The Effect Of Icariin On MiRNAs Expressionin Profile Of Rat Femoral Head Microvascular Endothelial Cells Against Steroids-induced Lesion

BackgroundThe early diagnosis of femoral head osteonecrosis of femoral head is difficult and the disability rate is high,which would affects the quality life of the patients.The incidence of steroid femoral head necrosis has risen to the leading of non-traumatic femoral head necrosis.Related research thinks,high-dose glucocorticoids application can impact something such as function protein,bone structure?the function of circulating endothelial cells and so on,which will cause ischemic necrosis of bone tissue.Studies have shown that icariin(ICA)has certain protective effects on endothelial cell.miRNA,as an important gene regulation factor,plays an important role in organism.Some studies have confirmed that miRNA plays an important role in the pathogenesis of femoral head necrosis.In the early stage,our team find the expression of miRNAs in microvascular endothelial cells of the femoral head can be significantly changed after using hormone.There is a certain regulation effect of icariin on partial imbalanced miRNAs.Therefore,we hypothesized that icariin may regulate the expression of related miRNA to interfere with the pathogenesis of steroid-induced femoral head necrosis.At this stage,our team use animal experiment to verify early views and conclusions,and to further explore the pathogenesis of glucocorticoid-induced ONFH,and the prevention and control mechanism of icariin.Objective1.To establish the model of steroid-induced ONFH and to evaluate the preventive effect of icariin on the occurrence of steroid-induced ONFH.2.Studies the effect of glucocorticoid on miRNAs expression profile of rat femoral head microvascular endothelial cells,and to explore miRNAs related to femoral head necrosis in the microvascular endothelial cells of the femoral head.Study on the effect of icariin on the mi-RNAs expressionin profile of rat femoral head microvascular endothelial cells against steroids-induced lesion,and to explore the target of icariin.Method1.30 SD rats were divided into 3 groups(contral group,model group and intervention group).Model group:intraperitoneal injection of two times lipopolysaccharide(20?g/kg),each time interval of 24 h,24 h later bilateral gluteal muscle alternating injection of methylprednisolone(40 mg/kg),continuous injection 3 times,each time interval of 24 h.Then using saline irritates the stomach for 4 weeks.Intervention group:the method of giving lipopolysaccharide and methylprednisolone is the same with model group,then using icariin(60mg/kg)irritates the stomach for 4 weeks.Blank control group:the same amount of physiological saline intraperitoneal injection,intramuscular injection and gavage were given at the same time point as the model group and intervention group.Once a week,weight changes were observed in rats,4 weeks later ELISA method was used to detect the rat plasma endothelin 1(ET-1),vascular endothelial growth factor(VEGF),blood clots regulatory proteins(TM),fibrinolytic enzyme activators inhibitor 1(PAI-1),th e original tissue fibrinolytic enzyme activation factor(t-PT),determination of nitric oxide(NO)content.The pathological HE staining was used to evaluate whether the model was successful and whether icariin had a preventive effect on the occurrence of steroid femoral head necrosis.2.Using mechanical shock,enzyme digestion and density gradient centrifugation to separate and cultivate microvascular endothelial cells.Immunofluorescence and flow cytometry were used to identify cell phenotype.Cell growth curve and tunnel experiment were used to evaluate cell function.3.15 SD rats were divided into 3 groups(contral group,model group and intervention group),5 in the blank group,5 in each group.The three groups of rats were treated with the method of experiment 1.After 4 weeks,2 rats were randomly selected from each group for pathological examination.Other rats were used to measure miRNA relative expression by high-througtput sequencing.Screen the different miRNA between the three groups.Bioinformatics analysis was performed on mirna-335.Results1.The incidence of femoral head necrosis in the model group was 90%,and 20%in intervention group(P=0.005).There was no significant difference about the weight in the three groups in the first two weeks.In the third week,the weight of the rats in the three groups was different.The weight of rats in contral group were the heaviest,followed by intervention group,and the weight of rats in model group were the lightest.At 4 weeks,the weight of control group and intervention group there was no obvious difference,which were heigher then model group.There was no significant difference about plasma ET-1,VEGF,TM and NO in three groups of rats.There was a statistically significant difference in plasma PAI-1 content in three groups,and icariin had the ability to down the level of PAI-1.t-PA level in the model group was significantly higher than other group,and there was no significant difference between control group and intervention group.2.CD31 and vWF are highly expressed in these cells,and CD133 is not expressed.The cells extracted by this method are of high purity and good function.3.Compared with model group,miR-132-3p and miR-335 were up regulated,miR-466-2-3p and let-7c-1-3p were down regulated in model group.mir-132-3p and mir-335 were regulated by icariin.The expression trend of mir-132-3p and mir-335 in control group,model group and intervention group wrer increase and then decrease..4.We used KEGG pathway enrichment analysis to identify the biological pathways controlled by miR-335,such as other types of O-glycan biosynthesis?apoptosis-multiple species Wnt signaling pathway?Calcium signaling pathway and so on.Target gene functions of miR-335 are as follows:tube formation?ruffle organization?cellular metabolic process and so on.Conclusion1.Icariin has the function to reduce the incidence of steroid-induced ONFH.2.Glucocorticoids may induce the occurrence of ONFH by regulating the expression of mir-335 in the microvascular endothelial cells of the femoral head.3.In the early stage of ONFH,the body may initiate itself repair mechanism by up-regulating the expression of mir-132-3p in the microvascular endothelial cells of the femoral head.4.Icariin may interfere with the occurrence of steroid-induced ONFH by regulating the expression of mir-335 in the microvascular endothelial cells of the femoral head.