摘要
目的:探讨PD-L1对脓毒血症外周血多形核中性粒细胞(PMN)延迟凋亡的调控作用及可能机制。方法:收集12例脓毒血症患者外周血PMN,同时采集15例健康体检者PMN,流式细胞术测定PMN凋亡;Western blot检测PMN中PD-L1表达;另以SPF级成年雄性SD大鼠随机分为假手术组、脓毒血症组、实验组(PD-L1 siRNA),盲肠结扎穿孔法造模,测定PMN凋亡情况,Western blot检测PMN中Cleaved caspase-3、PKC-α及NF-κB水平。结果:与健康体检者相比,脓毒血症患者在6、12 h时PMN凋亡水平显著降低(P<0.05);脓毒血症患者PD-L1水平显著高于健康对照组(P<0.05),且与6、12 h PMN凋亡水平呈显著负相关(r=-0.693,P=0.012;r=-0.685,P=0.014);脓毒血症组、实验组PMN凋亡水平在6、12 h均低于假手术组,与脓毒血症大鼠相比,转染PD-L1 siRNA后大鼠在6、12 h PMN凋亡水平显著增加(P<0.05);与假手术组相比,模型组大鼠PMN内Cleaved caspase-3水平在6、12 h显著下降(P<0.05),转染PD-L1 siRNA后大鼠PMN内Cleaved caspase-3水平在6、12 h显著上升(P<0.05);与假手术组相比,模型组、实验组PMN内PKC-α、磷酸化NF-κB水平增加,转染PD-L1 siRNA后大鼠PMN内PKC-α、磷酸化NF-κB水显著下降(P<0.05)。结论:脓毒血症患者体内PMN凋亡延迟,与PD-L1表达呈显著负相关,提示PD-L1可能是调控脓毒血症PMN延迟凋亡的重要分子。在脓毒血症大鼠模型中,沉默PD-L1基因能够改善脓毒血症大鼠PMN的凋亡延迟,其机制可能与抑制脓毒血症大鼠PMN中的PKC-α-NF-κB信号通路激活有关。
Objective:To investigate regulation and possible mechanism of PD-L1 on delayed neutrophil apoptosis in peripheral blood poly morphonuclear neutrophil(PMN)of sepsis.Methods:PMN were collected from 12 patients with sepsis and 15 healthy subjects.Apoptosis of PMN was detected by flow cytometry.PD-L1 expression in PMN was detected by Western blot.Adult male SD rats of SPF grade were randomly divided into sham operation group,sepsis group and experiment group(PD-L1 siRNA).Cecal ligation and perforation method was used to determine apoptosis of PMN.Western blot was used to detect Cleaved caspase-3,PKC-αand NF-κB levels in PMN.Results:Compared with healthy subjects,level of PMN apoptosis was significantly lower in patients with sepsis at 6 h and 12 h(P<0.05).Level of PD-L1 in patients with sepsis was significantly higher than that in healthy controls(P<0.05),and was significantly negatively correlated with 6 h,12 h PMN apoptosis(r=-0.693,P=0.012;r=-0.685,P=0.014);PMN apoptosis level in sepsis group and experiment group at 6 h,12 h were lower than that in sham operation group,compared with sepsis group,apoptosis level of PMN was significantly increased at 6h and 12h after transfection with PD-L1 siRNA(P<0.05).Compared with sham operation group,level of Cleaved caspase-3 in PMN in rats was decreased significantly at 6h and 12 h(P<0.05)model group.After transfected with PD-L1 siRNA,level of Cleaved caspase-3 in PMN increased significantly at 6 h and 12 h(P<0.05).Levels of PKC-αand phosphorylated NF-κB in PMN of model group and experiment group were increased compared with sham operation group.PKC-αand phosphorylated NF-κB in PMN of the rats were significantly decreased after transfection with PD-L1 siRNA(P<0.05).Conclusion:PMN apoptosis was delayed in sepsis patients,which significantly negatively correlated with expression of PD-L1,suggesting that PD-L1 may be an important molecule regulating the delayed apoptosis of neutrophils in sepsis.In sepsis rats model,silencing PD-L1 gene can improve the delay of PMN apoptosis in sepsis rats.The mechanism may be related to inhibition of PKC-α-NF-κB signaling pathway activation in PMN of sepsis rats.
作者
罗玉娇
郑小莉(指导)
罗波
曾静媛
左英
LUO Yu-Jiao;ZHENG Xiao-Li;LUO Bo;ZENG Jing-Yuan;ZUO Ying(College of Basic Medicine,Southwest Medical University,Luzhou 646000,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2020年第17期2049-2052,2057,共5页
Chinese Journal of Immunology
基金
国家自然科学基金(31470041)资助。