Nrf2基因过表达对百草枯诱导的骨髓间充质干细胞损伤的影响

Effect of Nrf2 overexpression damaged bone marrow mesenchymal stem cells induced by Paraquat

目的观察核因子E2相关因子2转录因子（nuclear factor erythroid 2-related factor 2，Nri2）基因过表达对百草枯（paraquat，PQ）诱导的骨髓间充质干细胞（bone marrow mesenchymal stem ceils，BMSCs）损伤的影响。方法BMSCs随机（随机数字法）分为6组：正常BMSCs组、BMSCs-mCherry（红色荧光蛋白）组、BMSCs-Nrf2组、BMSCs＋PQ组、BMSCs．mCherry＋PQ组、BMSCs-Nrf2＋PQ组。正常BMSCs组、BMSCs—mCherry组、BMSCs-Nrf2组：正常培养基培养；BMSCs＋PQ组、BMSCs-mCherry＋PQ组、BMSCs—Nrf2＋PQ组：终浓度1．0mmol／L的PQ培养。24h之后收集细胞及上清液，蛋白免疫印迹法（westernblot）检测胞核内Nrf2蛋白量，CCK-8法检测细胞存活率，流式细胞仪检测细胞凋亡情况，化学比色法检测上清液脂质过氧化产物丙二醛（MDA）的含量及过氧化氢酶（CAT）的活性，酶联免疫吸附试验（ELISA）检测上清液白细胞介素_6（IL-6）、IL-10及肿瘤坏死因子-α（TNF-α）的含量。数据通过SPSS20．0进行统计学分析，组间比较采用单因素方差分析。结果BMSCs—mCherry＋PQ组与BMSCs＋PQ组比较，各测定数据差异无统计学意义（P〉0．05）。与BMSCs组比较，BMSCs＋PQ组细胞核Nrf2蛋白表达量明显上升（P=0．008）；与BMSCs＋PQ组比较，BMSCs—Nrf2＋PQ组Nrf2蛋白表达量升高（P=0．031）。与BMSCs组比较，BMSCs＋PQ组细胞存活率明显下降，细胞凋亡率明显升高（P=0．000）；与BMSCs＋PQ组比较，BMSCs-Nri2＋PQ组细胞存活率明显升高[（53．27±2．40）％掷．（75．20±2．92）％，P=0．000]，凋亡数量显著降低[（26．43±2．21）％郴．（16．30±1．73）％，P=0．000]。与BMSCs组比较，BMSCs＋PQ组MDA、IL-6、TNF．0L水平明显上升，CAT、IL-10水平明显降低（P=0．000）；与BMSCs＋PQ组比较，BMSCs—Nrf2＋PQ组MDA含量明显降低[（43．88±0．89）nmol／L138．（37．29±1．88）nmol／L，P=0．000]，上清液CAT活力明显升高[（22．82±0．63）U／mL掷．（38．45±1．24）U／mL，P=0．000]，IL-6蛋白含量显著降低[（75．57±11．63）pg／mLvs．（52．00±5．87）pg／mL，P=0．001]，IL-10的蛋白含量明显升高[（6．96±0．93）pg／mLvs．（15．59±1．73）pg／mL，P=0．000]，TNF-α的蛋白含量显著降低[（164．51±9．29）pg／mLvs．（124．62±2．95）pg／mL，P=0．000]。结论Nrf2基因可明显提高BMSCs细胞核Nrf2蛋白的基础表达量，上调Nrf2-ARE通路的表达活性，抵抗PQ对BMSCs的损伤作用。

Objective To observe the effects of nuclear factor erythroid 2-related factor 2 （Nrf2） overexpression on damaged bone marrow mesenchymal stem cells （BMSCs） induced by paraquat （PQ）. Methods BMSCs were randomly （random number） divided into six groups： normal BMSCs group, BMSCs-mCherry （Red flurescent protein） group, BMSCs-Nrf2 group, BMSCs ± PQ group, BMSCs- mCherry ＋ PQ group and BMSCs-Nrf2 ＋PQ group. The BMSCs group, BMSCs-mCherry group and BMSCs- Nrf2 group were cultured by medium. The BMSCs ＋ PQ group, BMSCs-mCherry ＋ PQ group and BMSCs- Nrf2 ＋ PQ group were cultured by 1.0 mmol/L final concentration PQ. All cells and supernatant were collected at 24 h after culture in medium or in PQ. The nucleus protein level of Nrf2 was measured by Western blot; the cell viability was measured by CCK-8 assay; apoptosis of cells was detected by flow cytometry; the levels of malondialdehyde （MDA）, catalase （CAT） in supernatant were determined by chemical colorimetry; the levels of interleukin （IL） -6, IL-10 and tumor necrosis factor （TNF） -α in supernatants were detected by Enzyme-linked immunosorbent assays （ELISA） . One-way analysis of variance （AVOVA） was employed for statistical analysis by using SPSS version 20. 0 to compare values among all groups. Results There were no significant differences in all biomarker variables between BMSCs ＋ PQ group and BMSCs-mCherry ＋ PQ group （ P 〉 0. 05 ）. Compared with BMSCs group, the nucleus protein level of Nrl2 in BMSCs ＋ PQ group was higher markedly （P = 0. 008） ; compared with BMSCs ＋ PQ group, the nucleus protein level of Nrf2 in BMSCs-Nrf2 ＋ PQ group was also higher （ P = 0. 031 ）. The cell viability was much lower in BMSCs ＋ PQ group than that in BMSCs group, while the cell apoptosis was much higher in BMSCs ± PQ group （ P = 0. 000 ） ; compared with BMSCs ± PQ group, the cell viability in BMSCs-Nrf2 ＋ PQ group increased remarkably [ （75.20 ± 2. 92） % vs. （ 53.27 ± 2.40） % （ P = 0. 000） ], and the cell apoptosis decreased significantly [ （ 16. 30 ± 1.73） % vs. （26. 43 ± 2. 21 ） % （P = 0. 000） ]. The levels of MDA, IL-6, TNF-α in supernatant were much higher in BMSCs ＋ PQ group than those in BMSCs group, while the levels of CAT and IL-10 were much lower in BMSCs ＋ PQ group （ P = 0. 000） ; compared with BMSCs ＋ PQ group, the MDA, IL-6, TNF-α levels decreased remarkably in BMSCs-Nrf2 ＋ PQgroup [MDA： （37.29±1.88） nmol/L vs. （43.88 ±0.89） nmol/L, P=0. 000; IL-6：（52.00 ± 5.87） pg/mL vs. （75.57 ± 11.63） pg/mL, P = 0. 001 ; TNF-α ： （ 124. 62 ± 2. 95） pg/mLvs. （ 164. 51 ± 9. 29） pg/mL, P =0. 000], whereas the CAT and IL-10 levels in BMSCs-Nrf2 ± PQ group were markedly increased [CAT： （38.45±1.24） U/mLvs. （22. 82±0.63） U/mL, P=0.000; IL-10： （15.59± 1.73） pg/mL vs. （6.96± 0. 93 ） pg/mL, P = 0. 000 ]. Conclusion The Nrf2 gene can increase the nucleus protein level of Nrf2 in BMSCs and up-regnlate Nrf2-ARE pathway, which have the obvious effects on protecting BMSCs against oxidative damages.