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 AE 0.11 um at 0 h, 4.08 AE 0.09 um at 12 h, 3.07 AE 0.07 um at 24 h, 2.31 AE 0.10 um at 48 h, and finally 1.15 AE 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.

Lithium prevents glucocorticoid‐induced osteonecrosis of the femoral head by regulating autophagy

“…In addition, Zhao et al reported that the autophagy flux in the rabbit osteoblasts increased and the ciliary length decreased in a timedependent manner after methylprednisolone treatment, so they speculated methylprednisolone inhibited primary cilia formation and promoted autophagy, which could be the pathological basis of GC-ONFH. 31 PENG et al's study also showed that the level of autophagy marker proteins was also significantly increased during the process of rat osteoblasts apoptosis induced by dexamethasone, while autophagy inhibitor 3-MA attenuated dexamethasonemediated apoptosis. 32 Both cell and animal experiments in this study demonstrated that lithium, as an autophagy regulator and osteogenic activator, can inhibit the excessive autophagy of osteoblasts induced by GC, while retaining the osteogenic activity of osteoblasts, and achieve the purpose of treating GC-ONFH in rats.…”

“… 11 In addition, Zhao et al reported that the autophagy flux in the rabbit osteoblasts increased and the ciliary length decreased in a time‐dependent manner after methylprednisolone treatment, so they speculated methylprednisolone inhibited primary cilia formation and promoted autophagy, which could be the pathological basis of GC‐ONFH. 31 PENG et al. 's study also showed that the level of autophagy marker proteins was also significantly increased during the process of rat osteoblasts apoptosis induced by dexamethasone, while autophagy inhibitor 3‐MA attenuated dexamethasone‐mediated apoptosis.…”

“…First, this study and some previously mentioned studies suggest that over‐activated autophagy may be the inducement of GC‐ONFH. 11 , 30 , 31 , 32 In contrast, some studies suggest that autophagy is a GC‐induced self‐protection mechanism in cells. Studies have shown that GC‐ONFH may be related to osteoblast apoptosis, and increasing the autophagy level of osteoblasts can prevent GC‐induced osteoblast apoptosis.…”

Effect and mechanism of propranolol on promoting osteogenic differentiation and early implant osseointegration

“…OBs isolation and culture. Isolation and culture of OBs were performed as described by Zhao et al (20). New Zealand rabbits were anesthetized by intravenous injection of pentobarbital sodium.…”

ACP5, TNF, and MMP8 were identified as potential biomarkers of steroid-induced osteonecrosis of the femoral head

“…A recent study demonstrated that the osteoclastic-related genes OPG and RANML were abnormally expressed in SONFH, and therefore, they are potential diagnostic markers [6] . However, other processes, including coagulopathy [7] , dysregulated apoptosis [8] , and disordered lipid metabolism [9] were also crucial for SONFH. In addition, inflammatory pathways such as the PDK1/AKT/mTOR signaling pathway and the PERK and Parkin pathways [10,11] are utilized for the function of dysregulated genes in these function pathways might act as potential biomarkers of SONFH.…”