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酒精性肝病(alcoholic liver disease,ALD),又称酒精性肝损伤。ALD是由于长期过量摄入乙醇导致的一种慢性疾病,其危害极大。由于长期过量饮酒后,超过肝脏代偿能力,从而导致肝脏的脂肪变、纤维变甚至最后造成的肝硬化,是现代世界范围严重的公共卫生问题之一。据统计,由于饮酒导致的肝硬化居我国肝硬化病因的次位[1]。众所周知,现嗜酒者数量迅速增加,ALD的患病率现在也是急剧上升,ALD的患病率的增加给国家社会和家庭各方面都带来严重负担[2]。然而目前对于ALD并没有特别有效的防治方法,因此探索发现有效并且机制明确的防治ALD的物质迫在眉睫。
N-乙酰半胱氨酸(NAC)是还原型谷胱甘肽的前体物质,是一种含有硫基的具有抗氧化性的乙酰基衍生物,广泛存在于真核生物体内[3],我们在日常饮食中即可补充,特别是洋葱类[4]这种富含有机硫化物的食物中含量比较丰富。NAC在临床上在呼吸系统和肝脏解毒方面已有应用,随着近年来大量的研究证明,NAC由于具有抗氧化性等广泛的生物活性,且在非酒精性脂肪肝[5]、阻塞性肺疾病(COPD)[6]及精神类疾病[7]治疗方面均有较好效果。
本研究对象人张氏肝细胞是来源于人体非恶性肝组织的一类细胞株,细胞株状态相对稳定,已经在很多实验中得到了广泛应用[9-11]。体外细胞实验一方面具有成本低、周期短等优点,另一方面,在无法进行人体实验以及尊重实验动物的原则之下,可以选择来源于人的正常肝细胞进行体外培养获得相关资料。现阶段并没有关于在细胞水平上探究NAC对乙醇诱导肝细胞损伤保护作用的研究,因此选取此细胞株探究NAC对于乙醇诱导人正常肝细胞损伤是否具有保护作用。
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本次浓度筛选共设置4组,分别为control组(不作任何处理);乙醇损伤组(各组浓度分别为200 mmol/L、400 mmol/L、500 mmol/L)。当乙醇浓度为400 mmol/L时,乙醇损伤24 h后细胞活力约在75%左右(P < 0.01)。并且在此浓度干预24 h时LDH浓度最高(P < 0.01)(见表 1),参照刘吉云等[12-13]建立乙醇诱导肝细胞损伤模型的方法,选取乙醇浓度400 mmol/L,作用24 h来建立乙醇诱导肝细胞损伤模型。
分组 n 细胞存活率/% LDH/(U/L) 对照组 3 100.00±2.36 182.25±11.91 Hurt组 200mmol/L 3 100.03±1.11## 185.31±12.29## 400 mmol/L 3 75.63±2.84** 321.71±9.19** 500 mmol/L 3 45.16±3.26**## 264.19±4.81**## F — 319.12 135.62 P — < 0.01 < 0.01 MS组内 — 6.384 100.122 q检验:与对照组比较**P < 0.01;与400 mmol/L组比较##P < 0.01 表 1 不同浓度乙醇对张氏肝细胞作用24 h后细胞存活率、LDH检测结果的影响(x±s)
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采用MTT检测细胞存活率, 不同处理方法处理张氏肝细胞24 h后, 预加入NAC的细胞存活率都相对高于Hurt组细胞存活率(P < 0.05~P < 0.01),1~5 mmol/L的NAC对乙醇诱导损伤组的细胞存活率都有提高作用(见表 2)。
分组 n 细胞存活率/% AST/(U/L) ALT/(U/L) 对照组 3 100.15±2.91 42.91±10.12 1.89±0.32 Hurt组 3 75.90±2.13** 82.13±5.82** 7.18±1.32** NAC-L组 3 80.37±2.07**# 62.08±9.64*# 4.47±1.68*# NAC-M组 3 82.01±2.17**## 59.19±5.97*## 3.11±0.20## NAC-H组 3 85.12±2.00**## 43.60±6.15## 2.03±0.90## F — 49.34 12.82 13.03 P — < 0.01 < 0.01 < 0.01 MS组内 — 5.203 60.564 1.103 q检验:与对照组比较*P < 0.05, **P < 0.01;与Hurt组比较#P < 0.05, ##P < 0.01 表 2 各组细胞存活率及细胞内AST、ALT含量(x±s)
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各组细胞于倒置显微镜下观察, 对照组细胞呈椭圆形或多角形, 明显可见突触, 贴壁生长。经乙醇处理后, 可见一些细胞开始变圆,边界遮光性差,贴壁状态差有细胞出现漂浮死亡。NAC处理组可见细胞死亡变圆细胞减少,存活细胞增多(见图 1)。
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NAC干预组与Hurt组相比细胞AST、ALT含量均下降(P < 0.05),说明NAC在1~5 mmol/L的范围内均可以降低AST和ALT的含量(见表 2)。
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NAC干预组细胞上清液中TNF-α、IL-6含量均低于Hurt组(P < 0.05),说明NAC在1~5 mmol/L的范围内都可以降低上清液中TNF-α和IL-6的含量,TNF-α、IL-6随NAC浓度的增高而降低(见表 3)。
分组 n TNF-α/(ng/L) IL-6/(ng/L) 相对ROS水平/% 对照组 3 51.58±1.59 132.36±3.87 100.00±9.83 Hurt组 3 83.33±5.82** 210.56±7.03** 535.36±28.27** NAC-L组 3 64.76±3.74**## 194.04±14.42**# 343.73±40.64**## NAC-M组 3 61.67±1.38**## 163.06±5.90**## 138.02±15.68## NAC-H组 3 54.17±1.76## 158.06±7.50**## 117.87±18.82## F — 42.45 39.40 204.70 P — < 0.01 < 0.01 < 0.01 MS组内 — 11.071 72.672 516.580 q检验:与对照组比较**P < 0.01;与Hurt组比较#P < 0.01, ##P < 0.01 表 3 各组细胞上清液中TNF-α、IL-6含量及细胞内相对ROS水平(x±s)
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NAC干预后的各组细胞内ROS水平均低于Hurt组(P < 0.05),NAC在1~5 mmol/L的范围内都可以降低细胞中由于乙醇损伤导致的ROS,随NAC剂量的升高,细胞内ROS的水平逐渐减少(P < 0.01)(见表 3)。
N-乙酰半胱氨酸对乙醇诱导张氏肝细胞损伤的保护作用
Protective effects of NAC on the ethanol-induced Chang hepatocellular injury
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摘要:
目的探讨N-乙酰半胱氨酸(NAC)对体外乙醇处理诱导的张氏肝细胞损伤的保护作用。 方法体外培养张氏肝细胞,实验分为对照组、乙醇处理组和NAC高、中、低剂量进行预处理的保护组,并测定NAC预处理对乙醇诱导的肝细胞损伤模型的细胞存活率,细胞内天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、活性氧(ROS)、细胞上清液中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平的影响,评价NAC是否对乙醇造成的肝细胞损伤具有保护作用。 结果NAC预处理后,细胞的存活率高于乙醇处理组(P < 0.05),细胞形态学观察结果显示,NAC的干预组细胞形态优于乙醇处理组;NAC预处理显著降低细胞内AST、ALT、ROS和细胞上清液中TNF-α和IL-6的含量(P < 0.05)。 结论NAC干预可减轻体外乙醇处理诱导的肝细胞损伤,并降低细胞内ROS和炎性因子的水平。 Abstract:ObjectiveTo investigate the protective effects of N-acetylcysteine(NAC) on ethanol-induced Chang hepatocellular injury. MethodsThe Chang hepatocytes were cultured in vitro, and divided into the control group, ethanol treatment group, and high, medium and low dose NAC protection groups.The cell survival rate of ethanol-induced hepatocellulars injury model by NAC pretreatment was determined, and the effects of aspartate aminotransferase(AST), alanine aminotransferase(ALT), intracellular reactive oxygen species(ROS), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) were evaluated.The protective effects of NAC on ethanol-induced Chang hepatocellular injury were analyzed. ResultsAfter NAC pretreatment, the cell survival rate was higher than that of the ethanol-treated group(P < 0.05).The results of cell morphology showed that the cellular morphology in NAC intervention group was better than that in ethanol-treated group.The NAC pretreatment could significantly reduce the contents of TNF-α and IL-6 in cell supernatant, and levels of AST, ALT and ROS in cells(P < 0.05). ConclusionsThe NAC intervention can alleviate the Chang hepatocellular injury induced by ethanol treatment in vitro, and reduce the levels of intracellular ROS and inflammatory factors. -
Key words:
- hepatocyte injury /
- N-acetylcysteine /
- ethanol
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表 1 不同浓度乙醇对张氏肝细胞作用24 h后细胞存活率、LDH检测结果的影响(x±s)
分组 n 细胞存活率/% LDH/(U/L) 对照组 3 100.00±2.36 182.25±11.91 Hurt组 200mmol/L 3 100.03±1.11## 185.31±12.29## 400 mmol/L 3 75.63±2.84** 321.71±9.19** 500 mmol/L 3 45.16±3.26**## 264.19±4.81**## F — 319.12 135.62 P — < 0.01 < 0.01 MS组内 — 6.384 100.122 q检验:与对照组比较**P < 0.01;与400 mmol/L组比较##P < 0.01 表 2 各组细胞存活率及细胞内AST、ALT含量(x±s)
分组 n 细胞存活率/% AST/(U/L) ALT/(U/L) 对照组 3 100.15±2.91 42.91±10.12 1.89±0.32 Hurt组 3 75.90±2.13** 82.13±5.82** 7.18±1.32** NAC-L组 3 80.37±2.07**# 62.08±9.64*# 4.47±1.68*# NAC-M组 3 82.01±2.17**## 59.19±5.97*## 3.11±0.20## NAC-H组 3 85.12±2.00**## 43.60±6.15## 2.03±0.90## F — 49.34 12.82 13.03 P — < 0.01 < 0.01 < 0.01 MS组内 — 5.203 60.564 1.103 q检验:与对照组比较*P < 0.05, **P < 0.01;与Hurt组比较#P < 0.05, ##P < 0.01 表 3 各组细胞上清液中TNF-α、IL-6含量及细胞内相对ROS水平(x±s)
分组 n TNF-α/(ng/L) IL-6/(ng/L) 相对ROS水平/% 对照组 3 51.58±1.59 132.36±3.87 100.00±9.83 Hurt组 3 83.33±5.82** 210.56±7.03** 535.36±28.27** NAC-L组 3 64.76±3.74**## 194.04±14.42**# 343.73±40.64**## NAC-M组 3 61.67±1.38**## 163.06±5.90**## 138.02±15.68## NAC-H组 3 54.17±1.76## 158.06±7.50**## 117.87±18.82## F — 42.45 39.40 204.70 P — < 0.01 < 0.01 < 0.01 MS组内 — 11.071 72.672 516.580 q检验:与对照组比较**P < 0.01;与Hurt组比较#P < 0.01, ##P < 0.01 -
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