目的 观察Unc-51样激酶(ULK)1选择性抑制剂SBI-0206965对缺氧/复氧的HTR-8/Svneo细胞自噬和相关功能的影响。方法 建立滋养细胞HTR-8/Svneo细胞缺氧/复氧培养模型,体外模拟子痫前期氧化应激的病理生理状态。将HTR-8/Svneo细胞分为对照组、缺氧/复氧组和观察组(缺氧/复氧细胞培养液中加入10 μM的SBI-0206965),各组细胞分别采用光泽精法检测细胞ROS活性,硫代巴比妥酸法检测细胞MDA水平,CCK-8法检测细胞活力,实时定量PCR检测细胞的Beclin1和LC3B mRNA表达,划痕实验检测细胞迁移力,Transwell小室实验检测细胞侵袭能力。结果 与正常对照组比较,缺氧/复氧组ROS活性(t=8.418,P<0.001)、MDA含量(t=5.788,P<0.001),以及细胞Beclin1(t=8.987,P<0.001)和LC3B(t=10.000,P<0.001)mRNA表达明显上升,而细胞活力(t=4.747,P<0.001)、细胞侵袭(t=5.923,P<0.001)和迁移力(t=12.306,P<0.001)明显下降,差异均有统计学意义。与缺氧/复氧组比较,观察组细胞的ROS活性(t=2.641,P=0.019)、MDA含量(t=2.216,P=0.043)以及细胞Beclin1(t=3.245,P=0.005)和LC3B(t=3.194,P=0.006)mRNA表达明显下降,而细胞活力(t=2.234,P=0.041)、细胞侵袭(t=2.847,P=0.012)和迁移力(t=4.549,P<0.001)明显上调,差异均有统计学意义。结论 ULK1选择性抑制剂SBI-0206965能下调子痫前期氧化应激状态下HTR-8/Svneo细胞的自噬水平,改善受损的细胞活性和功能。
Abstract
Objective To observe the effect of SBI-0206965,a selective inhibitor of unc-51-like kinase (ULK) 1,on autophagy and related functions of HTR-8/Svneo cells under hypoxia/reoxygenation. Methods A hypoxia/reoxygenation culture model of trophoblast HTR-8/Svneo cells was established to simulate the pathophysiological state of oxidative stress in preeclampsia in vitro.The HTR-8/Svneo cells were divided into the normal control group,the hypoxia/reoxygenation group and the observation group (the hypoxic/reoxygenated cells treated with 10 μM SBI-0206965).The ROS activity was detected by the glossy sperm method,the MDA levels were detected by the thiobarbituric acid method,the cell viability was detected by CCK-8 kit,the expression of Beclin1 and LC3B was detected by real-time quantitative PCR,cell migration was detected by scratching test,and cell invasion ability was detected by Transwell chamber assay. Results Compared with the control group,the ROS activity (t=8.418,P<0.001),MDA content (t=5.788,P<0.001),Beclin1 mRNA expression (t=8.987,P<0.001) and LC3B mRNA expression (t=10.000,P<0.001) were significantly increased,while cell viability(t=4.747,P<0.001),cell invasion (t=5.923,P<0.001) and cell migration (t=12.306,P<0.001) were significantly decreased in the hypoxia/reoxygenation group.Compared with the hypoxia/reoxygenation group,the ROS activity (t=2.641,P=0.019),MDA content (t=2.216,P=0.043),Beclin1 mRNA expression (t=3.245,P=0.005) and LC3B mRNA expression (t=3.194,P=0.006) were significantly decreased,while cell viability (t=2.234,P=0.041),cell invasion (t=2.847,P=0.012) and cell migration (t=4.549,P<0.001) were significantly increased in the observation group. Conclusion ULK1 inhibitor SBI-0206965 can down-regulate the autophagy level of HTR-8/Svneo cells under oxidative stress in preeclampsia,and improve the activity and function of the damaged cells.
关键词
子痫前期 /
滋养细胞 /
SBI-0206965 /
自噬 /
氧化应激
Key words
Preeclampsia /
Trophoblasts /
SBI-0206965 /
Autophagy /
Oxidative stress
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基金
福建省自然科学基金资助项目(2020J011102)
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