4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) alleviates lung injury by inhibiting SIRT6-HIF-1α signaling pathway activation through the upregulation of miR-212-5p expression,Molecular Biology Reports

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4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) alleviates lung injury by inhibiting SIRT6-HIF-1α signaling pathway activation through the upregulation of miR-212-5p expression
Molecular Biology Reports ( IF 2.6 ) Pub Date : 2024-01-18 , DOI: 10.1007/s11033-023-09039-3
Li Ai ₁ , Ran Li ₁ , Yu Cao ₁ , Zhijuan Liu ₁ , Xiaoqun Niu ₁ , Yongxia Li ₁

Affiliation

Objective

Obstructive sleep apnea is closely related to oxidative stress. 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) can scavenge reactive oxygen species (ROS) and ameliorate oxidative damage in the body. The mechanism by which Tempol alleviates chronic intermittent hypoxia-induced lung injury has rarely been reported. This study aimed to confirm the molecular mechanism by which Tempol alleviates lung injury.

Methods

The levels of miR-212-5p and Sirtuin 6 (SIRT6) in injured lungs were analyzed using bioinformatics. In vitro, intermittent hypoxia (IH) treatment induced hypoxia in BEAS-2B cells and we established a model of chronic intermittent hypoxia (CIH) in mouse using a programmed hypoxia chamber. We used HE staining to observe the morphology of lung tissue, and the changes in lung fibers were observed by Masson staining. The levels of inflammatory factors in mouse serum were detected by ELISA, and the levels of the oxidative stress indicators GSH, MDA, SOD and ROS were detected using commercially available kits. Moreover, a real-time qPCR assay was used to detect miR-212-5p expression, and Western blotting was used to detect the levels of SIRT6, HIF-1α and apoptosis-related proteins. CCK-8 was used to detect cell proliferation. Subsequently, we used flow cytometry to detect cell apoptosis. Dual-luciferase gene reporters determine the on-target binding relationship of miR-212-5p and SIRT6.

Results

SIRT6 was highly expressed in CIH-induced lung injury, as shown by bioinformatics analysis; however, miR-212-5p expression was decreased. Tempol promoted miR-212-5p expression, and the levels of SIRT6 and HIF-1α were inhibited. In BEAS-2B cells, Tempol also increased proliferation, inhibited apoptosis and inhibited oxidative stress in BEAS-2B cells under IH conditions. In BEAS-2B cells, these effects of Tempol were reversed after transfection with an miR-212-5p inhibitor. miR-212-5p targeted and negatively regulated the level of SIRT6 and overexpression of SIRT6 effectively reversed the enhanced influence of the miR-212-5p mimic on Tempol’s antioxidant activity. Tempol effectively ameliorated lung injury in CIH mice and inhibited collagen deposition and inflammatory cell infiltration. Likewise, the therapeutic effect of Tempol could be effectively reversed by interference with the miR-212-5p inhibitor.

Conclusion

Inhibition of the SIRT6-HIF-1α signaling pathway could promote the effect of Tempol by upregulating the level of miR-212-5p, thereby alleviating the occurrence of lung injury and providing a new underlying target for the treatment of lung injury.

中文翻译：

4-羟基-2,2,6,6-四甲基哌啶-1-氧基 (Tempol) 通过上调 miR-212-5p 表达抑制 SIRT6-HIF-1α 信号通路激活来减轻肺损伤

客观的

阻塞性睡眠呼吸暂停与氧化应激密切相关。 4-羟基-2,2,6,6-四甲基哌啶-1-氧基 (Tempol) 可以清除活性氧 (ROS) 并改善体内的氧化损伤。 Tempol缓解慢性间歇性缺氧引起的肺损伤的机制鲜有报道。本研究旨在证实 Tempol 减轻肺损伤的分子机制。

方法

使用生物信息学分析受损肺部中miR-212-5p和Sirtuin 6 ( SIRT6 ) 的水平。在体外，间歇性缺氧（IH）治疗诱导BEAS-2B细胞缺氧，我们使用程序化缺氧室在小鼠中建立了慢性间歇性缺氧（CIH）模型。采用HE染色观察肺组织形态，采用Masson染色观察肺纤维变化。采用ELISA法检测小鼠血清中炎症因子的含量,采用市售试剂盒检测氧化应激指标GSH、MDA、SOD、ROS的含量。此外，采用实时qPCR检测miR-212-5p的表达，采用Western blotting检测SIRT6、HIF-1α和凋亡相关蛋白的水平。 CCK-8用于检测细胞增殖。随后，我们使用流式细胞仪检测细胞凋亡。双荧光素酶基因报告基因确定miR-212-5p和SIRT6的靶向结合关系。

结果

生物信息学分析显示SIRT6在CIH引起的肺损伤中高表达；然而， miR-212-5p表达降低。 Tempol 促进miR-212-5p表达，并抑制 SIRT6 和 HIF-1α 水平。在 BEAS-2B 细胞中，Tempol 还可以增加 IH 条件下 BEAS-2B 细胞的增殖、抑制细胞凋亡并抑制氧化应激。在 BEAS-2B 细胞中，转染miR-212-5p抑制剂后，Tempol 的这些作用被逆转。 miR-212-5p靶向并负向调节 SIRT6 的水平， SIRT6的过表达有效逆转了miR-212-5p模拟物对 Tempol 抗氧化活性的增强影响。 Tempol 有效改善 CIH 小鼠的肺损伤，并抑制胶原沉积和炎症细胞浸润。同样，Tempol 的治疗效果可以通过干扰miR-212-5p抑制剂来有效逆转。