Abstract
OBJECTIVE: To investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro. METHODS: The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO (2), 20%O (2)); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO (2), 1%O (2)); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups. RESULTS: HE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A ( P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B ( P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D ( P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B ( P<0.05). CONCLUSION: Hypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.