Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats

Abstract

To explore the expression and significance of hypoxia-inducible factor 1α (HIF-1α) in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Eight Sprague Dawley rats were selected to obtain the L 1-5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O 2 condition (group A), and under 0.5% O 2 condition (group B). Cell morphology was observed by inverted phase contrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-1α was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-1α mRNA. HIF-1α specific RNAi sequence compound was constructed and transfected into cells. The transfected endplate chondrocytes at passage 3 were cultured under 0.5% O 2 condition in group C and group D (HIF-1α gene was silenced). After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-1α, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-1α, collagen type II (COL II), Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-1α, Bax, and Bcl-2 by Western blot. At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group A and group B; there was no significant difference in apoptosis rate between groups A and B ( t=1.026, P=0.471), and HIF-1α mRNA and protein expressions in group B were significantly higher than those in group A ( t=22.672, P=0.015; t=18.396, P=0.013), but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B ( t=0.594, P=0.781; t=1.251, P=0.342). The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1α positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C ( t=27.143, P=0.002). The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C ( t=21.097, P=0.015; t=34.829, P=0.002; t=18.673, P=0.022; t=31.949, P=0.007). The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C ( t=37.648, P=0.006; t=16.729, P=0.036), but the protein expression of Bax in group D was significantly higher than that in group C ( t=25.583, P=0.011). HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.