Objective To explore the expression and significance of extracellular adenosine triphosphate(eATP) in concanavalin A(ConA)-induced murine liver injury model.Methods A total of 96 healthy mice were classified randomly into a control group and an experimental group(48 each group).From the tail vein,the mice in the control group were injected with a certain amount of normal saline,while those in the experimental group were injected with the same amount of 20 mg/kg ConA.After injection,liver tissues and blood specimens were collected at 2,6,12,18,24,48 h to do some test.Hematoxylin-eosin(HE) dyeing was carried out to assess the pathological change of liver tissue by microscope.The expression of purinergic receptors P2(P2X7) protein in liver and eATP,alanine aminotransferase(ALT),interleukin 1 beta(IL-1β) in serum were measured by Western-blot,chemiluminescence,Peitman Frankel assay,enzyme-linked immunosorbent assay(ELISA).Results ConA-induced liver injury in mice model was successfully constructed.Visible under an optical microscope,the lobular structure of liver tissue of mice in the control group was clear,with obvious liver cell cord,normal liver cell size and structure;there were obvious pathological changes of liver tissue of mice in the experimental group.Compared with the control group, the P2X7 of the experimental group had no significant change in expression levels at various time points.At 2,6,12,18,24,48 h ALT[(346±67) U/L,(927±105) U/L,(2.414±198) U/L,(1.785±153) U/L,(1.864±148) U/L,(438±84) U/L],eATP[(15.342±3.564) nmol/L,(46.135±7.531) nmol/L,(141.285±17.024) nmol/L,(705.263±98.635) nmol/L,(88.324±6.103) nmol/L,(26.142±2.379) nmol/L], IL-1β[(62±8) ng/L,(79±9) ng/L,(94±10) ng/L,(84±10) ng/L,(76±9) ng/L,(48±7) ng/L] of the experimental group were higher than those of control group′s [(45±9) U/L,(42±8) U/L,(45±8) U/L,(44±9) U/L,(43±10) U/L,(41±8) U/L;(0.746±0.036) nmol/L,(0.823±0.025) nmol/L,(0.692±0.043) nmol/L,(0.753±0.027) nmol/L,(0.814±0.043) nmol/L,(0.649±0.038) nmol/L;(30±4) ng/L,(31±4) ng/L,(30±4) ng/L,(33±5) ng/L,(37±5) ng/L,(35±5) ng/L], the differences were statistically significant(P<0.01).Conclusion In ConA-induced murine liver injury model, the expression of eATP increases in the injury liver tissues,promoting the synthesis and secretion of IL-1β through the P2X7 pathway,eventually aggravating the process of liver injury.%目的 探讨胞外腺苷三磷酸(eATP)在刀豆蛋白A(ConA)诱导肝损伤小鼠模型中的表达及意义.方法 选取96只健康的昆明种小鼠,采用随机数字法分为对照组和实验组,每组48只.实验组小鼠尾静脉注入20 mg/kg ConA,而对照组小鼠则注入等量的0.9%氯化钠注射液.注射完成后,分别在第2、6、12、18、24、48小时收集两组小鼠的肝脏标本以及血液标本并进行相关指标的检测.对于肝脏组织的病理学变化,采用苏木精-伊红染色法在显微镜下观察;肝脏组织中嘌呤受体P2(P2X7)蛋白和血清中eATP、丙氨酸转氨酶(ALT)、白细胞介素1β(IL-1β)的表达情况,分别采用免疫印迹法、化学发光技术、赖氏法、酶联免疫吸附测定实验进行检测.结果 ConA诱导小鼠肝损伤的模型成功构建.在光学显微镜下可见对照组小鼠的肝组织中肝小叶结构较为清晰,肝细胞索明显,肝细胞大小、结构正常;实验组小鼠的肝组织出现明显的病理学变化.与对照组相比,实验组P2X7在各个时间点的表达水平无明显变化.实验组第2、6、12、18、24、48小时ALT[(346±67) U/L,(927±105) U/L,(2 414±198) U/L,(1 785±153) U/L,(1 864±148) U/L,(438±84) U/L]、eATP[(15.342±3.564) nmol/L,(46.135±7.531) nmol/L,(141.285±17.024) nmol/L,(705.263±98.635) nmol/L,(88.324±6.103) nmol/L,(26.142±2.379) nmol/L]、IL-1β[(62±8) ng/L,(79±9) ng/L,(94±10) ng/L,(84±10) ng/L,(76±9) ng/L,(48±7) ng/L]均高于对照组[(45±9) U/L,(42±8) U/L,(45±8) U/L,(44±9) U/L,(43±10) U/L,(41±8) U/L;(0.746±0.036) nmol/L,(0.823±0.025) nmol/L,(0.692±0.043) nmol/L,(0.753±0.027) nmol/L,(0.814±0.043) nmol/L,(0.649±0.038) nmol/L;(30±4) ng/L,(31±4) ng/L,(30±4) ng/L,(33±5) ng/L,(37±5) ng/L,(35±5) ng/L],差异有统计学意义(P<0.01).结论 ConA诱导小鼠肝损伤后,eATP在损伤的肝组织中表达水平明显增高,并且通过P2X7受体途径,促进IL-1β的合成和分泌,进而参与肝损伤的进程.
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