Correlation of matrix metalloproteinases and Kashin-Beck disease / 中华地方病学杂志

ABSTRACT

Objective To investigate the expressions of matrix metalloproteinases(MMPs) in Kashin-Beck disease(KBD) cartilage as well as in a KBD rat model of T-2 toxin poisoning under selenium deficient conditions, and to investigate the effect of T-2 toxin on MMP-13 expression in human chondrocytes in vitro in order to determine a possible mechanism underlying KBD. Methods Samples of articular cartilage were divided into 2 groupscontrols(samples from 5 normal children, traffic accident or operation), and KBD(samples from 5 children with KBD, auctopsy). Thirty-two Sprague-Dawley rats were divided into two groups by body weight using random number table normal diet group(n = 16) and selenium-deficient diet group(n=16). The selenium level in normal diet was 101.500μg/kg, and in selenium-deficient diet was 1.118μg/kg. Rats were fed for 4 weeks with selenium-deficient or normal diet, respectively. After successful build up of the low selenium rat model, normal diet group was then subdivided into 2 sub-groups normal group(n = 8) and normal diet plus low T-2 toxin group(n = 8);and selenium-deficient diet group was also subdivided into 2 sub-groups selenium-deficient group ( n = 8 ) and selenium-deficient diet plus T-2 toxin group ( n = 8 ) . T-2 toxin of 100 μg·kg-1·d-1 was administered by intragastric administration for 30 days. Then the rats were sacrificed, and their knee joints were processed for histopathological evaluation. MMP-1 and MMP-13 locations in cartilages were performed by inmmunohistochemistry. Human chondrocytes C28/I2 were cultured in vitro. The experiment was divided into 4 groups empty vector plasmid group, MMP-13 promoter plasmid group, MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40 μg/L T-2 toxin group. MMP-13-luciferase reporter plasmid and vector plasmid were transiently transfected into C28/I2 cells for 24 hours, and then treated with 20 - 40 μg/L T-2 toxin for 24 hours. Transactivation of human MMP-13 promoter was analyzed using luciferase reporter constructs containing sequences spanning-1602 to+20 bp in C28/I2 chondrocytes. Results The percentages of chondrocytes staining for MMP-1 in the superficial and middle zones of KBD samples [(29.73 ± 10.12)%, (28.27 ± 0.91)%] were significantly higher than those of controls[(2.47 ± 0.11)%, (0.00 ± 0.00)%, all P < 0.05]. The percentages of chondrocytes staining for MMP-13 in the superficial and middle zones of KBD samples [(13.21 ± 4.32)%, (41.85 ± 6.32)%] were significantly higher than those of controls[(5.72 ± 0.31)%, (0.00 ± 0.00)%, all P<0.05]. The percentages of chondrocytes staining for MMP-13 in the superficial and middle zones of rats fed with selenium-deficient diet plus T-2 toxin group[(13.21 ± 4.32)%, (61.85 ± 8.68)%] were significantly higher than those of the normal and selenium-deficient groups[(2.43 ± 0.22)%, (5.89 ± 0.69)%, (3.03 ± 0.29)%, (25.99 ± 0.57)%, all P < 0.05]. Moreover, T-2 toxin activated the MMP-13 promoter detected with luciferase reporter assays in C28/I2 cells. The luciferase activities in MMP-13 promoter plasmid plus 20 μg/L T-2 toxin group and MMP-13 promoter plasmid plus 40μg/L T-2 toxin group(0.082 78 ± 0.008 40, 0.103 35 ± 0.013 19) were significantly higher than those in empty vector plasmid group and MMP-13 promoter plasmid group(0.024 19 ± 0.000 96, 0.040 32 ± 0.003 56, all P < 0.05). Conclusions These data suggest that T-2 toxin induces cartilage matrix degradation through up-regulation of MMP-13 promoter expression. Increased MMPs staining intensity in KBD cartilage and the rat KBD model of T-2 toxin poisoning under selenium deficient conditions suggest that matrix degradation appear to be driven by MMPs activity.