Effects of stimulator of interferon gene on ferroptosis mediated by acyl-CoA synthetase long-chain family member 4 in mouse dendritic cells under sepsis
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摘要:
目的 探讨干扰素基因刺激因子（STING）对脓毒症状态下小鼠树突状细胞（DC）内酰基辅酶A合成酶长链家族成员4（ACSL4）介导铁死亡的影响,为改善由创面感染等因素引发的脓毒症免疫应答失调提供依据。 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,按随机数字表法（分组方法下同）分为内毒素/脂多糖（LPS）刺激0 h（不刺激）组、LPS刺激6 h组、LPS刺激12 h组、LPS刺激18 h组和LPS刺激24 h组,用1 μg/mL LPS（浓度下同）培养相应时间后,采用蛋白质印迹法检测细胞中磷酸化STING（p-STING）、STING和ACSL4的蛋白表达；将成功转染含STING基因小干扰RNA（下称siSTING）慢病毒的DC2.4分为siSTING+磷酸盐缓冲液（PBS）组、siSTING+LPS组,将成功转染空载慢病毒的DC2.4分为空载体+PBS组、空载体+LPS组,给予PBS或LPS刺激并培养24 h,同前检测细胞中p-STING、STING和ACSL4的蛋白表达,采用脂质过氧化检测试剂盒观察细胞脂质过氧化程度,采用流式细胞术检测细胞凋亡率。以上细胞实验中样本数均为3。将80只6~8周龄雄性C57BL/6J小鼠分为假手术+生理盐水组、盲肠结扎穿孔（CLP）+生理盐水组、假手术+C-176组、CLP+C-176组,每组20只。对2个C-176组小鼠先经腹腔注射C-176、2个生理盐水组小鼠先经腹腔注射等量生理盐水,1 h后对2个假手术组小鼠行假手术、对2个CLP组小鼠行CLP术构建脓毒症模型。术后24 h,将每组10只小鼠处死后提取脾脏DC,同前行蛋白表达、脂质过氧化程度、凋亡率检测（样本数均为3）；另行苏木精-伊红染色观察小鼠心、肝、肺、肾病理组织损伤。观察各组剩余10只小鼠术后7 d内存活情况。 结果 LPS刺激24 h组DC2.4中p-STING、STING、ACSL4的蛋白表达及p-STING/STING比值均明显高于LPS刺激0 h组（P<0.05）。培养24 h后,siSTING+LPS组和空载体+PBS组DC2.4中p-STING、STING和ACSL4的蛋白表达均明显低于空载体+LPS组（P<0.05）；siSTING+LPS组和空载体+PBS组DC2.4脂质过氧化程度均弱于空载体+LPS组；空载体+PBS组、空载体+LPS组、siSTING+PBS组与siSTING+LPS组DC2.4凋亡率分别为（15.7±3.0）%、（37.8±2.9）%、（13.1±2.1）%与（20.6±1.8）%,其中空载体+PBS组、siSTING+LPS组DC2.4凋亡率均明显低于空载体+LPS组（P<0.05）。术后24 h,CLP+生理盐水组小鼠脾脏DC中p-STING、ACSL4的蛋白表达及p-STING/STING比值均明显高于假手术+生理盐水组和CLP+C-176组（P<0.05）,CLP+生理盐水组小鼠脾脏DC中STING的蛋白表达明显高于假手术+生理盐水组（P<0.05）；CLP+C-176组和假手术+生理盐水组小鼠脾脏DC脂质过氧化程度均弱于CLP+生理盐水组；假手术+生理盐水组、CLP+C-176组小鼠脾脏DC凋亡率均明显低于CLP+生理盐水组（P<0.05）,CLP+C-176组小鼠脾脏DC凋亡率明显高于假手术+C-176组（P<0.05）；CLP+生理盐水组小鼠心、肝、肺、肾病理组织损伤均较假手术+生理盐水组明显加重,CLP+C-176组小鼠各脏器上述病理组织损伤均较CLP+生理盐水组明显减轻。CLP+生理盐水组小鼠术后7 d内存活比明显低于假手术+生理盐水组（χ2=8.30,P<0.05）。 结论 脓毒症状态下,小鼠DC内STING显著活化,ACSL4介导的铁死亡增强；抑制STING活化能够显著降低脓毒症时小鼠DC内ACSL4介导的铁死亡水平,从而改善脓毒症小鼠存活情况。 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. -
参考文献
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图 1 蛋白质印迹法检测的5组小鼠DC系DC2.4中STING及p-STING与ACSL4的蛋白表达
注：p-STING为磷酸化干扰素基因刺激因子,STING为干扰素基因刺激因子,ACSL4为酰基辅酶A合成酶长链家族成员4,LPS为内毒素/脂多糖,DC为树突状细胞；条带上方1、2、3、4、5分别指示LPS刺激0 h（不刺激）组、LPS刺激6 h组、LPS刺激12 h组、LPS刺激18 h组、LPS刺激24 h组
图 2 2个转染组小鼠树突状细胞（DC）系DC2.4转染后72 h与空白对照组细胞相应时间点荧光表达 绿色荧光蛋白×200。2A、2B、2C.分别为空白对照组、空载体组、siSTING组,图2A无荧光表现,图2B、2C携带明显绿色荧光
注：空载体组和干扰素基因刺激因子基因小干扰RNA（下称siSTING）组细胞分别转染空载慢病毒、含siSTING慢病毒,空白对照组细胞仅常规培养
图 3 蛋白质印迹法检测的4组小鼠DC系DC2.4培养24 h后STING及p-STING与ACSL4的蛋白表达
注：p-STING为磷酸化干扰素基因刺激因子,STING为干扰素基因刺激因子,ACSL4为酰基辅酶A合成酶长链家族成员4,PBS为磷酸盐缓冲液,LPS为内毒素/脂多糖,DC为树突状细胞；条带上方1、2、3、4分别指示空载体+PBS组、空载体+LPS组、STING基因小干扰RNA（下称siSTING）+PBS组、siSTING+LPS组；空载体+PBS组和空载体+LPS组细胞先转染空载慢病毒、siSTING+PBS组和siSTING+LPS组细胞先转染含siSTING慢病毒,然后对2个PBS组细胞另给予PBS刺激、2个LPS组细胞另给予LPS刺激
图 4 4组小鼠树突状细胞（DC）系DC2.4培养24 h后脂质过氧化程度 Alexa Fluor 581-Alexa Fluor 591-Hoechst 33342×630。4A、4B、4C、4D.分别为空载体+PBS组、空载体+LPS组、siSTING+PBS组、siSTING+LPS组,图4B中细胞脂质过氧化程度较图4A、4D明显增强,图4D细胞脂质过氧化程度与图4C相近
注：空载体+磷酸盐缓冲液（PBS）组和空载体+内毒素/脂多糖（LPS）组细胞先转染空载慢病毒、干扰素基因刺激因子基因小干扰RNA（下称siSTING）+PBS组和siSTING+LPS组细胞先转染含siSTING慢病毒,然后对2个PBS组细胞另给予PBS刺激、2个LPS组细胞另给予LPS刺激；各图中右上小图是呈红色的还原态细胞,右下小图是呈绿色的发生脂质过氧化的细胞,左侧大图为细胞核Hoechst 33342染色（蓝色）和右侧2张小图的合成图
图 5 蛋白质印迹法检测的4组小鼠术后24 h脾脏DC中STING及p-STING与ACSL4的蛋白表达
注：p-STING为磷酸化干扰素基因刺激因子,STING为干扰素基因刺激因子,ACSL4为酰基辅酶A合成酶长链家族成员4,CLP为盲肠结扎穿孔,DC为树突状细胞；条带上方1、2及3、4分别指示小鼠注射生理盐水后分别行假手术、CLP术的假手术+生理盐水组、CLP+生理盐水组及小鼠注射C-176后分别行假手术、CLP术的假手术+C-176组、CLP+C-176组
图 6 4组小鼠术后24 h脾脏树突状细胞（DC）脂质过氧化程度 Alexa Fluor 581-Alexa Fluor 591-Hoechst 33342×630。6A、6B、6C、6D.分别为假手术+生理盐水组、CLP+生理盐水组、假手术+C-176组、CLP+C-176组,图6B中细胞脂质过氧化程度较图6A、6D明显增强,图6D中细胞脂质过氧化程度与图6C相近
注：假手术+生理盐水组、盲肠结扎穿孔（CLP）+生理盐水组小鼠注射生理盐水后分别行假手术、CLP术,假手术+C-176组、CLP+C-176组小鼠注射C-176后分别行假手术、CLP术；各图中右上小图是呈红色的还原态细胞,右下小图是呈绿色的发生脂质过氧化的细胞,左侧大图为细胞核Hoechst 33342染色（蓝色）和右侧2张小图的合成图
图 7 4组小鼠术后7 d内存活比（样本数为10）
注：假手术+生理盐水组、盲肠结扎穿孔（CLP）+生理盐水组小鼠注射生理盐水后分别行假手术、CLP术,假手术+C-176组、CLP+C-176组小鼠注射C-176后分别行假手术、CLP术；与假手术+生理盐水组比较,aP<0.05
Table 1. 5组小鼠DC系DC2.4中STING及p-STING与ACSL4的蛋白表达比较（
组别 样本数 p-STING STING p-STING/STING比值 ACSL4 LPS刺激0 h组 3 0.58±0.12 0.72±0.10 0.792±0.056 0.74±0.10 LPS刺激6 h组 3 0.56±0.12 0.72±0.13 0.780±0.069 0.56±0.22 LPS刺激12 h组 3 0.63±0.08 0.74±0.16 0.867±0.126 0.39±0.19 LPS刺激18 h组 3 0.82±0.08 0.90±0.14 0.911±0.050 0.68±0.13 LPS刺激24 h组 3 1.15±0.29 1.14±0.26 1.036±0.012 1.30±0.30 F值 7.65 3.64 6.10 8.86 P值 0.004 0.044 0.009 0.003 P1值 0.999 0.999 0.999 0.664 P2值 0.980 0.999 0.547 0.171 P3值 0.235 0.516 0.203 0.987 P4值 0.004 0.036 0.007 0.020 注：DC为树突状细胞,STING为干扰素基因刺激因子,p-STING为磷酸化STING,ACSL4为酰基辅酶A合成酶长链家族成员4,LPS为内毒素/脂多糖；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LPS刺激6 h组、LPS刺激12 h组、LPS刺激18 h组、LPS刺激24 h组与LPS刺激0 h（不刺激）组各指标比较所得 
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Table 2. 4组小鼠DC系DC2.4培养24 h后STING及p-STING与ACSL4的蛋白表达比较（
组别 样本数 p-STING STING ACSL4 空载体+PBS组 3 0.67±0.12 0.67±0.18 0.70±0.51 空载体+LPS组 3 1.73±0.47 1.29±0.09 1.41±0.30 siSTING+PBS组 3 0.78±0.11 0.72±0.09 0.41±0.27 siSTING+LPS组 3 0.71±0.10 0.60±0.17 0.53±0.34 P1值 0.041 0.025 0.034 P2值 0.936 0.436 0.571 P3值 0.043 0.021 0.023 注：DC为树突状细胞,STING为干扰素基因刺激因子,p-STING为磷酸化STING,ACSL4为酰基辅酶A合成酶长链家族成员4,PBS为磷酸盐缓冲液,LPS为内毒素/脂多糖；空载体+PBS组和空载体+LPS组细胞先转染空载慢病毒、STING基因小干扰RNA（下称siSTING）+PBS组和siSTING+LPS组细胞先转染含siSTING慢病毒,然后对2个PBS组细胞另给予PBS刺激、2个LPS组细胞另给予LPS刺激；p-STING、STING、ACSL4的细胞转染类型的主效应,F值分别为8.81、10.42、51.23,P值分别为0.097、0.084、0.019；刺激类型的主效应,F值分别为10.94、5.07、17.04,P值分别为0.080、0.153、0.054；二者交互作用,F值分别为34.35、70.51、24.41,P值分别为0.027、0.013、0.038；P1值为空载体+LPS组与空载体+PBS组各指标比较所得,P2值为siSTING+LPS组与siSTING+PBS组各指标比较所得,P3值为siSTING+LPS组与空载体+LPS组各指标比较所得
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Table 3. 4组小鼠术后24 h脾脏DC中STING及p-STING与ACSL4的蛋白表达比较（
组别 样本数 p-STING STING p-STING/STING比值 ACSL4 假手术+生理盐水组 3 0.71±0.13 0.68±0.13 0.824±0.295 0.54±0.11 假手术+C-176组 3 0.58±0.10 0.88±0.22 0.679±0.099 0.35±0.20 CLP+生理盐水组 3 1.36±0.14 1.00±0.06 1.483±0.108 1.15±0.08 CLP+C-176组 3 0.68±0.15 1.06±0.23 0.628±0.173 0.46±0.18 P1值 0.049 0.033 0.008 0.032 P2值 0.763 0.094 0.604 0.538 P3值 0.048 0.499 0.004 0.025 注：DC为树突状细胞,STING为干扰素基因刺激因子,p-STING为磷酸化STING,ACSL4为酰基辅酶A合成酶长链家族成员4,CLP为盲肠结扎穿孔；假手术+生理盐水组、CLP+生理盐水组小鼠注射生理盐水后分别行假手术、CLP术,对假手术+C-176组、CLP+C-176组小鼠注射C-176后分别行假手术、CLP术；p-STING、STING、p-STING/STING、ACSL4的抑制剂类型的主效应,F值分别为15.41、2.88、7.64、11.22,P值分别为0.059、0.231、0.109、0.078；造模类型的主效应,F值分别为30.40、29.20、8.70、69.56,P值分别为0.031、0.032、0.098、0.014；二者交互作用,F值分别为17.55、6.64、182.90、25.42,P值分别为0.052、0.123、0.005、0.037；P1值为CLP+生理盐水组与假手术+生理盐水组各指标比较所得,P2值为CLP+C-176组与假手术+C-176组各指标比较所得,P3值为CLP+C-176组与CLP+生理盐水组各指标比较所得
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