全氟辛酸对大鼠肝细胞BRL-3A脂质蓄积的影响

Effect of perfluorooctanoic acid on lipid accumulation in rat liver cells BRL-3A and its possible mechanism

目的探讨全氟辛酸(perfluorooctanoic acid, PFOA)对大鼠肝细胞BRL-3A细胞活力及对转录因子核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)和精氨酸琥珀酸合酶(arginosuccinate synthase 1,Ass1)表达的影响。方法培养大鼠肝细胞BRL-3A,分为对照组(0μmol/L PFOA)、低剂量组(6.25μmol/L PFOA)、中剂量组(25μmol/L PFOA)和高剂量组(100μmol/L PFOA)。分别于染毒48 h后,用噻唑蓝比色法检测细胞活力,自由基指示剂2′,7′-二氯氢化荧光素乙二酯检测活性氧簇(reactive oxygen species, ROS)含量;试剂盒检测氧化应激相关酶活性;荧光免疫细胞化学、Western blot检测Nrf2、Ass1蛋白表达水平。结果与对照组比较,随着PFOA染毒浓度的增高,中、高剂量组的细胞活力有下降趋势,但差异无统计学意义(P>0.05);细胞内的ROS含量升高,中、高剂量组增高显著(P<0.05),平均荧光强度分别为(5417.66±161.09)和(5725.50±166.83)。与对照组相比,低、中剂量组细胞内甘油三酯(triglyceride, TG)、总胆固醇(total cholesterol, TC)和丙二醛(malondialdehyde, MDA)含量无明显变化,高剂量组TG、TC和MDA含量显著升高(P<0.05),分别为(0.21±0.05)mmol/L、(14.5±6.07)mmol/L和(1.23±0.33)nmol/mL。荧光免疫细胞化学检测法、Western blot结果显示,Nrf2蛋白的表达水平在高剂量组显著增高(P<0.05),Ass1蛋白的表达水平在低剂量组显著增高(P<0.05)。结论 PFOA染毒可以导致BRL-3A细胞内ROS的积累,Nrf2和Ass1在PFOA导致的大鼠肝脏细胞氧化损伤刺激下通过增加表达发挥清除ROS和氨解毒作用。

OBJECTIVE To investigate the effect of perfluorooctanoic acid on rat hepatocytes BRL-3 A cell viability and the expression of transcription factor nuclear factor erythroid 2-related factor 2(Nrf2) and arginosuccinate synthase(Ass1). METHODS Rat hepatocytes BRL-3 A were cultured and divided into control group(0 μmol/L PFOA), low-dose group(6.25 μmol/L PFOA), and medium-dose group(25 μmol/L PFOA), high-dose group(100 μmol/L PFOA). After 48 hours, cell viability was detected by MTT, ROS content was detected by free radical indicator H;DCFDA, enzyme activity related to oxidative stress was detected by the kit, Nrf2 and Ass1 protein expression level was detected by Western blot and immunocytochemistry(ICC). RESULTS Compared with the control group, with the increase of the PFOA concentration, the cell viability of the middle and high dose groups had a downward trend, but there was no statistical significance(P>0.05). The intracellular ROS content increased, among which in the middle and high dose groups significantly increased(P<0.05), and the average fluorescence intensity was(5417.66±161.09) and(5725.50±166.83), respectively. Compared with the control group, the content of intracellular TG, TC and MDA in the low and medium dose groups did not change significantly, and the content of TG, TC and MDA in the high dose group was significantly increased(P<0.05), which was(0.21±0.05) mmol/L,(14.5±6.07) mmol/L and(1.23±0.33) nmol/mL, respectively. According to the ICC and Western blot result, the expression level of Nrf2 protein increased significantly in the high-dose group(P<0.05), and the expression level of Ass1 protein increased significantly in the low-dose group(P<0.05). CONCLUSION Exposure to a certain dose of PFOA can lead to the accumulation of ROS in BRL-3 A cells. Nrf2 and Ass1 can play a certain role in eliminating ROS and ammonia detoxification by increasing their expression under the oxidative damage of rat liver cells caused by PFOA.