Orthopedics Department, Second Affiliated Hospital of Harbin Medical University, Harbin, China.
Pediatric Orthopedics Department, Second Affiliated Hospital of Inner Mongolia Medical University, Inner Mongolia, China.
Orthop Surg. 2020 Apr;12(2):645-652. doi: 10.1111/os.12630. Epub 2020 Feb 16.
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).
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.
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.
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.
研究初级纤毛形成障碍和成骨细胞自噬在激素性股骨头坏死（SANFH）发病机制中的作用。
从兔骨中分离出成骨细胞，用 1μM 甲基强的松龙处理 0、12、24、48 和 72 h。通过 PCR 和 Western blot 检测 Beclin1、MAP1LC3、Atg-5、Atg-12、IFT20 和 OFD1 mRNA 和蛋白的表达，并进行统计学分析。通过激光共聚焦显微镜测量成骨细胞纤毛的长度，并通过 GFP-RFP-LC3 慢病毒转染追踪自噬流。
甲基强的松龙呈时间依赖性显著上调 Beclin1、MAP1LC3、Atg-5、Atg-12 和 OFD1 mRNA 和蛋白的表达，同时下调 IFT20 的表达（P＜0.05）。此外，甲基强的松龙处理后，成骨细胞的自噬流也呈时间依赖性增加，纤毛长度逐渐变短。0 h 时纤毛长度为 5.46±0.11 μm，12 h 时为 4.08±0.09 μm，24 h 时为 3.07±0.07 μm，48 h 时为 2.31±0.10 μm，72 h 时最终为 1.15±0.04 μm。甲基强的松龙处理对培养物中初级纤毛数量也有影响，0-72 h 时纤毛数量逐渐减少。自噬调节基因 Beclin1、MAP1LC3、Atg-5 和 Atg-12 与 IFT20 呈负相关，平均相关系数为-0.81。OFD1 与 IFT20 也呈负相关，平均相关系数为-0.53。
甲基强的松龙抑制初级纤毛形成并促进自噬，这可能是 SANFH 的病理基础。确切的调节机制需要进一步在体内研究。
