The study was approved by the Ethics Committee of the Experimental Animal Center of the First Affiliated Hospital of Xinjiang Medical University (IACUC-20200318-04).
Female C57/BL6 mice (4–8 weeks old, with a body weight of 18 ± 2 g), obtained from the Experimental Animal Center of the First Affiliated Hospital of Xinjiang Medical University, were used as the experimental animals.
Crude hydatid cyst fluid was obtained from sheep livers (purchased from the Hualing Slaughterhouse in Urumqi, Xinjiang), infected with
Flowchart of the Extraction of
To assess the impact of pathway inhibitors on the
The control group A (n = 6): Mice, inoculated with approximately 2000
The inhibitor group B (n = 6): Similar to group A, these mice were inoculated with the protoscoleces and reared for three months. Post-inoculation, they were given a caudal vein injection of a PI3K inhibitor (5 µg/g), followed by 0.1 mL/g and 0.05 mL/g of hydatid cyst fluid intraperitoneally and intratracheally, respectively.
The inhibitor group C (n = 6): Following the same initial procedure as group B, these mice received a caudal vein injection of an AKT inhibitor (8 µg/g) and were then administered 0.1 mL/g and 0.05 mL/g of hydatid cyst fluid intraperitoneally and intratracheally.
The hydatid cyst fluid sensitization group D (n = 6): After inoculation with approximately 2000
Subsequently, blood samples were collected from the medial canthus vein of the mice through enucleation 15 minutes after the intraperitoneal injection of cyst fluid, and bilateral lung tissue samples were extracted for analysis.
The serum and frozen lung tissue samples from the mice were subjected to three rounds of testing using the qRT-PCR technique. This involved analyzing the mRNA expressions of inflammatory factors and pathway proteins. To quantitatively assess gene expression levels, the threshold cycle (CT) values were normalized using glyceraldehyde 3-phosphate dehydrogenase as an internal standard. The relative expression levels of the genes were estimated using the 2-ΔΔCT method.
The detection of PI3K/AKT/NF-κB pathway proteins was performed utilizing the Western blotting method.
The protein extraction process included lysis, centrifugation, and storage (see
Protein extraction process.
| Step | Detailed Operational Process |
|---|---|
| Lysis | The sample was placed in a centrifuge tube, and a lysis buffer was added. It was let to sit on ice for 30 minutes, with the tube being inverted and mixed evenly every 15 minutes. |
| Centrifugation | Performed at 4℃ for five minutes; the supernatant solution was extracted subsequently. |
| Storage | Stored long-term in a -80℃ freezer |
Lysis Buffer: A total of 350 μl of RIPA lysis buffer was used for the lysis process. The buffer was supplemented with PMSF to obtain a final concentration of 1 mM. A phosphatase inhibitor cocktail was added to achieve a final concentration of 1X, and a protease inhibitor was also added to a obtain final concentration of 1X.
The protein standards were completely dissolved at room temperature. Then, 20 μl of a 5 mg/mL BSA protein standard solution was diluted to 100 μl with PBS solution to achieve a final concentration of 1.0 mg/mL. The test sample was added to a microplate (diluted 10 times), and the volume was adjusted to 20 μl with PBS.
The BCA working solution was prepared by mixing reagent A and reagent B in a 50:1 volume ratio. For quantification, 200 μl of the BCA working solution was added to the microplate while ensuring thorough mixing. This was then incubated at 37°C for 30 minutes.
Note: Alternatively, the samples may be incubated for two hours at room temperature or for 30 minutes at 60°C. During protein concentration determination using the BCA method, the color of the protein solution intensifies gradually, with the colorimetric reaction accelerating as the temperature increases. In cases of low protein concentrations, it is advisable to incubate at higher temperatures or extend the incubation time as needed.
Absorbance was measured at 562 nm, and readings were recorded. Samples without BSA served as the control standard. Utilizing the BSA standard detection data, a standard curve was plotted, allowing for the calculation of protein concentration in each sample.
For each sample, the loading amount was set to 20 μg, supplemented with appropriate 5 X SDS (the final concentration of β-ME was 1%), and adjusted to a 10 μl volume with buffer. Samples were boiled for 10 minutes in a 95°C water bath. Using SDS-PAGE gel, a 12% separating gel and a 5% stacking gel were prepared for electrophoresis analysis of the protein samples. Post-electrophoresis, membrane transfer, blocking, and primary antibody incubation (using one target protein/GAPDH as an internal reference) were carried out, and the samples were incubated overnight on a shaker at 4°C. The membranes were then washed three times with 1X Tris buffered saline with Tween 20 (TBST), each wash lasting 10 minutes. For secondary antibody incubation, the process was conducted at room temperature for one hour, followed by three washes with 1X TBST, each lasting 10 minutes. Finally, ECL luminescent liquid was applied for color development and imaging.
The chemiluminescent signals obtained from ECL processing were analyzed using Image Lab software. These signals were converted into grayscale images to facilitate relative quantitative analysis of signal intensity.
Tissue Fixation: For fixation, one side of the C57/BL6 mouse lung tissue, ≤ 3 mm thick, was submerged in a 10% formalin solution for 24 hours, maintaining a tissue-to-fixative ratio of 1:10. Tissue Dehydration: Post-formalin immersion, the tissue was sequentially soaked in 75% ethanol for 60 minutes, 85% ethanol for 90 minutes, and finally in anhydrous ethanol I and II, each for 60 minutes. Tissue Clearing: The dehydrated lung tissue was subjected to clearing in xylene I, II, and III, each for 60 minutes. Wax Infiltration: The lung tissue was embedded using paraffin wax with a melting point of 56–60℃. Sectioning: Uniform sections, 4–6 um thick, were cut, ensuring that there were no wrinkles or knife marks. Slide Baking: The slides were typically baked in a 60℃ oven for 30 minutes. This was followed by routine gradient dewaxing for hematoxylin and eosin (HE) staining. HE Staining: Paraffin sections were subjected to hematoxylin staining for 0.5–1 minute, rinsed with tap water, differentiated in 1% hydrochloric acid alcohol briefly, washed again, and blued in 1% ammonia water for one minute. After washing under running water, they were stained with eosin for a few seconds, rinsed, and then dehydrated and mounted as per standard HE staining procedures.
Flow cytometry was utilized to analyze macrophage phenotypes. The specific operational procedures are detailed in
Flow cytometry procedure.
| Step | Detailed Operational Steps |
|---|---|
| Tissue Extraction | Lung tissue from C57/BL6 mice was processed into a single cell suspension using glass slide compression, settling, and filtering steps. |
| Centrifugation | Centrifuged at 4℃, 300–400 g, for 4–5 minutes; the supernatant was discarded and cell viability was assessed using trypan blue staining. Cells were resuspended in an appropriate buffer to a obtain concentration of 2×107 cells/mL. |
| Incubation | Flow cytometry antibodies (CD45, F4/80, CD11C, CD11B, and CD86 [0.5–1 μg/106 cells]) were added for cell surface marker staining and incubated at room temperature for 20 minutes. |
| Washing | Performed twice with PBS |
Statistical analysis was conducted using R software, version 4.3.1. The normality of data was assessed using the “shapiro.test” function, and the homogeneity of variances was checked using the “levene’s test” function in R4.3.1. Normally distributed data with homogeneous variances were expressed as the mean ± standard deviation. Intergroup comparisons were made using the “aov()” function for one-way analysis of variance (ANOVA). Significant results from the one-way ANOVA led to