目的 旨在结合转录组测序的结果,聚焦糖尿病视网膜病变中的氧化应激水平和炎症水平,探讨硒结合蛋白1(selenium binding protein 1,SELENBP1)在糖尿病视网膜病变中的作用.方法对12周龄的db/m和db/db小鼠的视网膜组织进行转录组测序分析筛选差异基因.实验分为两组:db/m小鼠、db/db小鼠,对视网膜组织切片进行活性氧荧光染色,应用蛋白免疫印迹(Western-blot)检测小鼠视网膜组织中的氧化应激反应相关指标还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NOX)2、NOX4、超氧化物歧化酶2(SOD2)和炎性反应相关指标核因子-κB(NF-κB)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β的变化趋势;用实时荧光定量聚合酶链反应(qRT-PCR)、Western-blot和免疫组化(IHC)分析组织中SELENBP1的水平.在体外,应用高糖干预人视网膜微血管内皮细胞(HRMEC),实验分为两组:正常糖组(NG组)、高糖干预组(HG组)检测活性氧水平及氧化应激和炎性因子的表达;在HRMEC细胞中转染SELENBP1质粒和SELENBP1小干扰RNA,分为正常糖对照组(NG+Vector)、SELENBP1 质粒组(NG+SBP1)、高糖对照组(HG+si-NC)、SELENBP1 小干扰 RNA 组(HG+si-SBP1),分析活性氧水平及氧化应激和炎性因子的表达水平.结果 与db/m小鼠相比,db/db小鼠视网膜组织中活性氧含量升高、氧化应激相关因子表达增加、抗氧化因子表达下降、炎性因子表达增加,且SELENBP1的mRNA和蛋白水平都明显升高.体外实验表明,高糖干预HRMEC细胞后,细胞中活性氧水平、氧化应激水平和炎症水平明显升高,SELENBP1的mRNA和蛋白表达均明显升高;在HRMEC细胞中过表达SELENBP1后氧化应激水平和炎症水平明显升高;在HRMEC细胞中敲低SELENBP1后氧化应激水平和炎症水平明显下降.结论SELENBP1通过促进视网膜的氧化应激水平和炎症水平加重了糖尿病视网膜病变,抑制该基因的表达可能会成为减轻糖尿病视网膜病变的有效治疗靶点.
SELENBP1 ameliorates diabetic retinopathy by reducing oxidative stress and inflammation
Objective Aiming to combine the results of transcriptome sequencing and focus on the levels of oxidative stress and inflammation in diabetic retinopathy,investigating the role of selenium binding protein 1(SELENBP1)in diabetic retinopathy.Methods Transcriptome sequencing analysis was per-formed on retinal tissues from 12-week-old db/m and db/db mice to screen for differential genes.The ex-periment was divided into two groups:db/m mice and db/db mice,reactivingoxygen species fluorescence staining of retinal tissue sections.Western-blot was used to detect the changing trend of oxidative stress re-sponse-related indicators such as NADPH oxidase 2(NOX2),NADPH oxidase 4(NOX4),superoxide dismutase 2(SOD2),and inflammatory response-related index nuclear factor kappa B(NF-κB),tumor nec-rosis factor-alpha(TNF-α),interleukin 1 beta(IL-1β)in mouse retinal tissue.The level of SELENBP1 in tissues was analyzed by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR),Western-blot,and immunohistochemistry.In vitro,high glucose was used to intervene with human retinal microvascu-lar endothelial cells(HRMEC).The experiment was divided into two groups:the normal glucose group(NG)and the high glucose intervention group(HG),the levels of ROS and the expression of oxidative stress and inflammation-related factors were detected;then HRMEC cells transfected with SELENBP1 plas-mid and SELENBP1 small interfering RNA,they were divided into normal glucose control group(NG+Vector),SELENBP1 plasmid group(NG+SBP1),high glucose control group(HG+si NC),SELENBP1 small interfering RNA group(HG+si-SBP1),were subjected to the same detection as HRMEC cells after high glucose intervention.Results Compared with db/m mice,the content of ROS increased,the expression of oxidative stress-related factors increased,the expression of antioxidant factors decreased,and the expression of inflammation-related factors increased in the retinal tissue of db/db mice,and the mR-NA and protein levels of SELENBP1 were significantly increased.In vitro experiments showed that after high glucose intervention in HRMES cells,the levels of ROS,oxidative stress,and inflammation were signifi-cantly increased,and the mRNA and protein expression of SELENBP1 were significantly increased;overex-pression of SELENBP 1 in HRMES cells significantly increased the levels of oxidative stress and inflamma-tion;The opposite was true after knockdown of SELENBP 1 in HRMEC cells.Conclusion SELEN-BP1aggravates diabetic retinopathy by promoting the level of oxidative stress and inflammation in cells and tissues.Inhibiting the expression of this gene may become an effective therapeutic target to alleviate diabetic retinopathy.
万方数据
维普
