Biochemical reagent
Verapamil (V111249), ABZ (A131023), albendazole sulfoxide (ABZ-SO, 35395) and pentobarbital sodium were purchased from Sigma (Missouri, US) and Aladdin (Shanghai, China), respectively. Antibody and PCR-primers were purchased from Abcam (Cambridge, US) and Beijing Genomics Institute (Beijing, China). ELISA kits of CaM and CaMK Ⅱ were purchased from CUSABIO (Wuhan, China). All culture reagents were purchased from Gibco (Wisent, Canada). Extraction kits of total RNA and PCR kits were purchased from Takala (Tokyo, Japan).
Separation and culture of E. granulosus PSCx in vitro
E. granulosus PSCx were obtained from naturally infected sheep liver in the slaughterhouse of Xining City, Qinghai Province, China, and then were rinsed with phosphate buffered saline (PBS) and resuspended with Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillinan- streptomycin (P-S), and then incubated in 24-well culture plates (100 PSCx per well) at 37 °C, 5% CO2. The morphological alterations of all PSCx were observed under an inverted microscope (BX43, Olympus, Japan), and the survival rate of PSCx was recorded daily.
Mouse infected with E. multilocularis
All experimental Kunming mice aged 6–8 weeks were purchased from the Laboratory Animal Center of Lanzhou University and maintained in a HEPA-filtered and temperature-controlled light/dark cycle environment at 22–25 ºC. The mice were fed with rodent diet (Beijing Keao, Beijing, China) ad libitum in specific pathogen-free (SPF) laboratory conditions. E. multilocularis PSCx were aseptically isolated from the anesthetized E. multilocularis infected gerbil in our lab as described previously [28], with purpose to establish the murine infection model by in situ surgical intrahepatic implantation. The healthy mice (n = 15) were infected with E. multilocularis PSCx (1500 PSCx per mouse) in SPF lab, and other mice (n = 5) merely with a sham operation, i.e., the uninfected group, were raised under same conditions.
Drug treatment in vitro
The experimental groups were divided into (i) the vehicle group with 0.1% DMSO (n = 3), (ii) the ABZ-SO group with 40 and 20 µg/mL, wherein the ABZ-SO was resolved in 0.1% DMSO (n = 3), and (iii) the verapamil group with different concentrations of 100, 80, 40, 20, 10, 5, 2, 1 and 0.5 µg/mL (n = 3). E. granulosus PSCx on post-treatment with drugs were stained with 0.4% trypan blue for 10 min to observe the morphological changes. In addition, other E. granulosus PSCx were fixed with 4% glutaraldehyde, rinsed with PBS (1×), stained with 2% osmium tetroxide for 2 hours and 1% uranyl acetate for 30 min. Subsequently, these specimens were dehydrated serially by increasing the concentrations of ethanol, dried naturally and then coated with gold as described [28]. Finally, the microstructure of PSCx was observed under a scanning electron microscope (SEM, JSM-5600LV, JEOL, Japan).
Drug treatment in vivo
On post-infection of E. multilocularis PSCx for 3 months, all E. multilocularis infected mice were grouped for orally administered drug therapy: (i) the infected mice were treated daily with only honey/PBS (1:1 v/v) (n = 5), (ii) ABZ mice were treated daily with 40 mg/kg ABZ in honey/PBS (1:1 v/v) (n = 5), (iii) verapamil mice were treated daily with 40 mg/kg verapamil in honey/PBS (1:1 v/v) (n = 5), and (iv) the uninfected mice were merely treated daily with honey/PBS (1:1 v/v) (n = 5). After 4 months post-treatment, E. multilocularis cysts, serum and liver were collected from the mice for testing the calcium content, and protein and mRNA expression of CaM and CamKⅡ genes.
Calcium content analysis by ICP-MS, SEM-EDS and alizarin red staining
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Inductively coupled plasma mass spectrometry (ICP-analysis: Equal tissues or serum/culture supernatant were prepared for calcium Tissue digestion was done in a microwave digestion system using UltraClave (Milestone, Sorisole, and the sample detection was processed as described previously [29].
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Scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-Analysis: The changes in calcium content in the E. granulosus PSCx and in germinal layer cells of E. multilocularis metacestodes after treatment of verapamil were observed by a LEO Gemini Field Emission Gun-Scanning Electron Microscope (FEG-SEM, JEOL, Japan) as described previously [30].
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Alizarin red staining analysis: E. multilocularis metacestodes and mice liver were fixed with 4% paraformaldehyde for 2 weeks, and then stained with alizarin red to detect the calcification as described previously [31]. The images were analyzed by using Image J software (National Institutes of Health, Bethesda, MD, USA) to calculate the percentage of positively stained calcium deposits in the image field.
CaM and CamKⅡ protein expression analysis by IHC-P
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Paraffin-immunohistochemistry (IHC-tests: 4 µm thick sections of E. multilocularis metacestodes and mouse liver were processed for evaluating CaM or CamK Ⅱ expression as described previously [32, 33], followed by immunoreaction using rabbit anti-CaM/CamK Ⅱ antibody (Bioss, Beijing, China) at 1:300/1:200 and secondary antibody (Goat anti-rabbit IgG, Bioss, Beijing, China) at 1:800. Finally, the slides were viewed under a fluorescence microscope (Olympus, Japan). The semi-quantified analysis was evaluated by using Image J software.
- ELISA tests: The serum and tissue samples were added to PBS (pH = 7.containing PMSF (10 mM/L), followed by rapid homogenization and centrifugation at 1000 ⋅ g for 10 min to investigate the concentrations of CaM and CamK Ⅱ proteins according to the ELISA kit protocols.
CaM and CamK Ⅱ mRNA analysis by RT-qPCR
The expression of CaM or/and CamK Ⅱ mRNA in E. granulosus PSCx, mice liver and E. multilocularis cysts were tested by real-time quantitative polymerase chain reaction (RT-qPCR), and β-actin mRNA was used as an internal standard. Nucleic acid in different tissues was isolated by using Trizol reagent (Invitrogen, San Diego, USA), and then reverse-transcribed to cDNA, and amplification of cDNA was performed by RT-qPCR as described by Takara kit protocols (No. RR036A). The primers used were as follow: Eg-CamK Ⅱ/Em-Camk Ⅱ (forward, 5’-TCGTTGTTCAAGTCGGTTCG-3’; reverse, 5’-GGTGCTGAGA
GACCCACTAG-3’), Eg-β-actin/Em-β-actin (forward, 5’-AGACATCAGGGAGTGATGGTT
-3’; reverse, 5’- GAGGACTGGATGCTCCTCAGG-3’); mouse-CamK Ⅱ (forward, 5’-GGCC
TGGACTTT-CATCGATTCTA-3’; reverse, 5’-CATCAGGTGGATGTGAGGGTTC-3’), mouse-CaM (forward, 5’-AAGCCGAGCTGCAGGATATGA-3’; reverse, 5’-CAGTTCTGCC
GCACTGATGTAA-3’), mouse-β-actin (forward, 5’-TTGTTACCAACTGGGACG-3’; reverse, 5’-GGCATAGAGGTCTTTACGG − 3’).
Statistical analysis
The data were presented as means ± standard deviation (SD). Statistical differences among different 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 statistical significant differences.