低硒对T-2毒素致大鼠关节软骨细胞死亡形式的影响

Apoptosis and necroptosis in articular cartilage in rat induced by T-2 toxin under selenium deficient conditions

目的探讨低硒条件下T-2毒素中毒大鼠关节软骨细胞死亡形式。方法选择32只雄性健康SD大鼠，按体质量采用随机数字表法分为2组，每组16只，分别给予硒含量为101．5、1．1μg／蚝的常规和低硒饮食。30d后，将常规饲料组分为对照组和T-2组，低硒饲料组分为低硒组和低硒＋T-2毒素组，每组8只大鼠。T-2毒素组和低硒＋T-2毒素组每日给予T-2毒素（100μg／kg）灌胃饲养。30d后处死大鼠，取左侧膝关节，HE和番红0固绿染色，光镜下观察大鼠膝关节软骨病理学改变；原位缺口末端（TUNEL）法检测软骨细胞凋亡，计算凋亡指数；免疫组织化学法检测大鼠关节软骨细胞活化caspase-3和RIP3的表达。结果光镜下，低硒＋T-2毒索组关节软骨深层可见片状坏死，软骨细胞死亡变为“红色影子”细胞或红染的无结构区域。TUNEL染色，低硒＋T-2组与对照组、低硒组、T．2毒素组比较，表、中层关节软骨细胞凋亡指数差异有统计学意义[（7．474-0_34）％比（4．68±0．54）％、（2．67±O．64）％、（2．56±O．54）％；（72．06±6．15）％比（16．10±3．00）％、（19．57±3.49）％、（19．33±5．19）％，P均〈0．05]；表、中层活化caspase．3阳性率差异有统计学意义[（4．75±0．67）％比（1．24±0．25）％、（0．00±0．00）％、（0．00±O．00）％；（51．13±4．18）％比（10．97±3．01）％、（15．36±4．37）％、（15．23±3．13）％，P均〈0．05]；中层RIP3阳性率差异有统计学意义[（25．91±13．39）％比（1．59±1．14）％、（4．32±2．91）％、（7．50±5．00）％，P均〈0．05]。4组大鼠关节软骨深层细胞凋亡指数及活化caspase-3、RIP3表达水平比较，差异无统计学意义（P均〉0．05）。结论低硒条件下T-2毒素中毒大鼠关节软骨中层软骨细胞的死亡形式为坏死性凋亡与凋亡共同存在。

Objective To investigate the death of chondrocytes in rats which feed with T-2 toxin under selenium （Se） deficient conditions. Methods Thirty two healthy male SD rats were divided into two groups by weight which were normal diet group and Se deficiency diet group, 16 rats in each group. Rats in normal diet group were fed with Se 101.5 μg/kg diet, and rats in Se deficiency diet group were fed with Se 1.1μg/kg diet for 30 d. Normal diet group was divided into control group and T-2 toxin group, and Se deficiency diet group was randomly divided into Se-deficiency group and Se-deficiency plus T-2 toxin group, 8 rats in each group. After that, rats in T-2 toxin and Se-deficiency plus T-2 toxin groups were administrated intragastrically with T-2 toxin （100 μg/kg） everyday for 30 d. Rats were put to death, the left knee was taken and stained with hematoxylin-eosin and Safranin- Fast green, pathological changes of rat＇s knee joint cartilage were observed under light microscopy, expression levels of active caspase-3 and receptor interacting protein 3 （RIP3） in rat＇s articular cartilage cells were determined via the immunohistochemical method. The apoptosis was also detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay （TUNEL）. Results Red ＂ghost＂ outlines of chondrocyte and multiple chondral cell clusters surrounded the non-cell areas in deep zone of articular cartilage of knee joint stained with hematoxyIin-eosin were seen in Se-deficiency plus T-2 toxin group under light microscope. In the superficial zone of cartilage, the positive percent of TUNEL and active caspase-3 in Se-deficiency plus T-2 toxin group was higher than those of control group, Se-deficiency group and T-2 toxin group [（7.47 ± 0.34）% vs （4.68 ± 0.54）%, （2.67 ± 0.64）%, （2.56 ±0.54）%; （4.75 ± 0.67）% vs （1.24 ±0.25）%, （0.00 ± 0.00）%, （0.00 ± 0.00）%, P 〈 0.05]. In the middle zone of cartilage, the positive percent of TUNEL, active caspase-3 and RIP3 in Se-deficiency plus T-2 toxin group was significantly higher than those of control group, T-2 toxin group and Se-deficiency group [（72.06 ± 6.15）% vs （16.10 ± 3.00）%, （19.57 ± 3.49）%, （19.33 ± 5.19）%; （51.13 ± 4.18）% vs （10.97 ± 3.01）%, （15.36 ± 4.37）%, （15.23 ± 3.13）%; （25.91 ± 13.39）% vs （1.59 ± 1.14）%, （4.32 ± 2.91）%, （7.50 ± 5.00）%, P 〈 0.05]. The positive percents of TUNEL, active caspase-3 and RIP3 were not significantly different in the deep zone （P 〉 0.05）. Con^luslon The death of the middle zone in the rat cartilage induced by T-2 toxin under selenium deficient conditions is apoptosis and necroptosis.