棕榈酸酯通过调控p16INK4a/Rb信号通路诱导AC16人心肌细胞发生衰老表型改变

Palmitic Acid Induced Ac16 Human Myocardial Aging Phenotype Changes Through p16Ink4a/Rb Pathway

目的探讨棕榈酸酯(palmitic acid,PA)在AC16人心肌细胞衰老表型改变中的作用及机制。方法AC16细胞分为(1)对照组PA浓度(0.08、0.19、0.3、0.8 mmol/L)组;(2)对照组,时间(24、36、48、72 h)刺激组。细胞计数试剂盒-8法测细胞总体活性,流式细胞术测细胞周期;活性氧(reactive oxygen species,ROS)试剂盒测细胞内ROS水平;β-半乳糖苷酶(SA-β-Gal)染色测染色阳性细胞率;酶联免疫吸附测定法(enzyme-linked immuno⁃sorbent assay,ELISA)测衰老相关分泌表型(senescence-associated secretory phenotype,SASP)相关因子白细胞介素(interleukin,IL)-1A、IL-6、IL-8和干扰素(interferon,IFN)-γ浓度;实时定量聚合酶链反应(quantitative real time polymerase chain reaction,qRT-PCR)及Western blot测SASP及p16^(INK4a)/视网膜母细胞瘤抑制蛋白(retinoblasto⁃ma protein,Rb)通路表达。为观察p16^(INK4a)/Rb通路对心肌衰老表型影响,细胞分为4组:对照组、PA组、si-con组和si-p16^(INK4a)组,qRT-PCR测p16^(INK4a) mRNA表达;Western blot测p16^(INK4a)、p-Rb、Rb及IL-1A、IFN-γ蛋白表达;β-半乳糖苷酶染色测阳性细胞率。结果随PA浓度及刺激时间增加,细胞活性不断下降,差异有统计学意义(P<0.05)。与对照组相比,0.19 mmol/L PA组在G1期比例显著升高,S期显著降低,差异有统计学意义(P<0.05)。0.08~0.3 mmol/L PA组ROS水平高于对照组,PA组中β-半乳糖苷酶染色阳性率均高于对照组,差异有统计学意义(P<0.05)。0.19 mmol/L PA组IL-1A、IL-6、IL-8、IFN-γ浓度显著高于对照组,差异有统计学意义(P<0.05)。0.19 mmol/LPA组IL-1A、IL-6、IL-8、IFN-γ及p16^(INK4a)、Rb mRNA及蛋白表达明显高于对照组,而p-Rb蛋白表达下降,差异有统计学意义(P<0.05)。si-p16^(INK4a)组IL-1A、IFN-γ蛋白表达及SA-β-Gal阳性率低于si-con组,差异有统计学意义(P<0.05)。结论0.19 mmol/L PA可能通过调控p16^(INK4a)/Rb通路诱导人心肌细胞衰老表型改变。

Objectives To investigate the role and mechanism of palmitic acid(PA)in aging phenotype changes in AC16 cells.Methods AC16 cells were divided into(1)control group,PA of different concentrations(0.08,0.19,0.3,0.8 mmol/L)groups;(2)control group,times(24,36,48,72 h)groups.The total cytoactivity was detectd by cell counting kit-8.Cell cycle was detected by flow cytometry and reactive oxygen species(ROS)level was measured by ROS kit.SA-β-gal positive cells rate was evaluated by SA-β-gal staining.Concentrations of senescence-associated secretory phenotype(SASP)related factors interleukin(IL)-1A,IL-6,IL-8 and interferon(IFN)-γwere measured by enzyme-linked immunosorbent assay(ELISA).Expressions of SASP and p16^(INK4a)/retinoblastoma protein(Rb)pathway were analyzed by quantitative real time polymerase chain reaction(qRT-PCR)and Western blot.To observe the effect of p16^(INK4a)/Rb pathway on myocardial aging,cells were divided into 4 groups:control group,PA group,si-con group and si-p16^(INK4a) group.p16^(INK4a) mRNA expression was detectd by qRT-PCR.Western blot was used to detect p16^(INK4a),p-Rb,Rb and IL-1A,IFN-γprotein expression.SA-β-gal positive cells rate was measured by chemical staining.Results Compared with control group,cytoactivity decreased in a dose-and time-dependent manner.In 0.19 mmol/L group,proportion of cells in G1 phase increased and S phase decreased,compared with control group(P<0.05).Compared with control group,ROS level increased in 0.08-0.3 mmol/L groups and SA-β-Gal positive cells rate increased in all PA groups(P<0.05).In 0.19 mmol/L group,concentrations of IL-1A,IL-6,IL-8,IFN-γand expressions of IL-1A,IL-6,IL-8,IFN-γ,p16^(INK4a),Rb increased,while the expression of p-Rb protein decreased,compared with control group(P<0.05).Compared with si-con group,IL-1A,IFN-γprotein expressions and SA-β-Gal positive cells rate decreased in si-p16^(INK4a) group(P<0.05).Conclusions p16^(INK4a)/Rb pathway might play important roles in PA-induced human myocardial aging phenotype changes.