Effect and mechanism of HIF-1α regulating wnt/β-catenin signaling pathway on senescence of nucleus pulposus cells in rats under normal oxygen

English Keywords:Hypoxia-inducing factor 1α  wnt/β-catenin signaling pathway  Oxygen concentration  Nucleus pulposus cell senescence

English Abstract:

  【Abstract】 Objectives: To investigate the effect and mechanism of hypoxia-inducing factor 1α(HIF-1α) regulating wnt/β-catenin signaling pathway on senescence of nucleus pulposus cells in rats under normal oxygen. Methods: The primary cells of caudal nucleus pulposus were extracted from 5 female SD rats of 4 weeks old. (1)The nucleus pulposus cells were divided into the following 5 groups: transfection control group[treated with phosphate buffered saline(PBS)], adenovirus unloaded group(treated with unloaded adenovirus), and HIF-1α over-expression group(treated with adenovirus carrying over-expressing HIF-1α plasmid), unloaded siRNA group(treated with unloaded siRNA), and HIF-1α knockdown group(treated with siRNA knocked HIF-1α). HIF-1α, p53, p21, and p16 were detected by Western blot(WB) after normal oxygen culture for 48h, and cell senescence was detected by β-gal staining to evaluate the effect of HIF-1α on nucleus pulposus cell senescence under normal oxygen culture. (2)Nucleus pulposus cells were extracted and divided into adenovirus unloaded group, HIF-1α over-expression group, unloaded siRNA group, and HIF-1α knockdown group, with same treatment as mentioned above for each group. And HIF-1α, GSK-3β, and β-catenin pathway expressions were detected by WB test. Nucleus pulposus cells were extracted and divided into control group(treated with PBS), HIF-1α over-expression group(treated with adenovirus carrying over-expressing HIF-1α plasmid), XAV-939 group(treated with XAV-939), XAV-939+HIF-1α over-expression group(treated with XAV-939+ adenovirus carrying over-expressing HIF-1α plasmid). WB test was used to detect HIF-1α, p53, p21, p16,and wnt/β-catenin signal expressions and β-gal staining was applied to detect cell senescence to investigate the relationship between HIF-1α and wnt/β-catenin signalpathway and the effect of HIF-1α on senescence of nucleus pulposus cells. Results: (1)After adenovirus transfection of HIF-1α, HIF-1α expression increased in HIF-1α over-expression group comparing with transfection control group and adenovirus unloaded group(P<0.05). After siRNA transfection of HIF-1α, HIF-1α expression decreased in HIF-1α knockdown group than transfection control group and siRNA unloaded group(P<0.05). WB result showed that comparing with adenovirus unloaded group, HIF-1α expression increased in HIF-1α over-expression group(P<0.05), while the expressions of p53, p21, and p16 decreased(P<0.05); Comparing with siRNA unloaded group, H1F-1α expression decreased in HIF-1α knockdown group(P<0.05), while the expressions of p53 and p16 increased(P<0.05). β-gal staining showed culture 48h under normal oxygen condition, the number of senescence cells decreased in HIF-1α over-expression group(P<0.05) than adenovirus unloaded group and increased than siRNA unloaded group(P<0.001). (2)In comparison with adenovirus unloadedgroup, HIF-1α over-expression group increased in β-catenin expression(P<0.01) and decreased in GSK-3β expression(P<0.05); In comparison with siRNA unloaded group, HIF-1α knockdown group decreased in β-catenin expression(P<0.0001) and increased in GSK-3β expression(P<0.05). Comparing with the control group, β-catenin expression in XAV-939 group decreased(P<0.001), and the expressions of p53, p21, and p16 increased; β-catenin expression in XAV-939+ HIF-1α over-expression group decreased(P<0.05) and p21 expression increased(P<0.05). The number of senescence cells in XAV-939+HIF-1α over-expression group was bigger than that in HIF-1α over-expression group(P<0.001), and β-catenin expression in HIF-1α over-expression group was more than the control group, while that in XAV-939 group and XAV-939+HIF-1α over-expression group was less(P<0.05). Conclusions: HIF-1α inhibits senescence of rat nucleus pulposus cells cultured under normal oxygen by activating wnt/β-catenin signaling pathway.