臭氧化盐水对肝细胞Keap1-Nrf2-ARE通路的激活作用

Effect of ozonized saline on signaling passway of Keap1-Nrf2-ARE in rat hepatocytes

目的 研究臭氧化盐水对肝组织细胞Keap1-核因子E2相关因子2（Nrf2）-抗氧化元件（ARE）通路中Nrf2的作用.方法 采用成年健康雄性Sprague-Dawley大鼠,随机分为正常对照组（NC组）、模型组、臭氧等渗盐水（OS）组,OS对照组（OSC组）.OS组、OSC组分别予5 ml/kg OS,模型组予5 ml/kg氧气盐水每日尾静脉注射,连续15 d,第16天分别予OS组及模型组50%CCl4橄榄油溶液2ml/kg腹腔注射造肝损伤模型.NC组及OSC组予植物油2ml/kg腹腔注射,24 h后,检测大鼠血清ALT、AST、肝组织总抗氧化能力（TAOC）、还原型谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GPx）、过氧化氢酶（CAT）.再提取处死大鼠肝组织的核蛋白,应用Western blot测定其细胞核中Nrf2的含量,免疫荧光组织化学技术检测细胞内Nrf2的分布.结果 与模型组比较,OS组大鼠ALT、AST降低[（1240.4±188.2）U/L、（1245.4±176.9）U/L对比（539.8±175.3）U/L、（546.0±130.2）U/L）],差异有统计学意义（P〈0.01）,TAOC、GSH,GPx,CAT 活性升高,分别为（0.72±0.24）U/mg、（1.05±0.21）mg/g,（676.9±115.1）U/mg、（45.2±14.3）U/mg对比（1.37±0.19）U/mg、（2.23±0.55）mg/g、（1024.6±162.9）U/mg、（68.2±9.9）U/mg,差异有统计学意义（P〈0.01）.与NC组比较,OSC组大鼠肝组织TAOC、GSH、GPx,CAT活性升高,差异有统计学意义（P〈0.01或P〈0.05）.Western blot及免疫荧光均显示O3能增强肝细胞核内Nrf2的表达,Keap1-Nrf2-ARE通路的激活在O3抗氧化过程中发挥了重要的作用.结论 臭氧化盐水静脉注射可减轻CCl4所致大鼠肝损伤.其机制可能通过激活Keap1-Nrf2-ARE通路及其下游基因,增强细胞抗氧化和抗自由基的能力.

Objective To study the effect of ozonized saline on the activation of the Keapl-Nrf2ARE signaling pathway in rat liver cells. Methods Twenty maleSprague-Dawley rats were randomly divided into ozonized saline（OS） group, model group, ozonized saline control （OSC） group and normal control （NC）group. The rats in OS group and model group were intravenously administered with OS or oxygen saline （5 ml/kg） respectively, once a day for 15 days, and then intraperitoneally injected with CCU dissolved in Oliver oil. The rats in OSC group were pretreated with OS for 15 days. The rats in NC group were fed normally for 15 days. On the 16th day, the rats in OSC group and NC group were intraperitoneally injected with Oliver oil （2 ml/kg） without CCU. After 24 hours of CCU or olive oil intraperitoneal injection, the serum levels of alanine transaminase （ALT） and aspertate aminotransferase （AST） were measured. The liver tissues were also collected for detection of total anti-oxygen capability （TAOC）, glutathione （GSH）, catalase （CAT）, Glutathione peroxidase （GPx）. Western Blot was used to detect Nrf2 and immunofluorescence staining assay to display intracelluar distribution of Nrf2. Results Compared with the rats in model group,the serum ALT and AST levels of rats in OS group were significantly lower （P 〈 0.01） ,which were （1240.4 ± 188.2） U/L and （1245.4 ± 176.9） U/L vs （539.8 ± 175.3） U/L and （546.0 ± 130.2） U/L, and the TAOC, CAT, GPx and GSH activity of rats in OS group were significantly higher, which were （0.72 ± 0.24） U/mg, （1.05 ±0.21） mg/g, （676.9 ± 115.1） U/mg and （45.2 ± 14.3） U/mg vs （1.37 ± 0.19） U/mg, （2.23 ± 0.55） mg/g,（1024.6 ± 162.9） U/mg and （68.2 ± 9.9） U/mg, respectively. In contrast with NC group, pretreatment of OS in OSC group elevated TAOC, CAT, GPx and GSH activity （P 〈 0.01 or P 〈 0.05）. Ozonized saline can strengthen the Nrf2 expression in liver cells. Conclusions Preconditioning injection of ozonized saline can reduce rat＇s liver injury induced by CCl4- The ozonized saline, as a novel Nrf2 activator, can reduce the oxidative damage of radical oxygen species （ROS） and the deleterious substance by activating the KeaplNrf2-ARE signaling pathway and its downstream genes expression.