p22phox和NOX5在缺氧诱导成骨细胞自噬及凋亡中的作用研究

Role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia

目的探讨p22phox和NOX5在缺氧诱导成骨细胞自噬和凋亡中的作用。方法将新生大鼠颅骨组织剪碎,采用组织块贴壁法及差速贴壁法分离纯化成骨细胞。取第1代细胞行HE、茜素红、ALP染色以及流式细胞仪鉴定。采用三气培养箱制备成骨细胞缺氧模型,于缺氧0、3、6、12、24 h时,采用Western blot检测p22phox、NOX5、LC3Ⅱ/Ⅰ表达情况,流式细胞仪检测活性氧簇(reactive oxygen species,ROS)水平以及细胞凋亡率,选取细胞内ROS水平最高时间点作为后续实验的缺氧时间点。将第1代成骨细胞分成正常组、si-p22phox缺氧处理组和si-NOX5缺氧处理组,行对应转染及缺氧处理后,采用RT-PCR检测si-p22phox和si-NOX5抑制效率。然后再将第1代成骨细胞分成正常组、si-NC缺氧处理组、si-p22phox缺氧处理组以及si-NOX5缺氧处理组,行对应转染及缺氧处理后,采用Western blot检测细胞p22phox、NOX5、自噬相关蛋白(LC3Ⅱ/Ⅰ、Beclin)、凋亡相关蛋白(Bcl-2、Bax)表达情况,流式细胞术检测细胞凋亡率和ROS水平。最后,将成骨细胞分成缺氧12 h组(缺氧组)和同时抑制si-p22phox及si-NOX5并缺氧12 h组(抑制+缺氧组),对应处理后免疫荧光染色观察Beclin及Bax表达。结果经鉴定,分离获得的细胞为成骨细胞。缺氧处理后,成骨细胞p22phox、NOX5、LC3Ⅱ/Ⅰ蛋白相对表达量以及细胞凋亡率均逐渐升高(P<0.05),ROS水平亦升高(P<0.05)且12 h达峰值;选择缺氧12 h模型进行后续实验。抑制p22phox基因不影响NOX5的表达,而抑制NOX5基因也不影响p22phox的表达;与单纯缺氧处理相比,抑制p22phox或NOX5基因表达后LC3Ⅱ/Ⅰ、Beclin、Bax蛋白相对表达量下降(P<0.05),Bcl-2蛋白相对表达量增高(P<0.05),细胞凋亡率及ROS水平亦下降(P<0.05);同时抑制p22phox和NOX5基因表达后,免疫荧光染色见Beclin、Bax荧光较弱。结论抑制p22phox和NOX5基因表达可以降低缺氧条件下成骨细胞内ROS水平,同时降低细胞自噬以及凋亡,尤其减弱了细胞早期向晚期凋亡的过渡,增强了缺氧成骨细胞的增殖能力。

Objective To investigate the role of p22 phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.Methods The skull tissue of newborn rats was cut into small pieces,and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method.The first generation cells were harvested and identified by HE staining,Alizarin red staining,alkaline phosphatase(ALP)staining,and flow cytometry.A three-gas incubator was used to prepare a hypoxia model of osteoblasts.At 0,3,6,12,and 24 hours of hypoxia,the expressions of p22 phox,NOX5,and LC3Ⅱ/Ⅰwere detected by Western blot,and the level of reactive oxygen species(ROS)and cell apoptosis rate were detected by flow cytometry.And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments.The first generation osteoblasts were divided into normal group,si-p22 phox hypoxia group,and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment.The inhibition efficiency of si-p22 phox and si-NOX5 were detected by RT-PCR.Then the osteoblasts were divided into normal group,si-NC hypoxia group,si-p22 phox hypoxia group,and si-NOX5 hypoxia group.After transfection and hypoxia treatment,Western blot was used to detect the expressions of p22 phox,NOX5,autophagyrelated proteins(LC3Ⅱ/Ⅰ,Beclin),and apoptosis-related proteins(Bcl-2,Bax),and flow cytometry was used to detect the cell apoptosis rate and level of ROS.The first generation osteoblasts were divided into a hypoxia group for 12 hours(hypoxia group)and a group that simultaneously inhibited si-p22 phox and si-NOX5 and hypoxia for 12 hours(inhibition+hypoxia group).The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.Results After identification,the isolated cells were osteoblasts.After hypoxia treatment,the relative expressions of p22 phox,NOX5,and LC3Ⅱ/Ⅰproteins and the apoptosis rate of osteoblasts gradually increased(P<0.05),and the level of ROS also significantly increased(P<0.05)and reached the peak value at 12 hours.The 12-hour hypoxia model was selected for subsequent experiments.Silencing the p22 phox gene did not affect the expression of NOX5,and silencing the NOX5 gene did not affect the expression of p22 phox.Compared with hypoxia treatment,the relative expressions of LC3Ⅱ/Ⅰ,Beclin,and Bax proteins after inhibiting the expression of p22 phox or NOX5 gene significantly decreased(P<0.05),the relative expression of Bcl-2 protein significantly increased(P<0.05),the cell apoptosis rate and level of ROS also significantly decreased(P<0.05).After silencing the expressions of p22 phox and NOX5 genes at the same time,the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.Conclusion Inhibiting the expressions of p22 phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions,and at the same time reduce autophagy and apoptosis,especially attenuate the excessive apoptosis of cells in the early to late stages,and strengthen the hypoxic osteoblasts proliferation.