2.1. Management of Animal Study

Wild-type C57BL/6J were obtained from the animal center of Lanzhou University, while CRP-deficient mice with C57BL/6J backgrounds were generously provided by Prof. Yi Wu [17]. The experimental mouse usage protocol had been approved by the Institution of Animal Care and Use Committee (approval: EAF2021009, Lanzhou University, China). In brief, the mice were housed with a 12-h light/dark cycle (light: 7:00–19:00), with 40%–50% relative humidity at 21°C–22°C, and with free access to food and water in an SPF-grade animal center from School of Life Sciences at Lanzhou University.

2.2. Mouse Model

For the UC mouse model, wild-type C57BL/6J or CRP-deficient male mice aged 8–10 weeks were randomly grouped and subjected to the induction of UC using a 2% dextran sulfate sodium (DSS) solution in their drinking water every 2 days for 7–10 days. The mice’s weight was measured during the experiments. At the endpoint of the experiments, the mice were euthanized using carbon dioxide. Then, spleen weight and colon length were measured. Colon samples were resected for histological assay and Western blot. Serum was collected from the orbital sinus for enzyme-linked immunosorbent assay (ELISA). Among these experiments, the causal roles of CRP were assayed in CRP-deficient mice and wild-type mice as controls. The number of mice for each experiment is variable according to the supply capacity of mice, with at least five mice for each group to reach adequate statistical power.

According to the reported method, a 5% DSS solution was used for survival analysis [18]. The experiment was terminated until the total number of mice killed reached 90%, and the remaining mice were euthanized using carbon dioxide. The survival function was estimated using the Kaplan–Meier method.

2.3. Antibiotic Experiments

For the antibiotic experiments, a concentration of 1 g/L ampicillin was added to the drinking water [19] 2 weeks prior to the commencement of the study. The effect of intestinal flora depletion was assayed through fecal microbiota culture, according to a previous report [20]. In brief, the collected fecal samples from the mice were suspended and diluted 2 × 10⁵-fold with sterile water. The diluent was inoculated, spread on a nutrient agar plate (0.5% peptone, 0.3% beef extract/yeast extract, 1.5% agar, and 0.5% NaCl), and aerobically incubated at 30°C for 24 h. The bacteria colonies in the nutrient agar plate were counted. The intestinal flora–depleted mice of wild-type or CRP-deficient C57BL/6J male mice were subjected to the induction of UC. The body weight, colon length, and spleen index were recorded during the experiment.

2.4. Hematoxylin and Eosin (HE) and PAS Staining

For the HE staining procedure, colon samples from mice were resected and subsequently fixed in a 4% paraformaldehyde solution overnight at 4°C. Dehydration and paraffin embedding procedures were carried out following established protocols. The paraffin-embedded tissues were rehydrated sequentially using xylene and ethanol, followed by staining with HE. For the PAS staining procedure, the tissues were fixed using a Carnoy fixative as an alternative to paraformaldehyde. The remaining steps were analogous to the HE staining procedure, including an additional step for PAS staining.

2.5. Gene Expression Analysis (Western Blot and ELISA)

The UC colon samples and serum were derived from mouse models with wild-type mice as controls. The macrodissected colon samples and serum (derived from the orbital sinus) were frozen in liquid nitrogen and stored in a refrigerator at −80° until detection. Total protein was extracted utilizing lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, and 1% SDS). Thirty micrograms of total protein quantified through the BCA method was loaded, which was performed according to the standard Western blot protocol. The antibodies against mouse Muc2 or Tff3 were purchased from ABclonal Technology (catalog: A4767 for Muc2) and Abcam (catalog: ab30042 for Tff3), respectively. Tubulin was used as the control (ABclonal Technology, catalog: WX06622).

The concentration of IL-1β, IL-6, and IL-17 in serum was evaluated through ELISA according to the instructions of the ELISA kit (Shanghai Kexing Trading, Co., Ltd.; catalog: F2040-A for IL-1β, F2163-A for IL-6, and F2170-A for IL-17).

2.6. Statistical Analysis

Data were presented as means ± standard deviation (SD). All experimental procedures were replicated a minimum of three times. The statistical analysis used the Student t-test in OriginPro software (OriginLab Corporation).

3.1. CRP Deficiency Elicits a Comparable Response to That Observed in Wild-Type Mice With UC

Our study in a mouse model of UC induced with DSS revealed significant weight loss in both CRP-deficient and wild-type mice (Figure 1). Despite more pronounced weight loss in CRP-deficient mice (Figure 1(a)), more precise measures of colitis severity, like colon length (Figure 1(b)) and spleen index (Figure 1(c)), showed comparable results. These results suggested that the expression of CRP in mice did not significantly impact the progression of UC.

To thoroughly investigate the potential impact of CRP on UC, we conducted a series of meticulous histochemical staining experiments. Our findings indicated that the administration of DSS exacerbated the pathology associated with UC. However, we observed no significant variation in this pathology between subjects with or without CRP (Figure 2(a)). Meanwhile, applying PAS staining to detect goblet cells suggested that CRP might not be associated with the pathological alterations observed in UC (Figures 2(b) and 2(c)). In addition, the expression levels of mucosal markers muc2 and Tff3 were comparable between the CRP-deficient and control groups (Figure 2(d)). The etiology of UC has been extensively studied and widely acknowledged as a predominant influence of immune system dysregulation. The results demonstrated no significant difference in the circulating protein expression of major inflammatory factors (IL-1β, IL-6, IL-17) between CRP-deficient mice and wild-type mice (Figure 2(e)).

3.2. The Lack of Efficacy of CRP in UC Is Not Influenced by the Composition of the Intestinal Flora

Inflammatory bowel disease typically results in the disturbance of the intestinal mucosal barrier system [21–26]. The intestinal microbiota play a significant role in the development and progression of inflammatory bowel disease through the disordered intestinal barrier system [27]. Additionally, reports indicate that CRP can bind to bacteria, serving as a significant opsonizing agent [28, 29]. In our comprehensive study, we provided additional clarification on the potential functional contributions of CRP by conducting a depletion of the intestinal flora using antibiotics (the depletion effect up to 95%, as shown in Figure 3(a)). The findings indicated that the mice, with or without CRP, did not yield a significant difference in UC (Figures 3(b), 3(c), and 3(d)). In a model of UC induced by high concentrations of DSS to assess the survival function, no significant differences were observed between CRP-deficient and wild-type mice (Figure 4). Altogether, our study suggests that CRP is more likely to be a bystander in UC.