丁酸对缺氧血管内皮细胞一氧化氮分泌的影响及机制研究

Effect of butyrate on NO secretion from hypoxic human umbilical vein endothelial cells and its underlying mechanism

目的研究缺氧血管内皮细胞分泌一氧化氮(NO)下降的机制,探讨丁酸的干预作用。方法人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)分为常氧组(21%O2,N)、缺氧组(1%O2,H)、缺氧+溶剂对照组(H+D)、缺氧+丁酸组(H+B)、缺氧+干扰对照组(H+Ci)和缺氧+siRNA干扰组(H+Si)。硝酸还原法检测HUVECs培养上清液NO水平;荧光定量PCR检测一氧化氮合酶(eNOS)的mRNA水平,Western blot检测eNOS蛋白水平、乙酰化水平以及组蛋白去乙酰化酶3(histone deacetylase 3,HDAC3)蛋白水平。免疫共沉淀技术检测HDAC3和eNOS的相互作用。结果缺氧6 h后,HUVECs培养上清中NO水平降低,与常氧组相比,差异具有统计学意义(P<0.05);缺氧组HUVECs中eNOS蛋白水平降低,但eNOS的mRNA水平没有明显变化。与常氧组相比,缺氧组HUVECs中,HDAC3的蛋白表达没有变化,但HDAC3与eNOS的结合增加,eNOS的乙酰化水平下降。与缺氧+干扰对照组相比,缺氧+siRNA干扰组细胞中eNOS的蛋白水平显著升高。缺氧+丁酸(4 mmol/L)处理组HUVECs培养上清中NO水平升高,与缺氧+溶剂对照组相比,差异具有统计学意义(P<0.05);同时,缺氧+丁酸(4 mmol/L)处理组HUVECs细胞中eNOS蛋白水平升高。缺氧+丁酸(4 mmol/L)处理组HUVECs中HDAC3与eNOS的结合减少,eNOS乙酰化水平降低。结论缺氧促进HDAC3和eNOS结合,抑制eNOS的乙酰化,可能通过降低eNOS蛋白的稳定性,抑制NO分泌,而丁酸可以抑制这一过程。

Objective To investigate the molecular mechanism of hypoxia-decreased NO secretion in vascular endothelial cells,and to explore the intervention role of butyrate(Bur)in the process.Methods Human umbilical vein endothelial cells(HUVECs)were divided into normoxia group(21% O2),hypoxia group(1% O2),hypoxia plus solvent control group,hypoxia plus Bur group(4 mmol/L Bur),hypoxia plus control siRNA group,and hypoxia plus HDAC3 siRNA group.The NO content in the supernatant was tested by NO Assay Kit.The mRNA expression of endothelial nitric oxide synthetase(eNOS)was detected by RT-PCR,and its protein level and that of histone deacetylase 3(HDAC3)were measured by Western blotting.The interaction of HDAC3 to eNOS was analyzed by co-immunoprecipitation.Results Compared with the normoxia group,hypoxia for 6 h resulted in significant decrease in the NO level in the supernatant(P<0.05),and reduced eNOS protein level though no change in its mRNA level.In the hypoxic HUVECs,HDAC3 protein level did not changed when compared with the normoxia group.Co-immunoprecipitation analysis showed that hypoxia induced the interaction of HDAC3 with eNOS,and suppressed the acetylation of eNOS protein.The level of eNOS protein was obviously increased in the hypoxia plus HDAC3 siRNA group than the hypoxia plus control siRNA group.Compared with the hypoxia plus solvent control group,the level of NO secretion and eNOS protein were increased significantly in hypoxia plus Bur group(both P<0.05),but the interaction between the HDAC3 and eNOS was decreased and acetylation of eNOS protein was increased.Conclusion Hypoxia induces HDAC3-eNOS binding and inhibits acetylation of eNOS protein,which might be due to its destruction in eNOS stability and suppression in NO secretion.While,Bur treatment can reverse above process in vascular endothelial cells.