Isolation of parasites
- granulosus protoscoleces (PSCs) were aseptically isolated from liver hydatid cysts obtained from infected sheep scheduled for routine slaughter in an abattoir located in Urumqi (Xinjiang Autonomous Region, China). PSCs were aspirated from liver hydatid cysts, followed by pepsin-activation and in vitro culture according to our previous description .
Evaluation of pharmaceutical efficiency of AS on PSCs
After culturing for 24 hrs, the PSCs were collected and washed with PBS thrice. PSCs with a viability of ≥95% were used for the subsequent analysis. About 250 PSCs were seeded onto each well of the 96-well plates by dividing into the following groups: control group, plates supplemented with cultivation medium; DMSO group, treated with dimethyl sulfoxide (DMSO, 1%, v/v); H2O2 group, treated with 50 μM H2O2 (as a positive control for ROS); ABZ group (25 μM, as positive drugs); and AS groups, PSCs were treated with different concentrations of AS (65 μM, 130 μM, and 325 μM), respectively. After treating for 4 days, PSCs were collected to evaluate the mortality via the eosin dye exclusion test, followed by monitoring the ultramicroscopic change by transmission electron microscopy (TEM, JEM-100CXII, Alignment, Japan) as previously described [16, 17].
Effect of AS on E. granulosus mortality in vitro
PSCs were divided into the following groups: DMSO group; H2O2 group; ABZ group (25 μM); and AS high dose group (325μM, AS-H); H2O2 plus Mannitol group, treated with 50 μM H2O2 and 100 μM mannitol (scavenger of ROS); AS-H plus Mannitol group, treated with 325 μM AS and 100 μM mannitol. After treating for 4 days, PSCs were collected to evaluate the mortality and observe the ultramicroscopic changes.
Detection of ROS content in PSCs of E. granulosus
Generation of ROS in PSCs was determined as previously described . Briefly, the dihydrorhodamine 123 (DHR123, D1054, Sigma-Aldrich) was added to the 96-well plates covered with PSCs to a final concentration of 10 μM. Then the mixture was incubated at 37°C for 30 min. Upon removal of the supernatant, the mixture was washed using PBS once, followed by incubating with agents at 37°C. The wavelength was set at 530 nm (λex = 488 nm). A multifunction reader (Thermo Fisher) was utilized for the observation for 5.5 hrs every 30 min.
Detection of DNA damage by comet assay
Optimized comet assay for E. granulosus was carried out according to method for Plasmodium falciparum described by Gopalakrishnan et al . The PSCs were treated for 24 hr, the culture medium was abandoned and washed using PBS. Normal-melting point agarose (NMA) was transferred onto the slide, and incubated overnight at 65°C to solidify the agarose. Diluted PSCs (100) were mixed with 100 µl low-melting point agarose (LMA) added onto the slides which were then immediately covered with coverslips. After agarose solidification at 4 °C for 5 min, the coverslips were removed and the slides were immersed for 4 hrs at 4 °C in freshly lysis solution. The slides were equilibrated in alkaline solution for 20 min. Electrophoresis was carried out for 20 min at 25 V and 300 mA. Afterwards, slides were neutralized by washing with Tris-HCl buffer for 15 min, and were stained with 60 μl propidium iodide (PI) solution in dark. Finally, the images were observed using a fluorescent microscope (Olympus IX73) connected through a CCD-IRIS Color Video Camera (Hitachi Denshi, Japan), under a magnification of 200×. The image for PSCs was acquired immediately after opening the microscope shutter to the computer monitor, employing the CASP Program.
Experimental infection and in vivo treatment
Female Kunming mice (6 weeks old) provided by the Animal Center, Xinjiang Medical University, were subject to adaptive feeding for one week before experiments. All protocols involving animals were approved by the Animal Welfare and Committee of First Affiliated Hospital of Xinjiang Medical University (IACUC-20150225-70). Mice were infected by intraperitoneal injection 25 small vesicles with a diameter of 250-300 μm cultured from PSCs of E. granulosus in vitro as previously described [20, 21]. Six months later, selection of infected mice was performed under ultrasonographic examinations.
The infected mice were randomly divided into: (i) model group (n=10): treated with Tween-80/0.4 % CMC-Na; (ii) ABZ positive control group (n=10): treated with ABZ (200 mg/kg); and (iii) AS groups (n=30): treated with 50 mg/kg(1/20LD50), 100 mg/kg (1/10LD50) and 200 mg/kg (1/5LD50) of AS. Uninfected mice treated with an equal volume of 0.4 % CMC-Na served as control group (n=10). The solution was given by intragastric administration lasting for 6 weeks.
Sample collection and detection
Six weeks later, the blood samples were obtained by cardiac puncture after anesthesia under anesthesia with pentobarbital sodium administered intraperitoneally . Liver function was assessed by determining the content of total bilirubin (TBIL) and direct bilirubin (DBIL) levels, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) using the commercial kits (Jiancheng Biotech, Nanjing, China), according the manufacturer’s instructions. Moreover, serum TNF-α and GSH/GSSG was determined to evaluate the oxidative stress status. The mice were sacrificed by cervical dislocation immediately after blood collection, and then the cysts were isolated and subject to weighing the wet weight of cysts. The cystic fluid was obtained and then the content of superoxide dismutase (SOD) and H2O2 was determined. The cystic wall was observed using TME.
Data analysis was performed using SPSS 18.0, and measurement data were presented as mean ± standard error, and numeration data were presented as percentages. The comparison was analyzed with one-way ANOVA and χ2 test, respectively. P < 0.05 was considered to be statistically significant.