to elucidate the impact of SW on microRNA(miRNA)expression profile of BMECs derived from patients with glucocorticoid-induced ONFH.Method:BMECs were isolated from excised femoral heads of patients with glucocorticoid-induced ONFH and then the 3rd generation of BMECs were divided into the intervention group and the control group:in the former group,these cells were exposed to focused extracorporeal shockwave(0.11mJ/mm2,400 times)and in the latter group cells left untreated served as control.At 24h after treatment,miRNAs expression profiles were analyzed using the Illumina HiSeq2500 platform.Differentially-expressed miRNAs were identified,and their targeted genes and involved signaling pathways were predicted and appraised by bioinformatics analysis such as Gene Oncology and Pathway assay.Results:Three patients were included in this investigation.Cells from same individuals are divided into the treatment group 1(T1)and the control group 1(C1),T2 and C2 as well as T3 and C3.153 miRNAs showed differential expression in the comparison between T1 and C1 groups,218 miRNAs showed differential expression in the comparison between T2 and C2 groups,and 135 miRNAs showed differential expression in the comparison between T3 and C3 groups.After combining these data,a total of 54 differentially expressed miRNAs(41 up-regulated ones and 13 down-regulated ones)in bone microvascular endothelial cells were identified in more than one pairwise comparison after the shockwave therapy,among which 13 miRNAs were reported to be involved in the function regulation of endothelial cells.Only one of them was confirmed to be upregulated in all three pairwise comparisons.Conclusions:Extracorporeal shockwave-induced alteration of miRNAs in bone microvascular endothelial cells may play an important role in the treatment of glucocorticoid-induced osteonecrosis of the femoral head,but there are significant differences among different individuals.The exact mechanism of shock wave therapy for the intervention of bone microvascular endothelial cells of the femoral head remains to be determined.