Biochemical reagents
Verapamil (V111249), ABZ (A131023), albendazole sulfoxide (ABZ-SO, 35395) and pentobarbital sodium were purchased from Sigma (Missouri, US) and Aladdin (Shanghai, China). Antibodies and PCR primers were purchased from Abcam (Cambridge, US) and Beijing Genomics Institute (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for CaM and CaMKⅡ were purchased from CUSABIO (Wuhan, China). All culture reagents were purchased from Gibco (Wisent, Canada). Extraction kits for total RNA and PCR kits were purchased from Takara (Tokyo, Japan).
Separation and culture of E. granulosus PSC in vitro
E. granulosus PSC were obtained from the livers of naturally infected sheep from the slaughterhouse of Xining City, Qinghai Province, China, rinsed with phosphate buffered saline (PBS), resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin-streptomycin (P-S), and then incubated in 24-well culture plates (100 PSC per well) at 37 ℃, 5% CO2. Morphological alterations in PSC were observed under an inverted microscopy (BX43, Olympus, Japan), and the survival rate of PSC was recorded daily.
Mouse infection with E. multilocularis
Kunming mice 6-8 weeks of age were purchased from the Laboratory Animal Center of Lanzhou University and housed in a HEPA-filtered and temperature-controlled environment on a light/dark cycle at 22-25 ℃. The mice were fed a rodent diet (Beijing Keao, Beijing, China) ad libitum under specific pathogen-free (SPF) laboratory conditions. E. multilocularis PSC were aseptically isolated from anaesthetized E. multilocularis-infected gerbils in our laboratory as described previously [28] to establish a murine infection model by in situ surgical intrahepatic implantation. Healthy mice (n = 15) were infected with E. multilocularis PSC (1,500 PSC per mouse) in the SPF laboratory, and other mice (n = 5), which were used as the uninfected group, were subjected to a sham procedure; both groups were housed under the same conditions.
Drug treatment in vitro
The experimental animals were divided into (i) the vehicle group, which was treated with 0.1% dimethylsulfoxide (DMSO) (n = 3); (ii) the ABZ-SO group, which was treated with 40 and 20 μg/mL ABZ-SO dissolved in 0.1% DMSO (n = 3); and (iii) the verapamil groups, which were treated with 100, 80, 40, 20, 10, 5, 2, 1 or 0.5 μg/mL verapamil (n = 3). After drug treatment, E. granulosus PSC were stained with 0.4% trypan blue for 10 min to observe morphological changes. In addition, E. granulosus PSC were fixed with 4% glutaraldehyde, rinsed with PBS (1×), and stained with 2% osmium tetroxide for 2 hours and 1% uranyl acetate for 30 min. Subsequently, these specimens were dehydrated in increasing concentrations of ethanol, air-dried and coated with gold as described previously [28]. Finally, the microstructure of the PSC was observed under a scanning electron microscopy (SEM, JSM-5600LV, JEOL, Japan).
Drug treatment in vivo
Three months after infection with E. multilocularis PSC, all infected mice were divided into the following groups for oral drug administration: (i) the group of infected mice treated daily with honey/PBS (1:1 v/v) (n = 5); (ii) the group of ABZ mice treated daily with 40 mg/kg ABZ in honey/PBS (1:1 v/v) (n = 5); (iii) the group of mice were treated daily with 40 mg/kg verapamil in honey/PBS (1:1 v/v) (n = 5); and (iv) the group of uninfected mice treated daily with honey/PBS (1:1 v/v) (n = 5). After four months of treatment, E. multilocularis cysts, sera and livers were harvested from the mice to measure the calcium content and CaM and CamKⅡ protein and mRNA expression.
Calcium content analysis by ICP-MS, SEM-EDS and Alizarin red staining
(i) For inductively coupled plasma mass spectrometry (ICP-MS) analysis, equal amounts of tissue or serum/culture supernatant samples were prepared for calcium analysis. Tissue digestion was performed with a microwave digestion system using UltraClave (Milestone, Sorisole, Italy), and sample analysis was performed as described previously [29].
(ii) For scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) analysis, changes in the calcium content in E. granulosus PSC and in the germinal layer cells of E. multilocularis metacestodes were observed after treatment with verapamil by a LEO Gemini field emission gun scanning electron microscopy (FEG-SEM, JEOL, Japan) as described previously [30].
(iii) For Alizarin red staining, E. multilocularis metacestodes and mouse livers were fixed with 4% paraformaldehyde for 2 weeks, and then stained with Alizarin red to detect calcification as described previously [31]. The percentage of positively stained calcium deposits in the images was calculated by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Analysis of CaM and CamKⅡ protein expression analysis by IHC-P and ELISA
(i) For paraffin-immunohistochemistry (IHC-P), 4 μm thick sections of E. multilocularis metacestodes and mouse livers were processed to evaluate the expression of CaM or CamKⅡ as described previously [32, 33]; immunostaining was performed using rabbit anti-CaM/CamKⅡ antibodies (Bioss, Beijing, China) at a 1:300/1:200 dilution and secondary antibodies (goat anti-rabbit IgG, Bioss, Beijing, China) at a 1:800 dilution. Finally, the slides were imaged under a fluorescence microscopy (Olympus, Japan). Semiquantitative analysis was performed by using ImageJ software.
(ii) For ELISA, PBS (pH = 7.2) containing PMSF (10 mmol/L) was added to serum and tissue samples, which were then rapidly homogenised and centrifuged at 1,000 ´ g for 10 min to measure the protein concentrations of CaM and CamKⅡ according to the ELISA kit protocols.
Analysis of CaM and CamKⅡ mRNA expressionby RT-qPCR
The expression of CaM or/and CamKⅡ mRNA in E. granulosus PSC, mouse livers and E. multilocularis cysts was measured by real-time quantitative polymerase chain reaction (RT-qPCR), and β-actin mRNA was used as an internal standard. Nucleic acid was isolated from various tissues by using TRIzol reagent (Invitrogen, San Diego, USA), and reverse-transcribed into cDNA; amplification of cDNA was performed by RT-qPCR as described by the Takara kit protocols (no. RR036A). The following primers were used: Eg-CamKⅡ/Em-CamkⅡ (forward, 5’-TCGTTGTTCAAGTCGGTTCG-3’; reverse, 5’-GGTGCTGAGAGACCCACTAG-3’), Eg-β-actin/Em-β-actin (forward, 5’-AGACATCAGGGAGTGATGGTT-3’; reverse, 5’-GAGGACTGGATGCTCCTCAGG-3’); mouseCamKⅡ (forward, 5’-GGCCTGGACTTT-CATCGATTCTA-3’; reverse, 5’-CATCAGGTGGATGTGAGGGTTC-3’), mouse CaM (forward, 5’-AAGCCGAGCTGCAGGATATGA-3’; reverse, 5’-CAGTTCTGCCGCACTGATGTAA-3’), and mouse β-actin (forward, 5’-TTGTTACCAACTGGGACG-3’; reverse, 5’-GGCATAGAGGTCTTTACGG-3’).
Statistical analysis
The data are presented as the mean ± standard deviation (SD). Statistical differences between the groups were assessed by t-test and paired comparisons. Statistical analysis was performed by SPSS version 22.0 (IBM Corp., Chicago, USA) and GraphPad Prism version 7.0 (GraphPad Software, Inc., San Diego, CA, USA). P < 0.05 indicated significant differences.