【摘要】 目的检测对乙酰氨基酚(acetaminophen,APAP)诱导小鼠急性肝损伤中p62-keap1-Nrf2抗氧化信号通路变化。方法 40只C57/BL6小鼠建立急性肝损伤剂量-反应模型,随机分为4组:对照组、APAP低剂量组(180 mg/kg)、APAP中剂量组(300 mg/kg)和APAP高剂量组(500 mg/kg),每组10只。禁食12 h后,腹腔注射0.9%氯化钠溶液或APAP溶液,24 h后摘眼球取血。另取60只小鼠建立时间-反应模型,对照组给0.9%氯化钠溶液,染毒组腹腔注射300 mg/kg APAP后,分别于3、6、12、24和48 h各取10只小鼠摘眼球取血测生化指标。以上小鼠均剪取肝同一部位做病理切片,HE染色检测病理损伤。并匀浆肝组织提取蛋白,利用Western blot检测p62-keap1-Nrf2信号通路相关蛋白的表达水平,荧光定量PCR技术检测抗氧化基因的相对表达。结果剂量-反应模型中,APAP处理组与对照组相比,肝脏系数明显增大(P<0.05);ALT、AST水平显著升高(P<0.05);肝病理切片显示APAP处理组出现不同程度肝细胞坏死;p62-keap1-Nrf2通路相关蛋白p62、p-p62、Keap1、Nrf2胞浆及胞核蛋白表达增加;而抗氧化基因HO-1、GCLC相对表达量下降。时间-反应模型中,APAP处理组小鼠ALT、AST水平明显高于对照组,呈时间反应趋势,24 h达到峰值;抗氧化基因HO-1、GCLC在3和6 h表达明显增加(P<0.05),12、24和48 h急剧下降至较低水平。结论对乙酰氨基酚诱导的小鼠急性肝损伤中,p62-keap1-Nrf2信号通路激活,Nrf2可能在APAP肝损伤早期通过激活抗氧化相关基因表达发挥作用。更多还原

【Abstract】 Objective To investigate the change of p62-keap1-Nrf2 antioxidant signaling pathway in APAP-induced acute liver injury. Methods Forty male C57/BL6 mice were randomly divided into 4 groups( n = 10) : control group,APAP low-dose group( 180 mg/kg),APAP middle-dose group( 300 mg/kg) and APAP high-dose group( 500 mg/kg). After fasting 12 h,mice were intraperitoneally( ip) injected with saline or APAP solution. And they were sacrificed at 24 h. The other 60 mice were selected to establish the time-course model. After APAP( 300 mg/kg) intraperitoneal injection,ten mice were sacrificed at 3,6,12,24,48 h respectively.Serum and liver tissue in all groups were collected for further analysis. Serum was used to detect the levels of ALT and AST,and liver tissue was used for HE staining to evaluate pathological damage. And then we examined the expression of p62-keap1-Nrf2 signaling pathway by Western blot,measured the expression of antioxidant related genes by RT-PCR. Results In the dose-response model,APAP groups showed larger liver coefficient,higher ALT and AST levels compared with control group( P < 0. 05). The morphology of hepatocytes in the control group was normal,however,APAP groups performed different pathological damage. Additionally,APAP increased the expressions of p62,pp62,keap1 and the nuclear translocation of nrf2,while reduced the mRNA levels of HO-1 and GCLC. In the time-course model,the levels of ALT and AST were significantly increased by time in APAP groups compared with control group,and reached the peak at 24 h( P < 0. 05). The expressions of HO-1 and GCLC were significantly increased at 3 and 6 h( P < 0. 05) while decreased to a lower level after6 h. Conclusion The p62-keap1-Nrf2 signaling pathway was activated in APAP-induced acute liver injury,and Nrf2 might play an important role in the early stage of APAP liver injury by increasing the expression of antioxidant related genes.更多还原