Mechanism of Methylprednisolone‐Induced Primary Cilia Formation Disorder and Autophagy in Osteoblasts

Abstract

Objective To study the role of primary cilia formation disorder and osteoblasts autophagy in the pathogenesis of steroid‐induced avascular necrosis of the femoral head (SANFH). Methods Osteoblasts were isolated from rabbit bones and treated with 1 μM Methylprednisolone for 0, 12, 24, 48, and 72 h. The Beclin1, MAP1LC3, Atg‐5, Atg‐12, IFT20 and OFD1 mRNAs and proteins were detected by PCR and Western blotting, and their correlation was statistically analyzed. The lengths of osteoblast cilia were measured under a laser confocal microscope, and the autophagy flux was tracked by transfecting the osteoblasts with GFP‐RFP‐LC3 lentivirus. Results Methylprednisolone significantly upregulated Beclin1, MAP1LC3, Atg‐5, Atg‐12 and OFD1 mRNAs and proteins in a time‐dependent manner, and decreased that of IFT20 (P < 0.05). In addition, the autophagy flux in the osteoblasts also increased and the ciliary length decreased in a time‐dependent manner after Methylprednisolone treatment. The length of the cilia were 5.46 ± 0.11 um at 0 h, 4.08 ± 0.09 um at 12 h, 3.07 ± 0.07 um at 24 h, 2.31 ± 0.10 um at 48 h, and finally 1.15 ± 0.04 um at 72 h. Methylprednisolone treatment also affects primary cilium numbers in cultures, for 0 to 72 h. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg‐5 and Atg‐12, were found to be negatively correlated with IFT20, with an average correlation coefficient of −0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of −0.53. Conclusion Methylprednisolone inhibits primary cilia formation and promotes autophagy, which could be the pathological basis of SANFH. The exact regulatory mechanism needs to be further studied in vivo.

Keywords

• Autophagy
• Methylprednisolone
• Osteoblasts
• Osteonecrosis
• Primary cilia