白藜芦醇对自由脂肪酸诱导肝细胞凋亡的保护作用及机制研究

Anti-apoptotic effect of resveratrol on free fatty acid-induced human hepatic L02 cells and its mechanisms

目的研究表明白藜芦醇可改善非酒精性脂肪性肝病,但其作用机制尚不清楚。文中旨在观察白藜芦醇对自由脂肪酸(free fatty acids,FFAs)诱导人肝细胞L02凋亡的保护作用,探讨其可能的分子机制。方法 FFAs混合物、白藜芦醇与人肝细胞L02共同孵育,将细胞随机分为3组:对照组,自由脂肪酸组(2 mmol/L FFAs,油酸∶棕榈酸=2∶1),白藜芦醇组(2 mmol/L FFAs+50μmol/L白藜芦醇)。生物化学法检测肝细胞三酰甘油含量、caspase3活力、还原型谷胱甘肽(Glutathione,GSH)、丙二醛(malondialdehyde,MDA)含量;流式细胞术测定肝细胞凋亡情况;Western blotting测定肝细胞沉默信息调节因子1(silent information regulator 1,SIRT1)蛋白表达情况;荧光实时定量PCR检测过氧化氢酶(catalase,CAT)、锰超氧化物歧化酶(Mn superoxidedismucase,Mn SOD)、Bcl-2、Bax基因表达情况。结果自由脂肪酸组及白藜芦醇组肝细胞内三酰甘油含量与对照组比较,白藜芦醇组与自由脂肪酸组比较,差异均有统计学意义(P<0.05)。自由脂肪酸组肝细胞caspase3活力[(5.97±0.78)U/g]较对照组[(2.56±0.49)U/g]显著升高(P<0.05);白藜芦醇组肝细胞caspase3活力[(3.60±0.73)U/g]较自由脂肪酸组显著降低(P<0.05)。白藜芦醇组肝细胞早凋率[(4.94±0.44)%]和晚凋率[(6.52±0.61)%]与对照组[(3.38±0.33)%、(2.75±0.19)%]及自由脂肪酸组[(6.75±0.81)%、(8.52±0.54)%]比较,差异均有统计学意义(P<0.05)。自由脂肪酸组GSH含量[(73.8±13.1)nmol/g]、MDA含量[(3.77±0.92)mg/g]、SIRT1蛋白相对表达(0.61±0.07)与对照组[(113.7±13.8)nmol/g、(1.85±0.41)mg/g、(0.90±0.02)]、白藜芦醇组[(100.2±8.8)nmol/g、(2.36±0.82)mg/g、(0.84±0.04)]比较,差异均有统计学意义(P<0.05)。白藜芦醇组Mn SOD、CAT、Bax基因的相对表达与对照组及自由脂肪酸组比较,差异均有统计学意义(P<0.05)。结论白藜芦醇激活SIRT1,减轻肝细胞氧化应激,是其抑制FFAs诱发肝细胞凋亡的机制之一。

Objective Resveratrol can improve nonalcoholic fatty liver disease, but its action mechanisms remain unclear. This study aimed to investigate the protective effect of resveratrol against the free fatty acid （FFA）-induced apoptosis of human hepaticL02 ceils and its possible mechanisms. Methods Human hepatic L02 cells were incubated with FFA and resveratrol for 24 hours. The prepared cells were divided into a blank control, an FFA （2 mmol/ L）, and a resveratrol group （50 μmol/L resveratrol ＋ 2 mmol/L FFA）. After treatment, we measured the triglyceride （TG）, glutathi- one （GSH）, and malonaldchyde （MDA） contents and caspase3 ac- tivity in the hepatocytes, determined the apoptosis of the ceils by flow cytometry, and detected the protein expression of silent informationregulator 1 （SIRT1） by Western blot as well as the mRNA expressions of catalase （CAT）, Mn superoxide dismucase （MnSOD）, Bcl- 2, and Bax by qRT-PCR. Results The TG content and caspase3 activity in the hepatocytes were significantly increased in the FFA （ [ 351.8±64.2] μmol/L and [ 5.97±0. 78 ] U/g） and the resveratrol group （ [201.1±60.1 ] μmol/L and [ 3.60± 0.73 ] U/g） as compared with those of the blank control （ [ 40.2 ± 7.4 ] μmol/L and [ 2.56 ± 0. 49 ] U/g） （ both P 〈 0. 05 ）, but the caspase3 ac- tivity was markedly decreased in the resveratrol group in comparison with that of the FFA group （ P 〈 0.05 ）. Both early and late apop- tosis rates of the hepatocytes were remarkably higher in the FFA （ [ 6.75 ± 0. 81 ] % and [ 8.52±0.54 ] % ） and the resveratrol group （[4.94±0.44]% and [6.52±0.611%） than those in the blank control （[3.38±0.33]% and [2.72±0.191%） （bothP〈 0. 05 ）, with statistically significant differences between the former two groups （ P 〈 0.05 ）. The resveratrol group showed significant differences in the GSH content （ [ 100.2± 8.8 ] nmol/g）, the MDA level （ [ 2.36 ± 0.82 ] mg/g）, and the relative expression of SIRT1 （0.84±0.04） from the FFA group （[73.8±13.1] nmol/g, [3.77±0.92] mg/g, and0.61 ±0.07） and the control （ [ 113.7 ± 13.8 ] nmol/g, [ 1.85±0. 41 ] mg/g, and 0.90 ± 0.02） （ all P 〈 0. 05 ）. The resveratrol group also exhibited statistically significant differences in the relative expressions of the MnSOD, CAT, and Bax genes from the FFA and control groups （P 〈 0.05）. Conclusion Resveratrol attenuates FFA-induced apoptosis of human hepatic L02 cells by activating SIRT1 and reducing the oxidative stress of hepatocytes.