Hypoxia induced by CoCl2 influencing the expression and the activity of matrix metalloproteinase-2 in rat hepatic stellate cells / 中华肝脏病杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effects of hypoxia induced by cobalt chloride on the expression and the activity of matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC-T6) and to clarify the possible mechanisms.</p><p><b>METHODS</b>HSC-T6 cell line was grown in Dulbecco's modified Eagle medium with 10% fetal calf serum at 37 degrees C and 5% CO2. When reaching confluence, the cells were incubated with serum-free medium in the presence of cobalt chloride (0, 50, 100, 200 micromol/L) for six hours, and then the supernatant and the cells were harvested. The expression of the MMP-2 mRNA and HIF-1alpha protein in HSC-T6 cells was detected using RT-PCR and Western blot respectively. The activity of the MMP-2 in the supernatant was detected by zymography. The binding reaction between HIF-1a protein and MMP-2 gene sequence was investigated by electrophoresis mobility shift assay.</p><p><b>RESULTS</b>When the concentration of CoCl2 increased from 0 micromol/L to 200 micromol/L, the expressions of MMP-2 mRNA (the rate of light density) were increased from 0.53+0.12 to 1.57+0.11 and the differences among these four groups were significant (F=34.21). The activity of MMP-2 (the value of light density*band area) decreased gradually from 84.49+5.38 to 53.70+3.42, and the differences among these four groups were also significant (F=29.54). The expressions of HIF-1a were increased gradually with the increase of the CoCl2 concentration. The shift band in the lane of the nuclear protein extraction and the MMP-2 probe containing hypoxia response element showed delays when compared with the lane of the sole probe, and the binding was partially abolished when competing sense oligonucleotides were used.</p><p><b>CONCLUSIONS</b>Our results suggest that chemical hypoxia can up-regulate the expression of MMP-2 mRNA and decrease the activity of the enzyme. HIF-1alpha may play a part in the regulation of MMP-2 transcription under hypoxic conditions.</p>