Ⅱ型胶原在T-2毒素诱导大鼠关节软骨早期损伤中的干预作用

Role of type II collagen in protecting and preventing articular cartilage damage induced by T-2 toxin in rats

目的观察Ⅱ型胶原在T-2毒素诱导大鼠关节软骨早期损伤中的干预作用，在分子水平上寻找软骨损伤及修复的分子学生物标志，为探讨关节软骨损伤疾病的防治措施提供理论依据。方法Wistar大鼠80只．按体质量随机分为4组：阴性对照组、阳性对照组、高剂量干预组、低剂量干预组，每组20只。阴性对照组食用常规成品颗粒饲料，其他3组食用含100ng／kg T-2毒素染毒饲料；阴性对照组和阳性对照组饮自来水；低、高剂量干预组饮用含Ⅱ型胶原0．5、5．0g／L的自来水。在3、5个月时处死大鼠，光镜下观察大鼠透明软骨的组织病理学改变，用酶联免疫吸附试验（ELISA法）检测大鼠血清Ⅱ型胶原羧基末端肽（CTX—II）、软骨寡聚基质蛋白（COMP）及尿中吡啶啉（DPD）含量。结果光镜下阳性对照组大鼠关节软骨细胞排列紊乱，软骨细胞变形、变性，可见大面积的软骨细胞坏死，而高、低剂量干预组表现为软骨表面原纤维形成，表层软骨细胞肿胀变圆，扁平的软骨细胞减少，软骨细胞簇集等骨关节炎早期病理改变。在3、5个月时，阴性对照组、阳性对照组、高剂量干预组、低剂量干预组大鼠血清CTX-Ⅱ含量分别为（18．77±4．61）、（25．07±9．17），（24．43±5．23）、（39．17±10．49）。（21．11±5．02）、（33．20±9．74），（19．87±4．53）、（29．73±9．32）ug／L；血清COMP含量分别为（5．43±2．75）、（6．38±2．23），（21．37±4．72）、（24．52±4．26），（17．27±4．77）、（20．32±4．74），（20．13±5．07）、（19．44±4．92）ug／L。其中，3个月时，与阴性对照组比较，阳性对照组血清CTX-Ⅱ含量明显升高（P〈0．05），而低、高剂量干预组未见明显改变（P均〉0．05）；5个月时．与阴性对照组比较，其他3组血清CTX—II含量明显升高（P均〈0．05），而高、低剂量干预组明显低于阳性对照组（p均〈0．05）。3个月时，与阴性对照组比较，其他3组血清COMP含量明显升高（P均〈0．05），而与阳性对照组比较，高剂量干预组血清COMP含量明显降低（P〈0．05）；5个月时，与阴性对照组比较，其他3组血清COMP含量明显升高（P均〈0．05），而与阳性对照组比较，高、低剂量干预组血清COMP含量明显降低（P均〈0．05）。3、5个月时，上述4组大鼠尿液DPD含量分别为（3．47±2．20）、（4．14±1．06），（4．09±2．48）、（4．33±3．43），（3．86±2．31）、（5．72±3．89），（3．58±2．77）、（4．23±2．90）ug／L，组间比较，差异无统计学意义（F值分别为2．608、2,436，P均〉0．05）。结论Ⅱ型胶原能拮抗T-2毒素的软骨损伤作用，延缓关节软骨的破坏进程，降低大鼠血清中CTX—II及COMP水平。

Objective To observe the preventive effect of type II collagen on experimental rat articular cartilage damage induced by T-2 toxin, to explore molecular biomarkers of articular cartilage damage and repair, and to provide a theoretical basis for control of articular cartilage damage. Methods Eighty Wistar rats were randomly divided into 4 groups according to their body weights： negative control, positive control, high-dose intervention, and low-dose intervention groups, 20 rats in each group. Animals in negative control group were fed with standard rat chow, and animals in other three groups were fed with T-2-toxin-contaminated chow （100 ng/kg feed）. Animals in negative and positive control groups drank distilled water, animals in high-dose intervention and low-dose intervention groups drank water containing type II collagen（0.5, 5.0 g/L, respectively）. These rats were sacrificed after 3 and 5 months, respectively, and bilateral knee joints were collected. Histopathologic changes in hyaline cartilage were examined by light microscope, serum levels of type II collagen ＂carboxyl terminal peptide （CTX-H）, cartilage oligomeric matrix protein （COMP） and urinary deoxypyridinoline（DPD） were determined by enzyme-linked immunosorbent assay （ELISA）. Results HE staining showed, that the positive control articular chondroeytes were disarranged, deformated, degenerated, with necrosis and extensive areas of chondrocyte loss; but the two intervention groups only showed fibril formation and swelling and surface cartilage cells became round, fiat cartilage cells decreased in number, and cartilage cells clustered and so on early pathological changes of osteoarthritis. At the ends of 3 month and 5 month experiment, the levels of serum CTX- H in different groups were, negative control[ （18.77 ± 4.61）, （25.07 ± 9.17）ug/L], high-dose intervention[（21.11 ± 5.02）, （33.20 ± 9.74） ug/L], low-dose intervention[ （19.87 ± 4.53）, （29.73 ± 9.32）ug/L] and positive control[ （24.43 ± 5.23）, （39.17 ± 10.49）ug/L] ; the levels of serum COMP were, negative control group[（5.43 ± 2.75）, （6.38 ± 2.23）ug/L], high- dose intervention group [ （ 17.27 ± 4.77）, （20.32 ± 4.74） ug/L ], low-dose intervention group [ （20.13 ± 5.07）, （19.44 ± 4.92）ug/L] and positive control group[ （21.37 ± 4.72）, （24.52 ± 4.26）ug/L]. At the end of 3 month, compared with negative control group, the level of serum CTX- II in other three groups increased, but only positive control group increased signifieantly（P 〈 0.05 ）; at the end of 5 month, compared with negative control group, the level of serum CTX-II in other three groups increased significantly, and the difference was statistically significant （all P 〈 0.05 ）, and the level of CTX- II in the two intervention groups was significantly lower compared with that of positive control group（all P 〈 0.05）. Compared with negative control group, the level of serum COMP in other groups increased significantly at the end of 3 month（all P 〈 0.05） and only the level of serum COMP in high-dose intervention group was significantly lower compared with that of positive control group（P 〈 0.05）. At the end of 5 month, compared with negative control group, the level of serum COMP in other three groups increased significantly, the difference were statistically significant （all P 〈 0.05） ; the levels of serum COMP in the two intervention groups were significantly lower than that of positive control group（all P 〈 0.05）. At the ends of 3 month and 5 month, the content of urinary DPD in negative control group were [ （3.47 ± 2.20）, （4.14 ± 1.06）ug/L], positive control group [ （4.09 ± 2.48）, （4.33 ± 3.43）ug/L], high-dose intervention group [ （3.86 ± 2.31）, （5.72 ± 3.89）ug/L] and low-dose intervention group[ （3.58 ± 2.77）, （4.23 ± 2.90）ug/L]. The difference between the 4 groups were not statistically significant （F = 2.608, 2.436, all P 〉 0.05）. Conclusions Type II collagen could effectively reduce the level of serum CTX- II and COMP in experimental rats and delay the process of articular cartilage damage induced by T-2 toxin.