OPN regulates hypoxia-induced autophagy and proliferation in pulmonary artery smooth muscle cells through PI3K/AKT/mTOR signaling pathway
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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。 -
关键词:
- 低氧 /
- 骨桥蛋白 /
- 细胞自噬 /
- 细胞增殖 /
- PI3K/AKT/mTOR信号通路
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. -
Key words:
- hypoxia /
- osteopontin /
- autophagy /
- proliferation /
- PI3K/AKT/mTOR signaling pathway
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图 2 各组细胞PI3K、AKT、mTOR、OPN及自噬相关蛋白表达量(
$\bar x \pm s $ ，n=4)aP＜0.05，vs 常氧对照组；bP＜0.05，vs 低氧对照组
Figure 2. Expression levels of PI3K, AKT, mTOR, OPN and autophagy-related proteins in each group (
$\bar x \pm s $ , n=4)aP＜0.05, vs Normoxia group; bP＜0.05, vs Hypoxia group
图 3 各组细胞PI3K、AKT、mTOR、OPN及自噬相关蛋白表达量(
$\bar x \pm s $ ，n=4)aP＜0.05，vs 常氧对照组；bP＜0.05，vs 低氧对照组
Figure 3. Expression levels of PI3K, AKT, mTOR, OPN and autophagy-related proteins in each group (
$\bar x \pm s $ , n=4)aP＜0.05, vs normoxia；bP＜0.05, vs hypoxia
图 4 细胞免疫荧光观察各组PASMCs LC3B表达情况
aP＜0.05，vs 常氧对照组；bP＜0.05，vs 低氧对照组
Figure 4. LC3B expression of PASMCs in each group was observed by immunofluorescence
a P ＜0.05, vs Normoxia group; b P ＜0.05, vs Hypoxia group
图 5 细胞免疫荧光观察各组PASMCs Beclin1表达情况
aP＜0.05，vs 常氧对照组；bP＜0.05，vs 低氧对照组
Figure 5. Beclin1 expression of PASMCs in each group was observed by immunofluorescence
aP＜0.05, vs Normoxia group; bP＜0.05, vs Hypoxia group
图 6 电镜观察各组PASMCs超微结构变化(20000×)
N：细胞核；Mi：线粒体；RER：粗面内质网；GB：高尔基体；Atg：自噬小体
Figure 6. Ultrastructural changes of PASMCs in each group were observed by electron microscopy (20000×)
N: nucleus; Mi: mitochondrion; RER: rough endoplasmic reticulum; GB: Golgi body; Atg: autophagosome
图 7 EdU检测细胞增殖(
$\bar x \pm s $ ，n=5)aP＜0.05，vs 常氧对照组；bP＜0.05，vs 低氧对照组
Figure 7. Cell proliferation detected by EdU assay (
$\bar x \pm s $ , n=5)aP＜0.05, vs Normoxia group; bP＜0.05, vs Hypoxia group
表 1 干扰序列
Table 1. Interference sequence
名称 序列 (5'-3') OPN干扰序列 GATGTCCCTGACGGCCGAGGT ACCTCGGCCGTCAGGGACATC 
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