Animals

C57BL/6J mice, aged 7 weeks, were obtained from Changzhou Cavens laboratory animal Co. Ltd. (Chang-zhou, China). All mice were subsequently housed at the Experimental Animal Center of Xuzhou Medical University and housed in an air-conditioned room (RT 24℃ with a 12 h dark/light cycle). After a 1-week acclimation period, mice were used for experiments according to the guidelines of the Chinese Animal Care Committee and approved by the Institutional Animal Care Committee of Xuzhou Medical University.

T. gondii Infection

T. gondii cysts (TgCtWh6 isolate, a low virulent strain and usually causes chronic infection in mice [36]), were used to establish the infection model. Two groups (8 mice in each group) were randomly assigned. The control group (Con) received phosphate buffer saline (PBS) via gavage as vehicle control, while the T. gondii-infected group (Tg) was given 10 cysts of TgCtWh6 per mouse through gavage. The infection method followed the previously described protocol [37]. The mice were executed with CO2 four weeks after infection, and fresh colonic and blood samples were collected for further analysis.

DQ Intervention Experiment

The combination of DQ is one of classic therapies to decrease senescent cells in vivo [38]. Mice were randomly assigned to 4 groups (8 mice in each group): (I) Con group received 10% PEG400 by oral gavage; (II) Con + DQ group received oral gavage of dasatinib and quercetin; (III) Tg group mice were gavaged with TgCtWh6 cysts (10 cysts per mouse). (IV) Tg + DQ group were administered dasatinib and quercetin by oral gavage after T. gondii infection. For all DQ treatments, 5 mg/kg dasatinib (D-3307-500MG, LC Laboratories, Woburn, MA, USA) and 50 mg/kg quercetin (Q4951-10G, Sigma-Aldrich, St. Louis, USA) were administrated via oral gavage in 10% PEG400 (IP9000, Solarbio, Beijing, China). Administration of DQ or vehicle control begun at the 5th week post-infection and lasted 4 weeks (3 consecutive days every 2 weeks). These mice were executed with CO2, and fresh colonic and blood samples were collected for further analysis.

RNA Extraction and Quantitative (q) Real-time Polymerase Chain Reaction

RNA was extracted from the colonic tissues using the RNA isolator (R401, Vazyme Biotech Co., Ltd, Nanjing, China). The concentration and quantity of isolated RNA was measured at wavelength of 260 nm and 280 nm using a DU800 spectrophotometer (Beckman Coulter Inc., Brea, CA, USA). Subsequently, one μg purified RNA underwent reverse transcription into cDNA using HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (R223, Vazyme Biotech Co., Ltd). qPCR was performed using the ChamQ SYBR qPCR Master Mix (Q311, Vazyme Biotech Co., Ltd) on a real-time PCR detection platform (Roche, Switzerland). Relative expression levels were calculated by the 2−ΔΔCt method, and normalized to the expression of β-actin [39]. Primer sequences are provided in Table 1.

Table 1 The primer sequences used for q-PCR in the studyWestern Blotting

The colonic tissues were homogenized in ice-cold RIPA lysis buffer containing protease inhibitor cocktail and Phosphatase inhibitor cocktail (P1045, Beyotime). The supernatant was collected, and the protein concentration was quantified using a BCA assay (G2026-200 T, Servicebio). Twenty μg proteins (each sample) were subjected to separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (G2302-10, Servicebio) and transferred onto polyvinylidene difluoride (PVDF) membranes (1620177, BIO-RAD). The membranes were incubated with 5% non-fat milk at room temperature for 1 h to achieve blocking, and then incubated overnight at 4 °C with primary antibodies: p21CIP1/CDKN1A (YP-Ab-16762, 1:700, UpingBio technology), p16INK4A/CDKN2A (sc-1661,1:200, Santa Cruz), Occuldin (sc-133256, 1:200, Santa Cruz), ZO-1 (sc-33725, 1:100, Santa Cruz), TNF-α (A11534, 1:2000, Abclonal), IL-1β (A1112, 1:2000, Abclonal), T. gondii SAG1 (sc-52255, 1:500, Santa Cruz) and β-actin (81115–1-RR, 1:20,000, Proteintech). Following 3 washes in Tris Buffered Saline Tween (TBST, G2150-1L, Servicebio), the membrane was incubated with HRP-linked anti-rabbit IgG secondary antibody (GB23303, 1:3000, Servicebio), HRP-linked anti-mouse IgG secondary antibody (GB23301, 1:3000, Servicebio) and HRP-linked anti-rat IgG secondary antibody (GB23302, 1:3000, Servicebio) at room temperature for 1 h. After washing 3 times with TBST, the protein bands were detected with Clarity™ ECL western blot substrate (1,705,060, Bio-Rad) and visualized using the ChemiDoc Touch imaging system (Bio-Rad). Expression levels for each sample were standardized relative to β-actin.

Immunofluorescence Analysis

Immunofluorescence staining with zonula occludens-1 (ZO-1) antibody was performed to evaluate the gut barrier function. Following dissection at the cecum colon junction, colonic tissues were immediately preserved in a 4% paraformaldehyde solution. Next, the tissues were embedded in paraffin and cut into 5 μm- sections. These sections were sequentially deparaffinized in an environmentally friendly deparaffinizing solution (G1128, Servicebio) and anhydrous ethanol. We then utilized the epitope-unmasking solution (10 mM citric acid at pH 6.0) for antigen repair. After natural cooling the slides were washed in PBS. Subsequent staining steps followed established protocol [40]. These colonic sections were incubated with a primary anti-ZO-1 antibody (GB11195, 1:200 dilution, Servicebio) and a secondary goat-anti-rabbit CY3 conjugated antibody (GB21303, 1:300 dilution, Servicebio), followed by nuclear staining with DAPI (G1012, Servicebio) The photographs were captured under a fluorescence microscope (OLYMPUS IX51, Japan). ImageJ was used to calculate the numbers of ZO+ cells and mean fluorescence intensity of ZO-1 in the colon [41]. In detail, the images (20 × magnification) were taken to calculate the indexes. The average fluorescence intensity of ZO-1 was analyzed after the target channel separated by ImageJ. The mean fluorescence intensity was evaluated by using the formula: the Integrated Density (IntDen)/the image area (Area). Furthermore, ImageJ was used to adjust the images (uniform thresholding, noise removal) and then counted the numbers of ZO-1+ cells to obtain positive rate of ZO-1 (DAPI-stained cells were used as controls).

Alcian Blue Staining and Image Analysis

After being fixed in Carnot's fixative (Servicebio, G1120) for 4 h, colon samples were embedded in paraffin and sectioned at 5 μm for Alcian blue staining, following a previously described method [42]. The paraffin sections were deparaffinized in xylene and hydrated with distilled water. Subsequently, sections were incubated with alcian blue solution (G1027, Servicebio) for 30 min, followed by a 2 min wash in running tap water. Once rinsed in distilled water, the sections were dehydrated twice and treated with absolute alcohol twice, with each treatment lasting 3 min. The sections were cleared in xylene three times, each time for 3 min. Finally, the side of the slide with cells was sealed with rhamsan gum (WG10004160, Servicebio) and covered the coverslip. The thickness of the mucosal layer was quantitatively assessed using ImageJ.

Lipopolysaccharide Determination (LPS)

Serum LPS levels were determined by enzyme-linked immunosorbent assay (Limulus assay kit, Cat.18110115, Xiamen Bioendo Technology Co, Ltd, Xiamen, China) according to the protocol provided by the manufacturer. The absorbance was gauged at a wavelength of 545 nm with a spectrophotometer (Asuragen ClinBio128, USA) [40].

Statistical Analysis

Data were analyzed using GraphPad Prism 8.0 and expressed as mean ± standard deviation (SD). After the Shapiro–Wilk normality test was performed, the Student's t-test was used to compare the two groups; and the One-way analysis of variance (ANOVA) was used for the comparions of four groups, followed by the post hoc Tukey–Kramer test for multiple comparisons. A P-value < 0.05 was considered to indicate statistical significance.