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核因子相关因子2和超氧化物歧化酶mRNA表达与异烟肼致肝损伤的实验研究

Experimental study on the expression of nuclear factor erythroid 2-related factor-2 and superoxide dismutase during the isoniazid-induced liver injury

摘要:

目的 研究异烟肼致小鼠肝损伤进程中核因子相关因子-2 (Nrf-2)及超氧化物歧化酶(SOD) mRNA的表达情况.方法 昆明小鼠64只,通过随机数字表中的简单随机分组法将小鼠分为8组,实验组予以异烟肼90 mg· kg-1·d-1(按成人用药量换算得到)连续灌胃1d、3d、5d、7d、2周、3周和4周,每个时间点小鼠8只.对照组给予等容积蒸馏水灌胃2周.全自动生化检测仪检测血清ALT、AST水平;比色法检测肝组织中SOD活力和丙二醛含量;应用赛绿博实时荧光定量PCR法检测肝组织中Nrf-2、铜-锌超氧化物歧化酶(Cu-ZnSOD)、锰超氧化物歧化酶(MnSOD) mRNA表达情况.多组间比较方差齐性者采用单因素方差分析,SNK法分析组间差异;方差不齐的组间比较采用多个独立样本的秩和检验.结果 随着用药时间的延长,2周、3周、4周组小鼠血清ALT高于对照组(F=13.425,P=0.000);4周组血清AST高于对照组(F=3.498,P=0.004).总SOD活力在5d、7d、2周时分别为(272.63±22.43)、(276.14±20.90)、(271.00±27.43) U/mg,显著低于对照组的(317.95±19.32) U/mg(F=3.769,P=0.002);用药2周后Cu-ZnSOD活力为(242.53±21.48) U/mg,低于对照组(281.92±24.52)U/mg,但4周组活力为(277.11±24.52) U/mg,显著高于2周组(F=3.499,P=0.004);丙二醛含量在7d、2周、4周时分别为(0.94±0.28)、(0.91±0.23)、(0.80±0.15) nmol/mg,显著高于对照组的(0.54±0.09) nmol/mg(F=3.816,P=0.002).实时荧光定量PCR显示,用药4周后Nrf-2 mRNA表达比值增至对照组的6.24倍(F=2.987,P=0.014);Cu-ZnSOD mRNA在3周、4周分别增至2.29和3.99倍(F=8.261,P<0.01);MnSOD在3周时表达增至4.85倍(F=4.026,P=0.002).结论 在异烟肼致肝损伤发生、发展过程中,可上调SOD基因的表达,抵抗氧化损伤,同时可见Nrf-2基因的上调.

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abstracts:

Objective To observe the expressions of nuclear factor erythroid 2-related factor-2 (Nrf2) and superoxide dismutase (SOD) mRNA in mouse liver injury induced by isonicotinic acid hydrazide (isoniazid; INH).Methods A total of 64 Kunming mice were randomly divided into 8 groups using random number table:7 experimental groups and one control group.The mice in experimental groups were given isoniazid by intragastric administration at 90 mg/kg body weight for 1 d,3 d,5 d,7 d,2 weeks,3 weeks and 4 weeks with 8 mice at each time point.The mice in control group were given distilled water by intragastric administration for 2 weeks.Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic biochemical analyzer,and liver SOD activity and methane dicarboxylic aldehyde (MDA) content were detected by colorimetric methods.SYBR green real-time polymerase chain reaction was used to test the expressions of Nrf2,Cu-ZnSOD and MnSOD mRNA in the liver.Comparison among multiple groups with homogeneity of variance was analyzed using analysis of variance and the difference between groups was analyzed by SNK method.Comparison between groups with heterogeneity of variance was analyzed using independent-samples rank sum test.Results The ALT levels at 2 weeks,3 weeks and 4 weeks in INH groups were significantly higher than that in control group (F=13.425,P=0.000); the AST level at 4 weeks in INH group was significantly higher than that in control group (F=3.498,P=0.004).The total activities of SOD at 5 d,7 d and 2 weeks in INH groups were (272.63±22.43),(276.14±20.90) and (271.00±27.43) U/mg,respectively,which were significantly lower compared with control group ([317.95 ± 19.32] U/mg,F=3.769,P=0.002).The activity of Cu-ZnSOD at 2 weeks in INH group ([242.53±21.48] U/mg) was significantly lower than that in control group ([281.92±24.52] U/mg),while it was significantly higher than that at4 weeks ([277.11±24.52] U/mg,F=3.499,P=0.004).TheMDAlevelsat7 d,2 weeks and 4 weeks were (0.94±0.28),(0.91±0.23) and (0.80±0.15) nmol/mg,which were significantly higher than that in control group ([0.54± 0.09] nmol/mg,F=3.816,P=0.002).Compared with control group,the Nrf-2 mRNA expression ratio in INH group had 6.42-fold increase at 4 weeks (F=2.987,P=0.014); the Cu-ZnSOD mRNA expression ratios at 3 weeks and 4 weeks were increased to 2.29 and 3.99 folds,respectively (F=8.261,P<0.01) ; the MnSOD mRNA expression ratio was upregulated to 4.85 fold at 3 weeks (F=4.026,P=0.002).Conclusion During the development of liver injury induced by INH,the SOD gene expression is up-regulated to resist oxidative damage.Meanwhile,Nrf-2 gene is also up-regulated.

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