日本血吸虫感染对小鼠肝脏铁分布及铁蛋白表达的影响

Effect of Schistosoma japonicum infection on iron distribution and ferritin expression in mouse liver

目的研究日本血吸虫感染对小鼠肝脏铁分布及铁蛋白表达的影响。方法24只昆明小鼠随机分为对照组、感染6、8、10周组,每组6只。感染组经腹部皮肤接种尾蚴(20±2)只,待小鼠染虫6、8、10周时,处死小鼠留取肝脏,采用苏木精-伊红(HE)染色和马松(Masson)染色观察小鼠肝脏病理变化,采用二氨基联苯胺(DAB)增强的普鲁士蓝(Perl’s)铁染色观察肝组织铁分布,采用免疫组织化学染色检测肝脏铁蛋白的表达,采用逆转录聚合酶链反应(RT-qPCR)检测小鼠肝脏铁蛋白轻链和重链mRNA的表达。结果与对照组小鼠比较,血吸虫感染6、8、10周组小鼠肝脏颜色逐渐变深,质地逐渐变硬。HE和Masson染色显示,感染组小鼠肝组织内有虫卵沉积,围绕虫卵形成肉芽肿及纤维化,而对照组未出现虫卵肉芽肿。DAB增强的Perl’s铁染色显示,与对照组比较,感染6、8、10周组肝细胞铁染色强度降低,差异均有统计学意义(t=5.94、7.72、9.67,P均<0.05),肝血窦内有铁色素沉积,且随感染时间延长逐渐增多(t=3.89、8.59、12.50,P均<0.05),虫卵壳铁染色较强。免疫组织化学染色显示,感染6、8、10周组的肝脏铁蛋白表达均低于对照组(t=5.59、7.35、9.66,P均<0.05),且随着感染时间延长逐渐减少。RT-qPCR结果显示,感染6、8、10周组的铁蛋白轻链和重链mRNA表达水平均低于对照组(t_(轻链)=4.01、7.21、9.97,t_(重链)=4.50、5.23、5.22,P均<0.05)。铁蛋白轻链mRNA随着感染时间的延长逐渐减少,与感染6周组比较,感染8周、10周组差异均有统计学意义(t=3.20、5.96,P均<0.05)。重链mRNA随着感染时间的延长无明显变化,感染6、8、10周组间差异均无统计学意义(t=0.73、0.72、0.01,P均>0.05)。结论日本血吸虫感染可导致小鼠肝细胞铁含量减少,铁蛋白表达降低,这间接反映了血吸虫感染导致机体缺铁的状态,是血吸虫感染所致贫血的机制之一。

Objective To study the effect of Schistosoma japonicum infection on iron distribution and ferritin expression in mouse liver.Methods A total of 24 Kunming mice were randomly divided into control group,infected for 6 weeks group,infected for 8 weeks group and infected for 10 weeks group,with 6 mice in each group.The mice were infected with cercariae(20±2)through abdominal skin.After 6,8 and 10 weeks of infection,the mice were sacrificed and the liver was collected.The pathological changes of liver were observed by hematoxylin⁃eosin(HE)staining and Masson staining.The iron distribution of liver was showed by diaminobenzidine(DAB)enhanced Perl's iron staining.The expression of ferritin in liver were detected by immunohistochemical staining.The expression of ferritin light chain mRNA and heavy chain mRNA in mouse liver was detected by quantitative reverse transcription PCR(RT⁃qPCR).Results Compared with control group,the liver of mice infected with Schistosoma japonicum became darker and harder.HE and Masson staining showed that there were egg deposits in the liver tissue of infected mice,and the eggs were surrounded by granulomas and collagen fibers.The control group had no worm egg granulomas.DAB enhanced Perl's iron staining showed that the iron distribution of hepatocytes in the 6,8 and 10 weeks infected group mice was lower than that in the control group(t=5.94,7.72,9.67;all P<0.05).There were iron pigment deposits in the hepatic sinusoids,and gradually increased with the extension of infection time(t=3.89,8.59,12.50;all P<0.05),and the iron staining of the egg shell was strong.Immunohistochemical staining showed that the expression of liver ferritin in the 6,8 and 10 weeks infected groups were lower than that in the control group(t=5.59,7.35,9.66;all P<0.05),and gradually decreased with the extension of infection time.RT⁃qPCR results showed that the expression levels of ferritin light chain and heavy chain mRNA in the 6,8 and 10 weeks infected groups were lower than those in the control group(t_(light chain)=4.01,7.21,9.97,t_(heavy chain)=4.50,5.23,5.22;all P<0.05).Ferritin light chain mRNA gradually decreased with the prolongation of infection time,compared with the 6 weeks infected group,there were significant differences between 8 and 10 weeks infected groups(t=3.20,5.96;all P<0.05),while ferritin heavy chain mRNA did not change significantly,there were not significant differences in 6,8 and 10 weeks infected groups(t=0.73,0.72,0.01;all P>0.05).Conclusion Schistosoma japonicum infection could reduce the iron content and ferritin expression in liver cells of mice,which suggested that iron deficiency caused by schistosomiasis infection might be one of the mechanisms of schistosomiasis anemia.