葛根素通过AMPK-mTOR通路激活自噬改善大鼠激素性股骨头坏死的研究

目的 探讨葛根素通过自噬信号通路对激素性股骨头坏死早期血管内皮细胞自噬的干预作用。方法 48只雄性SD大鼠随机分成4组（每组12只）。对照组：单纯肌肉注射生理盐水，2ml/只/次，共3次，间隔24h；模型组：肌肉注射甲基强的松龙，20mg/kg共3次，间隔24h。葛根素干预组：同模型组方法造模，在最后一次臀肌注射完甲基强的松龙后给予葛根素（100mg/kg?d)腹腔注射。雷帕霉素干预组：同模型组方法造模，在最后一次臀肌注射完甲基强的松龙后同时给予雷帕霉素（1.2mg/kg?d，自噬特异性激动剂)腹腔注射。造模后二周、四周后处死实验动物，取出双侧后肢的股骨头及主要供血血管。HE染色光镜下观察股骨头坏死情况；利用免疫组化法和RT-PCR方法检测Beclin1、LC3、P62、AMPK,mTOR和ULK1的mRNA与蛋白的表达；采用ELISA法检测血管内皮损伤标记物6-keto-PGF1a的表达。结果 与对照组比较，模型组大鼠骨细胞坏死加重，血管内皮细胞6-keto-PGF1a的表达增加（P<0.05），血管内皮细胞中自噬相关蛋白AMPK、ULK1、Beclin1、LC3表达降低（P<0.05）、mTOR、P62表达增高（P<0.05）；与模型组比较，葛根素干预组大鼠的骨坏死程度明显改善，6-keto-PGF1a的表达降低（P<0.05），自噬相关蛋白AMPK、ULK1、Beclin1、LC3表达增高（P<0.05）、mTOR、P62表达降低（P<0.05）。结论 葛根素可能通过AMPK-mTOR-Ulk1 信号通路激活血管内皮细胞的自噬，从而减轻激素所造成的股骨头坏死。

PUERARIN ACTIVATES AUTOPHAGY THROUGH AMPK-MTOR PATHWAY TO IMPROVE STEROID INDUCED FEMORAL HEAD NECROSIS IN RATS

Abstract: Objective To investigate the intervention effect of puerarin on autophagy of vascular endothelial cells in the early stage of steroid-induced femoral head necrosis through AMPK-mTOR-ULK1 signaling pathway.Methords Forty-eight male Sprague-Dawley rats were randomly divided into 4 groups (12 rats per group).Control group: normal saline was injected intramuscularly, 2ml/ time, 3 times in total, with a week interval of 24h;Model group: intramuscular injection of methylprednisolone 20mg/kg for 3 times at an interval of 24h.Model + puerarin group: The model was made by the same method as model group, and puerarin (100mg/kg?d) was injected intraperitoneally after the last injection of methylprednisolone into gluteus muscle.Model + rapamycin group: The model was made by the same method as model group, and rapamycin (1.2mg/kg?d, autophagy specific agonist) was injected intraperitoneally after the last injection of methylprednisolone into gluteus muscle. Two and four weeks after modeling, the experimental animals were sacrificed, and the femoral head and the main blood supply vessels of the bilateral hind limbs were removed.The necrosis of femoral head was observed under light microscope with HE staining.The mRNA and protein expressions of Beclin1, LC3, P62, AMPK,mTOR and ULK1 were detected by immunohistochemistry and RT-PCR.The expression of 6-Keto-PGF1A, a marker of vascular endothelial injury, was detected by ELISA.Results Compared with the control group, osteocyte necrosis in model group was aggravated, the expression of 6-Keto-PGF1A in vascular endothelial cells was increased (P<0.05), the expression of autophagy related proteins AMPK, ULK1, Beclin1 and LC3 in vascular endothelial cells was decreased (P<0.05), and the expression of mTOR and P62 were increased (P<0.05).Compared with model group, the degree of osteonecrosis of rats in puerarin group was significantly improved, the expression of 6-Keto-PGF1A was decreased (P<0.05), the expression of autophagy related proteins AMPK, ULK1, Beclin1, LC3 were increased (P<0.05), and the expression of mTOR and P62 were decreased (P<0.05). Conclusion Puerarin may activate autophagy of vascular endothelial cells through AMPK-mTOR-ULK1 signaling pathway, so as to reduce hormone induced necrosis of the femoral head.