纤维蛋白原Bβ15-42肽在细胞水平对缺血再灌注急性肾损伤线粒体自噬的影响
Effects of Fibrin-derived Peptide Bβ15-42 on Mitophagy in AKI with Ischemia-reperfusion at the Cellular Level
张紫婷 1贾琚丹 1仇文芳 1乔玉峰1
作者信息
- 1. 030012 太原,山西医科大学第五临床医学院、山西省人民医院
- 折叠
摘要
目的 探讨纤维蛋白原Bβ15-42肽(fibrin-derived peptide Bβ15-42,FgBβ15-42)在细胞水平对缺血再灌注急性肾损伤(acute kidney injury,AKI)线粒体自噬的影响.方法 体外培养人肾小管上皮细胞,随机分为5组,即正常对照组(Con-trol 组)、缺氧复氧(hypoxia/reoxygenation,H/R)模型组(H/R 组)、H/R 模型+9μmol/L FgBβ15-42 肽组(Fg 组)、H/R 模型+20μmol/L 氯喹组(CQ 组)、H/R 模型+9μmol/L FgBβ15-42 肽+20µmol/L 氯喹组(Fg+CQ 组).Control 组:将同质化细胞置于完全培养基中,在有氧(5％CO2,21％O2)条件下培养28h.H/R组:细胞用无糖、无血清DMEM/F12培养基在缺氧(5％CO2,1％O2和94％N2)条件下培养24h,再更换含血清的DMEM/F12培养基置于有氧(5％CO2,21％O2)环境中4h.Fg组:在复氧时加入FgBβ15-42肽9µmol/L进行干预.CQ组:在进行同质化处理时加入氯喹进行预处理(20µmol/L),后序步骤同H/R组.Fg+CQ组:按照上述CQ组及FgBβ15-42组在不同时间段的加药顺序、剂量,及H/R组的造模时间进行干预.干预结束后采用CCK-8法检测HK2细胞活力;电子显微镜观察线粒体形态、线粒体自噬体结构及数量;Western blot法检测自噬相关蛋白p62、LC3的表达水平.结果 与Control组比较,H/R组细胞的存活率下降(P＜0.01);线粒体损伤明显,出现了肿胀、棘断裂等,且线粒体自噬体的数目增多;自噬标志性蛋白LC-3 Ⅱ/Ⅰ表达上调(P＜0.05),自噬底物蛋白p62表达下调(P＜0.01).与H/R组比较,Fg组、CQ组、Fg+CQ组细胞的存活率回升(P＜0.01),线粒体自噬体减少,LC-3 Ⅱ/Ⅰ表达下调(P＜0.01),p62表达上调(P＜0.05),细胞自噬被抑制.结论 FgBβ15-42肽干预后可降低H/R-AKI细胞中自噬及线粒体自噬水平,对HK2细胞H/R损伤有保护作用.
Abstract
Objective To investigate the effects of fibrin-derived peptide Bβ15-42(FgBβ15-42)on mitophagy in acute kidney injury(AKI)with ischemia-reperfusion at the cellular level.Methods Human kidney-2(HK2)cells were cultured in vitro and ran-domly divided into five groups:normal control group(Control group),hypoxia/reoxygenation(H/R)model group(H/R group),H/R+9μmol/L FgBβ15-42 peptide group(Fg group),H/R+20μmol/L chloroquine group(CQ group),and H/R+9μmol/L FgBβ15-42 peptide+20μmol/L chloroquine group(Fg+CQ group).Control group:cells were homogenized and replaced with complete medium and incubated in an aerobic incubator(5％CO2,21％O2)for 28hours.H/R group:cells were added to sugar-free and serum-free DMEM/F12medium and then placed in a hypoxic incubator(5％CO2,1％O2 and 94％N2)for 24hours,washed with PBS and added to DMEM/F12medium containing serum and then placed in a reoxygenated environment(5％CO2,21％O2)for 4hours.Fg group:mod-eled according to the above method,intervention was performed by adding FgBβ15-42 peptide 9μmol/L to the complete medium when reoxygenation was performed after the completion of hypoxia.CQ group:pre-treatment with chloroquine(20μmol/L)in serum-free medium at the time of homogenization,with the same post-procedural steps as in the H/R group.Fg+CQ group:the intervention was performed according to the dosing sequence of the CQ group and FgBβ15-42 group at different time periods as mentioned above,and the modeling time of the H/R group.After the intervention,CCK-8 was used to detect the cell viability of HK2.The mitochondrial morphol-ogy,mitophagosome structure and number were observed by electron microscope.The expression levels of autophagy related proteins p62 and LC3 were detected by Western blot.Results Compared with the control group,the survival rate of H/R group was decreased(P＜0.01);the mitochondrial damage was obvious,such as swelling and spinous rupture,and the number of mitophagosomes increased.The expression of autophagy protein LC-3 Ⅱ/Ⅰ was up-regulated(P＜0.05),and the expression of autophagy substrate protein p62 was down-regulated(P＜0.01).Compared with H/R group,the survival rate of Fg,CQ and Fg+CQ groups increased(P＜0.01),mito-phagosome was decreased.the expression of LC-3 Ⅱ/Ⅰ was down-regulated(P＜0.01),the expression of p62 was up-regulated(P＜0.05),and autophagy was inhibited.Conclusion The intervention of FgBβ15-42 peptide can reduce the level of autophagy and mitophagy in H/R-AKI cells,and has a protective effect on H/R injury in HK2 cells.
关键词
线粒体自噬/缺血再灌注损伤/缺氧复氧损伤/FgBβ15-42肽/急性肾损伤Key words
Mitophagy/Ischemia-reperfusion injury/Hypoxia reoxygenation injury/Fibrin-derived peptide Bβ15-42/Acute kidney injury引用本文复制引用
基金项目
山西省自然科学基金项目(面上项目)(201901D111438)
山西省"136"专项科研课题项目(SZ2019015)
出版年
2024
NETL
NSTL
万方数据