关键词: 细粒棘球蚴, 巨噬细胞, 肿瘤坏死因子α, 天冬氨酸蛋白水解酶3, 共培养

Abstract:

Objective To explore the cytotoxic effect of mouse immune cells on the Echinococcus granulosus protoscolex and understand the types of immune cells involved and the cytokines secretion changes. Methods Healthy C57BL/6 mice were used to extract splenocytes and peritoneal macrophages. The protoscoleces from sheep E. granulosus cysts were collected and grouped (3 000 per group). To select the immune cell type exhibiting a stronger inhibitory effect on the protoscoleces, macrophage co-culture group and splenocyte co-culture group were co-cultured with 6 × 10⁶ macrophages or splenocytes, respectively. To choose an optimal cell count, co-culture group 1-5 were co-cultured with 1.2 × 10⁶, 2.4 × 10⁶, 4.8 × 10⁶, 7.2 × 10⁶ and 9.6 × 10⁶ immune cells, respectively. The co-culture system was established. The E. granulosus cyst fluid and tumor necrosis factor-α (TNF-α) inhibitor were added into the co-culture system respectively, and the activity of protoscoleces and the changes in concentration of TNF-α, interleukin 6 (IL-6), IL-10 and transforming growth factor-β (TGF-β) in the co-culture supernatant were observed. Eosin staining was used to detect the activity of protoscoleces, the dichlorodihydrofluorescein diacetate (DCFH-DA) method was used to measure the of reactive oxygen species levels, the JC-1 method was used to assess mitochondrial membrane potential, Western blotting was performed to detect the expression levels of the Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 3 (caspase-3), and ELISA was used to measure the concentration of cytokines in the culture supernatant. Independent samples t-test was used for comparisons between two groups and one-way ANOVA was used for multiple groups. Results On day 6 of co-culture, the protoscoleces activity in the macrophage co-culture group and the splenocyte co-culture group was (25.07 ± 0.40)% and (76.18 ± 0.31)%, respectively. The protoscoleces activity in the macrophage co-culture group was lower than that in the splenocyte co-culture group at all time points (F = 564.20, P < 0.05). On day 4 of co-culture, the relative fluorescent intensity of reactive oxygen species in the protoscoleces of the macrophage co-culture group was 32.20 ± 7.85, which was higher than that of the splenocyte co-culture group (12.44 ± 2.93) (t = 7.07, P < 0.05). On day 6 of co-culture, the relative expression levels of caspase-3 protein in the macrophage co-culture group and the splenocyte co-culture group were 1.28 ± 0.02 and 1.16 ± 0.02, respectively, and the relative expression levels of Bax protein were 1.29 ± 0.01 and 0.46 ± 0.01, respectively. The relative expression levels of caspase-3 and Bax protein in the macrophage co-culture group were higher than those in the splenocyte co-culture group at all time points (F = 55.87, 167.20; both P < 0.05). Macrophages exhibited a stronger inhibitory effect on protoscoleces than splenocytes. On day 6 of co-culture, the relative fluorescent intensities of mitochondrial membrane potential in protoscoleces from co-culture groups 1-5 were 20.15 ± 8.96, 24.40 ± 9.71, 48.41 ± 10.20, 94.62 ± 8.72 and 112.85 ± 24.23, respectively, all of which were higher than that of the protoscolex control group (2.50 ± 1.02) (F = 26.18, P < 0.01). On day 6 of co-culture, the relative expression levels of caspase-3 protein in protoscoleces from co-culture group 4 and 5 were 1.35 ± 0.03 and 1.49 ± 0.05, respectively, which were higher than that of the protoscolex control group (0.28 ± 0.01) (t = 17.03, 10.60; both P < 0.05). The relative expression levels of Bax protein in protoscoleces from co-culture groups 4 and 5 were 1.34 ± 0.01 and 1.38 ± 0.04, respectively, which were higher than that of the protoscolex control group (0.78 ± 0.04) (t = 6.68, 6.46; both P < 0.05). On day 4 of co-culture, the concentrations of TNF-α, IL-6 and TGF-β in the supernatants of co-culture group 4 were (240.90 ± 17.29), (435.90 ± 12.33) and (137.10 ± 6.62) pg/ml, respectively, all of which were higher than those in the cell control group [(42.02 ± 0.52), (65.72 ± 1.91), (24.72 ± 1.78) pg/ml] (t = 54.52, 15.97, 17.59; all P < 0.05). The concentration of IL-10 in the supernatant of co-culture group 4 was (42.16 ± 1.45) pg/ml, which was not significantly different from that of the cell control group [(45.64 ± 1.03) pg/ml] (t = 1.29, P > 0.05). The concentrations of TNF-α, IL-6, IL-10 and TGF-β in the supernatants of the co-culture groups at all time points were higher than those in the cell control group (F = 294.66, 450.50, 687.72, 660.15; all P < 0.05). On days 1, 3, 5 and 7 of co-culture, the relative fluorescent intensities of mitochondrial membrane potential in protoscoleces from the cyst fluid group were 4.46 ± 1.25, 4.33 ± 0.39, 4.89 ± 0.77 and 7.97 ± 0.62, respectively, all of which were lower than those in the macrophage group (5.67 ± 1.72, 13.60 ± 0.50, 35.28 ± 5.65, 77.50 ± 9.60) (F = 115.90, P < 0.01). On day 6 of co-culture, the concentrations of TNF-α, IL-6, IL-10 and TGF-β in the supernatant of the cyst fluid group were (64.12 ± 2.65), (1 049.65 ± 25.70), (230.30 ± 12.98) and (138.57 ± 13.71) pg/ml, respectively, and those in the macrophage group were (41.61 ± 1.31), (68.00 ± 0.42), (56.15 ± 6.43) and (32.94 ± 4.90) pg/ml, respectively. The concentrations of TNF-α, IL-6, IL-10 and TGF-β in the supernatants of the cyst fluid group at all time points were higher than those in the macrophage group (F = 289.80, 366.50, 145.40, 32.94; all P < 0.05). On day 7 of co-culture, the protoscoleces activity in the macrophage group and the inhibitor group was (21.18 ± 1.61)% and (94.31 ± 2.58)%, respectively. The protoscoleces activity in the inhibitor group was higher than that in the macrophage group at all time points (F = 1 810.00, P < 0.05). On days 2, 4 and 6 of co-culture, the concentrations of TNF-α in the inhibitor group were (33.55 ± 7.48), (13.78 ± 4.96) and (19.20 ± 0.69) pg/ml, respectively, all of which were lower than those in the macrophage group [(209.24 ± 9.90), (209.47 ± 10.55), (211.36 ± 13.66) pg/ml] (t = 33.16, 30.46, 23.76; all P < 0.05). Conclusion Macrophages co-cultured in vitro with the E. granulosus protoscoleces could express cytokines such as TNF-α, which inhibits the activity of the protoscoleces and promotes their apoptosis. Cyst fluid from E. granulosus and TNF-α inhibitors could reduce the secretion of TNF-α by macrophages, thereby alleviating the killing effect of macrophages on the protoscoleces.

Key words: Echinococcus granulosus, Macrophages, TNF-α, Caspase-3, Co-culture