关键词: 全反式维甲酸, 多房棘球蚴原头节, 增殖活性, 凋亡, 活性氧

Abstract:

Objective To investigate the effect of all-trans retinoic acid (ATRA) on the activity and growth of Echinococcus multilocularis protoscoleces in vitro. Methods The cysts were dissected aseptically from the gerbils and the active E. multilocularis protoscolices were obtained by grinding, filtering, and cleaning. The protoscolices were divided into different concentrations of the ATRA groups (10, 25, 50, and 100 μmol/L groups with the corresponding final concentration of ATRA), the dimethyl sulfoxide (DMSO) group (the final concentration of DMSO was 0.1%), and the blank group (the same amount of complete medium was added). After 9 days of intervention, eosin staining was used to observe the activity and morphological changes of the groups under the microscope. The survival rate of the protoscolices was calculated, and the survival curve was drawn. E. multilocularis microcysts were obtained by co-culturing the protoscolices with rat hepatocellular carcinoma RH35 cells for 5-6 weeks, and the microcysts were treated with different final concentrations of ATRA (10, 100 μmol/L) for 9 days. The activity and morphological changes of the microcysts were observed under a microscope, and the DMSO group and blank group were set up. After 48 h of intervention, the EdU positive rate was detected by EdU cell imaging and the relative expression of Caspase-3 in protoscolices was detected using the Caspase-3 kit. After 24 hours of intervention, a ROS detection kit was used to quantify the levels of reactive oxygen species (ROS) in each group. An independent sample t-test was used to compare the two groups of samples, and a one-way analysis of variance (ANOVA) was used to compare the difference between the ATRA and DMSO groups of different concentrations. Results The volume of dead protoscoleces in the 10, 25, 50, and 100 μmol/L groups significantly decreased and could be stained by eosin dye. With the increase in time and drug concentration, the outline blurred, light transmission weakened, and small hook detachment increased. On day 4, the survival rates of the protoscoleces in the 10, 25, 50, and 100 μmol/L groups were (87.33 ± 4.90) %, (74.00 ± 2.08) %, (64.33 ± 2.03) % and (53.33 ± 1.86) %, respectively. They were all lower than those in the DMSO group [(95.67 ± 1.20) %] (F = 98.41, P < 0.05). On day 9, the survival rates of protoscoleces in the 10, 25, and 50 μmol/L groups were (62.00 ± 2.64)%, (36.33 ± 2.52)% and (7.67 ± 1.53)%, which were lower than those in the DMSO group [(85.67 ± 2.08)%] (F = 1154.34, P < 0.05). When different concentrations of ATRA were co-cultured with multilocular echinococcus microcysts for 9 days, with the increase in ATRA concentration, the vesicles grew slowly, and the intracapsular structure became cloudy. The germinal layer and stratum corneum of microcysts were intact in the blank group and the DMSO group. EdU cell imaging showed red and blue fluorescence in ATRA groups with different concentrations. The EdU positive rates in the 10, 25, 50, and 100 μmol/L groups were (51.63 ± 3.09) %, (42.09 ± 1.36) %, (38.46 ± 0.65) %, and (25.23 ± 1.32) %, respectively. All of them were lower than those in the DMSO group [(58.32 ± 0.91)%] (F = 168.59, P < 0.05). The caspase-3 activity of the 10, 25, 50, and 100 μmol/L groups was (19.23 ± 2.27), (26.27 ± 3.45), (43.29 ± 2.10) and (72.80 ± 1.40) U/L, respectively, showing a dose-dependent increase. All of them were higher than the DMSO group [(14.22 ± 0.52) U/L] (F = 404.08, P < 0.05). The green fluorescence of ROS in the ATRA group was stronger than that in the DMSO group. The fluorescence intensity of the 10, 25, 50, and 100 μmol/L groups was 260.96 ± 2.52, 282.10 ± 7.40, 330.30 ± 12.46 and 346.10 ± 6.39, respectively. All of them were higher than the DMSO group (236.03 ± 6.89) (F = 80.53, P < 0.05). Conclusion ATRA can inhibit the activity and growth of E. multilocularis protoscoleces in vitro, induce significant ROS accumulation in protoscolices, inhibit protoscolices proliferation, and promote apoptosis.

Key words: All-trans retinoic acid, Protoscolices of Echinococcus multilocularis, Proliferation activity, Apoptosis, ROS