多房棘球蚴囊液对小鼠肝细胞凋亡与自噬影响的研究

Effect of polylocular Echinococcus multilocularis cyst fluid on apoptosis and autophagy of mouse hepatocytes

背景多房棘球蚴病是一种流行于北半球的人兽共患寄生虫病,由于不能形成完整的“囊壁”,其病灶中的囊液持续外渗,不断刺激宿主肝细胞,使肝细胞出现“丢失”的情况。目的初步探究多房棘球蚴囊液(hydatid cyst fluid,HCF)对小鼠肝AML-12细胞凋亡与自噬的影响。方法CCK-8法筛选合适的HCF浓度后,将小鼠AML-12细胞分为对照组、HCF 0.4 mg/mL组和HCF 0.8 mg/mL组。CCK-8法检测各组小鼠肝AML-12细胞活性(浓度选择);流式细胞术检测小鼠肝AML-12细胞凋亡情况;Western blot法检测DDIT4、Beclin-1、Caspase-3、PCNA、LC3等凋亡自噬相关蛋白的表达情况;单丹磺酰尸胺(monodansyl cadaverine,MDC)法检测细胞自噬的情况,透射电镜下观察HCF对小鼠肝AML-12细胞超微结构的影响。结果CCK-8结果显示24 h、48 h、72 h时0.8 mg/mL、1.6 mg/mL、3.2 mg/mL、6.4 mg/mL HCF组和48 h、72 h时0.4 mg/mL HCF组的AML-12细胞增殖抑制率均较对照组明显升高(P<0.01);流式细胞计数检测结果显示0.4 mg/mL、0.8 mg/mL HCF作用于细胞48 h后细胞凋亡率明显升高(P<0.01);MDC法结果显示0.4 mg/mL、0.8 mg/mL HCF作用于细胞48 h后出现大量高亮度类圆形团块,平均荧光强度明显升高(P<0.01);Western blot法检测结果显示,与对照组相比,0.4 mg/mL、0.8 mg/mL HCF组DDIT4、PCNA、Caspase-3、Beclin-1、LC3-Ⅱ蛋白表达显著升高(P<0.01);与0.4 mg/mL HCF组相比,0.8 mg/mL HCF组LC3-Ⅱ、Beclin-1蛋白表达显著升高(P<0.05)。透射电镜下可见对照组细胞质未见明显肿胀,线粒体基质较均匀,嵴少量减少,自噬水平低;0.4 mg/mL HCF组线粒体损伤程度较低,轻微肿胀,嵴少量断裂,胞内可见较多自噬溶酶体;0.8 mg/mL HCF组细胞线粒体损伤程度高,线粒体膜破损、基质外溢,嵴缺失,胞内可见较大面积自噬溶酶体。结论HCF对体外培养的AML-12细胞的增殖有明显抑制作用,DDIT4表达增高,并促进AML-12细胞自噬的发生,诱导细胞凋亡。

Background Alveolar echinococcosis is a zoonotic parasitosis that is prevalent in the Northern Hemisphere.The focus of echinococcus multilocularis does not form a complete“cyst wall”,and the cyst fluid continues to extravasate,which constantly stimulates the host hepatocytes,resulting in the“loss”of hepatocytes.Objective To investigate the effect of Hydatid cyst fluid(HCF)on apoptosis and autophagy of mouse liver cell line AML-12.Methods The mouse AML-12 cells were divided into control group,0.4 mg/mL HCF group and 0.8 mg/mL HCF group after screening the appropriate HCF concentrations by CCK8 method.CCK-8 method was used to detect the activity of HCF on AML-12 cells at different time points and concentrations.Flow cytometry was used to detect the apoptosis of mouse liver AML-12 cells.Western blot was used to detect the expression of apoptosis-and autophagy-related proteins,including DDIT4,Beclin-1,Caspase-3,PCNA and LC3.Autophagy was detected by Mon sulfonyl cadaver amide(MDC)and the changes in ultrastructure of AML-12 cells induced by HCF were observed under transmission electron microscope.Results CCK-8 method showed that compared with the control group and the 0.4 mg/mL HCF group at 48 h and 72 h,the 0.8 mg/mL,1.6 mg/mL,3.2 mg/mL and 6.4 mg/mL HCF groups at 24 h,48 h and 72 h could significantly inhibit the proliferation of AML-12 cells(all P<0.01).The results of flow cytometry showed that the apoptosis rate of cells showed a significant increase after treating with 0.4 mg/mL and 0.8 mg/mL HCF for 48 hours(P<0.01).MDC method showed that a large number of round-like masses with high brightness appeared after cells were treated with 0.4 mg/mL and 0.8 mg/mL HCF for 48 hours,and the average fluorescence intensity increased significantly(P<0.01).Western blot assay showed that the protein expression levels of DDIT4,PCNA,Caspase-3,Beclin-1 and LC3-Ⅱin 0.4 mg/mL and 0.8 mg/mL HCF groups were significantly higher than those in the control group(all P<0.01),and the expression level of LC3-ⅡBeclin-1 in 0.8 mg/mL HCF group was higher compared with 0.4 mg/mL HCF group(P<0.05).Under transmission electron microscope,there was no obvious swelling in the cytoplasm,while mitochondria displayed a homogeneous matrix and a small decrease of cristae,indicating a low level of autophagy in control group;and in 0.4 mg/mL HCF group,the mitochondria slightly swelled and a small amount of cristae ruptured with more autophagy lysosomes existed,showing a low degree of mitochondria damage.In 0.8 mg/mL HCF group,the mitochondrial damage deteriorated,as indicated by mitochondrial membrane damage,matrix leakage,cristae loss,and a large area of autophagy lysosomes.Conclusion HCF can significantly inhibit the proliferation,increase the expression level of DDIT4,and promote the autophagy and apoptosis of AML-12 cells.