The influence of stimulator of interferon genes on lipopolysaccharide-induced necroptosis of mouse dendritic cells

Li Jinru; Duan Yu; Ling Hua; Li Qiong; Yao Yongming; Dai Xingui

doi:10.3969/j.issn.1002-1949.2024.07.011

Abstract：Objective To investigate the influence of stimulator of interferon genes(STING)on lipopolysaccharide(LPS)-induced necroptosis of dendritic cells(DCs).Methods The DC 2.4 cell line of the 3rd to 10th passage in the logarithmic growth stage stimulated by 1 µg/mL LPS were divided into Control group,LPS 6 h group,LPS 12 h group,LPS 24 h group,LPS 72 h group.Western blot was used to determine the expression of phosphorylated(P)-STING,STING,phosphorylated-mixed lineage kinase domain-like protein(P-MLKL),MLKL in DCs.DCs transfected with lentivirus containing siRNA sequence of STING gene were divided into STING-knockdown+phosphate buffer solution(PBS)group and STING-knockdown+LPS group,and DCs transfected with empty lentivirus were divided into empty vector+PBS group and empty vector+LPS group.After LPS stimulation and cultivation for 12 h,the apoptotic rate of cells was determined by flow cytometry,the situation of apoptosis was observed by SYTOX Green staining and the apoptotic rate was calculated,the protein expression of P-STING,STING,P-MLKL and MLKL was detected by using Western blot.Results Compared with those in control group,the protein expression of STING of cells in LPS 12 h group was obviously increased(P＜0.05),and the ratios of P-MLKL/MLKL of cells in LPS 12 h group and LPS 24 h group were significantly increased(P＜0.05),therefore 12 hours were used as the subsequent LPS stimulation duration.After 12 hours of culture,compared with PBS group,LPS group showed increased necroptosis rate.Furthermore,cells in LPS group exhibited general organelle swelling,the abundant vacuoles in the cytoplasm,the disruption of cell membrane integrity,and cellular disintegration.After 72 hours of transfection,compared with empty vector group,sh2-STING-knockdown group and sh3-STING-knockdown group,the expression of STING determined by Western blot in DCs was significantly decreased in sh1-STING-knockdown group(P＜0.05).After 12 hours of culture,flow cytometry assay revealed that the apoptotic rate of cells in empty vector+PBS group,empty vector+LPS group,sh1-STING-knockdown+PBS group and sh1-STING-knockdown+LPS group were(36.34±24.42)％,(52.57±35.77)％,(37.81±28.9)％,(46.45±32.44)％,respectively.And again,the SYTOX Green staining showed the apoptotic rate of cells in empty vector+PBS group,empty vector+LPS group,sh1-STING-knockdown+PBS group and sh1-STING-knockdown+LPS group were(13.4±6.3)％,(67.6±24.0)％,(2.0±1.1)％and(7.1±4.2)％.After 12 hours of culture,compare with empty vector+LPS group,the apoptotic rates of cells and the ratio of P-MLKL/MLKL were significantly decreased in sh1-STING-knockdown+LPS group(P＜0.05);compared with empty vector+PBS group,the ratio of P-MLKL/MLKL was remarkably increased in empty vector+LPS group(P=0.011);there was no statistical difference in P-MLKL/MLKL between STING+PBS group and STING+LPS group(P=0.889).Conclusions The expression of STING in mice DCs is raised under LPS stimulations and reached its peak at 12 h,and the activated STING has an obvious stimulative effect on cell necroptosis.