The Experimental Study On Kaschin Beck Disease Induced By Deoxynivalenol

ObjectiveTo observe injury mechanism of articular chondrocytes caused by deoxynivalenol (DON),the injury differences of knee bone and articular cartilage between Wistar adult rats and young rats and the expression of Collagen typeⅡ,iNOS and MMP-13 in articular chondrocytes;and further explore the relationship and Kashin-Beck disease pathogenesis, to search for the risk factors and control measures which developed to provide the scientific basis for Kashin-Beck disease。Methods1. Through animal experiments, animal model of KBD is copied to study the DON of pathogenesis mechanisms in the Kashin-Beck disease .1.1 The specimens of articular cartilage were examined by light microscope and electron microscopy pathological morphology.1.2 The level of Collagen typeⅡ,iNOS,MMP-13 was observed by immunohistochemical technique.2. Chondrocytes culture in vitro2.1 MMP-13 and PGE2 were detected by ELISA Kits; NO was measured by Griess assay with spectrophotometer.2.2 Cell apoptosis was assayed by flow cytometry(FCM)with Annexin V-FITC/PI staining;Inducible nitric oxide synthase(iNOS) and collagenⅡin cells were detected by FCM.2.3 The expression levels of iNOS and mRNA and collagenⅡmRNA were measured with RT-PCR.Result1. light microscope and electron microscopy result:The articular cartilage matrix in the control groups were well-distributed and dense,the structure of epiphyseal cartilage was intact from epiphysis to metaphyseal and the cartilage matrix was weakly eosinophilic with a good level of cell arrangement. Compared with the control groups, in the experimental group of the young rats, the degeneration, necrosis and disordered arrangement of the cartilage cells were obvious. Healthy cells and damaged cells were interactively arranged with the appearance of DLD. The number of bone trabeculae was significantly decreased and the bone trabeculae became thin, discontinuous with destroyed network structures. In the experimental group of the adult rats, the degeneration and necrosis of the cartilage cells could be observed and some cartilage cells vanished with nuclear dissolution. By transmission electron microscope, the smaller and even-sized granular material with higher electronic density could be observed. The swelling and pycnosis of mitochondria both exist. Parts of the mitochondria were degenerated into vacuole and the ridge was damaged or even disappeared. The shape of the cell nucleus was irregular with nuclear pyrosis and aggregation at the periphery of the nucleons. The collagen network of articular cartilage was damaged.2. immunohistochemical technique result:The results showed that the effect of DON was observed, such as: (1) The expression of Collagen typeⅡin DON groups was(4.79±0.96)%,(2.82±0.68)%which all were significantly higher than control(7.42±1.12)%,(P<0.05); (2) The level of iNOS had statistical significance in comparision between 3 groups(P<0.05);low and high-dosage of DON treatment groups(8.52%±1.90%,11.78%±2.51%)were significantly higher than control(s6.90%±1.61%),(LSD-t test:t=1.587, P=0.127;t=4.781,P=0.000)。moreover,hign-dosage groups compared with low-dosage groups(t=3.193,P=0.004); (3) The effection of MMP-13 had statistical significance in comparision between 3 group(sP<0.05);low and high- dosage of DON treatment groups(6.45%±1.56%,12.47%±2.45%) were significantly higher than controls(3.06%±1.13%),(LSD-t test:t=3.964, P=0.000; t=11.005,P=0.000).3. FCM,RT-PCR,ELISA result:(1)The rates of cell apoptosis in DON groups were 6.78%～19.05%, which were significantly higher than control 1.20%(P<0.05)(.2) The level of NO in DON groups was 20.8μmol/L～40.7μmol/L, which was significantly higher than control 10.2μmol/L(P<0.05);The level of MMP-13 in DON groups was 0.25μmol/L～0.56μmol/L,which was significantly higher than control 0μmol/L(P<0.05);The level of PGE2 in DON groups was 3.2μmol/L～20.6μmol/L, which was significantly higher than control1.6μmol/L(P<0.05).(3)The proportions of cells with positive iNOS in DON groups were 14.8%～56.8 %,which were significantly higher than controls 7.1 % (P<0.05);The proportions of in dose of 0.4μg/ml and 1.0μg/ml DON groups were 56.7 % and 52.7 %,which were significantly lower than control 62.2 % (P<0.05).(4)The IOD of iNOS mRNA in DON groups was 1.07～1.33, which was significantly higher than control 0.62(P<0.05);The level of collagenⅡmRNA in dose of 0.4μg/ml and 1.0μg/ml DON groups was 0.83 and 0.84, which was significantly lower than control 1.14 (P<0.05).ConclusionDON could promote the increase of iNOS in articular cartilage cells, iNOS could promote anabolism of NO,and moreover,NO increased expression of MMP-13,which both promoted resolution of articular cartilage matrix such as Collagen typeⅡ;DON induced apoptosis in articular cartilage cells. By transmission electron microscope, DON can lead to the impairment of the bone and articular cartilage and the injury outcome is related to the developmental stage of the bone. Before the closure of diaphysis-epiphysis, DON can cause cartilage damage, dysostosis and even stagnation of the bone development. After its closure of diaphysis-epiphysis, the injury was present only in cartilage. This indicates that DON may be prompted to KBD which cause bone and joint diseases.