原花青素B2通过AKT-FoxO3拮抗脂质诱导的小鼠肝细胞衰老研究

Procyanidin B2 inhibits hepatocyte senescence induced by free fatty acid via activating AKT-FoxO3

目的研究原花青素B2(PCB2)对小鼠肝细胞衰老的调控作用,探讨其可能的分子机制。方法将小鼠胚胎肝细胞株BNL CL.2以梯度浓度棕榈酸(PA)及PCB2处理后,采用噻唑蓝(MTT)检测肝细胞活力,确定PA及PCB2最适浓度。将BNL CL.2细胞分为对照组、PA组和PCB2组,其中PA组用于建立肝细胞衰老模型,PCB2组用最适浓度的PCB2和PA共同作用于细胞。以β-半乳糖苷酶(SA-β-Gal)染色检测细胞衰老情况,反转录实时定量PCR(RT-qPCR)检测细胞周期相关基因表达情况,免疫荧光检测细胞丝氨酸/苏氨酸蛋白激酶-叉头转录因子3(AKT/FoxO3)通路的蛋白表达情况。结果MTT筛选得到PA最适浓度为100μmol/L,PCB2为12.5μg/ml,并用于后续实验。SA-β-Gal染色结果显示,PA组染色阳性细胞数较对照组明显增加;经PCB2处理后,染色阳性细胞数较PA组减少,差异有统计学意义(P<0.001)。RTqPCR结果提示,与对照组比较,PA处理后细胞周期相关基因CDK2表达下调,p21表达上调;经PCB2干预后,CDK2表达较PA组增加,p21表达较PA组下降,差异有统计学意义(P<0.05)。免疫荧光检测结果显示,与对照组比较,PA处理后AKT表达在胞核中减少,在胞质中增加,FoxO3在胞核中明显增加;与PA组比较,PCB2处理后AKT表达在胞核中增加,在胞质中减少,FoxO3在胞核中明显减少。结论PCB2对PA诱导的小鼠肝细胞衰老具有明显保护作用,其机制可能是通过AKT/FoxO3信号通路发挥调控作用。

Objective To explore the effects and potential molecular mechanism of proantho cyanidin B2(PCB2)on hepatocyte senescence.Methods BNL CL.2 cells were treated with palmitic acid(PA)or PCB2 titrations.Untreated cells served as normal control.MTT was used to evaluate cell proliferation activity to identify optimal concentrations of PA and PCB2.The senescence of cells was detected by SA-β-Gal staining.Real-time quantitative PCR(qPCR)was used to quantify the expression of cell cycle related genes.The protein expression of the AKT/FoxO3 pathway was analyzed by immunofluorescence.Results The optimum concentration of PA and PCB2 was 100μmol/L and 12.5μg/ml,respectively.The results of SA-β-Gal staining indicated that the number of positive cells increased significantly after PA treatment.Compared with PA treatment,the number of positive cells decreased after PCB2 treatment,with statistically significant differences.The results of qPCR indicated that the expression of cell cycle related gene CDK2 was down-regulated and p21 was up-regulated after PA treatment.After PCB2 intervention,CDK2 expression was increased and p21 expression was decreased,with statistically significant differences.Immunofluorescence showed that the expression of AKT decreased in the nucleus and increased in the cytoplasm after PA treatment,while FoxO3 increased significantly in the nucleus.After treatment with PCB2,AKT expression increased in the nucleus,decreased in the cytoplasm,and FoxO3 expression decreased significantly in the nucleus.Conclusions PCB2 has a protective effect on PA-induced senescence of mouse hepatocytes,possibly through the AKT/FoxO3 signaling pathway.