SIRT1/FoxO3α信号通路在小檗碱预先给药减轻肝实质细胞缺氧复氧损伤中的作用  

Role of SIRT1/FoxO3α signaling pathway in berberine pretreatment-induced reduction of hypoxia/reoxygenation-caused injury to hepatic parenchymal cells

作　　者：盛明薇[1] 林元邦[2] 杜洪印[1] 喻文立[1] 贾莉莉[1] 

机构地区：[1]天津市第一中心医院麻醉科,300192 [2]天津医科大学总医院普通外科,300052

出　　处：《中华麻醉学杂志》2017年第4期504-507,共4页Chinese Journal of Anesthesiology

基　　金：基金项目：国家高技术研究发展计划（863计划,2012AA021001）;天津市卫生局科技基金（16KG101,13KG105）

摘　　要：目的评价沉默信息调节因子1（SIRT1）/叉头框蛋白3α（FoxO3α）信号通路在小檗碱预先给药减轻肝实质细胞缺氧复氧损伤中的作用。方法培养AML12小鼠肝实质细胞，以1×106个/ml密度接种于6孔板（2 ml/孔）和96孔板（200 μl/孔），采用随机数字表法分为4组（n＝36）：对照组（C组）常氧条件下（5%CO2-21%O2-74%N2）培养，缺氧复氧组（H/R组）、小檗碱预先给药组（BP组）及SIRT1-siRNA（SS组）缺氧（5%CO2-1%O2-94%N2）12 h、复氧条件下（5%CO2-21%O2-74%N2）培养6 h，SS组缺氧前24 h加入SIRT1靶向小干扰RNA（SIRT1-siRNA），BP组和SS组缺氧前2 h加入小檗碱（终浓度5 μmol/L）。于复氧6 h时采用MTT法测定细胞活力，ELISA法测定MDA含量和SOD活性，流式细胞术检测细胞凋亡率，Western blot法检测SIRT1及FoxO3α表达，免疫沉淀法检测FoxO3α的乙酰化水平。结果与C组比较，H/R组、BP组和SS组细胞活力降低，MDA含量升高，SOD活性降低，细胞凋亡率升高，SIRT1表达及FoxO3α核/胞浆比值增加，细胞核FoxO3α乙酰化水平升高（P〈0.05）；与H/R组比较，BP组细胞活力升高，MDA含量降低，SOD活性升高，细胞凋亡率降低，SIRT1表达及FoxO3α核/胞浆比值增加，细胞核FoxO3α乙酰化水平升高（P〈0.05）；与BP组比较，SS组细胞活力降低，MDA含量升高，SOD活性下降，细胞凋亡率升高，SIRT1表达及FoxO3α核/胞浆比值降低，细胞核FoxO3α乙酰化水平降低（P〈0.05）。结论小檗碱预先给药减轻缺氧复氧肝实质细胞损伤的机制与促进细胞SIRT1表达，抑制细胞核内FoxO3α乙酰化水平有关。Objective To evaluate the role of silent information regulator factor 2-related enzyme 1（SIRT1）/Forkhead Box O3（FoxO3a）signaling pathway in berberine pretreatment-induced reduction of hypoxia/reoxygenation（H/R）-caused injury to hepatic parenchymal cells.Methods Hepatic parenchymal cells obtained from AML12 mice were cultured and seeded in 6-well plates（2 ml/well）and in 96-well plates（200 μl/well）at the density of 1×106 cells/ml.The cells were divided into 4 groups（n=36 each）using a random number table： control group（group C）, group H/R, berberine pretreatment group（group BP）and SIRT1-siRNA group（group SS）. The cells were cultured in normal culture atmosphere（5% CO2-21% O2-74% N2）in group C. In H/R, BP and SS groups, the cells were exposed to hypoxic air（5% CO2-1% O2-94% N2）for 12 h, followed by 6 h reoxygenation in normal culture atmosphere（5% CO2-21% O2-74% N2）. In group SS, small interference RNA targeting SIRT1（SIRT1-siRNA）was added to the culture medium at 24 h prior to hypoxia.Berberine（final concentration 5 μmol/L）was added at 2 h prior to hypoxia in BP and SS groups.At the end of reoxygenation, the cell viability was measured by methyl thiazolyl tetrazolium assay, the malondialdehyde（MDA）content and superoxide dismutase（SOD）activity were determined using enzyme-linked immunosorbent assay, cell apoptosis was detected by flow cytometry, the expression of SIRT1 and FoxO3α was detected by Western blot, and the acetylation of FoxO3α was measured by using immunoprecipitation.Apoptotic rate was calculated.Results Compared with group C, the cell viability was significantly decreased, the MDA content was increased, the SOD activity was decreased, apoptotic rate was increased, the expression of SIRT1 and ratio of FoxO3α expression in nucleus/in cytoplasma were increased, and the acetylation of FoxO3α in the nucleus was increased in H/R, BP and SS groups（P〈0.05）. Compared with group H/R, the cell viability was significantly increased, the

关 键 词：小檗碱 肝细胞 再灌注损伤 抗衰老酶 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]