NLRP3对小鼠肝脏缺血再灌注损伤的影响  

Effect of NLRP3 in liver tissues of mice with hepatic ischemia- reperfusion injury

作　　者：闫非易 朱志军 李世朋 张海明 孙丽莹 伍海锐 王学斌 郭伟[1] 韩威[3] Yan Feiyi;Zhu Zhijun;Li Shipeng;Zhang Haiming;Sun Liying;Wu Hairui;Wang Xuebin;Guo Wei;Han Wei(Liver Transplantation Center, Capital Medical University, National Clinical Research Center for Digestive Disease, Department of General Surgery, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China;Department of General Surgery, Jiaozuo People’s Hospital, Xinxiang Medical University, Jiaozuo 454000, China;Department of General Surgery, Beijing Luhe Hospital, Capital Medical University, Beijing 101100, China)

机构地区：[1]首都医科大学肝脏移植中心国家消化系统疾病临床医学研究中心首都医科大学附属北京友谊医院普外科,100050 [2]新乡医学院附属焦作市人民医院普外科,454000 [3]首都医科大学附属北京潞河医院普外科,101100

出　　处：《国际外科学杂志》2019年第5期300-305,F0003,共7页International Journal of Surgery

基　　金：国家自然科学基金项目(81570586);首都临床特色应用研究(Z181100001718220).

摘　　要：目的通过探讨NLR家族Pyrin域蛋白3(NLRP3)在小鼠肝脏缺血再灌注损伤(HIRI)肝组织中的表达,来初步判断NLRP3在HIRI过程中的作用。方法纯系C57BL/6雄性小鼠共42只,7~8周龄,体重为20~25 g,建立C57BL/6小鼠HIRI模型,采用完全随机化分组的方法将小鼠分为空白对照组,假手术组,2 h、6 h、12 h、24 h HIRI组及NLRP3抑制剂组(CY-09组),每组各6 只。收集各组小鼠肝脏标本。各组小鼠血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平使用全自动生化分析仪检测;各组小鼠血清白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、肿瘤坏死因子-α(TNF-α)含量使用酶联免疫吸附实验(ELISA)检测;用苏木精-伊红染色及氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)染色病理学检测方法观察各组小鼠肝脏标本细胞凋亡情况;用Western blotting检测各组小鼠炎症因子NLRP3、凋亡蛋白-1(Caspase-1)表达水平。计量资料以均数±标准差(Mean±SD)表示,组间比较采用单因素方差分析,如方差不齐则采用Dunnett C检验。结果各HIRI组小鼠血清ALT、AST水平和血清IL-1β、IL-18、TNF-α含量显著高于空白对照组及假手术组(P<0.05);各HIRI组的NLRP3和Caspase-1蛋白相对表达量显著增加。CY-09组小鼠血清ALT、AST水平和血清IL-1β、IL-18、TNF-α含量显著低于各HIRI组(P<0.05);CY-09组肝脏组织苏木精-伊红染色坏死区域明显少于各HIRI组;TUNEL染色提示CY-09组小鼠肝细胞凋亡率显著低于12 h HIRI组(P<0.05);CY-09组NLRP3蛋白相对表达量较各HIRI组小鼠显著下降,Caspase-1蛋白水平较各HIRI组显著下降。结论 HIRI可引起NLRP3表达增加,抑制NLRP3的表达后,HIRI的程度相对减轻。Objective According investigate the expression of NLRP3 in liver tissues of mice with hepatic ischemia-reperfusion injury (HIRI), to determin the role of NLRP3 in the process of HIRI. Methods Established mice model of partial HIRI. Forty-two male C57BL/6 mice (aged 7 to 8 weeks, weight 20 to 25 g) were respectively divided into 7 groups: no-treatment control group, sham operation group, HIRI groups (2、6、12、24 h) and CY-09 group, 6 mice in each group. The injury of the hepatic tissues in the 7 groups was analyzed based on detecting the levels of alanine transaminase (ALT), aspartate transaminase (AST), interleukin-1β(IL-1β), interleukin-18 (IL-18), tumor necrosis factor-α(TNF-α) by ELISA. HE and TUNEL staining were used to observe the pathological changes of liver tissues after HIRI. Western blotting assay were carried out to detect the expressions of NLRP3 and Caspase-1. Measurement data were expressed as mean±standard deviation (Mean±SD), and one-way variance analysis was used for comparison between groups. If the variance was not uniform, Dunnett C test was used. Results Serum ALT, AST, IL-1β, IL-18 and TNF-α of mice detected in HIRI groups were higher than no-treatment control group and sham operation group at all endpoints (P<0.05). The relative expression of NLRP3 and Caspase-1 in the liver tissues of mice in the HIRI groups were significantly higher than that in the no-treatment control group and sham operation group. Serum ALT, AST, IL-1β, IL-18 and TNF-α of mice detected in CY-09 group were lower than HIRI groups at all endpoints (P<0.05). Less hepatocellular necrosis were exhibited in CY-09 group, comparing to HIRI groups. The hepatocyte apoptosis rate of mice in the CY-09 group was significantly lower than that in the 12 h HIRI group (P<0.05). The relative expression of NLRP3 in the liver tissues of mice in the CY-09 group was significantly lower than that in other groups. The relative expression of Caspase-1 in the liver tissues of mice in the CY-09 group was significantly lower than th

关 键 词：肝疾病 再灌注损伤 缺血 小鼠 MR家族Pyrin域蛋白3 

分 类 号：R657.3[医药卫生—外科学]