白细胞介素-1β对胰岛β细胞胰岛素原剪切的作用及机制研究  

Effect and mechanism of interleukin-1β on proinsulin processing of islets β cells

作　　者：温媛媛 史会连[2] 黄凯玥 覃元 程昱淅 张伟 袁栎[1] 陈园园[3] Wen Yuanyuan;Shi Huilian;Huang Kaiyue;Qin Yuan;Cheng Yuxi;Zhang Wei;Yuan Li;Chen Yuanyuan(Department of Biochemistry and Molecular Biology,Nanjing Medical University,Nanjing 210029,China;Department of Infectious Diseases,Jiangsu Province Hospital of Chinese Medicine,Nanjing 210004,China;Key Laboratory of Human Functional Genomics of Jiangsu Province,Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学基础医学院生物化学与分子生物学系,南京210029 [2]江苏省中医院感染科,南京210004 [3]南京医科大学、江苏省人类功能基因组学重点实验室,南京210029

出　　处：《中华糖尿病杂志》2023年第9期835-843,共9页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金(31101011, 82070804, 81903974);江苏省自然科学基金(BK20221421)。

摘　　要：目的探讨白细胞介素-1β(IL-1β)对胰岛β细胞中胰岛素原剪切的影响及作用机制。方法将原代小鼠胰岛分为对照组(正常培养)和IL-1β处理组, 将大鼠β细胞系INS-1细胞分为对照组、IL-1β处理组、15 μmol/L SKF9636处理组、15 μmol/L SKF96365+IL-1β处理组、1 μmol/L Go6976处理组、1 μmol/L Go6976+IL-1β处理组、10 μmol/L U0126处理组、10 μmol/L U0126+IL-1β处理组、30 μmol/L磷脂酰肌醇3-激酶(PI3K)通路抑制剂LY294002处理组、30 μmol/L LY294002+IL-1β处理组、2 μmol/L核因子-κB(NF-κB)通路抑制剂BAY117082处理组、2 μmol/L BAY117082+IL-1β处理组。每组设置3个平行组。通过酶联免疫吸附试验法检测胰岛素原和总胰岛素浓度, 并以胰岛素原与总胰岛素的比值来评价胰岛β细胞中胰岛素原的剪切水平。通过实时荧光定量聚合酶链反应和Western blotting法检测IL-1β处理后胰岛素原剪切关键酶激素原转化酶(PC)1/3、PC2蛋白及其编码基因Pcsk1和Pcsk2的表达。对IL-1β作用的信号通路进行筛选检测, 通过qPCR和Western blotting法确定IL-1β调控Pcsk1和Pcsk2表达的信号通路。两组间比较采用独立样本t检验, 多组间比较采用单因素方差分析。结果在原代小鼠胰岛中, 与对照组相比, IL-1β处理组使得低糖孵育时原代小鼠胰岛的上清液中胰岛素原占比由5.5%±0.8%升高为31.5%±2.1%(P<0.01)。INS-1细胞培养上清中胰岛素原占比由7.2%±0.5%升高为22.9%±3.2%(P<0.01);在原代小鼠胰岛中, IL-1β处理组Pcsk1和Pcsk2 mRNA水平与对照组相比均显著降低(Pcsk1 mRNA分别为0.40±0.06和1.00, Pcsk2 mRNA分别为0.37±0.02和1.00);IL-1β处理组PC1/3和PC2蛋白水平与对照组相比也显著降低(PC1/3分别为0.38±0.08和0.87±0.05, PC2分别为0.28±0.04和0.85±0.03), 差异均具有统计学意义(P<0.01)。在INS-1细胞中, IL-1β处理组Pcsk1和Pcsk2 mRNA水平与对照组相比均显著降低(Pcsk1 mRNA分别为0.52±0.06和1.00, Pcsk2 mRNA�Objective To investigate the effect and mechanism of interleukin-1β(IL-1β)on proinsulin processing in isletsβcells.Methods Mouse islets were divided into normal group and IL-1βgroup,ratβ-cell line INS-1 cells were divided into control group(normal culture),IL-1βgroup,15μmol/L SKF9636 group,15μmol/L SKF96365+IL-1βgroup,1μmol/L Go6976 group,1μmol/L Go6976+IL-1βgroup,10μmol/L U0126 group,10μmol/L U0126+IL-1βgroup,30μmol/L phosphatidylinositol 3-kinase(PI3K)pathway inhibitor LY294002 group,30μmol/L LY294002+IL-1βgroup,2μmol/L nuclear factor-κB(NF-κB)inhibitors pathway BAY117082 group,2μmol/L BAY117082+IL-1βgroup.Three parallels were established for each group.The concentration of proinsulin and total insulin were determined by enzyme-linked immunosorbent assay,then the ratio of proinsulin to total insulin was calculated to analyze the proinsulin processing in isletsβcells.The mRNA levels of Pcsk1 and Pcsk2(encoding key enzymes for proinsulin processing)and the protein levels of prohormone convertase(PC)1/3 and PC2 were then detected by quantitative real-time PCR and Western blotting,respectively.In addition,the signal pathway of IL-1βwas detected through using of signal pathway inhibitors with quantitative polymerase chain reaction(qPCR)and Western blotting.Independent sample t test was used for comparison between two groups,and one-way analysis of variance was used for comparison between multiple groups.ResultsCompared with the control group,IL-1βgroup increased the ratio of proinsulin to total insulin from 5.5%±0.8%to 31.5%±2.1%in the supernatant of isolated mouse islets in low-glucose incubation and from 7.2%±0.5%to 22.9%±3.2%in the supernatant of INS-1 cells(both P<0.01).In mouse islets,Pcsk1 and Pcsk2 mRNA levels were both significantly decreased in the IL-1βgroup compared with the control group(Pcsk1 mRNA:0.40±0.06 vs.1.00,Pcsk2 mRNA:0.37±0.02 vs.1.00,respectively);PC1/3 and PC2 protein levels were also significantly decreased in the IL-1βgroup compared with the control group(P

关 键 词：胰岛 Β细胞 白细胞介素-1Β 胰岛素原剪切 激素原转化酶基因 

分 类 号：R587.1[医药卫生—内分泌]