Nrf2抗氧化损伤通路在CCl_4所致小鼠肝毒性中的保护作用（英文）

Genetically graded activation of Nrf2 protects mice against carbon tetrachloride-induced hepatotoxicity

目的观察核转录相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)抗氧化损伤通路对四氯化碳(CCl4)所致小鼠肝毒性的保护作用。方法选取Nrf2-null、Wild-type、Keap1-KD、Keap1-HKO小鼠,按基因型分为4组,分别给予40 mg/kg CCl4腹腔注射,腹腔注射生理盐水(10 m L/kg)作对照,16 h后收集血浆和肝组织样本,检测血中谷丙转氨酶(alanine transaminase,ALT)和乳酸脱氢酶(lactic dehydrogenase,LDH)活性以及肝组织中丙二醛(malondialdehyde,MDA)含量;HE染色观察肝组织病理学变化,Real-time PCR和Western blot法检测相关基因表达。结果 CCl4增加Nrf2-null组和Wild-type组血中ALT、LDH活性及肝组织MDA含量,同时造成肝组织病理损伤,而在Keap1-KD组和Keap1-HKO组影响不明显;Nrf2因子靶基因(Nqo1和Gclc)在CCl4的诱导表达逐渐增高(顺序为Nrf2-null、Wild-type、Keap1-KD、Keap1-HKO),继而使炎症因子、内质网应激基因、细胞凋亡基因、细胞坏死基因的表达(m KC、MIP-2、IL-1β、TNFα、Gadd45、Chop10、Bax、Caspase 3、Mcl、Noxa)按Nrf2-null、Wild-type、Keap1-KD、Keap1-HKO顺序逐渐下降。结论 Nrf2因子激活保护CCl4所致小鼠肝损伤反应。

Objective To investigate the protective role of nuclear factor erythroid 2 - related factor 2 （Nrf2） in CCl4 - induced hepatotoxicity in mice. Methods Nrf2 - null, wild - type （WT） , Keapl - knock down （ Keapl -KD） and Keapl -hepatocyte knockout （Keapl -HKO） mice with maximum Nrf2 activation, were respective- ly treated with CCl4（40 mg/kg, i.p. ） or 10 ml/kg normal saline （control）. Blood and liver samples were col- lected in 16 h after the treatment. Serum enzyme activities, such as alanine transaminase （ALT） and lactic dehy- drogenase （LDH）, liver lipid peroxidation （malondialdehyde, MDA） and histopathology （HE staining） were determined as biochemical indicators of hepatocellular necrosis. RT - PCR and Western blot analysis were used to measure the expression of Nrf2 - targeted molecules, inflammation and cell death - related molecules at mRNA and protein levels. Results CCl4 increased the activities of serum ALT and LDH and content of liver MDA, andcaused liver pathological damage in Nrf2 - null and WT mice, but had no such effect in Keapl - KD and Keapl - HKO mice. The expression of the Nrf2 - targeted genes/proteins, namely Nqol and Gclc, was gradually in- creased with Nrf2 activation in Keapl - KD and Keapl - HKO mice . The expression of pro - inflammatory genes, such as neutrophil -specific chemokines mKC and MIP -2, IL- 115 and TNFα, was increased by CCl4 challenge in Nrf2 -null and WT mice but attenuated in Keapl -KD and Keapl -HKO mice. The expression of endoplasmic reticulum （ER） stress gene Gadd45 and Chop10 was markedly increased in Nrf2 -null mice and at- tenuated with Nrf2 activation. Nrf2 also prevented CCl4 - increased expression of cell death gene implicated in apoptosis （Bax, Caspase 3 and Mcl） and necrosis （Noxa）. Conclusion Nrf2 activation in genetic models pro- tects against CCl4 - induced oxidative stress and liver injury through induction of antioxidant genes.