虾青素对小鼠肝脏原代细胞氧化应激的影响

摘要

[目的]研究虾青素对过氧化氢(H2O2)诱导小鼠肝脏原代细胞产生氧化应激的影响.[方法]原位?步灌流法提取ICR小鼠的肝脏原代细胞,用丙?醛(Malondialdehyde,MDA)作为指标、采用H2O2处理建立氧化应激模型.采用流式细胞仪测定细胞中活性氧(Reative oxygen species,ROS)含量及凋亡水平变化,生物化学法测定细胞中MDA和谷胱甘肽(Glutathione,GSH)的含量、超氧化物歧化酶(Superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)的活性,用荧光定量PCR检测抗氧化酶SOD、GSH-px mRNA的相对表达量,Western-blot检测细胞核中Nrf2蛋白相对表达量.[结果]使用5μg·mL–1虾青素预保护细胞3 h,10μmol·L–1 H2O2刺激3 h的情况下氧化应激模型效果最好.虾青素降低了H2O2诱导后小鼠肝脏原代细胞的细胞凋亡率以及ROS、MDA和GSH含量,提升了SOD、GSH-px的活性以及SOD、GSH-px的mRNA相对表达量,抑制了Nrf2蛋白的核转移.[结论]虾青素对H2O2诱导产生氧化应激的肝脏原代细胞具有保护作用.%[Objective]To investigate the effect of astaxanthin on H2O2-induced oxidative stress response in primary mouse hepatocytes.[Method]Primary mouse hepatocytes were isolated by an in situ two-step perfusion technique. The model of oxidative stress response in primary mouse hepatocytes was established using H2O2 treatment and malondialdehyde(MDA) as an indicating index. The content of reative oxygen species (ROS) and change in apoptosis rate were measured by flow cytometry. The contents of MDA and glutathione(GSH), and the activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-px) were measured by biochemical methods. The relative mRNA expression levels of antioxidant enzymes including SOD and GSH-px were measured by fluorescence quantitative PCR. The relative expression of Nrf2 protein was measured by Western-blot.[Result]The model of oxidative stress response performed the best with the primary mouse hepatocytes pre-protected by 5 μg·mL–1 astaxanthin for 3 h before being exposed to 10μmol·L–1H2O2 for 3 h. Astaxanthin decreased the apoptosis rate and the contents of ROS, MDA and GSH in primary mouse hepatocytes induced by H2O2. Astaxanthin increased the activities of SOD and GSH-px as well as the relative mRNA expression levels of SOD and GSH-px, and inhibited the nuclear transfer of Nrf2 protein.[Conclusion]Astaxanthin has protective effects on H2O2-induced primary mouse hepatocytes with oxidative stress.