Promotion of the articular cartilage proteoglycan degradation by T-2 toxin and selenium protective effect

Abstract

Objective: To identify the relationship between T-2 toxin and Kashin-Beck disease (KBD), the effects of T-2 toxin on aggrecan metabolism in human chondrocytes and cartilage were investigated in vitro.

Methods: Chondrocytes were isolated from human articular cartilage and cultured in vitro. Hyaluronic acid (HA), soluble CD44 (sCD44), IL-1beta and TNF-alpha levels in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). CD44 content in chondrocyte membrane was determined by flow cytometry (FCM). CD44, hyaluronic acid synthetase-2 (HAS-2) and aggrecanases mRNA levels in chondrocytes were determined using reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemical method was used to investigate expressions of BC-13, 3-B-3(-) and 2-B-6 epitopes in the cartilage reconstructed in vitro.

Results: T-2 toxin inhibited CD44, HAS-2, and aggrecan mRNA expressions, but promoted aggrecanase-2 mRNA expression. Meanwhile, CD44 expression was found to be the lowest in the chondrocytes cultured with T-2 toxin and the highest in control plus selenium group. In addition, ELISA results indicated that there were higher sCD44, IL-1beta and TNF-alpha levels in T-2 toxin group. Similarly, higher HA levels were also observed in T-2 toxin group using radioimmunoprecipitation assay (RIPA). Furthermore, using monoclonal antibodies BC-13, 3-B-3 and 2-B-6, strong positive immunostaining was found in the reconstructed cartilage cultured with T-2 toxin, whereas no positive staining or very weak staining was observed in the cartilage cultured without T-2 toxin. Selenium could partly inhibit the effects of T-2 toxin above.

Conclusion: T-2 toxin could inhibit aggrecan synthesis, promote aggrecanases and pro-inflammatory cytokines production, and consequently induce aggrecan degradation in chondrocytes. These will perturb metabolism balance between aggrecan synthesis and degradation in cartilage, inducing aggrecan loss in the end, which may be the initiation of the cartilage degradation.

Fig. 1

H & E staining of cartilage reconstructed in vitro. (a) and (b) Staining of cartilage reconstructed without T-2 toxin. 5~10 layers of chondrocytes were grown on BMG, and the cell was intact with clear red in cytoplasm and blue in nuclei; (c) and (d) Staining of cartilage reconstructed with T-2 toxin. Only 1~5 layers of cells were grown on BMG. Nuclei staining became lighter, nuclear fragmentations and many dots as well as pellets of necrosis could be seen. The magnification of (a) and (c) was 10× by light microscope and (b) and (d) was 40×

Fig. 1

H & E staining of cartilage reconstructed in vitro. (a) and (b) Staining of cartilage reconstructed without T-2 toxin. 5~10 layers of chondrocytes were grown on BMG, and the cell was intact with clear red in cytoplasm and blue in nuclei; (c) and (d) Staining of cartilage reconstructed with T-2 toxin. Only 1~5 layers of cells were grown on BMG. Nuclei staining became lighter, nuclear fragmentations and many dots as well as pellets of necrosis could be seen. The magnification of (a) and (c) was 10× by light microscope and (b) and (d) was 40×

Fig. 1

H & E staining of cartilage reconstructed in vitro. (a) and (b) Staining of cartilage reconstructed without T-2 toxin. 5~10 layers of chondrocytes were grown on BMG, and the cell was intact with clear red in cytoplasm and blue in nuclei; (c) and (d) Staining of cartilage reconstructed with T-2 toxin. Only 1~5 layers of cells were grown on BMG. Nuclei staining became lighter, nuclear fragmentations and many dots as well as pellets of necrosis could be seen. The magnification of (a) and (c) was 10× by light microscope and (b) and (d) was 40×

Fig. 1

H & E staining of cartilage reconstructed in vitro. (a) and (b) Staining of cartilage reconstructed without T-2 toxin. 5~10 layers of chondrocytes were grown on BMG, and the cell was intact with clear red in cytoplasm and blue in nuclei; (c) and (d) Staining of cartilage reconstructed with T-2 toxin. Only 1~5 layers of cells were grown on BMG. Nuclei staining became lighter, nuclear fragmentations and many dots as well as pellets of necrosis could be seen. The magnification of (a) and (c) was 10× by light microscope and (b) and (d) was 40×

Fig. 2

RT-PCR analysis of T-2 toxin effects on the mRNA expression of aggrecan metabolism related biomacromolecules in human chondrocytes. (a) Representative RT-PCR results. Total RNA was extracted from human chondrocytes which had been cultured for 5 d with or without T-2 toxin and selenium; (b) Relative mRNA levels of CD44H, HAS-2, aggrecan, aggrecanase-2 and GAPDH. Results were normalized to GAPDH level. Results are presented as mean±SEM (n=4)

Fig. 2

RT-PCR analysis of T-2 toxin effects on the mRNA expression of aggrecan metabolism related biomacromolecules in human chondrocytes. (a) Representative RT-PCR results. Total RNA was extracted from human chondrocytes which had been cultured for 5 d with or without T-2 toxin and selenium; (b) Relative mRNA levels of CD44H, HAS-2, aggrecan, aggrecanase-2 and GAPDH. Results were normalized to GAPDH level. Results are presented as mean±SEM (n=4)

Fig. 3

DNA content in different groups. Chondrocytes were cultured for 5 d with or without T-2 toxin and selenium. DNA content in T-2 toxin group was significantly lower than that in control group. ^* P=0.002, T-2 toxin group vs control group; ^# P=0.003, T-2 toxin+Se group vs control+Se group (mean±SEM, n=6)

Fig. 4

HA levels in culture media. The chondrocytes were cultured for 5 d and the culture medium was collected. HA levels were normalized to DNA content. HA level in T-2 group was the highest and the lowest in the control group. ^* P=0.001, T-2 toxin group vs control group; ^# P=0.01, T-2 toxin+Se group vs control+Se group (mean±SEM, n=5)

Fig. 5

IL-1β and TNF-α levels in culture media. Chondrocytes were cultured for 5 d, and the culture media were collected to investigate IL-1β and TNF-α levels by ELISA. The samples were normalized to DNA content of the same flask in order to remove the errors caused by inequality in chondrocytes number. ^*Control group vs T-2 group, P<0.05; ^#Control+Se group vs T-2+Se group, P<0.05 (mean±SEM, n=5)

Fig. 6

sCD44 levels in culture media. Chondrocytes were cultured for 5 d, and the culture media were collected to investigate sCD44 levels. sCD44 levels were normalized to DNA content to remove the errors caused by inequality in chondrocytes number. ^*Control group vs T-2 group, P<0.05 (mean±SEM, n=5)

Fig. 7

Immunostaining for neoepitope NITEGE on the cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 7

Immunostaining for neoepitope NITEGE on the cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 7

Immunostaining for neoepitope NITEGE on the cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 7

Immunostaining for neoepitope NITEGE on the cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 8

Immunostaining with monoclonal antibody 3-B-3(−) on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 8

Immunostaining with monoclonal antibody 3-B-3(−) on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 8

Immunostaining with monoclonal antibody 3-B-3(−) on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 8

Immunostaining with monoclonal antibody 3-B-3(−) on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 9

Immunostaining with monoclonal antibody 2-B-6 on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 9

Immunostaining with monoclonal antibody 2-B-6 on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 9

Immunostaining with monoclonal antibody 2-B-6 on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×

Fig. 9

Immunostaining with monoclonal antibody 2-B-6 on cartilage reconstructed in vitro. Chondrocytes were cultured for 18 d after seeded on the BMG. (a) Control group; (b) Control+Se group; (c) T-2 group; (d) T-2+Se group. NITEGE epitope was stained red and nuclei were blue. The magnification was 20×