本研究探索了原花青素B2对BNL.CL2肝细胞衰老的拮抗作用。应用棕榈酸(PA)诱导肝细胞衰老细胞模型，通过MTT实验测定PA和PCB2的细胞增殖效应；利用衰老特异性β-半乳糖苷酶试剂盒检测PCB2对细胞衰老特异性β-半乳糖苷酶表达的影响；通过实时定量PCR检测PCB2对细胞周期相关基因CDK6和p53 mRNA表达水平的影响；采用免疫荧光染色法检测PCB2对P53和GATA4蛋白表达以及细胞核大小的影响。结果表明，PA浓度为100 μM时能够有效抑制细胞增殖（control：1.289，PA：0.655，p<0.001），而PCB2浓度为12.5 μg/mL时可明显改善细胞增殖作用（0.766；p<0.01）；与PA组比，PCB2组中表达β-半乳糖苷酶的阳性细胞明显降低；PA诱导细胞核增大（control：1973.9 vs PA：7995.1, p<0.05），而PCB2可降低细胞核大小（3848.8，p<0.05）；PA能够诱导衰老相关基因p53表达上调（3.36，p<0.05）和CDK6基因表达下调（0.49，p<0.05），PCB2可有效改善PA诱导的肝细胞衰老（p53：1.11；CDK6：1.01，p<0.05）。免疫荧光染色显示，PA诱导的肝细胞衰老使p53和GATA4共定位于细胞核；经PCB2处理后，p53和GATA4蛋白在核表达减少，且共定位于细胞质中。因此PCB2能够拮抗肝细胞衰老，其可能通过GATA4/p53信号通路发挥作用。

The alleviation of BNL.CL2 hepatocyte senescence by procyanidin B2 was investigated. Palmitic acid (PA) was used to induce hepatocyte senescence. The MTT assay was used to identify the cell viability treated by PA and PCB2. The effect of PCB2 on cell senescence-specific β-galactosidase (SA-β-Gal) expression was detected by senescence-specific β-galactosidase assay kit. The effect of PCB2 on the expression of cell cycle-related genes CDK6 and p53 mRNA was examined by real-time quantitative PCR. The effect of PCB2 on P53 and GATA4 protein expression and nuclear size was detected by immunofluorescence staining. The results showed that the 100 μM PA inhibited cell proliferation (control: 1.289 vs PA: 0.655, p<0.001), 12.5 μg/mL PCB2 improved the cell proliferation (0.766, p<0.01) which was inhibited by PA. Compared with PA group, the number of positive cells expressing β-galactosidase in PCB2 group is significantly decreased. The nucleus size was increased after PA treatment (PA: 7995.1, p <0.05), while PCB2 reversed the effect (3848.8, p <0.05). Senescence associated gene p53 expression was raised (3.36, p <0.05), CDK6 gene expression decreased (0.49, p <0.05) in the PA group; PCB2 could effectively improve the senescence of pa induced liver cells (p53: 1.11; CDK6: 1.01, p <0.05). Immunofluorescence staining showed that p53 and GATA4 were co-localized in the nucleus of the PA-induced senescent cells. After the PCB2 treatment, expression of p53 and GATA4 proteins in nucleus decreased, and p53 and GATA4 proteins co-occurred in the cytoplasm. Therefore, we believe that PCB2 can alleviate hepatocyte senescence, which may be through the GATA4/p53 signaling pathway.

国家自然科学基金项目（81673675）