Efficacy Of Icariin On Intervention Of Steroid-induced Osteonecrosis Of The Femoral Head Via Regulating MiR-23b:an In Vivo Study

BackgroundThe prevention and intervention for steroid-induced osteonecrosis of the femoral head(SONFH)have presented a great clinical cost-effectiveness.Icariin(ICA)could play a role in regulating the expression of microRNAs like miR-23b,impacting on how endothelial cells work and intervening the progression of SONFH.Impairment of bone microvascular endothelial cells(BMECs)by glucocorticoid is a key cause for SONFH.ICA could improve vascular roles and relieve the dysfunction of BMECs.miR-23b is involved in the process of angiogenesis and apoptosis of endothelial cells.ObjectiveA serial of rat models of SONFH were constructed to evaluate whether ICA could prevent and treat SONFH by improving blood circulation in vivo.The purpose of this study is to investigate the molecular mechanism by which ICA and miR-23b intervene the progression of SONFH in rat models,and to find out the target mRNA of miR-23b.Methods1.Seventy eight-week-old SD rats,were randomly assigned into the following seven groups:A)SONFH+miR-23b group,B)SONFH+miR-23b sponge group,C)SONFH+ICA group,D)SONFH+ICA+miR-23b group,E)SONFH+ICA+miR-23b sponge group,F)miR-23b NC group,and G)Control group,with ten rats in each group.At 6 weeks from the final injection of methylprednisolone,all rats were killed.Femoral heads and blood were collected,then Micro-CT,Haematoxylin and eosin staining(HE),Immunohistochemical staining(ISH),RT-qPCR and Flow cytometry were used to assess the efficacy of ICA on SONFH in rat models.2.Thirty eight-week-old SD rats,were randomly assigned into the following three groups:A)Control group,B)SONFH group,and C)SONFH+ICA group,with ten rats in each group.BMECs were isolated and cultured from the femoral head issue and identified by immunofluorescence staining.CCK-8 assay was used to check the cell proliferation.BMECs from the SONFH group and the SONFH+ICA group were stimulated with Ad-miR-23b or Ad-miR-23b sponge respectively.BMECs from the Control group were stimulated with FBS or Ad-miR-23b NC respectively.Then Western blot was used to determine the alterations in their corresponding proteins such as SEMA6A,VEGF,PI3K,Bax,Bcl-2,Sprouty2,RAF,MEK,ERK.Luciferase reporter was used to detect the direct target mRNA of miR-23b.Results1.All SD rats survived during the experiment.Micro-CT showed that the subchondral bone density of the femoral head of rats in the SONFH+miR-23b sponge group was lowest,the thickness was so,the bony microstructure was impaired,bone density was significantly decreased especially when compared with SONFH+miR-23b group and SONFH+ICA+miR-23b group.HE staining showed that compared with SONFH+miR-23b sponge group,there were less empty lacunae and necrotic bone marrow cells in the SONFH+miR-23b group and SONFH+ICA+miR-23b group,no empty lacunae and necrotic bone marrow cells in the miR-23b NC group and Control group.ICH staining showed that the miR-23b NC group or the Control group had the most CD31+cells,while the SONFH+miR-23b sponge group had no vascular structure and the least CD31+cells.RT-qPCR showed ICA may upregulate the expression of miR-23b,then suppress the expressions of SEMA6A and Sprouty2 mRNA,consequently resulting in downregulation of VEGF mRNA.Flow cytometry showed ICA may elevate the potential of immune system in rat model of SONFH.2.The isolated cells presented the typical endothelial cells morphology.CD31and vWF were highly expessed by immunofluorescence.CCK-8 results showed that three groups of BMECs had no significant difference in ability of proliferation.Western blot showed that ICA and miR-23b suppressed SEMA6A and Sprouty2 expressions,and promoted VEGF,PI3K expression and ERK,MEK expression respectively.On top of that,ICA and miR-23b enhanced Bcl-2 and diminished Bax expression.Luciferase reporter assay showed SEMA6A may be the target mRNA of miR-23b.ConclusionIn vivo,we found ICA can upregulate the expression of miR-23b,and intervene the progress of SONFH in the rat model and improve the blood supply of femoral head.ICA may provide a rise in the proportion of assited T cells and an improvement in the immune system.ICA and miR-23b may have an impact on SEMA6A,VEGF,PI3k,and Sprouty2 proteins involving in the process of angiogenesis and apoptosis in translation level.ICA and miR-23b may play a key role in activating the cell signaling pathways of AKT/PI3K,MAPK/ERK and apoptosis.ICA can upregulate of the expression of miR-23b in BMECs deriving from rat model of SONFH,then curb the expression of SEMA6A that can suppress the VEGF and PI3K,downregulate the expression of Sprouty2 that can suppress the ERK and MEK.SEMA6A may be the target mRNA of miR-23b.