大花红景天介导PI3K/AKT-HDAC2轴对人胚肺成纤维二倍体细胞衰老的调控作用

Regulatory Effect of Rhodiola Crenulata on Senescence of Human Embryo Lung Fibroblasts Diploid Cells Through PI3K/AKT-HDAC2 Axis

关键词:  细胞衰老  大花红景天  衰老相关β-半乳糖苷酶  组蛋白去乙酰化酶2  PI3K/AKT

Key words:  cellular senescence  Rhodiola crenulata  senescence associated-beta-galactosidae  histone deacetylase 2  PI3K/AKT

摘要:

目的 探究大花红景天中杞柳苷（salipurposide，Sali）、红景天苷（salidroside，Sal）、异柳糖苷（isotachioside，Isota）对人胚肺成纤维二倍体细胞（2BS）衰老调控作用及可能机制。方法 显微镜下观察并选取年轻28代龄2BS （28PD）细胞和衰老50代龄2BS （50PD）细胞。MTT试验测定1.25，2.5，5.0，7.5，10.0 μmol·L^–1 Sal，2.5，5.0，7.5，10.0，12.5 μmol·L^–1 Sali以及Isota对50PD细胞活性的影响。根据MTT结果，5.0 μmol·L^–1 Sal和10.0 μmol·L^–1 Sali、Isota用于进行后续实验。28PD和50PD细胞分为28PD组、50PD组、50PD+Sali组、50PD+Sal组、50PD+Isota组，各组相应药物干预24 h。测定细胞衰老相关β-半乳糖苷酶（senescence associated-beta-galactosidae，SA-β-gal）表达情况，细胞丙二醛（malondialdehyde，MDA）、活性氧（reactive oxygen species，ROS）含量以及超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化酶（glutathione peroxidase，GSH-PX）活性情况，实时荧光定量PCR和蛋白质印迹法分别测定细胞磷脂酰肌醇三羟基激酶（phosphatidylinositol-3-kinase，PI3K）、蛋白激酶B （AKT）、组蛋白去乙酰化酶2（histone deacetylase 2，HDAC2）的mRNA表达以及PI3K、磷酸AKT （p-AKT）、HDAC2蛋白表达情况。结果 与28PD组比较，50PD组细胞SA-β-gal表达增加，MDA、ROS含量增加，SOD、GSH-PX活性降低，PI3K、AKT、HDAC2的mRNA以及相应蛋白表达降低（P<0.01）。50PD细胞经Sali、Sal、Isota干预后，SA-β-gal表达显著降低（P<0.01），MDA、ROS含量显著降低（P<0.01），SOD、GSH-PX活性显著提高（P<0.01），PI3K、AKT、HDAC2的mRNA以及相应蛋白表达均显著增加（P<0.05或P<0.01）。结论 大花红景天中Sali、Sal、Isota能降低50PD细胞SA-β-gal表达，其作用可能与抑制氧化应激、调控PI3K/AKT-HDAC2轴表达有关。

Abstract:

OBJECTIVE To explore the regulatory effect and mechanism of salipurposide(Sali), salidroside(Sal) and isoflavone(Isota) from Rhodiola crenulata on senescence of human embryo lung fibroblast diploid cells(2BS). METHODS Observing and selecting young 28 generations 2BS(28PD) cells and senescence 50 generations 2BS(50PD) cells under the microscope. MTT method was used to determine the effects of 1.25, 2.5, 5.0, 7.5, 10.0 μmol·L^-1 Sal, 2.5, 5.0, 7.5, 10.0, 12.5 μmol·L^-1 Sali and Isota on the activity of 50PD cells. The 5.0 μmol·L^-1 Sal, 10.0 μmol·L^-1 Sali and Isota were used for the following experiments according to MTT results. 28PD and 50PD cells were divided into 28PD group, 50PD group, 50PD+Sali group, 50PD+Sal group, 50PD+Isota group, intervention for 24 h. The senescence associated-beta-galactosidae(SA-β-gal); malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD), glutathione peroxidase(GSH-PX) levels of cells were measured with the test kits; the mRNA expression of phosphatidylinositol-3-kinase(PI3K), protein kinase B(AKT), histone deacetylase 2(HDAC2) and the protein expression of PI3K, p-AKT, HDAC2 were detected by qRT-PCR and Western blotting respectively. RESULTS Compared with the 28PD group, 50PD group cell SA-β-gal expression was increased, the content of MDA and ROS were increased, the activity of SOD and GSH-PX were decreased, the mRNA expression of PI3K, AKT, HDAC2 and related protein expression were decreased(P<0.01). 50PD cells after intervention with Sali, Sal, and Isota, SA-β-gal expression were decreased(P<0.01), the content of MDA and ROS were decreased significantly(P<0.01), the activity of SOD and GSH-PX were increased(P<0.01), the mRNA expression of PI3K, AKT, HDAC2 and related protein expression were significantly increased(P<0.05 or P<0.01). CONCLUSION Sali, Sal and Isota from Rhodiola crenulata can reduce the expression of SA-β-gal of 50PD cells, its effect may related to the inhibition of oxidative stress and regulation of expression of PI3K/AKT-HDAC2 axis.