高碘对金属硫蛋白Ⅰ／Ⅱ敲除小鼠甲状腺线粒体超氧化物生成的影响

Effects of high concentrations of iodide exposure on mitochondrial superoxide production in the thyroid of metallothionein Ⅰ/Ⅱ knockout mice

目的观察高碘对金属硫蛋白Ⅰ／Ⅱ敲除（MT-Ⅰ／ⅡKO）小鼠甲状腺线粒体超氧化物生成的影响。方法利用6-8周龄健康雄性MT-Ⅰ／ⅡKO小鼠和同源对照（WT）小鼠的甲状腺组织，制备甲状腺细胞悬液，分别用10-4、10-3、10-2mol／L碘化钾（KI）和10-3mol／L过氧化氢（H2O2）处理2h，并以无KI或H2O2处理作为对照。利用甲基噻唑基四唑（MTT）法检测细胞活力，酶联免疫吸附试验（ELISA）法检测培养液中乳酸脱氢酶（LDH）含量．利用MitoSOX探针通过流式细胞术检测甲状腺细胞线粒体超氧化物生成。结果与对照组[（100．00±0．00）％、（100．00±0．00）％]比较，10-4、10-3、10-2mol／LKI组和10-3mol／LH2O2组WT、MT-Ⅰ／ⅡKO小鼠甲状腺细胞活力[（73．63±2．05）％、（72．41±2．26）％、（69．63±2．29）％、（44．90±2．93）％和（65．40±2．39）％、（64，51±2．27）％、（61．48±2．33）％、（40．80±2．76）％]均显著下降（P均〈0．05），且MT-Ⅰ／ⅡKO小鼠与WT小鼠相比，细胞活力下降更加明显（P均〈0．05）。与对照组[（1995．28±30．52）、（2004．96±19．71）U／L]比较，10-4、10-3、10-2mol／LKI组和10-3mol／LH2O2组WT和MT-Ⅰ／ⅡKO小鼠甲状腺细胞培养液中LDH含量[（2809．22±156．53）、（2850．80±137．83）、（2920．45±152．92）、（4487．49±130．67）U／L和（3261．06±120．44）、（3474．19±142．15）、（3597．08±150．86）、（4706．64±148．57）U／L]均显著升高（P均〈0．05），且MT-Ⅰ／ⅡKO小鼠与WT小鼠相比，LDH含量升高更加明显（P均〈0．05）。与对照组（26．49±7．66、37．11±8．48）比较，10-2mol／LKI组和100mol／LH202组WT、MT-Ⅰ／ⅡKO小鼠甲状腺细胞线粒体超氧化物生成（58．96±5．1l、87．95±4．25和71．21±5．55、99．76±4．42）明显增加（P均〈0．05）；且MT-Ⅰ／ⅡKO小鼠与WT小鼠相比，线粒体超氧化物生成增加更加明显（P均〈0．05）。结论高碘（10-2mol／L）和10-3mol／LH2O2可诱导WT和MT-Ⅰ／ⅡKO小鼠甲状腺细胞线粒体超氧化物生成增多，细胞活力下降．LDH分泌增加：MT-Ⅰ／ⅡKO小鼠较WT小鼠甲状腺线粒体超氧化物生成增加更明显。提示MT-Ⅰ／Ⅱ在对抗氧化应激方面具有一定作用。

Objective To investigate the effects of high concentrations of iodide exposure on mitochondrial superoxide production, cell viability and cell damage in the thyroid of metallothionein Ⅰ/Ⅱ knockout （MT-Ⅰ/Ⅱ KO） mice and corresponding wild type （WT） mice. Methods Thyroid cell suspension of six to eight weeks old healthy male MT-Ⅰ/Ⅱ KO mice and WT mice were prepared. The thyroid cells were treated with high concentrations （ 10-4, 10-3, 10-2 mol/L） of potassium iodide （KI）, or 10-3 mol/L hydrogen peroxide （H2O2） for 2 hour, respectively. Cell viability was evaluated with methyl thiazolyl tetrazolium （MTT） assay. Lactate dehydrogenase （LDH） level in cell culture medium was detected by enzyme-linked immunosorbent assay （ELISA）. Mitochondrial superoxide production in the thyroid cells was measured by flow cytometry using a fluorescent probe, mitochondrial superoxide（MitoSOX）. Results Compared to the control group[（100.00 ± 0.00）%, （100.00 ± 0.00）%], the cell viability of 10-4, 10-3, 10-2 mol/L KI and 10-3 mol/L H2O2 exposure groups were significantly decreased in the thyroid cells of both WT [（73.63 ± 2.05）%, （72.41 ± 2.26）%, （69.63 ± 2.29）%, （44.90 ± 2.93）%] and MT- Ⅰ/Ⅱ KOmice[（65.40 ± 2.39）%,（64.51 ± 2.27）%,（61.48 ± 2.33）%, （40.80± 2.76）%, all P〈 0.05]. Compared to the control group[ （1 995.28 ± 30.52）, （2 004.96 ± 19.71）U/L], significantly increased LDH activities were detected in the thyroid cells of WT [（2 809.22 ± 156.53）, （2 850.80 ± 137.83）, （2 920.45 ± 152.92）, （4 487.49 ± 130.67）U/L] and MT-Ⅰ/ⅡKO mice[（3 261.06± 120.44）,（3 474.19 ± 142.15）, （3 597.08 ± 150.86）, （4 706.64 ± 148.57）U/L, all P 〈 0.05]. Compared to the control group （26.49 ± 7.66, 37.11 ± 8.48）, the MitoSOX red fluorescence intensities of 10-2 mol/L KI and 10-3 mol/L H2O2 groups were significantly increased in WT mice（58.96±5.11, 87.95 ± 4.25） and MT- Ⅰ/ⅡKO mice（71.21 ± 5.55, 99.76 ± 4.42） by flow cytometry （all P 〈 0.05）. Compared to the thyroid cells in WT mice, significantly decreased cell viability （all P 〈 0.05）, significantly increased LDH activity（all P 〈 0.05） and significantly increased MitoSOX red fluorescence intensity by flow cytometry （all P 〈 0.05） were detected in the thyroid cells of MT-Ⅰ/Ⅱ KO mice following treatment with KI or H2O2. Conclusions High concentrations of iodide（ 10-2 mol/L） and 10-3 mol/L H2O2 may lead to significant increase of mitochondrial superoxide production and LDH activity, decrease of cell viability in both WT and MT-Ⅰ/Ⅱ KO mice. More significant increase of superoxide production is detected in MT- Ⅰ/Ⅱ KO mice, indicating the potential protective role of metallothionein in the thyroid cells of WT mice.