Keap1/Nrf2/ARE通路相关基因在热诱导的氧化应激小鼠肝脏中的表达

为了研究热应激对小鼠肝脏抗氧化功能及Keap1 (kelch-like ECH-associated protein- 1)/Nrf2（NF-E2-related factor 2）/ARE (antioxidant response element)通路相关基因表达的影响，选用30只8周龄雄性小鼠随机分成6组，每 d连续42 ℃热处理2 h，分别在热处理0 d(对照组)、1 d、2 d、4 d、8 d和12 d时观察肝脏组织形态学和免疫组织化学分析，另取一部分肝脏组织保存于-80 ℃用于后续荧光定量PCR实验，检测肝脏抗氧化指标及Keap1/Nrf2/ARE通路相关基因的表达．结果显示：小鼠的体表温度和直肠温度在热处理后都极显著高于热处理前．组织形态学观察发现，热处理导致小鼠肝脏组织充血和肝细胞水肿．小鼠肝脏氧化应激指标 MDA (malondialdehyde)含量在热处理第2 d较对照组显著升高，GSH (glutathione)含量、GSH-PX (glutathione peroxidase)活力和总SOD (superoxide dismutase)活力在第4 d和12 d都有升高．免疫组织化学发现，与对照组和第12 d组相比，Nrf2蛋白在第1 d，2 d，4 d，8 d表达明显，其中Nrf2蛋白在第4 d表达最为显著. 荧光定量RT PCR结果表明，与对照组比较Keap1基因的表达量从热处理第1 d开始显著降低，Nrf2基因的表达量在第4 d和12 d显著升高，HO-1 (Heme oxygenase-1)基因的表达量在第1 d显著升高，NQO1 (Quinone oxidoreductase)和GCLC (Glutamate cysteine ligase catalytic)基因的表达量在第1 d和4 d显著升高．上述结果表明，热应激引起了小鼠肝脏氧化损伤， Keap1/Nrf2/ARE通路可能参与了肝脏自身缓解热应激的过程.

Expression of Keap1/Nrf2/ARE Pathway-related Genes in Protective Response to Heat-induced Oxidative Stress in the Liver of Mice

To investigate the effect of heat stress on the antioxidant function in relation to the mRNA expression of Keap1 (kelch-like ECH-associated protein-1)/Nrf2（NF-E2-related factor 2）/ARE (antioxidant response element) in mouse liver, male mice of 8 weeks were placed in 42 ℃ for 2 hours of 12 successive days. The mice were sacrificed on days 0 (control), 1, 2, 4, 8 and 12 immediately following heat stress. The body surface temperature and rectal temperature were found markedly increased, together with severe damage of the liver with the hyperemia and cellular edema. Hepatic MDA increased significantly at day 2 comparing with the level of control. Hepatic GSH level, GSH-PX and T-SOD activities increased at day 4 and 12. Immunohisto chemistry showed Nrf2 protein expression on day 1 through day 8, with a peak at day 4. Comparing to the control, the Keap1 mRNA expression decreased significantly at day 1, whereas the Nrf2 mRNA level increased at days 4 and 12. The HO-1 mRNA expression increased at day 1; NQO1 and GCLC increased at days 1 and 4. The results suggest that heat could induce oxidative stress in the liver with the involvement Keap1/Nrf2/ARE signal pathway.