摘要
目的:观察小分子热休克蛋白HSPB1是否减轻过氧化氢(hydrogen peroxide,H_(2)O_(2))诱导的人血管内皮细胞早衰,并从沉默信息调节因子1(silent information regulator 1,SIRT1)信号通路来探讨其可能的作用机制。方法:将常规培养的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)随机分为空白对照组、50μmol/L H_(2)O_(2)组和H_(2)O_(2)+HSPB1过表达组。通过RT-qPCR法检测p16和p21的mRNA表达;衰老相关β-半乳糖苷酶(senescence-associatedβ-galactosidase,SA-β-Gal)活性检测试剂盒检测SA-β-Gal活性;Western blot检测p53、p16、p21、血管细胞黏附分子1(vascular cell adhesion molecule-1,VCAM-1)和细胞间黏附分子1(intercellular adhesion molecule-1,ICAM-1)的蛋白水平;流式细胞术检测细胞内活性氧(reactive oxygen species,ROS)水平。同时,通过沉默SIRT1基因,观察SIRT1对HSPB1减缓H_(2)O_(2)诱导HUVECs衰老的影响。结果:与H_(2)O_(2)组比较,HSPB1过表达可减轻H_(2)O_(2)诱导的HUVECs损伤,包括p16和p21 mRNA水平降低、SA-β-Gal活性及ROS水平显著降低(P<0.05或P<0.01)。同时,HSPB1过表达可使p53、p16、p21、VCAM-1和ICAM-1蛋白水平显著降低(P<0.05或P<0.01)。沉默SIRT1基因可逆转HSPB1对p16和p21 mRNA表达及组蛋白H2AX磷酸化的抑制作用(P<0.05或P<0.01)。同时,沉默SIRT1基因导致HSPB1抑制SA-β-Gal活性作用也显著减弱(P<0.01)。另外,SIRT1 siRNA逆转了HSPB1对p53、p21及p16蛋白表达的抑制作用(P<0.05或P<0.01)。结论:小分子热休克蛋白HSPB1能够有效减轻H_(2)O_(2)诱导的HUVECs早衰,其机制与激活SIRT1信号通路,抑制氧化应激反应有关。
AIM:To observe the effect of small heat shock protein HSPB1 on the premature senescence of human vascular endothelial cells induced by hydrogen peroxide(H_(2)O_(2)),and to explore its possible mechanism based on silent information regulator 1(SIRT1)signaling pathway.METHODS:Human umbilical vein endothelial cells(HUVECs)were randomly divided into blank control group,H_(2)O_(2)(50μmol/L)group,and H_(2)O_(2)+HSPB1 over-expression group.The mRNA expression of p16 and p21 was detected by RT-qPCR.Senescence-associatedβ-galactosidase(SA-β-Gal)activity assay kit was used to detect SA-β-Gal activity.The protein levels of p53,p16,p21,vascular cell adhesion molecule-1(VCAM-1)and intercellular adhesion molecule-1(ICAM-1)were detected by Western blot.The level of reactive oxygen species(ROS)was determined by flow cytometry.Furthermore,the role of HSPB1 and its possible downstream signaling pathway in HUVECs was determined by SIRT1 gene silencing.RESULTS:Compared with H_(2)O_(2) group,over-expression of HSPB1 reduced H_(2)O_(2)-induced injury of HUVECs,decreased the mRNA expression of p16 and p21(P<0.05 or P<0.01),and decreased the activity of SA-β-Gal(P<0.05)and the level of ROS(P<0.01).Meanwhile,over-expression of HSPB1 increased SIRT1 protein level(P<0.05),but decreased p53,p16,p21,VCAM-1 and ICAM-1 protein levels(P<0.05).Silencing of SIRT1 gene reversed the inhibitory effect of HSPB1 on p16 and p21 mRNA expression and H2AX phosphorylation in HUVECs with H_(2)O_(2)-induced premature senescence(P<0.05 or P<0.01).Silencing of SIRT1 gene also significantly attenuated the inhibitory effect of HSPB1 on SA-β-Gal activity(P<0.01).In addition,SIRT1 siRNA reversed the inhibitory effect of HSPB1 on the protein levels of p53,p21 and p16(P<0.05 or P<0.01).CONCLUSION:Small heat shock protein HSPB1 attenuates H_(2)O_(2)-induced premature senescence of HUVECs,and its mechanism may be related to the activation of SIRT1 signaling pathway and inhibition of oxidative stress response.
作者
侯常苗
刘更盛
蒋宇
刘艳娟
邹联洪
陈芳
刘协红
HOU Chang-miao;LIU Geng-sheng;JIANG Yu;LIU Yan-juan;ZOU Lian-hong;CHEN Fang;LIU Xie-hong(Hunan Provincial Key Laboratory of Emergency and Critical Care Metabonomics,Institute of Emergency Medicine,Hunan Provincial People's Hospital(The First Affiliated Hospital of Hunan Normal University),Changsha 410005,China;Clinical Medical College of Hunan University of Traditional Chinese Medicine,Brain Hospital of Hunan Province,Chang-sha 410007,China;Otorhinolaryngology Head and Neck Surgery,Yiyang Central Hospital,Yiyang 413099,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2022年第11期1929-1937,共9页
Chinese Journal of Pathophysiology
基金
湖南省自然科学基金青年项目(No.2020JJ5302)
湖南省教育厅优秀青年项目(No.20B374)。
关键词
细胞早衰
HSPB1蛋白
SIRT1信号通路
氧化应激
血管内皮细胞
Cellular premature senescence
HSPB1 protein
SIRT1 signaling pathway
Oxidative stress
Vascular endothelial cells