Analysis Of The Correlation Between HIF-1α And Autophagy In Rat Nucleus Pulposus Cells Under Hypoxia

Objective:To control the oxygen content to simulate the hypoxic environment of normal nucleus pulposus cells,to investigate whether hypoxia can induce autophagy in rat nucleus pulposus cells,and to further study the hypoxia-inducible factor-1αin nucleus pulposus cells under hypoxia.Whether the expression level of the molecule has a certain correlation with the expression level of autophagy marker molecules such as LC3 and Beclin1.Methods:Healthy adult SD rats with uniform body weight and no degeneration of the intervertebral disc were taken.After execution,their tail bones were taken and nucleus pulposus cells were extracted.The extracted nucleus pulposus cells were purified by differential adherence and then subcultured.At the third generation,observe the cell morphology through a microscope,and then stain the third generation nucleus pulposus cells with hematoxylin-eosin staining and type II collagenase immunofluorescence staining to observe the cell morphology and staining results.For the identification of nucleus pulposus cells.After the identified cells were identified as rat nucleus pulposus cells,the third generation of nucleus pulposus cells were taken and randomly divided into 4 groups of A,B,C,and D.Group A was placed in Culture at normal oxygen content（37°C,5%CO2,20%O2）;group B was cultured under low oxygen（37°C,5%CO2,1%O2）;group C cells were first treated with HIF-1α-siRNA Transfection and culture under the same hypoxia conditions as group B;group D nucleus pulposus cells were added with autophagy inhibitor 3-MA^[1],and then placed in the same hypoxia as group B Culture under oxygen environment.After culturing the above four groups of cells for 8 hours,extract the protein and RNA of each group,and then use Western blot technology.Hypoxia and qRT-PCR technology to detect the amount of induced expression of autophagy-related genes LC3 and hypoxia-inducible factor-1αResults:By observing the morphology and staining results of the extracted cells,it was confirmed that the purified cells were rat nucleus pulposus cells.Western blot and qRT-PCR can detect hypoxia-inducible factor-1αand Expression of phagocytic marker molecules.Compared with group A,the expression levels of hypoxia-inducible factor-1αand autophagy marker molecules^[2]LC3 and Beclin1 in group B increased significantly,and the difference was statistically significant.（P<0.05）.Compared with group B,the expression levels of hypoxia-inducible factor-1αand autophagy marker molecules in nucleus pulposus cells in group C were significantly decreased,and the difference was statistically significant（P<0.05）.Compared with group B,In comparison,the expression of LC3 and other autophagy markers LC3 in group D was significantly reduced,and the difference was statistically significant（P<0.05）;the expression of hypoxia-inducible factor-1αwas almost the same as that in group B.（P>0.05）.Conclusion:Hypoxic environment can induce autophagy in rat nucleus pulposus cells and hypoxia-inducible factor-1αhas a certain correlation with autophagy in hypoxia environment,that is,the expression of hypoxia-inducible factor-1αDecreasing the level of autophagy in rat nucleus pulposus cells,while reducing the expression of autophagy marker molecules has little effect on the expression of hypoxia-inducible factor-1α.