Role of apurinic/apyrimidinic endodeoxyribonuclease 1 in ferroptosis of mouse dendritic cells under simulated sepsis
10.3760/cma.j.cn501225-20240430-00159
- VernacularTitle:脱嘌呤/脱嘧啶脱氧核糖核酸内切酶1对模拟脓毒症状态下小鼠树突状细胞铁死亡的作用
- Author:
Qiyuan ZHOU
1
;
Jingyan LI
;
Yongming YAO
;
Yingping TIAN
Author Information
1. 河北医科大学第二医院急诊医学科，石家庄 050000
- Keywords:
Sepsis;
Immunity;
Dendritic cells;
Oxidative stress;
Ferroptosis;
Apurinic/apyrimidinic endodeoxyribonuclease 1
- From:
Chinese Journal of Burns
2024;40(10):930-939
- CountryChina
- Language:Chinese
-
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.
