包虫囊液对干扰素信号TRIF/IFITM3通路的影响

The impact of hydatid cyst fluid on the interferon signaling TRIF/IFITM3 pathway

目的 探索包虫囊液干预人源肝细胞(L02)过程中TRIF/IFITM3通路相关蛋白的变化。方法 从感染细粒棘球蚴病的羊肝中获取囊液,用不同比例囊液浓度(1∶10、1∶100和1∶1000)干预肝细胞并设置阴性对照组(未加囊液干预)。采用囊液浓度(1∶1000)干预肝细胞后分别在0、24、48和72 h收集细胞。实时荧光定量PCR检测TRIF/IFITM3通路相关mRNA的表达,Western blot检测TRIF、IRF3、IFITM3、Foxp3的蛋白表达变化。免疫荧光检测IFITM3、Foxp3的表达和定位。Spearman分析TRIF、IFITM3及Foxp3的相关性。结果 Western blot显示囊液浓度(1∶1000)促进TRIF、IRF3、IFITM3蛋白表达,TRIF的相对表达量分别是(0.45±0.09)、(0.54±0.05)、(1.08±0.24)、(1.75±0.31);IFITM3的相对表达量分别是(1.02±0.41)、(1.00±0.16)、(1.24±0.12)、(1.84±0.20)。囊液浓度(1∶1000)刺激肝细胞0、24、48和72 h后,Western blot显示TRIF、IRF3、IFITM3蛋白表达量逐渐下降,而Foxp3表达量增高。TRIF的相对表达量分别是(1.00±0.31)、(1.08±0.19)、(0.90±0.13)、(0.57±0.05);IFITM3的相对表达量分别是(0.80±0.26)、(0.94±0.09)、(0.82±0.19)、(0.54±0.18)。Foxp3的相对表达量分别是(0.70±0.11)、(0.80±0.02)、(0.83±0.03)、(1.08±0.04)。免疫荧光显示,在泡球蚴感染60 d时,小鼠肝脏IFITM3和Foxp3发生共定位。Spearman分析TRIF与IFITM3存在正相关性(r=0.704,P<0.01),TRIF与Foxp3存在负相关性(r=-0.859,P<0.01),IFITM3与Foxp3存在负相关性(r=-0.686,P<0.01)。结论 较低浓度的包虫囊液刺激TRIF/IFITM3通路相关分子表达,进而激活天然免疫的抗病原体功能;随着囊液干预时间增加TRIF/IFITM3受抑制,而Foxp3高表达,促进包虫在宿主体内慢性寄生。

Objective This study investigates the modulation of TRIF/IFITM3 pathway-associated proteins in human liver cells(L02)following exposure to hydatid cyst fluid.Methods Hydatid cyst fluid was harvested from the livers of sheep infected by Echinococcus granulosus.L02 cells were treated with varying concentrations of this fluid(1∶10,1∶100,1∶1000)alongside a negative control group that did not receive cyst fluid.Samples were collected at 0,24,48,and 72 hours post-treatment for the 1∶1000 dilution.The expression levels of mRNAs related to the TRIF/IFITM3 pathway were quantified using real-time fluorescence quantitative PCR,while protein expression changes in TRIF,IRF3,IFITM3,and Foxp3 were analyzed through Western blot.Immunofluorescence was employed to examine the localization and expression of IFITM3 and Foxp3 proteins.Spearman's rank correlation coefficient was calculated to evaluate the relationships among TRIF,IFITM3,and Foxp3 expressions.Results Western blot showed that hydatid cyst fluid(1∶1000)promoted TRIF,IRF3,and IFITM3 protein expression,and the expression of TRIF were(0.45±0.09),(0.54±0.05),(1.08±0.24),(1.75±0.31);the expression of IFITM3 were(1.02±0.41),(1.00±0.16),(1.24±0.12),(1.84±0.20).The hydatid cyst fluid(1∶1000)stimulated the hepatocytes for 0,24,48 and 72 h,Western blot showed a gradual decrease in the protein expression of TRIF,IRF3,IFITM3 and an increase in the expression of Foxp3.The expression of TRIF were(1.00±0.31),(1.08±0.19),(0.90±0.13),(0.57±0.05);the expression of IFITM3 were(0.80±0.26),(0.94±0.09),(0.82±0.19),(0.54±0.18);the expression of Foxp3 were(0.70±0.11),(0.80±0.02),(0.83±0.03),(1.08±0.04).Immunofluorescence confirmed the co-localization of IFITM3 and Foxp3 in liver cells at 60 days post-infection.Spearman correlation analysis indicated a strong positive correlation between TRIF and IFITM3 expressions(r=0.704,P<0.01),a strong negative correlation between TRIF and Foxp3(r=-0.859,P<0.01),and a negative correlation between IFITM3 and Foxp3(r=-0.686,P<0.01).Conclusion Low concentrations of hydatid cyst fluid elicit an upregulation of TRIF/IFITM3 pathway-related molecules,thereby activating the host's innate immune response against the pathogen.Over time,this response is suppressed,coinciding with an increase in Foxp3 expression,which may facilitate the chronic parasitism of hydatid cysts within the host.