Graduate School of Inner Mongolia Medical University, Hohhot, China.
Department of Bone Tumor, The Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China.
Orthop Surg. 2022 Oct;14(10):2669-2681. doi: 10.1111/os.13369. Epub 2022 Sep 2.
To study the relationship between vascular endothelial cells (VEC) and autophagy, and its regulatory mechanism in steroid-induced avascular necrosis of the femoral head (SANFH).
In cell experiment, VEC were isolated and cultured from the femoral head of Sprague-Dawley rats and divided into three groups: blank control group (Ctrl), methylprednisolone group (MP), and methylprednisolone+mTOR-shRNA group (MP + shmTOR). The autophagy formation was observed by transmission electron microscope. The mRNA expression of PI3K, Akt, mTOR, Beclin1 and MAP1LC3 was detected by RT-PCR and the protein expression was detected by Western blot and immunofluorescence. Expression of the damage marker 6-keto-PGF1α was detected by the ELISA method. In vivo experiment, after establishing the model, the grouping method was the same as cell experiment. Autophagosomes were observed by same method, and the expression of related factors was detected by the same method in cell experiment.
In the cell experiment, autophagosomes in the MP group were significantly lower than in the Ctrl group, and the autophagosomes in the MP + shmTOR group were intermediate between two groups (P < 0.05). The mRNA expression levels of PI3K, Akt and mTOR in the MP group were significantly higher than in the Ctrl group, while the MP+ shmTOR group presented intermediate levels between these groups (average gray value were 3837.90, 2996.30, 3005.60, F = 428.64, P < 0.05). MRNA expression levels of Beclin1 and MAP1LC3 in the MP group were significantly lower than that in Ctrl group (P < 0.05). The content of 6-keto-PGF1α in the MP + shmTOR group was higher than in the Ctrl group and lower than in the MP group at the evaluated time intervals (average absorbance value were 104.98, 206.83, 145.91, F = 352.83, P < 0.01). In vivo experiment, the content of 6-Keto-PGF1α in the hormone group increased as time went on; the mTOR-si group was higher than that in control group, but lower than that in the hormone group (P < 0.01). The mRNA expressions of Beclin1 and MAP1LC3 in the control group were higher than those in the hormone group, while the mRNA expressions of PI3K, Akt and mTOR were lower than those in the mTOR-si group (P < 0.05).
The steroid inhibited the physiological protective effect of autophagy on SANFH by increasing the expression of PI3K/Akt/mTOR signaling pathway related factors and decreasing the expression of Beclin1 and MAP1LC3 in the femoral head VEC.
研究血管内皮细胞（VEC）与自噬之间的关系及其在激素诱导性股骨头坏死（SANFH）中的调控机制。
在细胞实验中，从 SD 大鼠股骨头中分离和培养 VEC，并将其分为三组：空白对照组（Ctrl）、甲泼尼龙组（MP）和甲泼尼龙+mTOR-shRNA 组（MP+shmTOR）。通过透射电镜观察自噬体的形成。采用 RT-PCR 检测 PI3K、Akt、mTOR、Beclin1 和 MAP1LC3 的 mRNA 表达，采用 Western blot 和免疫荧光法检测蛋白表达。采用 ELISA 法检测损伤标志物 6-酮-PGF1α 的表达。在体内实验中，建立模型后，分组方法与细胞实验相同。采用相同方法观察自噬体，采用相同方法检测细胞实验中相关因子的表达。
在细胞实验中，MP 组的自噬体明显低于 Ctrl 组，而 MP+shmTOR 组的自噬体处于两组之间（P<0.05）。MP 组的 PI3K、Akt 和 mTOR 的 mRNA 表达水平明显高于 Ctrl 组，而 MP+shmTOR 组的表达水平处于两组之间（平均灰度值分别为 3837.90、2996.30、3005.60，F=428.64，P<0.05）。MP 组的 Beclin1 和 MAP1LC3 的 mRNA 表达水平明显低于 Ctrl 组（P<0.05）。MP+shmTOR 组的 6-酮-PGF1α 含量在各时间点均高于 Ctrl 组，低于 MP 组（平均吸光度值分别为 104.98、206.83、145.91，F=352.83，P<0.01）。在体内实验中，激素组的 6-Keto-PGF1α 含量随时间的推移而增加；mTOR-si 组高于对照组，但低于激素组（P<0.01）。对照组的 Beclin1 和 MAP1LC3 的 mRNA 表达水平高于激素组，而 PI3K、Akt 和 mTOR 的 mRNA 表达水平低于 mTOR-si 组（P<0.05）。
激素通过增加股骨头 VEC 中 PI3K/Akt/mTOR 信号通路相关因子的表达，降低 Beclin1 和 MAP1LC3 的表达，抑制自噬对 SANFH 的生理性保护作用。
