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-Ⅱ in 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-Ⅱ Beclin-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.
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