Effects of low-dose T-2 toxin on cultured cartilage cells and molecular mechanism

Abstract

Objective Through observing the effect of low-dose T-2 toxin on chondrocyte, to study the molecular mechanism of cartilage damage. Methods The primary chondrocytes were isolated from articular cartilage of d 1-2 Wistar neonate rats through enzymatic digestion. Different doses (0.005, 0.010, 0.100 μg/L) of T-2 toxin were added after 24 h in vitro culture. The survival rate of chondrocytes was detected with Trypan blue staining. Echylosis (matrix metalloproteinase, MMP1) was analyzed by immunohistochemistry. The damage of articular chondrocyte was observed by transmission electron microscope. Results ①Cell morphology of in vitro cultured chondrocyte: the newly isolated chondrocytes were spherical. After 24 hours, the adherent cells gradually began to stretch the triangle or polygon; the nucleus was large and round; the cell was clear and transparent, containing secretory granules. ②Cell proliferation: T-2 toxin had a significant inhibitory effect on chondrocyte proliferation, the higher the concentration of T-2 toxin, the significant the inhibitory effects [0.000 μg/L(control) group: 3.45 × 108/L, 0.005 μg/L T-2 toxin group: 3.45 × 108/L, 0.010 μg/L T-2 toxin group: 2.06 × 108/L, χ2 = 9.554, P< 0.05]. ③Immunohistochemical observation: dysplasia, nucleus condensation and membrane rupture were observed in T-2 toxin treated group, brown staining was observed in all groups at varying degrees. The deepest staining was in 0.005 μg/L T-2 toxin group, with the strongest secretion of MMP1; with increasing doses of the toxin, the damage to cartilage cells was severe, MMP1 secretion was less, staining was weak, and the weakest staining was in the 0.100 μg/L T-toxin group. ④Under transmission electron microscopy: in control group, cytoplasm was rich in rough endoplasmic reticulum, nuclear membrane and cell membrane were clear; in 0.005 μg/L T-2 toxin group, the cell nucleus showed pyknosis, organelles were decreased in cytoplasm; in 0.100 μg/L T-2 toxin group, the microvilli was dropped out of cartilage surface, nuclear changes were obvious, and mitochondria was myeloid degeneration; rough endoplasmic reticulum was degranulation and expansion into cystiform, chondrocytes were apoptosis occasionally, the cell nucleus showed pyknosis, and the formation of high-density plaque. Conclusion Low dose of T-2 toxin could damage the primary cultured articular chondrocyte in vitro. The results have showed that there are damaged cytostasis, chondrocyte degeneration, necrosis and apoptosis. Key words: T-2 toxin; Chondrocyte; Injury mechanism; Cell culture in vitro