摘要
目的:利用Cre/iDTR系统特异性剔除Kupffer细胞,探究Kupffer细胞在对乙酰氨基酚(APAP)诱导的药物性肝损伤中的作用。方法:腹腔注射1μg白喉毒素(DT),流式细胞术检测注射后肝脏Kupffer细胞的清除效率。接着将Clec4fCre/iDTR小鼠分为4组,分别是对照组、注射DT组、注射APAP组及注射DT和APAP组,饥饿18 h后腹腔注射300 mg/kg APAP,建立急性药物性肝损伤模型。酶联免疫吸附试验(ELISA)和细胞因子微球检测(CBA)技术检测血清中丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)的释放和炎症因子的分泌;苏木精-伊红(HE)染色和原位末端转移酶标记法(TUNEL)染色检测肝组织的坏死和肝细胞的凋亡;免疫组织化学检测肝脏炎性细胞的浸润;免疫印迹法检测肝组织中信号通路的活化;定量聚合酶链反应(qPCR)检测肝脏中炎症因子的mRNA表达水平。结果:清除Kupffer细胞,降低APAP模型血清中ALT、AST转氨酶的释放,减轻肝组织坏死和肝细胞凋亡。与对照组相比,清除Kupffer细胞抑制血清中TNF-α、IL-6等炎症因子的分泌,减少肝脏F4/80^(+)、Ly6G^(+)细胞的浸润,降低TNF-α、IL-6等炎性因子的mRNA水平以及JNK炎症通路的活化。结论:清除Kupffer细胞可显著缓解APAP诱导的药物性肝损伤,明确了Kupffer细胞在APAP致肝损伤中的重要作用。
Objective:To explore the role of Kupffer cells in APAP-induced liver injury by using the Cre/iDTR syste to specifically depleteKupfer cells.Methods:ClefrviDTRm ice were injected intraperitoneally with 1μg DT(Diphtheria Ttoxin,diphtheria toxin),and the depletion efficiency of Kupffer cells in the liver was detected by flow cytometry,.Then,CleferviDTRm ice were divided into four groups,namely control group,DT group,APAP group,DT and APAP group.300 mg/kg APAP was injected intraperitoneally after 18 h of starvation to establish an acute liver injury model.ELISA and CBA analysis were used to detect the release of ALT and AST and the secretion of inflammatory factors in serum.H&E staining and TUNEL staining were used to detect the necrosis of liver tissue and the apoptosis of hepatocytes.Immunohistochemistry was used to detect inflammatory cell infiltration in the liver and immunoblotting to detect activation of the JNK signaling pathway.qPCR was used to detect the transcriptional expression of inflammatory factors in the liver.Results:Hepatic Kupffer clls in Clec4fcre/iDTRmice were almost completely depleted at 12 and 24 h after injection of 1μg DT.Depletion of Kupffer cells potently ameliorated APAP-induced liver injury,as manifested by decreased serum ALT and AST levels,less hepatic necrotic area and hepatocyte apoptosis,reduced inflammatory cell infiltration and production of inflammatory cytokines,and abrogated activation of the JNK pathway.Conclusion:This study demonstrated that removal of Kupfer cells significantly attenuated APAP-induced liver injury,suggesting an essential contribution of Kupffer cells to APAP hepatotoxicity.
作者
刘慧茹
王婷
李亚婷
向慎思
詹轶群
任广明
杨晓明
LIU Huiru;WANG Ting;LI Yating;XIANG Shensi;ZHAN Yiqun;REN Guangming;YANG Xiaoming(School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;Faculty of Environment and Life Sciences,Beijing University of Technology,Beijing 100124,China;Beijing Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2024年第6期68-77,共10页
China Biotechnology
基金
北京市自然科学基金(7222122)资助项目。
