Effect and mechanism of N-acetylcysteine on methylnitrosourea-induced precancerous lesions of gastric cancer
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摘要:目的
探讨N-乙酰半胱氨酸(NAC)对甲基硝基亚硝基胍(MNNG)诱导的胃癌癌前病变(PLGC)的影响并分析其作用机制。
方法体外培养胃上皮细胞系GES-1, 并使用不同浓度的MNNG进行处理。使用MNNG诱导建立大鼠PLGC模型。通过显微镜观察细胞形态学变化，并采用划痕实验和Transwell实验分别评估细胞迁移和侵袭能力。采用免疫印迹法(Western blot)分析上皮间质转化[上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)蛋白表达]及活性氧(ROS)/磷脂酰肌醇3-激酶(PI3K)/丝裂原活化蛋白激酶(AKT)/核因子-κB(NF-κB)通路相关蛋白[磷酸化(p-)PI3K、p-AKT及核内p65蛋白]表达。采用DCFH-DA染色检测ROS水平。采用苏木精-伊红(HE)染色观察胃组织病理变化。
结果体外实验结果表明, NAC处理可减轻MNNG诱导的GES-1细胞形态及生长特性改变，抑制细胞迁移、侵袭。体内实验结果表明, NAC处理可减少MNNG诱导的体内病变、增生。NAC可抑制体内外MNNG诱导的上皮间质转化进程(E-cadherin表达增加, N-cadherin表达下降)并抑制ROS/PI3K/AKT/NF-κB通路(p-PI3K、p-AKT及核内p65蛋白表达下降)。激活ROS/PI3K/AKT/NF-κB通路可部分逆转NAC对MNNG诱导的GES-1细胞迁移、侵袭、上皮间质转化以及对ROS/PI3K/AKT/NF-κB通路相关蛋白的作用。
结论NAC对MNNG诱导的胃癌癌前病变的保护作用可能与ROS/PI3K/AKT/NF-κB通路的失活相关。
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关键词:
- 乙酰半胱氨酸 /
- 甲基硝基亚硝基胍 /
- 胃癌癌前病变 /
- 活性氧/磷脂酰肌醇3-激酶/丝裂原活化蛋白激酶/核因子-κB通路
Abstract:ObjectiveTo investigate the effects of N-acetylcysteine (NAC) on methylnitrosourea (MNNG)-induced precancerous lesions of gastric cancer (PLGC) and analyze its underlying mechanisms.
MethodsGastric epithelial cell line GES-1 was cultured in vitro and treated with different concentrations of MNNG. The rat PLGC model was established by MNNG induction. The morphological changes of the cells were observed by microscope, and the migration and invasion ability of the cells were evaluated by wound healing and transwell tests, respectively. Western blot analysis was conducted to evaluate epithelial-mesenchymal transition (E-cadherin and N-cadherin proteins expression) and related proteins expression of reactive oxygen species (ROS)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor-κB (NF-κB) pathway [phosphorylated PI3K (p-PI3K), p-AKT and nuclear p65 protein]. ROS levels were detected using DCFH-DA staining. Histopathological changes in gastric tissues were observed using hematoxylin and eosin (HE) staining.
ResultsIn vitro experiments demonstrated that NAC treatment alleviated MNNG-induced alterations in the morphology and growth characteristics of GES-1 cells, inhibiting cell migration and invasion. In vitro experiments showed that NAC treatment reduced MNNG-induced lesions and hyperplasia. NAC inhibited the EMT process (increased E-cadherin expression and decreased N-cadherin expression) and suppressed the ROS/PI3K/AKT/NF-κB pathway (decreased expression of p-PI3K, p-AKT and nuclear p65 protein) both in vitro and in vivo. Activation of the ROS/PI3K/AKT/NF-κB pathway partially reversed the effects of NAC on MNNG-induced cell migration, invasion, epithelial-mesenchymal transition and related proteins in the ROS/PI3K/AKT/NF-κB pathway in GES-1 cells.
ConclusionThe protective effect of NAC on MNNG-induced PLGC may be associated with the inactivation of the ROS/PI3K/AKT/NF-κB pathway.
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图 1 MNNG诱导建立胃上皮细胞GES-1癌前转变模型
A: GES-1细胞形态学变化; B: 各组细胞迁移能力比较; C: 各组细胞侵袭能力比较;
D: 细胞中E-cadherin、N-cadherin蛋白表达比较。与MNNG 0 h比较, ***P < 0.001。![]()
图 2 MNNG诱导ROS/PI3K/AKT/NF-κB通路在胃上皮细胞中的表达
A: DCFH-DA探针检测ROS水平; B: 细胞中p-PI3K/PI3K、p-AKT/AKT、核内p65蛋白表达比较。
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图 3 NAC通过抑制ROS/PI3K/AKT/NF-κB信号减轻MNNG诱导的GES-1细胞癌前转变
A: CCK-8检测细胞活力, 与NAC 0 mmol/L比较, ***P < 0.001; B: DCFH-DA探针检测ROS水平;
C: 细胞中p-PI3K/PI3K、p-AKT/AKT、核内p65蛋白表达比较; D: GES-1细胞形态学变化; E: 各组细胞迁移能力比较;
F: 各组细胞侵袭能力比较; G: 细胞中E-cadherin、N-cadherin蛋白表达比较。与对照组相比, ***P < 0.001;
与MNNG组相比, ###P < 0.001; 与MNNG + NAC组比较, △△△P < 0.001。![]()
图 4 NAC抑制MNNG诱导的体内癌前病变进展
A: 不同周龄大鼠体质量; B: HE染色检测胃组织病理变化; C: 组织中E-cadherin、N-cadherin蛋白表达比较。
与对照组比较, ***P < 0.001; 与MNNG组比较, ###P < 0.001。表 1 MNNG诱导后细胞中E-cadherin、N-cadherin蛋白表达比较(x±s)(n=3)
时点 E-cadherin表达 N-cadherin表达 MNNG 0 h 1.00±0.04 1.00±0.06 MNNG 6 h 0.80±0.06*** 1.92±0.37*** MNNG 12 h 0.56±0.10*** 2.49±0.26*** MNNG 24 h 0.36±0.05*** 3.51±0.37*** 与MNNG 0 h比较, * * * P < 0.001。 
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表 2 MNNG诱导后细胞中p-PI3K/PI3K、p-AKT/AKT、核内p65蛋白表达比较(x±s)(n=3)
时点 ROS水平 p-PI3K表达 PI3K表达 p-AKT表达 AKT表达 p65 (核) MNNG 0 h 1.00±0.12 1.00±0.03 1.00±0.02 1.00±0.06 1.00±0.03 1.00±0.03 MNNG 6 h 2.43±0.31 1.81±0.35 0.96±0.03 2.29±0.93 1.00±0.02 1.75±0.11*** MNNG 12 h 10.31±1.55*** 2.80±0.09** 0.93±0.02 2.99±1.11** 0.98±0.04 2.60±0.04*** MNNG 24 h 27.32±5.37*** 3.23±1.10*** 0.95±0.02 3.64±1.16*** 0.99±0.03 3.23±0.17*** 与MNNG 0 h比较, * * P < 0.01, * * * P < 0.001。
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表 3 NAC降低MNNG诱导的GES-1细胞ROS水平(x±s) (n=3)
组别 ROS水平 对照组 1.00±0.13 MNNG组 35.81±2.51*** MNNG+NAC 2.5 mmol/L组 13.79±1.66### MNNG+NAC 5.0 mmol/L组 3.43±0.38### MNNG+NAC 10.0 mmol/L组 2.27±0.20### 与对照组相比, * * * P < 0.001; 与MNNG组相比, ###P < 0.001。
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表 4 740Y-P可逆转NAC对MNNG诱导的GES-1细胞上皮间质转化x±s) (n=3)
组别 p-PI3K表达 PI3K表达 p-AKT表达 AKT表达 p65 (核) E-cadherin表达 N-cadherin表达 对照组 1.00±0.04 1.00±0.03 1.00±0.03 1.00±0.02 1.00±0.05 1.00±0.04 1.00±0.07 MNNG组 3.15±0.49*** 0.96±0.03 4.09±0.78*** 0.98±0.03 3.50±0.09*** 0.32±0.05*** 4.03±0.28*** MNNG+NAC组 1.77±0.17### 0.98±0.02 2.18±0.43### 0.97±0.06 2.12±0.10### 0.68±0.04### 2.14±0.22### MNNG+NAC+740Y-P组 2.47±0.37△△ 0.93±0.02 3.02±0.53△ 0.99±0.07 2.99±0.13△△△ 0.48±0.03△△△ 3.31±0.35△△△ 与对照组比较, * * * P < 0.001; 与MNNG组比较, ###P < 0.001; 与MNNG+NAC组比较, △P < 0.05, △△P < 0.01, △△△P < 0.001。
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表 5 NAC抑制MNNG诱导体内上皮间质转化(x±s) (n=6)
组别 E-cadherin表达 N-cadherin表达 对照组 1.00±0.04 1.00±0.02 MNNG组 0.38±0.07*** 2.52±0.23*** MNNG + NAC组 0.78±0.09### 1.61±0.09### 与对照组比较, * * * P < 0.001; 与MNNG组比较, ###P < 0.001。
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表 6 NAC抑制MNNG诱导的PI3K/AKT/NF-κB通路表达(x±s) (n=6)
组别 p-PI3K表达 PI3K表达 p-AKT表达 AKT表达 p65表达(核) 对照组 1.00±0.04 1.00±0.02 1.00±0.05 1.00±0.03 1.00±0.06 MNNG组 2.16±0.13*** 0.99±0.03 2.51±0.51*** 0.99±0.04 2.16±0.35*** MNNG+NAC组 1.72±0.08### 0.99±0.04 1.88±0.55## 1.00±0.05 1.63±0.17# 与对照组比较, * * * P < 0.001; 与MNNG组比较, #P < 0.05, ##P < 0.01, ###P < 0.001。
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