Role of apurinic/apyrimidinic endodeoxyribonuclease 1 in ferroptosis of mouse dendritic cells under simulated sepsis
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摘要:
目的 探讨脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1（APE1）对模拟脓毒症状态下小鼠树突状细胞（DC）铁死亡的作用,为改善创面感染等引起的脓毒症免疫抑制提供依据。 方法 该研究为实验研究。取第3~10代对数生长期的小鼠DC系DC2.4进行研究（样本数均为3）,采用1 μg/mL内毒素/脂多糖（LPS,浓度下同）处理DC 0（未处理）、6、12、24、48、72 h构建脓毒症模型,采用蛋白质印迹法检测细胞中APE1及抗铁死亡蛋白谷胱甘肽过氧化物酶4（GPX4）、溶质载体家族7成员11（SLC7A11）蛋白表达,采用流式细胞术检测细胞中活性氧水平,采用活细胞成像技术检测细胞脂质过氧化水平；将成功转染含APE1基因短发夹RNA序列慢病毒的DC分为敲减APE1+磷酸盐缓冲液（PBS）组、敲减APE1+LPS组,将成功转染空载慢病毒的DC分为空载体+PBS组、空载体+LPS组,给予PBS或LPS刺激并培养24 h后同前行相应检测；将成功转染含APE1基因过表达RNA序列慢病毒的DC分为过表达APE1+PBS组、过表达APE1+LPS组,将成功转染空载慢病毒的DC分为空载体+PBS组、空载体+LPS组,给予PBS或LPS刺激并培养24 h后同前行相应检测。将88只6~8周龄雄性C57BL/6J小鼠按随机数字表法分为玉米油+假伤组、玉米油+盲肠结扎穿孔（CLP）组、抑制剂+假伤组、抑制剂+CLP组,每组22只。对2个抑制剂组小鼠按照40 mg/kg每日灌胃1 mg/mL APE1抑制剂E3330,对2个玉米油组小鼠每日灌胃等量玉米油,2周后对2个CLP组小鼠行CLP术构建脓毒症模型,对2个假伤组小鼠行假手术。从4组小鼠中各选取16只,观察术后7 d内存活情况；术后24 h采用CD11c阳选磁珠提取4组分别剩余的6只小鼠脾脏DC同前行相应检测（样本数均为3）。 结果 与LPS处理0 h比较,LPS处理6 h时细胞中APE1蛋白表达显著升高（P<0.05）,LPS处理24、48、72 h时细胞中APE1与GPX4蛋白表达及LPS处理24 h时细胞中SLC7A11蛋白表达均显著降低（P<0.05）,LPS处理24、48、72 h时细胞中活性氧水平（P<0.05）与细胞脂质过氧化水平均显著升高。培养24 h后,敲减APE1+LPS组细胞中GPX4蛋白表达较敲减APE1+PBS组显著降低（P<0.05）,细胞中活性氧水平（P值均<0.05）及细胞脂质过氧化水平显著高于敲减APE1+PBS组与空载体+LPS组。培养24 h后,过表达APE1+LPS组细胞中APE1、SLC7A11、GPX4蛋白表达均较空载体+LPS组显著升高（P<0.05）,细胞中活性氧水平（P<0.05）及细胞脂质过氧化水平显著低于空载体+LPS组。术后24 h,抑制剂+CLP组小鼠细胞中APE1与GPX4蛋白表达水平均显著低于抑制剂+假伤组、玉米油+CLP组（P<0.05）；玉米油+CLP组小鼠细胞中活性氧水平（12 693±913）显著高于玉米油+假伤组（4 205±805,P<0.05）,抑制剂+CLP组小鼠细胞中活性氧水平（18 085±223）显著高于抑制剂+假伤组（4 381±787）和玉米油+CLP组（P值均<0.05）；抑制剂+CLP组小鼠细胞脂质过氧化水平显著高于抑制剂+假伤组与玉米油+CLP组。术后7 d内,抑制剂+CLP组小鼠存活比显著低于抑制剂+假伤组（χ2=22.67,P<0.05）。 结论 模拟脓毒症状态下,小鼠DC中APE1表达降低,氧化应激及铁死亡增强；敲减APE1会加重DC铁死亡,过表达APE1则有效减轻DC铁死亡；抑制DC中APE1表达与脓毒症预后不良密切相关。 Abstract:Objective To investigate the role of apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) in ferroptosis of mouse dendritic cells (DCs) under simulated sepsis, providing evidence for improving immunosuppression in sepsis caused by wound infection. Methods This study was an experimental research. The mouse DC line DC2.4 in the logarithmic growth phase (with passages 3-10) was used for the experiments (with each sample size of 3). The sepsis model was established by treating DCs with 1 μg/mL lipopolysaccharide (LPS, same concentration throughout) for 0 (untreated), 6, 12, 24, 48, and 72 h. Western blotting was used to detect the protein expressions of APE1 and anti-ferroptosis proteins glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) in cells, flow cytometry was employed to detect reactive oxygen species (ROS) levels in cells, and live-cell imaging technology was used to measure cell lipid peroxidation levels. DCs successfully transfected with lentivirus containing APE1 short hairpin RNA sequence were divided into APE1-knockdown+phosphate buffer solution (PBS) group and APE1-knockdown+LPS group. DCs successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. After stimulation with PBS or LPS and 24 h of culture, corresponding assays were conducted as above. DCs transfected with lentivirus containing APE1 overexpression RNA sequence were divided into APE1-overexpression+PBS group and APE1-overexpression+LPS group. DCs transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. After stimulation with PBS or LPS and 24 h of culture, corresponding assays were conducted as above. A total of 88 male C57BL/6J mice aged 6-8 weeks were divided into corn oil+sham injury group, corn oil+cecal ligation and puncture (CLP) group, inhibitor+sham injury group, and inhibitor+CLP group (n=22) according to the random number table. Mice in the two inhibitor groups were gavaged with APE1 inhibitor E3330 (1 mg/mL in concentration) at 40 mg/kg per day, while mice in the two corn oil groups were gavaged with an equal amount of corn oil daily. Two weeks later, mice in the two CLP groups underwent CLP surgery to establish a sepsis model, while mice in the two sham injury groups underwent sham injury. Sixteen mice from each group were selected to observe survival within 7 d post-surgery. At 24 h post-surgery, CD11c-positive magnetic beads were used to extract spleen DCs from the remaining six mice in each group for corresponding assays (n=3) as above. Results Compared with that of LPS treatment at 0 h, APE1 protein expression significantly increased in cells at 6 h of LPS treatment (P<0.05), APE1 and GPX4 protein expressions significantly decreased at 24, 48, and 72 h of LPS treatment, SLC7A11 protein expression significantly decreased at 24 h of LPS treatment (P<0.05), while the ROS level in cells (P<0.05) and cell lipid peroxidation level significantly increased at 24, 48, and 72 h of LPS treatment. After 24 h of culture, the GPX4 protein expression of cells in APE1-knockdown+LPS group was significantly lower than that in APE1-knockdown+PBS group (P<0.05), while the ROS level in cells (P<0.05) and cell lipid peroxidation level were significantly higher than those in APE1-knockdown+PBS group and empty vector+LPS group. After 24 h of culture, APE1, SLC7A11, and GPX4 protein expressions of cells in APE1-overexpression+LPS group were significantly higher than those in empty vector+LPS group (P<0.05), while the ROS level in cells (P<0.05) and cell lipid peroxidation level were significantly lower than those in empty vector+LPS group. At 24 h post-surgery, APE1 and GPX4 protein expressions of mice cells in inhibitor+CLP group were significantly lower than those in inhibitor+sham injury group and corn oil+CLP group (P<0.05); the ROS level of mice cells in corn oil+CLP group (12 693±913) was significantly higher than that in corn oil+sham injury group (4 205±805, P<0.05), while the ROS level of mice cells in inhibitor+CLP group (18 085±223) was significantly higher than that in inhibitor+sham injury group (4 381±787) and corn oil+CLP group (with P values all<0.05); the cell lipid peroxidation level of mice in inhibitor+CLP group was significantly higher than that in inhibitor+sham injury group and corn oil+CLP group. Within 7 d post-surgery, the survival ratio of mice in inhibitor+CLP group was significantly lower than that in inhibitor+sham injury group (χ²=22.67, P<0.05). Conclusions Under simulated sepsis, the APE1 expression in mouse DCs is decreased, and oxidative stress and ferroptosis are enhanced; knocking down the APE1 exacerbates DC ferroptosis, while APE1 overexpression effectively reduces DC ferroptosis. The inhibition of APE1 expression in DCs is closely associated with poor prognosis in sepsis. -
Key words:
- Sepsis /
- Immunity /
- Dendritic cells /
- Oxidative stress /
- Ferroptosis /
- Apurinic/apyrimidinic endodeoxyribonuclease 1
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参考文献
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图 1 采用蛋白质印迹法检测的LPS处理各时间点小鼠DC系DC2.4中APE1及SLC7A11与GPX4蛋白表达
注：APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4,DC为树突状细胞；条带上方1、2、3、4、5、6分别指示内毒素/脂多糖（LPS）处理0（未处理）、6、12、24、48、72 h
图 2 LPS处理各时间点小鼠DC系DC2.4脂质过氧化水平 BODIPY™ 581/591 C11-Hoechst 33342×126。2A、2B、2C、2D、2E、2F.分别为LPS处理0（未处理）、6、12、24、48、72 h,图2A、2B、2C中细胞脂质过氧化水平低,图2D、2E、2F中细胞脂质过氧化水平高于图2A、2B、2C
注：LPS为内毒素/脂多糖,DC为树突状细胞；未发生过氧化脂质染色为红色,过氧化脂质染色为绿色,细胞核染色为蓝色
图 3 采用蛋白质印迹法检测的各组小鼠DC系DC2.4培养24 h后APE1及SLC7A11与GPX4蛋白表达。3A.基因敲减实验中各组；3B.基因过表达实验中各组
注：APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4,DC为树突状细胞；图3A与图3B条带上方1、2均分别指示细胞转染空载慢病毒并分别经磷酸盐缓冲液（PBS）、内毒素/脂多糖（LPS）处理的空载体+PBS组、空载体+LPS组,图3A条带上方3、4分别指示细胞转染含APE1基因短发夹RNA序列慢病毒并分别经PBS、LPS处理的敲减APE1+PBS组、敲减APE1+LPS组,图3B条带上方3、4分别指示细胞转染含APE1基因过表达RNA序列慢病毒并分别经PBS、LPS处理的过表达APE1+PBS组、过表达APE1+LPS组
图 4 各组小鼠树突状细胞（DC）系DC2.4培养24 h后脂质过氧化水平 BODIPY™ 581/591 C11-Hoechst 33342×126。4A、4B、4C、4D.分别为基因敲减实验中空载体+PBS组、空载体+LPS组、敲减APE1+PBS组、敲减APE1+LPS组,图4A、4C脂质过氧化水平低,图4B脂质过氧化水平高于图4A与4C,图4D脂质过氧化水平显著高于图4A与4C；4E、4F、4G、4H.分别为基因过表达实验中空载体+PBS组、空载体+LPS组、过表达APE1+PBS组、过表达APE1+LPS组,图4E、4G、4H脂质过氧化水平低,图4F脂质过氧化水平显著高于图4E、4G、4H
注：未发生过氧化脂质染色为红色,过氧化脂质染色为绿色,细胞核染色为蓝色；空载体+磷酸盐缓冲液（PBS）组、空载体+内毒素/脂多糖(LPS)组细胞转染空载慢病毒并分别经PBS、LPS处理,敲减脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1（APE1）+PBS组、敲减APE1+LPS组细胞转染含APE1基因短发夹RNA序列慢病毒并分别经PBS、LPS处理,过表达APE1+PBS组、过表达APE1+LPS组细胞转染含APE1基因过表达RNA序列慢病毒并分别经PBS、LPS处理
图 5 采用蛋白质印迹法检测的4组小鼠术后24 h脾脏树突状细胞中APE1及SLC7A11与GPX4蛋白表达
注：APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4；条带上方1、2及3、4分别指示小鼠灌胃玉米油2周后分别行假手术、盲肠结扎穿孔（CLP）术的玉米油+假伤组、玉米油+CLP组及小鼠灌胃E3330 2周后分别行假手术、CLP术的抑制剂+假伤组、抑制剂+CLP组
图 6 4组小鼠术后24 h脾脏树突状细胞脂质过氧化水平 BODIPY™ 581/591 C11-Hoechst 33342×126。6A、6B、6C、6D.分别为玉米油+假伤组、玉米油+CLP组、抑制剂+假伤组、抑制剂+CLP组,图6A、6C脂质过氧化水平低,图6B脂质过氧化水平稍高于图6A、6C,图6D脂质过氧化水平显著高于图6A、6B、6C
注：未发生过氧化脂质染色为红色,过氧化脂质染色为绿色,细胞核染色为蓝色；玉米油+假伤组、玉米油+盲肠结扎穿孔（CLP）组小鼠灌胃玉米油2周后分别行假手术、CLP术,抑制剂+假伤组、抑制剂+CLP组小鼠灌胃E3330 2周后分别行假手术、CLP术
图 7 4组小鼠术后7 d内存活比（样本数为16）
注：玉米油+假伤组、玉米油+盲肠结扎穿孔（CLP）组小鼠灌胃玉米油2周后分别行假手术、CLP术,抑制剂+假伤组、抑制剂+CLP组小鼠灌胃E3330 2周后分别行假手术、CLP术；与玉米油+假伤组比较,aP<0.05；与抑制剂+假伤组比较,bP<0.05
Table 1. LPS处理各时间点小鼠DC系DC2.4中APE1及SLC7A11与GPX4蛋白表达比较（
LPS处理时间点 样本数 APE1 SLC7A11 GPX4 0 h 3 0.96±0.10 1.00±0.11 0.93±0.12 6 h 3 1.50±0.16 0.94±0.10 1.00±0.03 12 h 3 1.07±0.13 0.80±0.05 0.86±0.05 24 h 3 0.50±0.18 0.67±0.11 0.53±0.16 48 h 3 0.39±0.19 0.95±0.08 0.32±0.04 72 h 3 0.34±0.21 1.03±0.05 0.17±0.08 F值 22.79 7.82 43.92 P值 <0.001 0.002 <0.001 P1值 0.019 0.927 0.917 P2值 0.964 0.100 0.992 P3值 0.048 0.004 0.002 P4值 0.012 0.974 <0.001 P5值 0.007 0.999 <0.001 注：LPS为内毒素/脂多糖,DC为树突状细胞,APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4；F值、P值为各时间点间各指标总体比较所得；P1值、P2值、P3值、P4值、P5值分别为LPS处理6、12、24、48、72 h与LPS处理0 h（未处理）各指标比较所得 
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Table 2. 基因敲减实验中4组小鼠DC系DC2.4培养24 h后APE1及SLC7A11与GPX4蛋白表达比较（
组别 样本数 APE1 SLC7A11 GPX4 空载体+PBS组 3 1.01±0.06 1.05±0.08 1.00±0.17 空载体+LPS组 3 0.70±0.07 0.67±0.18 0.31±0.06 敲减APE1+PBS组 3 0.53±0.14 0.60±0.09 0.84±0.11 敲减APE1+LPS组 3 0.50±0.11 0.51±0.12 0.49±0.08 F值 16.43 17.53 12.22 P值 <0.001 <0.001 0.002 P1值 0.023 0.003 0.013 P2值 0.987 0.633 0.022 P3值 0.002 0.003 0.367 P4值 0.143 0.695 0.563 注：DC为树突状细胞,APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4,PBS为磷酸盐缓冲液,LPS为内毒素/脂多糖；空载体+PBS组、空载体+LPS组细胞转染空载慢病毒并分别经PBS、LPS处理,敲减APE1+PBS组、敲减APE1+LPS组细胞转染含APE1基因短发夹RNA序列慢病毒并分别经PBS、LPS处理；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为空载体+LPS组与空载体+PBS组、敲减APE1+LPS组与敲减APE1+PBS组、敲减APE1+PBS组与空载体+PBS组、敲减APE1+LPS组与空载体+LPS组各指标比较所得
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Table 3. 基因过表达实验中4组小鼠DC系DC2.4培养24 h后APE1及SLC7A11与GPX4蛋白表达比较（
组别 样本数 APE1 SLC7A11 GPX4 空载体+PBS组 3 0.40±0.03 0.98±0.16 1.08±0.23 空载体+LPS组 3 0.24±0.04 0.67±0.04 0.59±0.10 过表达APE1+PBS组 3 1.15±0.30 1.03±0.10 1.10±0.08 过表达APE1+LPS组 3 1.07±0.27 0.98±0.11 1.06±0.12 F值 15.04 6.32 8.65 P值 0.001 0.017 0.007 P1值 0.768 0.043 0.014 P2值 0.961 0.968 0.985 P3值 0.009 0.946 0.998 P4值 0.005 0.038 0.017 注：DC为树突状细胞,APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4,PBS为磷酸盐缓冲液,LPS为内毒素/脂多糖；空载体+PBS组、空载体+LPS组细胞转染空载慢病毒并分别经PBS、LPS处理,过表达APE1+PBS组、过表达APE1+LPS组细胞转染含APE1基因过表达RNA序列慢病毒并分别经PBS、LPS处理；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为空载体+LPS组与空载体+PBS组、过表达APE1+LPS组与过表达APE1+PBS组、过表达APE1+PBS组与空载体+PBS组、过表达APE1+LPS组与空载体+LPS组各指标比较所得
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Table 4. 4组小鼠术后24 h脾脏树突状细胞中APE1及SLC7A11与GPX4蛋白表达比较（
组别 样本数 APE1 SLC7A11 GPX4 玉米油+假伤组 3 0.93±0.04 0.91±0.07 0.94±0.05 玉米油+CLP组 3 0.50±0.12 0.66±0.12 0.77±0.06 抑制剂+假伤组 3 0.83±0.10 0.91±0.08 0.90±0.06 抑制剂+CLP组 3 0.24±0.10 0.73±0.07 0.54±0.06 F值 33.75 6.62 26.64 P值 <0.001 0.015 <0.001 P1值 0.002 0.031 0.034 P2值 <0.001 0.117 <0.001 P3值 0.584 >0.999 0.845 P4值 0.043 0.769 0.008 注：APE1为脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1,SLC7A11为溶质载体家族7成员11,GPX4为谷胱甘肽过氧化物酶4,CLP为盲肠结扎穿孔；玉米油+假伤组、玉米油+CLP组小鼠灌胃玉米油2周后分别行假手术、CLP术,抑制剂+假伤组、抑制剂+CLP组小鼠灌胃E3330 2周后分别行假手术、CLP术；F值、P值为组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为玉米油+CLP组与玉米油+假伤组、抑制剂+CLP组与抑制剂+假伤组、抑制剂+假伤组与玉米油+假伤组、抑制剂+CLP组与玉米油+CLP组各指标比较所得
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