[Effects of icariin on autophagy and exosome production of bone microvascular endothelial cells].

OBJECTIVE: To evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).

METHODS: BMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10 -5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β 1 (TGF-β 1 ), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.

RESULTS: With the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually ( P <0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin ( P <0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group ( t =-10.191, P =0.001); and there was no significant difference in exosome diameter and total protein content between the two groups ( P >0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β 1 /CD9 proteins in the intervention group were significantly higher than those in the non-intervention group ( P <0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups ( P <0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group ( P <0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group ( P <0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group ( P <0.05); the 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: Icariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.