2.1 Parasites and animal experiments
Echinococcus multilocularis was obtained from the Key Laboratory of Echinococcosis in Qinghai Province (Qinghai Province, China)and had been maintained in the laboratory. Eight to ten-week-old male SD rats were used as final hosts. SD rats were purchased from the Nanjing Qinglongshan Laboratory Animal Breeding Centre, China. We inoculated 1200–1500 E. multilocularis in the liver of each SD ratto establish HAE rat model. 6-month-old HAE rats were used for experiments. PBN (N-tert-Butyl-α-phenylnitrone; Saint Louis, Missouri, USA) was used as a ROS scavenger in vivo experiments. The efficiency of PBN[20, 21]was tested by treating HAE rats (three rats per group) with three different concentrations—20, 50, and 100 mg/(kg.d)—for 30 days by continuous intraperitoneal injection. Subsequently, PBN 50 mg/(kg.d) group, 50 mg/(kg.d) normal saline (ns) group and control group were established to explore ROS-mediated activation of NLRP3 inflammasome in HAE rats, 10 rats per group. We administered daily intraperitoneal injection to reduce the impact of ROS generation. The feeding and housing were performed in specific-pathogen free (SPF) conditions All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals established by National Health Commission of China.
2.2 Patient tissues
Tissue specimens were collected from 60 patients with HAE who underwent surgery in the Department of Hepatobiliary and Pancreatic Surgery of the Affiliated Hospital of Qinghai University from January 2019 to June 2019. All patients had not received clinical adjuvant therapy, such as radiotherapy, chemotherapy, and radiofrequency ablation before surgery. All specimens were confirmed to be HAE by pathological examination. The pathological stage and degree of differentiation of the lesions were based on PNM classification criteria. In all patients, the marginal zone of the lesion (less than 0.5 cm away from the lesion) and the tissue adjacent to the lesion (more than 3 cm away from the lesion) were quickly frozen in liquid nitrogen within 15 minutes of surgical resection, and stored in -80 °C. All patients were followed up regularly after surgery. Blood AFP and B-ultrasound were routinely reviewed, and CT was performed if necessary. All patients signed an informed consent and all protocols involving human and animals were approved by Ethics Committee of the Affiliated Hospital of Qinghai University. The experiment started after the approval by Ethics Committee of the Affiliated Hospital of Qinghai University. The Ethical approval number is P-SL-2019054.
2.3 Immunohistochemical staining
Paraffin sections were incubated in 3% H2O2 at room temperature for 5–10 min to eliminate the activity of endogenous peroxidase. The sections were rinse with phosphate-buffered saline (PBS) for 5 min. Respective primary antibodies (NLRP3, Abcam, Shanghai, China; Caspase-1, Thermo Scientific, Waltham, MA, USA; IL-18, IL-1β, Boster Biotechnology, Wuhan, China) were added and incubated at 4 ℃ overnight. The sections were rinsed with PBS and further incubated with appropriate secondary antibody (goat anti-rabbit IgG, goat anti-mouse IgG; Beyotime Biotechnology, Shanghai, China) at 20–37℃ for 10–30 min. The optical density and area of all the images were measured by using an Image-Pro Plus 6.0 Image Analysis System (Media Cybernetics, Inc., Bethesda, MD, USA) and the mean density was calculated.
2.4 immunofluorescence staining for cell localization
Paraffin sections were incubated in 3% H2O2 to eliminate the activity of endogenous peroxidase. Normal goat serum blocking solution was added to eliminate background staining. The appropriate primary antibodies (CD68, Proteintech, Wuhan, China; NLRP3, Abcam, Shanghai,China;Caspase-1, Thermo Scientific, Waltham, MA, USA) were added and incubated at 4 °C overnight. Sections were washed with PBS and incubated with fluorescent secondary antibody (CY3 Conjugated AffiniPure Goat anti-rabbit IgG, Boster Bio, Wuhan, China) for 2 h. DAPI (Beyotime Biotechnology, Shanghai, China) was added and incubate in the dark for 30 min to stain the nuclei. Cells was observed and images collected under fluorescence microscope (OLYMPUS BX33, JPN).
2.5 Blood biochemical testing
Tail vein blood of the SD rat was collected and WBC, lymphocytes, monocytes levels were measured using a standard hematology analyzer (IDEXX Laboratories Inc., Westbrook, USA)
2.6 Analysis of reactive oxygen species expression and apoptosis by flow cytometry
To detect ROS, Kupffer cells were resuspended with diluted 50 µM DCFH-DA from a reactive oxygen species assay kit (Nanjin Jiancheng Bioengineering Institute, Nanjing, China ), and incubated at room temperature for 30 min. For tissue samples, we initially prepared single cell suspension. Liver was washed with 20 mL PBS, and incubated in 20 mL of type IV collagenase digestion solution (Beyotime Biotechnology, Shanghai, China) at 37 °C. The liver was cut to 2–3 mm3 with scissors and washed twice with PBS. The single cell suspension were collected for ROS detection.Cells were washed twice with PBS. ROS relative expression was detected through the FITC detection channel (Ex = 500 nm). For apoptosis assay, cells were stained with annexin-V-FITC (MultiSciences,Hangzhou, China) and propidium iodide (PI) according to the manufacturer’s instructions.1–10 × 105 cells were washed with PBS and resuspended in 500 µl 1 × Binding Buffer. Next, 5 µl Annexin V-FITC and 10 µl PI to each tube. After vortexing gently, the cells were incubated at room temperature in the dark for 5 min. Apoptosis assay was detected through the FITC detection channel and the PI detection channel. Flow cytometry was performed on a FACS calibur (Becton and Dickson, USA). FlowJo (V10.5, Becton and Dickson, USA)was used to analyze the data.
2.7 Co-cultivation experiments
For co-cultivation experiments, normal rat liver cell BRL were purchased from the Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China). BRL cells (3 × 105) were plated in the top chamber (6-well; 0.4 µm Pore Polycarbonate Membrane Insert; Corning Life Sciences, Shanghai, China). E. multilocularis (1 × 103) and rat liver macrophages (1 × 106 ) were placed in the lower chamber. Rats BRL hepatocytes, liver macrophages, and E. multilocularis were cultured in DMEM (Gibco, Burlington, ON, Canada) medium containing 10% FBS (FBS; Gibco Burlington, ON, Canada). We chose N-Acetyl-L-Cysteine (NAC, Macklin Biochemical, Shanghai, China)as ROS scavenger. After centrifugation at 4,000 rpm for 10 min, the medium supernatant was collected and stored at -80℃, and the co-cultured liver macrophage protein extract was collected. We detected the expression of IL-1β and IL-18 in the supernatant, the expression levels of NLRP3 and Caspase-1 protein in Kupffer cells, and the expression level of ROS in Kupffer cells.
2.8 Western blotting analysis
Protein samples were quantified with the BCA protein assay kit (BIO-RAD, Mississauga, ON, Canada) and subjected to electrophoresis in reducing conditions with poly-acrylamide gels. The proteins were transferred to PVDF membranes. The membranes were blocked for 1 h in TBST (TBS with 0.1% Tween-20) with 5% non-fat milk and incubated with the appropriate antibodies (NLRP3, Abcam, Shanghai, China; Caspase-1, Thermo Scientific, Waltham, MA, USA) overnight at 4℃. After washing, the membranes were blotted with the appropriate HRP conjugated secondary antibody; the blots were developed with ECL reagent from Sangon Biotech (Shanghai, China) in an ImageQuant LAS 4000 image system (GE Healthcare, USA) and analyzed with the Image-Pro Plus 6.0 (Media Cybernetics,, Bethesda, MD, USA). The intensity of bands was normalized to the corresponding values of β-actin.
The ELISA kit (Rat IL-18 ELISA Kit, MultiSciences, Hangzhou, China; Rat IL-1β ELISA Kit, Abcam, Shanghai, China) was used, according to the manufacturer’s instructions to determine IL-18 and IL-1β levels in serum and co-cultured cell supernatant. Each sample was assayed in duplicate and the optical density (OD value) of each well was measured immediately with microplate reader (BIO-RAD iMark, USA).
2.10 Extraction and identification of SD rat Kupffer cell
SD rat liver tissues were washed with 20 mL HBSS (Thermofisher, Shanghai, China) and incubated in 20 mL of type IV collagenase digestion solution (Beyotime Biotechnology, Shanghai, China) at 20–37 °C. Incompletely digested tissues were filtered out and the centrifuged to precipitate liver cells. The supernatant was collected and resuspended in 30% Percoll (Macklin Biochemical, Shanghai, China). The cells in the interface layer were carefully pipetted, diluted with HBSS solution, and the cells collected. For identification, cells were prepared on glass slide cover, fixed with 4% paraformaldehyde for 15 min, and blocked with normal goat serum room temperature for 30 min. Diluted (1:100) primary antibody (CD68 antibody, Proteintech, Wuhan, China) was added and incubated at 4 °C overnight. The slides were washed with PBS and incubated with fluorescent secondary antibody (CY3 Conjugated AffiniPure Goat anti-rabbit IgG, BosterBio, Wuhan, China) at 20–37℃ for 1 h. DAPI (Beyotime Biotechnology, Shanghai, China) was added with further incubation in the dark for 5 min to stain the nuclei. Cells were observed and images collected under fluorescence microscope (OLYMPUS BX33,JPN) at magnifications of 200x and 400x.
2.11 Statistical analysis
All statistical analyses were performed by the Graphpad prism 6. Data were analyzed by Student’s t-test, variance (ANOVA) with Tukey’s multiple comparisons test or Wilcoxon test as appropriate. Pearson was used for correlation analysis and Chi-square test was used to analyze the relationship between NLRP3 expression and clinicopathological characteristics of patients with HAE. Quantitative data are presented as the mean ± standard deviation. All P values are two-sided; P value < 0.05 was considered statistically significant;*P < 0.05, **P < 0.01, ***P < 0.001,****P < 0.0001; ns, not significant.