Effects of stimulator of interferon gene on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 in mouse dendritic cells under sepsis

Wu Mengyao; He Pengyi; Duan Yu; Zheng Liyu; Yao Renqi; Zhou Qiyuan; Chen Yu; Dong Ning; Wu Yao; Yao Yongming

doi:10.3760/cma.j.cn501225-20240518-00184

Volume 40 Issue 10

Oct. 2024

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2024 > 40(10): 920-929

Wu MY,He PY,Duan Y,et al.Effects of stimulator of interferon gene on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 in mouse dendritic cells under sepsis[J].Chin J Burns Wounds,2024,40(10):920-929.DOI: 10.3760/cma.j.cn501225-20240518-00184.

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Effects of stimulator of interferon gene on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 in mouse dendritic cells under sepsis

doi: 10.3760/cma.j.cn501225-20240518-00184

1.
Medical Innovation Research Division and the Fourth Medical Center of PLA General Hospital, Beijing 100853, China
2.
Department of General Surgery, the First Medical Center of PLA General Hospital, Beijing 100853, China

Funds:

Key Program of National Natural Science Foundation of China 82130062, 82241062

The Youth Independent Innovation Science Fund Support Project of PLA General Hospital 22QNFC017

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Corresponding author: Yao Yongming, Email: c_ff@sina.com
Received Date: 2024-05-18

Abstract

Abstract

Objective To investigate the effects of stimulator of interferon gene (STING) on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 (ACSL4) in mouse dendritic cells (DCs) under sepsis, providing a basis for improving the dysregulation of immune response in sepsis caused by factors such as wound infection. Methods This study was an experimental research. The mouse DC line DC2.4 in the logarithmic growth phase (with passages 3-10) were divided into lipopolysaccharide (LPS) stimulation 0 h (unstimulated) group, LPS stimulation 6 h group, LPS stimulation 12 h group, LPS stimulation 18 h group, and LPS stimulation 24 h group according to the random number table (the same grouping method below), which were cultured with 1 μg/mL LPS (the same concentration below) for the corresponding time. The protein expressions of phosphorylated STING (p-STING), STING, and ACSL4 in cells were determined by Western blotting. DC2.4 successfully transfected with lentivirus containing STING gene small interfering RNA (hereinafter referred to as siSTING) were divided into siSTING+phosphate buffer solution (PBS) group and siSTING+LPS group. DC2.4 successfully transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group. After being stimulated with PBS or LPS and cultured for 24 hours, the protein expressions of p-STING, STING, and ACSL4 in cells were determined as above. Cell lipid peroxidation degrees were observed using the lipid peroxidation assay kit, and cell apoptosis rates were detected using flow cytometry. The sample numbers in the above cell experiments were all 3. Eighty male C57BL/6J mice aged 6 to 8 weeks were divided into sham surgery+normal saline (NS) group, cecal ligation and puncture (CLP)+NS group, sham surgery+C-176 group, and CLP+C-176 group, with 20 mice in each group. Mice in the two C-176 groups were intraperitoneally injected with C-176, while mice in the two NS groups were intraperitoneally injected with an equivalent volume of NS. One hour later, sham surgery was performed on the mice in the two sham surgery groups, and CLP surgery was performed on the mice in the two CLP groups to establish a sepsis model. At 24 h post-surgery, 10 mice from each group were sacrificed to extract spleen DCs, and protein expression, lipid peroxidation, and apoptosis rates were detected as above (n=3). Hematoxylin-eosin staining was performed to observe pathological damage in the heart, liver, lung, and kidney tissue. The remaining 10 mice in each group were observed for survival within 7 days after surgery. Results The protein expressions of p-STING, STING, and ACSL4, as well as the p-STING/STING ratio in DC2.4 in LPS stimulation 24 h group were significantly higher than those in LPS stimulation 0 h group (P<0.05). After 24 h of culture, the protein expressions of p-STING, STING, and ACSL4 in DC2.4 in siSTING+LPS group and empty vector+PBS group were significantly lower than those in empty vector+LPS group (P<0.05); the lipid peroxidation degrees of DC2.4 in siSTING+LPS group and empty vector+PBS group were weaker than those in empty vector+LPS group. The apoptosis rates of DC2.4 in empty vector+PBS group, empty vector+LPS group, siSTING+PBS group, and siSTING+LPS group were (15.7±3.0)%, (37.8±2.9)%, (13.1±2.1)%, and (20.6±1.8)%, respectively. The apoptosis rates of DC2.4 in empty vector+PBS group and siSTING+LPS group were significantly lower than that in empty vector+LPS group (P<0.05). At 24 h post-surgery, the protein expressions of p-STING and ACSL4, and the p-STING/STING ratio in spleen DCs of mice in CLP+NS group were significantly higher than those in sham surgery+NS group and CLP+C-176 group (P<0.05); the protein expression of STING in spleen DCs of mice in CLP+NS group was significantly higher than that in sham surgery+NS group (P<0.05); the lipid peroxidation degrees of spleen DCs of mice in CLP+C-176 group and sham surgery+NS group were weaker than that in CLP+NS group. The apoptosis rates of spleen DCs of mice in sham surgery+NS group and CLP+C-176 group were significantly lower than that in CLP+NS group (P<0.05), and the apoptosis rate of spleen DCs of mice in CLP+C-176 group was significantly higher than that in sham surgery+C-176 group (P<0.05). Pathological tissue damage in the heart, liver, lung, and kidney of mice in CLP+NS group was significantly worse than that in sham surgery+NS group, while such damage in the above organs of mice in CLP+C-176 group was significantly alleviated compared with that in CLP+NS group. The survival ratio of mice in CLP+NS group within 7 days after surgery was significantly lower than that in sham surgery+NS group (χ²=8.30, P<0.05). Conclusions Under sepsis, STING activation in mouse DCs is significant, which enhances ACSL4-mediated ferroptosis. Inhibiting STING activation can significantly reduce ACSL4-mediated ferroptosis level in mouse DCs under sepsis, thereby improving the survival rate of septic mice.
- Sepsis,
- Immunomodulation,
- Cell death,
- Dendritic cells,
- Ferroptosis,
- Stimulator of interferon gene,
- Acyl-CoA synthetase long-chain family member 4

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References

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