摘要
目的 初步观察脓毒症小鼠脾脏树突状细胞(DC)焦亡情况及其与炎性因子水平、DC免疫功能的相关性。方法 70只BALB/c小鼠随机分为假手术组(n=20)、脓毒症模型组(CLP组,n=30)与胱天蛋白酶(CASP)-1抑制剂干预组(CLP+YVAD组,n=20),CLP组和CLP+YVAD组按术后不同时间点分为CLP 12 h组、CLP 24 h组、CLP 48 h组、CLP72 h组及CLP+YVAD24 h组、CLP+YVAD72 h组。所有小鼠于术后预定时间眼眶取血、处死并取脾脏组织。流式细胞仪测定脾脏DC焦亡率及表面标志物(CD80、CD86、MHC-Ⅱ)表达水平,激光共聚焦显微镜观察CASP-1在小鼠脾脏DC中的活化情况,Western blotting检测DC中CASP-1的表达情况,ELISA法检测血清中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-12、IL-1β、IL-6浓度;观察CLP术后小鼠死亡情况。提取BALB/c小鼠T细胞与各组小鼠脾脏DC共培养,采用流式细胞仪测定T细胞增殖率,ELISA法检测共培养上清中γ干扰素(IFN-γ)、IL-4、IL-10浓度。结果 与假手术组比较,DC焦亡率在CLP术后12 h升高(P<0.05),于24 h达高峰(P<0.01),随后呈下降趋势,至72 h仍高于假手术组(P<0.01)。激光共聚焦显微镜观察显示,CLP术后小鼠脾脏DC CASP-1活化明显。Western blotting检测结果显示,DC中焦亡蛋白CASP-1表达在脓毒症早期(12 h和24 h)较假手术组明显上调(P<0.01)。而给予CASP-1特异性抑制剂Ac-YVAD-cmk后,CLP+YVAD 24 h组DC焦亡率较CLP 24 h组下降(P<0.01),CLP+YVAD 72 h组DC表面标志物CD80、MHC-Ⅱ表达水平较CLP 72 h组上调(P<0.01),术后7 d小鼠生存率提高(P<0.01)。ELISA法检测结果显示,CLP小鼠血清TNF-α、IL-12、IL-1β和IL-6浓度在脓毒症早期(24 h)和后期(72 h)均较假手术组明显升高(P<0.01),而CLP+YVAD组血清内上述因子浓度明显下降(P<0.01)。共培养实验结果显示,与DC-CLP 24 h组比较,DC-CLP+YVAD 24 h组T细胞增殖率明显增高(P<0.01),共培养上清中IFN-γ水平下降,而IL-4、IL-10水平上升(P<0.01)。结论 DC焦亡在脓毒症早期启动并持续存在,可能是脓毒症状态下炎性因子水平异常增高和DC免疫功能抑制的病理生理机制之一,且与脓毒症预后不良相关。
Objective To preliminarily observe the pyroptosis of splenic dendritic cells(DC)in septic mice and its correlation with the levels of inflammatory factors and DC immune function.Methods Seventy BALB/c mice were randomly divided into sham group(n=20),sepsis model group(CLP group,n=30)and caspase(CASP)-1 inhibit group(CLP+YVAD group,n=20).The CLP group and CLP+YVAD group were then divided into CLP 12 h group,CLP 24 h group,CLP 48 h group and CLP 72 h group,and CLP+YVAD 24 h group and CLP+YVAD 72 h group at different time points after operation.Orbital blood was collected from all mice at a predetermined time after operation,and the mice were sacrificed and spleen tissues were extracted.The pyroptosis rate of DC and the expression levels of DC surface markers(CD80,CD86,MHC-Ⅱ)were determined by flow cytometry.Activation of CASP-1 in mouse spleen DC was observed using confocal laser microscope.Western blotting was used to determine the expression of CASP-1 in DC.ELISA was performed to measure the levels of tumor necrosis factor(TNF)-α,interleukin(IL)-12,IL-1β,and IL-6 in serum.Observe the death of mice after CLP operation.BALB/c mouse T cells were extracted and co-cultured with the mouse spleen DC of each group,the T cell proliferation rate was measured by flow cytometry,and the concentrations of interferon(IFN)-γ,IL-4 and IL-10 in the co-cultured supernatants were detected by ELISA.Results The pyroptosis rate of DC was increased 12 h after CLP compared to sham group(P<0.05),peaked at 24 h(P<0.01),and then progressively declined,but remained higher than sham group at 72 h after CLP(P<0.01).Confocal laser microscopy revealed that the activation of DC CASP-1 in spleen of mice after CLP was obvious.Western blotting showed the expression of CASP-1 in the early stage of sepsis(12 h and 24 h)was significantly higher than that in sham group(P<0.01).After administration of CASP-1 specific inhibitor Ac-YVAD-cmk,the DC pyroptosis rate at CLP+YVAD 24 h group was lower than that in CLP 24 h group(P<0.01).The expression levels of DC surface markers CD80 and MHC-Ⅱat CLP+YVAD 72 h group were up-regulated compared with CLP 72 h group(P<0.01),and the survival rate of mice at 7 d after operation was improved(P<0.01).The results of ELISA showed that the concentrations of serum TNF-α,IL-12,IL-1βand IL-6 of CLP mice in the early stage(24 h)and late stage(72 h)of sepsis were significantly higher than those in sham group(P<0.01),while the serum concentrations of the above factors in CLP+YVAD group decreased significantly(P<0.01).In the co-culture experiment,compared with DC-CLP 24 h group,the T cell proliferation rate of DC-CLP+YVAD group significantly increased(P<0.01),the level of IFN-γdecrease in co-cultured supernatants,and the levels of IL-4 and IL-10 increased(P<0.01).Conclusions Pyroptosis of DC appears to be activated at the early stage upon septic challenge,might be an important pathophysiological mechanism with regard to the extensive release of inflammatory cytokines as well as immune suppression of DC,which is associated with poor prognosis of sepsis.
作者
贺鹏翼
董宁
吴瑶
郝济伟
祝筱梅
姚咏明
He Peng-Yi;Dong Ning;Wu Yao;Hao Ji-Wei;Zhu Xiao-Mei;Yao Yong-Ming(Medical College of PLA,Beijing 100853,China;Medical Innovation Research Division of Chinese PLA General Hospital,Beijing 100853,China;Fourth Medical Center of Chinese PLA General Hospital,Beijing 100048,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2023年第5期537-544,共8页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(81871557,82130062,82241062)。
关键词
脓毒症
炎症反应
免疫抑制
树突状细胞
焦亡
sepsis
inflammatory response
immunosuppression
dendritic cells
pyroptosis
