Abstract:

Objective To understand the affect of aminoalcohol compound HT24 on the expression of tubulin in Echinococcus multilocularis protoscoleces. Methods Protoscoleces were isolated from BALB/c mice infected with E. multilocularis metacestode, and cultured in vitro for 1 day. Then they were assigned into groups of low, medium and high concentration of HT24 treatment, which were added with 0.5% DMSO (control group) and 25 μmol/L, 50 μmol/L or 100 μmol/L HT24, respectively. Each group was set with 3 replicates, and 2 000 protoscoleces in each well. The viability and morphological changes of the protoscoleces were observed in first 3 days after addition of HT24. Samples were collected on the third day and subjected to transmission electron microscopy (TEM) to observe the ultrastructural changes of flame cells. Samples were collected at 3, 5, and 7 days for real-time fluorescence quantitative PCR (qPCR) to examine the mRNA expression levels of α9-, β2-, β4- and β6-tubulin, and Western blotting to examine the protein levels of β-tubulin. Two-way ANOVA was applied for between-group comparisons. Results After 3 days of HT24 treatment, in the low-concentration group, the E. multilocularis protoscoleces had unclear outline, swollen body, decreased transparency, and detachment of some hooks. In the medium-concentration group, the protoscoleces were obviously enlarged and hooks were detached. All protoscoleces in the high-concentration group died and dwindled in size. TEM showed that in the medium- and high-concentration groups, the membranes of flame cells were broken, ciliary in the cytoplasm ruptured with large notches or fracture openings, and some cilia spilled out and were free outside the cells. qPCR showed that compared to the control group, the mRNA expression of α9-tubulin on day 7 in the low-, medium- and high-concentration groups was 8.01 ± 1.54, 9.84 ± 0.40 and 7.85 ± 2.60, respectively, there was significant difference between the 3 groups (P < 0.01); the mRNA expression of β2-tubulin in the low-, medium- and high-concentration group on day 7 was 10.97 ± 0.69,11.83 ± 0.97 and 6.48 ± 1.52, respectively, there was significant difference between the 3 groups (P < 0.01); the mRNA expression of β4-tubulin on day 7 in the low-, medium- and high-concentration groups was 10.61 ± 1.24, 8.31 ± 1.05 and 7.47 ± 0.96, respectively, there was significant difference between the 3 groups (P < 0.01); the mRNA expression of β6-tubulin on day 7 was 8.83 ± 0.80, 7.89 ± 1.30 and 5.63 ± 2.25 in the low-, medium- and high-concentration groups, respectively, there was significant difference between the 3 groups (P < 0.01). Western blotting results showed that on day 3 after treatment, the β-tubulin protein levels at the low-, medium- and high-concentration groups were 0.36 ± 0.06, 0.23 ± 0.03, and 0.61 ± 0.06, respectively, all lower than the control group (0.67 ± 0.05) (P < 0.01); on day 5 after treatment, the β-tubulin protein levels in the low-, medium- and high-concentration groups were 1.12 ± 0.03, 0.55 ± 0.04, and 0.30 ± 0.02, respectively, all higher than the control group (0.26 ± 0.03) (P < 0.01); on day 7 after treatment, they were 0.73 ± 0.05, 0.36 ± 0.04, and 0.40 ± 0.04, respectively, The latter two were lower than control group (0.73 ± 0.05) (P < 0.01). Conclusion HT24 can lower the mRNA and protein levels of β-tubulin and affect the function of microtubules, thereby suppressing the viability of E. multilocularis metacestodes.

Key words: Echinococcus multilocularis, Amino alcohols, Protoscoleces, Tubulin