Experimental Study Of Icariin On The Prevention And Treatment Of Glucocorticoid-Induced Osteonecrosis Of The Femoral Head By Interfering With Bone Microvascular Endothelial Cells

Part ?Study on the function changes of bone microvascular endothelial cells in patients with glucocorticoid-induced osteonecrosis of the femoral head Objective:Femoral head microcirculation obstacle caused by a dysfunction of the bone microvascular endothelial cells(BMECs)could be significant in the development of glucocorticoid-induced osteonecrosis of the femoral head(ONFH).This study investigated the differences of the angiogenic and apoptotic activities of BMECs isolated from patients with glucocorticoid-induced ONFH and femoral neck fractures.Methods:This study enrolled a total of 12 patients,six of whom were assigned to the ONFH group whereas the other six served as the control group.The ONFH group was composed of patients with glucocorticoid-induced ONFH while the control group had femoral neck fractures.The ONFH group included 3 females and 3 males(mean age 51.7± 5.2 years),while the control group included 5 females and 1 male(mean age 65.3±2.9 years).BMECs were isolated from the subchondral region of the femoral head in the both groups and identified by immunofluorescent staining.Cell proliferation,cell viability,tube formation assay,Transwell assay,TUNEL assay,and Western blot analysis were performed.Results:The cells in the both groups developed the typical cobblestone morphology of endothelial cells.The isolated cells of both groups showed the expression of CD31 and vWF.These results indicated that these cells were BMECs.No significant differences were noticed in BMECs proliferation between the two groups.However,compared to the control,cell viability,tube formation,and migration of BMECs were significantly decreased and the number of TUNEL positive cells was markedly increased in the ONFH group.In the ONFH group,it was also noted that the amount of Bax and cleaved-caspase3 was elevated while that of Bcl-2 was reduced.Conclusion:The findings of our study revealed that BMECs obtained from the glucocorticoid-induced ONFH patients had decreased angiogenic and increased apoptotic activities,which could explain the pathogenesis and progression of glucocorticoid-induced ONFH.Part ?Effects of Icariin on bone microvascular endothelial cells in the glucocorticoid-induced conditionObjective:Glucocorticoid is widely used in the treatment of autoimmune and inflammatory diseases,which is the most common cause of osteonecrosis of the femoral head(ONFH).The dysfunction of the bone microvascular endothelial cells(BMECs)is closely related with the pathogenesis of glucorticoid-induced ONFH.Reports indicate that icariin(ICA)can enhance vascular roles and also inhibit endothelial cell dysfunction.However,whether ICA can promote angiogenesis glucocorticoids-induced BMECs is not clear.In this experiment,we explored this hypothesis in vitro experiments.Methods:BMECs were isolated and cultured from the subchondral region of the femoral head and identified by immunofluorescence staining.CCK-8 assay was applied to check the cell proliferation.Wound-healing assay,transwell asssy test and tube formation assay were used to determine the angiogenic capacity in vitro.RT-qPCR was used to detect the mRNA expression of VEGF,CD31,vWF,and PDGF-B.Western blot was used to detect the protein expressions of VEGF,Akt,p-Akt,Bax and Bcl-2.Light microscopy showed that the isolated cells formed a typical cobblestone-like endothelial cell morphology,Results:The isolated cells developed the typical cobblestone-like endothelial cells morphology.CD31 and vWF were highly expressed by immunofluorescence,which indicated that isolated cells were BMECs.These results indicated that the cells were BMECs.CCK-8 results showed that 0.1 mg/mL hydrocortisone significantly suppressed BMECs proliferation while ICA at 10-5 M reversed this inhibition.RT-qPCR results observed that hydrocortisone obviously restrained the mRNA expression of CD31,while ICA significantly increased the mRNA expression of CD31.ICA obviously promoted BMECs migration and tube formation.Western blot showed that ICA obviously promoted VEGF expression and the activation of Akt.Furthermore,ICA enhanced Bcl-2 expression but diminished Bax expression.Conclusion:The findings of our study revealed that ICA can reduce the BMECs damage caused by hydrocortisone,improve the cell activity,migration ability,and angiogenesis ability and significantly promote BMECs angiogenesis in vitro experiment.Part ?Prevention and treatment of Icariin on ealy stage glucocorticoid-induced necrosis of femoral head in ratsObjective:It is of great clinical significance to study the prevention and treatment of glucocorticoid-induced osteonecrosis of the femoral head(ONFH).Icariin(ICA)could improve the cell activity,migration ability,and angiogenesis ability,reduce damages and promotes angiogenesis of BMECs in the glucocorticoid-induced condition.In order to further study whether ICA can prevent and treat hormonal ONFH by protecting blood circulation in vivo,we constructed glucocorticoid-induced ONFH in a rat model to evaluate.Methods:Thirty SD rats,eight-week-old and healthy,were randomly assigned into the following three groups:the control group,the Methylprednisolone(MP)group,and the MP+ICA group,with ten rats in each group.Rats from the MP group and MP+ICA group were used to establish the glucocorticoid-induced ONFH animal model by the sequential drug administration of lipopolysaccharide and MP.Rats in MP+ICA group were administrated with 60 mg/kg ICA by gavage every day,and rats in the control group and MP group were administrated with normal saline of the same dose of ICA by gavage.All rats were killed after 6 weeks,and the therapeutic effect of ICA was evaluated from gross specimens,Hematoxylin-eosin staining(HE),MicroCT,immunohistochemistry and femoral head angiography.Results:All 30 SD rats survived during the experiment.HE staining showed that compared with MP+ICA group,there were more empty lacunae and necrotic bone marrow cells in the MP group,but no empty lacunae and necrotic bone marrow cells in the control group.The incidence of ONFH in the control group,MP group,and MP+ICA group was 0(0/10),20%(2/10),and 80%(8/10),respectively.MicroCT results showed that the subchondral bone density of the femoral head of rats in the MP group was lower,the thickness was thinner,the bone microstructure was destroyed,and the bone density was significantly reduced compared with the MP+ICA group.The subchondral bone of the femoral head of rats in the control group was intact,evenly distributed,and the bone structure was not destroyed.Immunohistochemistry showed that the control group had the most CD31-positive cells,while the MP group had no intact vascular structure and the least CD31-positive cells.In the MP+ICA group,the complete vascular structure was found and the number of CD31-positive cells was significantly increased compared with the MP group.Femoral head angiography showed that the femoral head blood vessels in the control group were rich,while the femoral head blood vessels were seriously ruined and the number was significantly reduced in the MP group.Compared with the control group,the number of femoral head blood vessels in the MP+ ICA group was reduced,but much more than that of the MP the group.Conclusion:In vivo animal experiments,we found that ICA can delay the development of glucocorticoid-induced ONFH in the rat and protect the blood supply of the femoral head.This provides a new cognition for ICA to prevent and treat glucocorticoid-induced ONFH in the rat.