Han Xiao, Dong Xiao-Xuan, Shi Ming-Yu, Feng Li, Wang Xin-Ling, Zhang Jin-Song, Yan Qi-Chang
Department of Ophthalmology, the Fourth Affiliated Hospital of China Medical University; Key Laboratory of Lens Research of Liaoning Province; Eye Hospital of China Medical University, Shenyang 110005, Liaoning Province, China.
Department of Ophthalmology, the Fourth People's Hospital of Shenyang, Shenyang 110031, Liaoning Province, China.
Int J Ophthalmol. 2019 Sep 18;12(9):1371-1379. doi: 10.18240/ijo.2019.09.01. eCollection 2019.
To explore the effects of IκBα SUMOylation and NF-κB p65 deacetylation on NF-κB p65 activity induced by high glucose in cultured human lens epithelial cells (HLECs).
HLECs (SRA01/04) were cultured with 5.5, 25, and 50 mmol/L glucose media for 24h, and with 50 mmol/L glucose media for 0, 12, and 24h respectively. SUMO1 and SIRT1 expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (WB). IκBα and NF-κB p65 expressions were detected by WB. With NAC, DTT, MG132 or Resveratrol (RSV) treatment, SUMO1 and SIRT1 expressions were detected by WB. Protein expression localizations were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO1 or SIRT1 overexpression, as well as MG132 and RSV, on the nuclear expression and activity of IκBα and NF-κB p65 were analyzed by immunoblot and dual luciferase reporter gene assay.
SUMO1 and SIRT1 expressions were influenced by high glucose in mRNA and protein levels, which could be blocked by NAC or DTT. SUMO1 was down-regulated by using MG132, and SIRT1 was up-regulated under RSV treatment. IκBα nuclear expression was attenuated and NF-κB p65 was opposite under high glucose, while IκBα and NF-κB p65 location was transferred to the nucleus. SUMO1 or SIRT1 overexpression and MG132 or RSV treatment affected the nuclear expression and activity of IκBα and NF-κB p65 under high glucose condition.
IκBα SUMOylation and NF-κB p65 deacetylation affect NF-κB p65 activity in cultured HLECs under high glucose, and presumably play a significant role in controlling diabetic cataract.
探讨IκBα 小泛素样修饰（SUMO）化和核因子κB（NF-κB）p65去乙酰化对高糖诱导的培养人晶状体上皮细胞（HLECs）中NF-κB p65活性的影响。
将HLECs（SRA01/04）分别用5.5、25和50 mmol/L葡萄糖培养基培养24小时，以及用50 mmol/L葡萄糖培养基分别培养0、12和24小时。通过逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹法（WB）检测SUMO1和沉默信息调节因子1（SIRT1）的表达。通过WB检测IκBα和NF-κB p65的表达。用N-乙酰半胱氨酸（NAC）、二硫苏糖醇（DTT）、MG132或白藜芦醇（RSV）处理后，通过WB检测SUMO1和SIRT1的表达。通过免疫荧光和共免疫荧光检查蛋白质表达定位。通过免疫印迹和双荧光素酶报告基因检测分析SUMO1或SIRT1过表达以及MG132和RSV对IκBα和NF-κB p65核表达及活性的影响。
SUMO1和SIRT1的表达在mRNA和蛋白质水平受高糖影响，这可被NAC或DTT阻断。使用MG132可使SUMO1下调，RSV处理可使SIRT1上调。高糖条件下IκBα核表达减弱而NF-κB p65相反，同时IκBα和NF-κB p65的定位转移至细胞核。SUMO1或SIRT1过表达以及MG132或RSV处理在高糖条件下影响IκBα和NF-κB p65的核表达及活性。
IκBα SUMO化和NF-κB p65去乙酰化在高糖条件下影响培养的HLECs中NF-κB p65活性，可能在糖尿病性白内障的发生中起重要作用。
