关键词: 多房棘球蚴, 二甲双胍, 凋亡, 自噬

Abstract:

Objective To investigate the affect of different concentrations of metformin on autophagy and apoptosis of Echinococcus multilocularis cysts and protoscoleces cultured in vitro. Methods The E. multilocularis cysts and protoscoleces were co-cultured in vitro with 1, 5, and 10 mmol/L metformin for 24 and 48 h, respectively, while PBS was used in culture as control. The cysts were observed a microscopically for their viability, size and transparency, and the protoscoleces were stained with hematoxylin and eosin, and observed for viability to estimate survival rate. After co-cultured with metformin at 1, 5, and 10 mmol/L for 48 h, the cell apoptosis-related enzymatic activity of caspase3 and caspase9 in protoscoleces were determined using assay kit. The germinal layer cells in the cysts were collected for determination the apoptosis using flow cytometry. Proteins from the cysts and protoscoleces were extracted to analyze the expression of apoptosis related cleaved-caspase3, casapase9, and B-cell lymphoma/leukemia-2 using Western blotting. After the cysts and protoscoleces were co-cultured with 1, 5, 10 mmol/L metformin for 48 h, the changes in expression of adenosine 5′-monophosphate (AMP)-dependent protein kinase (P-AMPK), mammalian rapamycin target protein (mTOR), autophagy-related genes, and autophagy-related protein LC3-Ⅱ, were analyzed using Western blotting. The difference between the two groups was compared by t test, and the difference between multiple groups was compared by analysis of variance. Results After co-culturing of the cysts with metformin at concentration of 1, 5, and 10 mmol/L, the cyst viability gradually decreased with time, the internal structure was disordered, and the turbidity increased. Apoptotic body-like structures were observed in the cysts after 48 h co-culturing with 10 mm/L metformin. After 24 h of co-cultivation of protoscoleces with different concentrations of metformin, the survival rate in the 1, 5, and 10 mmol/L groups was 0.91 ± 0.10, 0.80 ± 0.12, 0.57 ± 0.11, respectively, which are lower than the control group (0.99 ± 0.02) (F = 95.829, P < 0.05). Afrer 48 h, the survival rate of the protoscoleces in the 1, 5, and 10 mmol/L groups was 0.68 ± 0.18, 0.46 ± 0.15, 0.04 ± 0.01, respectively, which are lower than the control group (0.80 ± 0.22) (F = 287.524, P < 0.05). After co-cultivation with 10 mmol/L metformin for 48 h, all the protoscoleces showed red stained. The relative expression levels of caspase3 in the 1, 5, and 10 mmol/L groups were 33.32 ± 2.94, 53.89 ± 1.01, 124.88 ± 26.44, respectively, which were higher than the control group (16.21 ± 6.20) (F = 36.628, P < 0.05). While the relative expression level of caspase9 were 47.48 ± 9.56, 54.50 ± 1.29, 165.09 ± 16.85, respectively, which was higher than that of the control group (25.29 ± 53.560) (F = 120.612, P < 0.05). The apoptosis rates of 1, 5, and 10 mmol/L group were (14.94 ± 1.35)%, (23.85 ± 2.97)%, (33.87 ± 4.93)%, respectively, which were higher than the control group (8.92 ± 1.95)% (F = 293.452, P < 0.05). The relative expression levels of cleaved-caspase3 and caspase9 in the 1, 5, and 10 mmol/L groups were higher than the control group (F = 144.574, 45.675, P < 0.05); the relative expression levels of Bcl-2 were all lower than the control group (F = 39.487, P < 0.05). After co-culturing the cysts with metformin at different concentrations for 48 h, the relative expressions of P-AMPK and mTOR in the 1, 5, and 10 mmol/L groups were higher than the control group (F = 33.342, 60.617, P < 0.05), and the relative expressions of Atg14 and LC3-Ⅱ was lower than the control group (F = 41.688, 41.375, P < 0.05). Following co-cultured of protoscoleces with metformin for 48 h, the relative expressions of P-AMPK, mTOR, Atg14, and LC3-Ⅱ were higher than the control group (F =25.853, 10.695, 12.528, 17.613, P < 0.05). Conclusion Metformin may suppress the viability of E. multilocularis cysts and protoscoleces and accelerate their apoptosis in vitro, showing concentration- and time-dependent pattern. It was demonstrated that AMPK-mTOR signaling pathway may contribute to initiate autophagy of the parasite.

Key words: Alveolar echinococcosis, Metformin, Apoptosis, Autophagy