缺氧诱导因子1α调控wnt/β-catenin信号通路对常氧条件下大鼠髓核细胞衰老的作用及机制

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

中文关键词:  缺氧诱导因子1α  wnt/β-catenin信号通路  氧气浓度  髓核细胞衰老

英文关键词:Hypoxia-inducing factor 1α  wnt/β-catenin signaling pathway  Oxygen concentration  Nucleus pulposus cell senescence

中文摘要:

  【摘要】 目的：探究缺氧诱导因子1α（HIF-1α）调控wnt/β-catenin信号通路对常氧培养下大鼠髓核细胞衰老的影响及作用机制。方法：取5只4周龄雌性SD大鼠，提取鼠尾髓核原代细胞进行研究。（1）将髓核细胞分为5组：转染对照组[用磷酸盐缓冲盐水（PBS）处理髓核细胞]、空载腺病毒组（用空载腺病毒处理髓核细胞）、过表达HIF-1α组（用载过表达HIF-1α质粒的腺病毒处理髓核细胞）、空载siRNA组（用空载siRNA处理髓核细胞）、敲减HIF-1α组（用siRNA敲减HIF-1α处理髓核细胞），在常氧下培养48h后，免疫蛋白印迹（western blot，WB）检测HIF-1α和衰老相关基因p53、p21、p16，β-gal染色检测细胞衰老，评估常氧条件下HIF-1α对于髓核细胞衰老的影响。（2）取髓核细胞，分为空载腺病毒组、过表达HIF-1α组、空载siRNA组、敲减HIF-1α组，处理方式同前，在常氧下培养48h后，WB检测HIF-1α、GSK-3β和β-catenin通路的表达。取髓核细胞，分为对照组（用PBS处理髓核细胞）、过表达HIF-1α组（用载过表达HIF-1α质粒的腺病毒处理髓核细胞）、XAV-939组（用XAV-939处理髓核细胞）、XAV-939+过表达HIF-1α组（用XAV-939+载过表达HIF-1α质粒的腺病毒处理髓核细胞），WB检测HIF-1α、p53、p21、p16和wnt/β-catenin的表达，β-gal染色检测细胞衰老，以检测HIF-1α与wnt/β-catenin信号通路的关系以及对髓核细胞衰老的影响。结果：（1）腺病毒转染HIF-1α后，与转染对照组和空载腺病毒组相比，过表达HIF-1α组HIF-1α表达增加（P<0.05）。小干扰RNA转染HIF-1α后，与转染对照组和空载siRNA组相比敲减HIF-1α组HIF-1α表达降低（P<0.05）。WB结果显示，与空载腺病毒组相比过表达HIF-1α组HIF-1α表达增加（P<0.05），而p53、p21和p16表达降低（P<0.05），与空载siRNA组相比敲减HIF-1α组HIF-1α表达降低（P<0.05），而p53和p16表达增加（P<0.05）。β-gal染色显示，在常氧条件下培养48h，与空载腺病毒组相比，过表达HIF-1α组衰老细胞降低（P<0.05），与空载siRNA组相比，敲减HIF-1α组衰老细胞数量则增加（P<0.001）。（2）与空载腺病毒组相比，过表达HIF-1α组β-catenin表达升高（P＜0.01），同时GSK-3β表达降低（P＜0.05）。与空载siRNA组相比敲减HIF-1α组β-catenin表达降低（P＜0.0001），同时GSK-3β表达升高（P＜0.05）。与对照组相比，XAV-939组β-catenin表达降低（P<0.001），p53、p21和p16的表达升高，XAV-939+过表达HIF-1α组β-catenin表达降低（P<0.05），p21表达升高（P<0.05）。与过表达HIF-1α组相比XAV-939+过表达HIF-1α组衰老细胞数增加（P<0.001），与对照组相比过表达HIF-1α组的细胞核内β-catenin表达更多，而XAV-939组和XAV-939+过表达HIF-1α组则相对较少（P<0.05）。结论：HIF-1α通过激活wnt/β-catenin信号通路抑制常氧条件培养下大鼠髓核细胞的衰老。

英文摘要:

  【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.