【作者】 赵岩；

【导师】 申艳娜；

【作者基本信息】 天津医科大学 ， 临床检验诊断学， 2023， 硕士

【摘要】 目的肺炎链球菌(Streptococcus pneumoniae,S.pneumoniae)是世界范围内的一种重要的临床病原体,可长期无症状定植于上呼吸道,其感染可引起严重的侵袭性疾病,如中耳炎、肺炎、脑膜炎甚至败血症等。NOD样受体家族含pyrin结构域3(NOD-like receptor family pyrin domain containing 3,NLRP3)炎症小体作为先天免疫系统的重要组成部分,是抵御病原体入侵宿主的第一道屏障。研究表明S.pneumoniae的溶血素参与激活NLRP3炎症小体,然而具体机制尚不完全清楚。活化的NLRP3炎症小体可触发细胞焦亡进而在胞膜上成孔,使炎性细胞因子分泌至胞外发挥其免疫防御功能,而细胞焦亡可否在S.pneumoniae感染过程中发挥作用还有待进一步研究。间变性淋巴瘤激酶(Anaplastic lymphoma kinase,ALK)主要参与调控非小细胞肺癌等癌症的发生发展过程,其在感染性疾病中发挥的作用尚不明确。曾有研究发现,ALK在脂多糖(Lipopolysaccharide,LPS)与三磷酸腺苷(Adenosine triphosphate,ATP)协同刺激巨噬细胞过程中可通过促进NEK7-NLRP3复合物形成进而激活NLRP3炎症小体,其在感染性疾病中发挥的作用尚不明确。由此,我们拟探究ALK在革兰阳性菌S.pneumoniae感染过程中的作用机制。此外,C-Jun氨基末端激酶(C-Jun N-terminal kinase,JNK)在抵抗多类型病原体感染过程中均发挥重要作用,且JNK在糖尿病心肌病中是激活NLRP3炎症小体的重要分子之一,然而其在感染性疾病诱发的细胞焦亡中可否发挥作用未见报道。基于以上研究成果,本研究旨在明确ALK、JNK与NIMA相关激酶7(NIMA-related kinase 7,NEK7)在S.pneumoniae感染激活NLRP3炎症小体及细胞焦亡过程中的作用机制,以期为S.pneumoniae感染性疾病的研究提供新的理论依据。方法1.C57BL雌性小鼠随机分为未感染组、未感染+ALK抑制剂处理组、未感染+JNK抑制剂处理组、感染组、感染+ALK抑制剂处理组、感染+JNK抑制剂处理组。菌落涂布法计数小鼠肺组织及支气管肺泡灌洗液菌落数量;ELISA检测小鼠支气管肺泡灌洗液中IL-1β及IL-6含量;H&E染色观察小鼠肺部组织结构。2.小鼠原代腹腔渗出巨噬细胞(Peritoneal Exudate Cells,PECs)经S.pneumoniae(MOI 5)分别感染0、2、4、6、8、10、12小时,Western Blot方法检测pro-caspase-1、p20、IL-1β、p17、NEK7、GSDMD及GSDMD-NT的蛋白表达水平。3.S.pneumoniae(MOI 5)感染PECs 6小时,RT-PCR检测Nek7的m RNA水平;Western Blot检测NEK 7的蛋白水平;Co-IP检测NEK7-NLRP3复合物的形成。4.S.pneumoniae(MOI 5)分别感染PECs 0、5、15、30、60分钟。Western Blot检测p-ALK、p-JNK的蛋白水平。5.使用ALK抑制剂预处理PECs 2小时,经S.pneumoniae(MOI 5)感染1小时,Western Blot检测JNK磷酸化水平;RT-PCR检测Jnk的m RNA表达水平。6.使用NEK7-siRNA或ALK/JNK抑制剂分别预处理PECs 48/2小时,经S.pneumoniae(MOI 5)感染6小时,Western Blot检测细胞内容物中IL-1β、p17、pro-caspase-1、p20、GSDMD、GSDMD-NT的蛋白水平变化,检测细胞上清液中IL-1βp17、caspase-1 p20的蛋白水平变化;Co-IP检测NEK7-NLRP3复合物的形成;RT-PCR检测Caspase-1、Il-1β、Nlrp3、Nek7、Gsdmd的m RNA水平变化;LDH试剂盒检测细胞上清液中LDH浓度;免疫荧光观察并计算ASC斑点蛋白数量;ELISA检测细胞的培养上清液中IL-1β的分泌程度;菌落涂布法计数PECs内活菌数量并计算细菌清除率。结果1.较未感染组小鼠,感染组小鼠肺组织及支气管肺泡灌洗液中菌落数量明显增多、支气管肺泡灌洗液中IL-1β及IL-6含量明显增多、肺部组织结构紊乱并伴随红细胞渗出等炎性病理变化;较感染组小鼠,抑制剂预处理感染组小鼠肺组织及支气管肺泡灌洗液中菌落数量显著增多、支气管肺泡灌洗液中IL-1β及IL-6含量明显减低、肺部炎性病理变化减轻。2.S.pneumoniae感染过程中p20、p17及GSDMD-NT的蛋白表达水平在感染6小时显著增高,NEK7的蛋白表达水平在感染6小时明显减低。3.感染组NEK7的表达水平较未感染组明显降低而NEK7-NLRP3复合物显著增多。4.P-ALK、p-JNK分别在S.pneumoniae感染PECs 5、15分钟时开始增高,且具有时间依赖性,其磷酸化水平分别于S.pneumoniae感染PECs 15、60分钟时达到峰值。5.S.pneumoniae感染诱导的JNK的磷酸化水平及m RNA高表达水平在经ALK抑制剂预处理后显著降低。6.感染组p20、p17及GSDMD-NT的蛋白水平较未感染组显著增高,而经NEK7-siRNA或ALK/JNK抑制剂分别预处理48/2小时后可显著降低已增高的蛋白水平;感染组Caspase-1、Il-1β及Gsdmd的m RNA水平较未感染组显著增高,而NEK7-si RNA或ALK/JNK抑制剂预处理可显著降低其已增高的转录水平;感染组LDH分泌水平较未感染组明显增高,而NEK7-si RNA或ALK/JNK抑制剂预处理可显著降低其分泌水平;ALK/JNK抑制剂预处理明显减少S.pneumoniae感染诱导的Nlrp3的m RNA表达水平、ASC斑点蛋白数量、IL-1β的分泌水平、NEK7-NLRP3复合物的形成及细菌清除率。结论1.NEK7在S.pneumoniae感染诱导的NLRP3炎症小体激活过程中至关重要。2.ALK/JNK信号通路通过调控S.pneumoniae感染过程中NLRP3的表达及NEK7-NLRP3复合物的形成参与激活NLRP3炎症小体。3.ALK/JNK/NEK7-NLRP3信号通路参与S.pneumoniae感染诱导的细胞焦亡。更多还原

【Abstract】 ObjectiveStreptococcus pneumoniae(S.pneumoniae),an important pathogen in clinic worldwide,colonizes the upper respiratory tract with no symptoms for a long period time.S.pneumoniae infection causes serious invasive diseases,such as otitis media,pneumonia,meningitis and sepsis.The NOD-like receptor family pyrin domain containing 3(NLRP3)inflammasome,an important component of the innate immune system,serves as the first line carrier in defense against pathogen infection.It has been shown that hemolysins of S.pneumoniae are involved in activating NLRP3inflammasome,however,the exact mechanism is not fully understood.Activate NLRP3 inflammasome triggers pyroptosis and the inflammatory cytokines release from the gasdermin pore for immune defense.The role of pyroptosis in S.pneumoniae infection needs to be further investigated.Anaplastic lymphoma kinase(ALK)is involved in regulating the development of cancers,such as non-small cell lung cancer.However,the function of ALK in infectious diseases is still unclear.ALK activates NLRP3 inflammasome by promoting the formation of NEK7-NLRP3complex during the stimulation of lipopolysaccharide and adenosine triphosphate in macrophages.Thus,we investigate the role of ALK during S.pneumoniae infection.In addition,JNK plays a key role in the resistance to multiple pathogens,and JNK is an important molecule in activating NLRP3 inflammasome in diabetic cardiomyopathy,whether JNK participated in infectious disease-induced pyroptosis has not been reported.Based on the above results,this study aims to clarify the role of ALK,JNK and NIMA-related kinase 7(NEK7)in the S.pneumoniae infection-induced NLRP3 inflammsome activation and pyroptosis.This study may provide a new theoretical basis of S.pneumoniae infectious diseases.Methods1.C57BL female mice were randomly divided into uninfected group,uninfected+ALK inhibitor pretreated group,uninfected+JNK inhibitor pretreated group,infected group,infected+ALK inhibitor pretreated group and infected+JNK inhibitor pretreated group.The number of colonies in lung tissues and bronchoalveolar lavage fluid of mice was counted by agar plating method;the secretion level of IL-1βand IL-6 in bronchoalveolar lavage fluid of mice were detected by ELISA;the structure of lung tissue was observed by H&E stain.2.Mouse peritoneal exudate cells(PECs)were infected with S.pneumoniae for 0,2,4,6,8,10 and 12 h at MOI of 5.The protein expression levels of pro-caspase-1,p20,IL-1β,p17,NEK7,GSDMD and GSDMD-NT were detected by Western Blot.3.PECs were infected with S.pneumoniae(MOI 5)for 6 h.The m RNA level of Nek7 was detected by RT-PCR;the protein level of NEK7 was detected by Western Blot;the formation of NEK7-NLRP3 complex was detected by Co-IP.4.PECs were infected with S.pneumoniae(MOI 5)for 0,5,15,30 and 60 min.The expression level of p-ALK and p-JNK were detected by Western Blot.5.PECs were pretreated with or without ALK inhibitor for 2 h and then infected with S.pneumoniae(MOI 5)for 1 h.The phosphorylation level of JNK was detected by Western Blot;the m RNA expression level of Jnk was detected by RT-PCR.6.PECs were pretreated with or without NEK7-si RNA or ALK/JNK inhibitor for48/2 h and infected with S.pneumoniae(MOI 5)for 6 h.The protein levels of IL-1β,p17,pro-caspase-1,p20,GSDMD and GSDMD-NT in cell lysis were detected by Western Blot;the protein levels of p17 and p20 in cell supernatant were detected by Western Blot;the formation of NEK7-NLRP3 complex were detected by Co-IP;the m RNA levels of Caspase-1,Il-1β,Nek7,Nlrp3 and Gsdmd were detected by RT-PCR;the concentration of LDH in cell supernatant were detedted by LDH kit;the formation of ASC specks were observed by immunofluorescence;the secretion of IL-1βwere detedted by ELISA;bacteria killing were calculate by agar plating method.Results1.The colonies in the lung tissue and bronchoalveolar lavage fluid(BALF)in S.pneumoniae infection group were much more than those in non-infection group;the secretion level of IL-1βand IL-6 in the BALF in the infection group were increased significantly;the infection group obtained a serious damage in the structure of lung tissues;the bacteria number in the lung tissues and BALF of the inhibitor pretreated infection group was increased significantly than those in infection group;the secretion level of IL-1βand IL-6 in the BALF inhibitor pretreated infection group decreased significantly;the inflammatory pathological changes in the lung tissues had a improvement with inhibitor pretreated during S.pneumoniae infection2.The protein expression levels of caspase-1 p20,IL-1βp17 and GSDMD-NT were significantly increased at 6 h during S.pneumoniae infection,and the protein expression level of NEK7 was decreased at the meanwhile.3.The protein expression level of NEK7 in the infected group was significantly decreased,while the NEK7-NLRP3 complex were increased obviously.4.The expression levels of p-ALK and p-JNK were increased at 5 and 15 min post S.pneumoniae infection and their phosphorylation levels reached a peak at 15 and 60min.5.The phosphorylation level and m RNA high expression level of JNK induced by S.pneumoniae infection were significantly reduced after pretreatment with ALK inhibitors.6.PECs pretreated with NEK7-si RNA or ALK/JNK inhibitors reversed the S.pneumoniae infection induced the protein levels of caspase-1 p20、IL-1βp17 and GSDMD-NT;the m RNA levels of Caspase-1、Il-1βand Gsdmd were significantly decreased by NEK7-si RNA or ALK/JNK inhibitors pretreated upon S.pneumoniae infection;the secretion levels of LDH were increased in the infection group than those in the non-infection group,while PECs pretreated with NEK7-si RNA or ALK/JNK inhibitors reduced the secretion level;ALK/JNK inhibitors downregulated the S.pneumoniae infection induced the m RNA expression of Nek7,the secretion level of inflammatory cytokines,the formation of the NEK7-NLRP3complex and ASC specks;ALK/JNK inhibitors reduced the bacterial clearance.Conclusions1.NEK7 is vital in S.pneumoniae infection-induced NLRP3 inflammasome activation.2.ALK/JNK signaling pathway activates NLRP3 inflammasome by regulating the expression of NLRP3 and the formation of NEK7-NLRP3 complex during S.pneumoniae infection.3.ALK/JNK/NEK7-NLRP3 axis is involved in S.pneumoniae induced pyroptosis.更多还原