摘要: |
目的 探讨达格列净通过p38丝裂原活化蛋白激酶(p38 MAPK)信号通路对D-葡萄糖诱导的人肾小球足细胞凋亡、自噬、炎症反应及氧化损伤的影响。方法 体外培养人肾小球足细胞(HGPCs),分为对照组(5 mmol/L D-葡萄糖)、D-葡萄糖组(30 mmol/L D-葡萄糖)、达格列净组(30 mmol/L D-葡萄糖+50 μmol/L达格列净)、抑制剂组(30 mmol/L D-葡萄糖+10 μmol/L p38 MAPK通路抑制剂SB 203580)、达格列净+抑制剂组(30 mmol/L D-葡萄糖+50 μmol/L达格列净+10 μmol/L SB 203580)和达格列净+激活剂组(30 mmol/L D-葡萄糖+50 μmol/L达格列净+10 μmol/L p38 MAPK通路激活剂C16-PAF)。对照组与D-葡萄糖组用D-葡萄糖干预24 h;其他组D-葡萄糖干预24 h后相应药物继续干预 24 h。采用细胞计数试剂盒-8(CCK-8)检测细胞活力;采用Hoechst 33258染色法检测细胞凋亡率;采用ELISA检测白细胞介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)、丙二醇(MDA)和超氧化物歧化酶(SOD)的表达水平;采用实时荧光定量PCR(RT-qPCR)检测酵母ATG6同源物(Beclin-1)、微管相关蛋白1轻链3 Ⅱ(LC3 Ⅱ)mRNA表达水平;采用Western印迹法检测Beclin-1、LC3 Ⅱ、p53、p38 MAPK及p-p38 MAPK蛋白表达。结果 与对照组相比,D-葡萄糖组细胞活力降低(P<0.05),达格列净组细胞活力升高(P<0.05),细胞凋亡率,IL-1β、IL-6、TNF-α、MDA、Beclin-1、LC3 ⅡmRNA和蛋白、p53和p-p38 MAPK蛋白水平升高(P<0.05),SOD水平降低(P<0.05)。与D-葡萄糖组相比,达格列净组和抑制剂组细胞凋亡率、IL-1β、IL-6、TNF-α、MDA、Beclin-1、LC3 ⅡmRNA和蛋白、p53和p-p38 MAPK蛋白水平降低(P<0.05),SOD水平升高(P<0.05)。与达格列净组相比,达格列净+抑制剂组细胞凋亡率、IL-1β、IL-6、TNF-α、MDA、Beclin-1、LC3 Ⅱ mRNA和蛋白、p53和p-p38 MAPK蛋白水平进一步降低(P<0.05),SOD水平进一步升高(P<0.05);达格列净+激活剂组与达格列净+抑制剂组变化趋势相反,与达格列净组差异有统计学意义(P<0.05)。结论 达格列净可抑制高糖诱导的人HGPCs的凋亡、自噬、炎症反应及氧化损伤,其作用机制可能与抑制p38 MAPK通路信号转导相关。 |
关键词: 糖尿病肾病 足细胞 达格列净 p38丝裂原活化蛋白激酶信号通路 |
DOI:10.12025/j.issn.1008-6358.2023.20230113 |
分类号:R 692 |
基金项目:广西省来宾市科学研究与技术开发计划项目(来科转220907). |
|
Dapagliflozin inhibits human glomerular podocytes damage through p38 mitogen-activated protein kinase |
LUO Ling-guang, LONG Xin-ping, WEI Shao-heng
|
Department of Endocrinology, the People's Hospital of Laibin, Laibin 546100, Guangxi, China
|
Abstract: |
Objective To explore the effects of dapagliflozin on D-glucose-induced apoptosis, autophagy, inflammation and oxidative damage in human glomerular podocytes through p38 mitogen-activated protein kinase (MAPK) signaling pathway. Methods The human glomerular podocytes (HGPCs) were divided into control group (5 mmol/L D-glucose), D-glucose group (30 mmol/L D-glucose), dapagliflozin group (30 mmol/L D-glucose+50 μmol/L dapagliflozin), inhibitor group (30 mmol/L D-glucose+ 10 μmol/L p38 MAPK pathway inhibitor SB 203580), Dapagliflozin+inhibitor group (30 mmol/L D-glucose+50 μmol/L dapagliflozin+10 μmol/L SB 203580) and dapagliflozin+activator group (30 mmol/L D-glucose+50 μmol/L dapagliflozin+10 μmol/L p38 MAPK pathway activator C16-PAF) . HGPCs were cultured for 24 hours in the control and D-glucose groups, and were intervened for 24 hours with corresponding drugs after cultured for 24 hours. Cell counting kit-8 (CCK-8) was used to detect the cell viability. Hoechst 33258 staining was used to detect the apoptosis rate. ELISA was used to detect the expression levels of inflammatory factors interleukin (IL) -1β, IL-6, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) and superoxide dismutase (SOD). Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expression levels of yeast ATG6 homolog (Beclin-1) and microtubule-associated protein 1 light chain 3 (LC3) Ⅱ. Western blotting was used to detect the protein expressions of Beclin-1, LC3Ⅱ, p53, p38 MAPK and p-p38 MAPK. Results Compared with the Control group, the cell viability was significantly decreased in the D-glucose group (P<0.05) and was increased in the dapagliflozin group (P<0.05). Compared with the control group, the apoptosis rate, IL-1β, IL-6, TNF-α, MDA, mRNA and protein levels of Beclin-1 and LC3 Ⅱ, protein levels of p53 and p-p38 MAPK were increased (P<0.05), and SOD level was decreased (P<0.05) in the D-glucose group. Compared with the D-glucose group, the apoptosis rate, IL-1β, IL-6, TNF-α, MDA, mRNA and protein levels of Beclin-1 and LC3 Ⅱ, protein levels of p53 and p-p38 MAPK were decreased in dapagliflozin group and inhibitor groups (P<0.05), SOD level was increased (P<0.05). Compared with the dapagliflozin group, the apoptosis rate, IL-1β, IL-6, TNF-α, MDA, mRNA and protein levels of Beclin-1 and LC3 Ⅱ, protein levels of p53 and p-p38 MAPK were further decreased in the dapagliflozin+inhibitor group (P<0.05), SOD level was further increased (P<0.05), and the change trend of these indicators in dapagliflozin+activator group was opposite to dapagliflozin+inhibitor group (P<0.05). Conclusions Dapagliflozin can inhibit the apoptosis, autophagy, inflammation and oxidative damage induced by D-glucose in HGPCs, and its mechanism may be related to the inhibition of p38 MAPK pathway signal transduction. |
Key words: diabetic nephropathy human glomerular podocyte dapagliflozin p38 mitogen-activated protein kinase signaling pathway |