日本血吸虫可溶性虫卵抗原诱导小鼠巨噬细胞凋亡机制

Soluble egg antigen of Schistosoma japonicum induces macrophage apoptosis in mice

目的观察日本血吸虫感染过程中巨噬细胞数量及其凋亡动态变化,并探讨血吸虫可溶性虫卵抗原(SEA)诱导巨噬细胞凋亡的可能机制。方法将C57BL/6小鼠(6~8周龄)随机分为4组,其中3组作为实验组,另1组作为正常对照组。实验组每只小鼠均经腹部皮肤感染(12±1)条日本血吸虫尾蚴,于感染后3、5周和8周各处死1组小鼠;正常对照组小鼠不感染血吸虫,于实验组小鼠感染当天处死。取各组小鼠肝组织和腹腔渗出细胞,检测肝脏和腹腔巨噬细胞数量及其凋亡动态变化。此外,体外用SEA、PBS及卵清蛋白(ovalbumin,OVA)处理纯化的小鼠腹腔巨噬细胞,流式细胞术检测巨噬细胞凋亡;以实时定量PCR和Western blotting技术检测巨噬细胞中BCL⁃2家族成员mRNA和蛋白表达水平;以流式细胞术和Western blotting技术检测caspase 3活化水平。同时,体外在caspase抑制剂、H_(2)O_(2)或N⁃乙酰⁃L⁃半胱氨酸(NAC)存在时用SEA处理巨噬细胞,以流式细胞术检测巨噬细胞凋亡水平。结果感染日本血吸虫尾蚴后3、5周和8周,小鼠肝脏[(0.873±0.106)×10^(6)、(2.737±0.460)×10^(6)个和(3.107±0.367)×10^(6)个;F=81.900,P<0.01]和腹腔中巨噬细胞总数[(5.282±1.136)×10^(5)、(7.500±1.200)×10^(5)个和(12.800±0.800)×10^(5)个;F=55.720,P<0.01]不断增加,且感染小鼠肝脏[(0.092±0.018)×10^(6)、(0.186±0.025)×10^(6)个和(0.173±0.027)×10^(6)个;F=57.780,P<0.0001]和腹腔中凋亡的巨噬细胞数量[(0.335±0.022)×10^(5)、(0.771±0.099)×10^(5)个和(1.094±0.051)×10^(5)个;F=49.460,P<0.01]亦不断增加。经SEA体外处理的巨噬细胞凋亡比例[(24.330±0.784)%]高于PBS[(18.500±1.077)%]及OVA[(18.900±1.350)%]处理组(P均<0.01)。经SEA和PBS处理的巨噬细胞中,Bcl⁃2[(1.662±0.943)vs.(1.00±0.00);t=1.215,P>0.05]、Bax[(0.711±0.200)vs.(1.00±0.00);t=2.507,P>0.05]、Bak[(1.255±0.049)vs.(1.00±0.00);t=0.897,P>0.05]、BCL⁃2[(0.068±0.004)vs.(0.070±0.005);t=0.699,P>0.05]、BAX[(0.089±0.005)vs.(0.097±0.003);t=2.232,P>0.05]、BAK[(0.439±0.048)vs.(0.571±0.091);t=2.231,P>0.05]表达水平差异均无统计学意义。在cas-pase抑制剂存在时,SEA仍能诱导巨噬细胞凋亡(F=0.411,P>0.05);而在H_(2)O_(2)或NAC存在时,SEA不能诱导巨噬细胞凋亡(F=11.880、9.897,P均<0.05)。结论在日本血吸虫感染过程中,SEA可能通过促进活性氧表达而诱导巨噬细胞凋亡。

Objective To investigate the dynamic changes of macrophage numbers and apoptosis during Schistosoma japonicum infection,and to investigate the possible mechanisms of macrophage apoptosis induced by S.japonicum soluble egg antigen(SEA).Methods C57BL/6 mice at ages of 6~8 weeks were randomly divided into 4 groups,including three experimental groups and a normal control group.Each mouse in the experimental groups was infected with(12±1)cercariae of S.japonicum via the abdominal skin,and all mice in an experimental group were sacrificed 3,5,8 weeks post⁃infection,respectively,while mice in the control group were not infected with S.japonicum cercariae and sacrificed on the day of S.japonicum infection in the experimental group.Mouse liver specimens and peritoneal exudation cells were sampled in each group,and the dynamic changes of macrophage numbers and apoptosis were detected.Mouse peritoneal macrophages were isolated,purified and treated with S.japonicum SEA,PBS and ovalbumin(OVA)in vitro,and the macrophage apoptosis was detected using flow cytometry.The mRNA and protein expression of BCL⁃2 protein family members were determined in macrophages using real⁃time quantitative PCR(qPCR)and Western blotting assays,and the activation of caspase 3 was determined using flow cytometry and Western blotting.In addition,macrophages were in vitro treated with S.japonicum SEA in presence of a caspase inhibitor,H_(2)O_(2) or N⁃acetyl⁃L⁃cyste⁃ine,and the apoptosis of macrophages was detected using flow cytometry.Results The total macrophage numbers continued to increase in mouse liver[(0.873±0.106)×10^(6),(2.737±0.460)×10^(6) and(3.107±0.367)×10^(6) cells,respectively;F=81.900,P<0.01]and peritoneal specimens[(5.282±1.136)×10^(5),(7.500±1.200)×10^(5) and(12.800±0.800)×10^(5) cells,respectively;F=55.720,P<0.01]3,5 and 8 weeks post⁃infection with S.japonicum,and the numbers of apoptotic macrophages also continued to increase in mouse liver[(0.092±0.018)×10^(6),(0.186±0.025)×10^(6) and(0.173±0.0270)×10^(6) cells;F=57.780,P<0.01]and peritoneal specimens[(0.335±0.022)×10^(5),(0.771±0.099)×10^(5)and(1.094±0.051)×10^(5)cells;F=49.460,P<0.01]3,5 and 8 weeks post⁃infection with S.japonicum.The apoptotic rate of SEA⁃treated macrophages[(24.330±0.784)%]was significantly higher than that of PBS⁃[(18.500±1.077)%]and OVA⁃treated macrophages[(18.900±1.350)%](both P values<0.01).There were no significant differences in the mRNA or protein expression of Bcl⁃2[Bcl⁃2 mRNA expression:(1.662±0.943)vs.(1.000±0.000),t=1.215,P>0.05;BCL protein expression:(0.068±0.004)vs.(0.070±0.005),t=0.699,P>0.05],Bax[Bax mRNA expression:(0.711±0.200)vs.(1.000±0.000),t=2.507,P>0.05;BAX protein expression:(0.089±0.005)vs.(0.097±0.003),t=2.232,P>0.05]and Bak[Bak mRNA expression:(1.255±0.049)vs.(1.00±0.00),t=0.897,P>0.05;BAK protein expression:(0.439±0.048)vs.(0.571±0.091),t=2.231,P>0.05]between in SEA⁃and PBS⁃treated macro⁃phages.S.japonicum SEA induced macrophage apoptosis in the presence of a caspase inhibitor(F=0.411,P>0.05);however,SEA failed to induce macrophage apoptosis in the presence of H_(2)O_(2) or NAC(F=11.880 and 9.897,both P values<0.05).Conclusion S.japonicum SEA may induce macrophage apoptosis through promoting reactive oxygen species expression during S.japonicum infections in mice.