骨桥蛋白通过PI3K/AKT/mTOR信号通路调控低氧诱导的肺动脉平滑肌细胞自噬和增殖的机制探讨

丁琦; 刘川川; 王泽; 周锐; 刘辉琦; 陈辛玲; 王生兰

doi:10.3969/j.issn.2095-5227.2023.05.015

骨桥蛋白通过PI3K/AKT/mTOR信号通路调控低氧诱导的肺动脉平滑肌细胞自噬和增殖的机制探讨

OPN regulates hypoxia-induced autophagy and proliferation in pulmonary artery smooth muscle cells through PI3K/AKT/mTOR signaling pathway

摘要

摘要:
背景骨桥蛋白(osteopontin，OPN)可参与低氧引起的血管重塑，使肺动脉压力升高。但OPN调控低氧诱导的肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells，PASMCs)自噬在低氧性肺动脉高压(hypoxic pulmonary hypertension，HPH)形成中的作用机制尚不明确。
目的探讨低氧条件下OPN对PASMCs增殖及通过PI3K/AKT/mTOR通路对PASMCs自噬的调控作用。
方法原代分离PASMCs，采用免疫细胞化学法进行平滑肌细胞鉴定。将细胞分为常氧对照组(Normoxia)、低氧对照组(Hypoxia)、低氧 + OPN干扰空病毒组(H + OPN EV)、低氧 + OPN干扰慢病毒组(H + OPN shRNA)、低氧 + PI3K抑制剂LY294002组(H + LY)。各组分别用EdU阳性标记率法检测细胞增殖能力；Western blot法检测各组PASMCs中的OPN、PI3K、AKT、mTOR及自噬相关蛋白Beclin1、LC3B的表达；透射电镜、免疫荧光法观察各组PASMCs自噬情况。
结果与Normoxia组相比，Hypoxia组OPN、PI3K、AKT、mTOR、Beclin1、LC3B蛋白表达量升高(P＜0.05)，细胞增殖能力增强(P＜0.05)，自噬小体数量增多，LC3B、Beclin1红色荧光强度增强(P＜0.05)。与Hypoxia组相比，H + OPN EV组各项指标无统计学差异(P＞0.05)，而H + OPN shRNA组和H + LY组OPN、PI3K、AKT、mTOR蛋白表达量降低(P＜0.05)，Beclin1、LC3B蛋白表达量升高(P＜0.05)，细胞增殖能力减弱(P＜0.05)，自噬小体数量增多，LC3B、Beclin1红色荧光强度增强(P＜0.05)。
结论在低氧条件下，OPN可促进PASMCs增殖并通过PI3K/AKT/mTOR信号通路抑制PASMCs自噬。

Abstract:
Background Osteopontin (OPN) can participate in the vascular remodeling induced by hypoxia and increase the pulmonary artery pressure. However, the mechanism of OPN regulating autophagy of hypoxic induced pulmonary artery smooth muscle cells (PASMCs) in the formation of hypoxic pulmonary hypertension (HPH) remains unclear.
Objective To study the role of OPN in PASMCs proliferation and autophagy through PI3K/AKT/mTOR signaling pathway at the hypoxia condition.
Methods PASMCs were isolated and smooth muscle cells were identified by immunocytochemistry. The cells were divided into Normoxia control group (Normoxia), Hypoxia control group (Hypoxia), Hypoxia + OPN interference empty virus group (H + OPN EV), Hypoxia + OPN interference lentivirus group (H + OPN shRNA), Hypoxia + PI3K inhibitor LY294002 group (H + LY). EdU positive labeling rate was used to detect cell proliferation. The expression levels of OPN, PI3K, AKT, mTOR and autophagy-related proteins Beclin1 and LC3B in PASMCs were detected by Western blot. The autophagy of PASMCs in each group was observed by transmission electron microscopy and immunofluorescence.
Results Compared with the Normoxia group, the expression levels of OPN, PI3K, AKT, mTOR, Beclin1 and LC3B significantly increased in the Hypoxia group (P<0.05), the proliferation of PASMCs was enhanced (P<0.05), the number of autophagosomes increased, and the red fluorescence intensity of LC3B, Beclin1 was also enhanced (P<0.05). Compared with the Hypoxia group, all indicators in H + OPN EV group had no statistically significant difference (P>0.05), while the expression levels of OPN, PI3K, AKT and mTOR in the H + OPN shRNA group and H + LY group decreased (P<0.05), the expression levels of Beclin1 and LC3B increased (P<0.05), the proliferation of PASMCs decreased (P<0.05), the number of autophagosomes increased, and the red fluorescence intensity of LC3B, Beclin1 was enhanced (P<0.05).
Conclusion Under hypoxia condition, OPN can promote the proliferation of PASMCs and inhibit autophagy in PASMCs through PI3K/AKT/mTOR signaling pathway.

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