谷氨酰胺对过氧化氢诱导氧化应激人结肠癌HT-29细胞损伤和凋亡的影响

Effects of Glutamine on Oxidative Damage and Apoptosis of Human Enterocyte-Like HT-29 Cells Induced by Hydrogen Peroxide

本试验旨在通过研究谷氨酰胺对过氧化氢(H_2O_2)诱导氧化应激人结肠癌HT-29细胞损伤和凋亡的影响,阐明谷氨酰胺的抗氧化效果和作用机理。HT-29细胞经不同浓度[0(对照组)、0.5、2.0、10.0 mmol/L]谷氨酰胺和0.35 mmol/L H_2O_2分别处理12、24、32 h后,测定细胞的超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,并采用荧光定量PCR方法分析谷氨酰胺对H_2O_2诱导的细胞凋亡相关基因的mRNA相对表达量,以及采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法对HT-29细胞染色,并用流式细胞仪检测细胞的凋亡情况。结果表明:1)处理24 h后,0.5、2.0 mmol/L Gln组SOD活性显著高于对照组(P<0.05);处理32 h后,0.5、2.0 mmol/L Gln组SOD活性显著高于对照组(P<0.05),MDA含量显著低于对照组(P<0.05)。2)处理12 h后,各组天冬氨酸蛋白水解酶-3(Caspase-3)和B淋巴细胞瘤-2相关X蛋白(Bax)mRNA相对表达量无显著差异(P>0.05)。与对照组比较,0.5 mmol/L Gln组核转录因子κB(NF-κB)mRNA相对表达量显著降低(P<0.05),0.5与2.0 mmol/L Gln组B淋巴细胞瘤-2(Bcl-2)mRNA相对表达量显著提高(P<0.05)。处理24 h后,与对照组比较,0.5、2.0和10.0 mmol/L Gln组Caspase-3、NF-κB和Bax mRNA相对表达量均显著降低(P<0.05),Bcl-2mRNA相对表达量显著升高(P<0.05)。处理32 h后,与对照组比较,2.0 mmol/L Gln组细胞表面诱导凋亡分子(FAS)、Caspase-3、NF-κB、Bax mRNA相对表达量均显著降低(P<0.05),Bcl-2mRNA相对表达量显著升高(P<0.05);10.0 mmol/L Gln组FAS、Caspase-3、NF-κB、Bax mRNA相对表达量均显著升高(P<0.05)。3)处理24 h后,与对照组比较,Gln处理使活细胞数量提高了5.32%~11.97%,坏死细胞数量降低了6.75%~12.66%。处理32 h后,与对照组比较,Gln处理使活细胞数量提高了1.39%~7.63%,坏死细胞数量降低了3.40%~4.57%。由此可见,谷氨酰胺可抑制氧化应激反应,降低H_2O_2诱导的HT-29细胞凋亡。

This experiment was conducted to study effects of glutamine （ Gln） on oxidative damage and apop-tosis of human enterocyte-like HT-29 cells induced by hydrogen peroxide （H2O2）, and to explore the antioxi-dant function of Gln and its mechanisms. HT-29 cells I were induced by different concentrations [ 0 （ control group）, 0.5, 2.0 and 10.0 mmol/L] Gln and 0.35 mmol/L H2O2 for 12, 24 and 32 h, the superoxide dis-mutase （ SOD） activity and malondialdehyde （ MDA） content in cells were measured, and the mRNA relative expression of related to cell apoptosis gene were also assessed by RT-qPCR. Furthermore, the HT-29 cells were dyed with the Annexin V-FITC/PI double staining method, and the apoptosis of cells was determined by the flow cytometer. The results showed as follows：1） after treated 24 h, the SOD activity in 0.5 and 2.0 mmol/L Gln groups was significantly higher than that in control group （ P〈0.05）; after treated 32 h, the SOD activity in 0.5 and 2.0 mmol/L Gln groups was significantly higher than that in control group （P〈0.05）, and the MDA content was significantly lower than that in control group （ P〈0.05） . 2） After treated 12 h, there were no significant differences on the mRNA relative expressions of cysteinyl aspartate specific proteinase-3 （Caspase-3） and B cell lymphoma/leukemia-2 associated X protein （Bax） among all groups （P〉0.05）. Compared with the control group, the mRNA relative expression of nuclear factor kappa B （ NF-κB ） in 0.5 mmol/L Gln group was significantly decreased （P〈0.05）, the mRNA relative expression of B cell lym-phoma/leukemia-2 （Bcl-2） in 0.5 and 2.0 mmol/L Gln groups was significantly increased （P〈0.05）. After treated 24 h, compared with the control group, the mRNA relative expressions of Caspase-3, NF-κB and Bax in 0.5, 2.0 and 10.0 mmol/L Gln groups were significantly decreased （P〈0.05）, while the mRNA relative expression of Bcl-2 was significantly increased （ P〈0. 05 ） . After treated 32 h, compared with the control group, the mRNA relative expressions of FAS, Caspase-3, NF-κB and Bax in 2.0 mmol/L Gln group were significantly decreased （P〈0.05）, while the mRNA relative expression of Bcl-2 was significantly increased （P〈0.05）; the mRNA relative expressions of FAS, Caspase-3, NF-κB and Bax in 10.0 mmol/L Gln group were significantly decreased （P〈0.05）. 3） After treated 24 h, compared with the control group, the living cells number in Gln groups increased by 5.32% to 11.97%, and the necrotic cells number decreased by 6.75%to 12.66%. After treated 32 h, compared with the control group, the living cells number in Gln groups in-creased by 1.39% to 7.63%, and the necrotic cells number decreased by 3.40% to 4.57%. It is concluded that glutamine can restrain the oxidant damage of HT-29 induced by H2O2 and decrease the cells apoptosis.