EGF enema and EGFR monoclonal antibody injection alleviate the inflammatory bowel disease in AMO/DSS induced mice model

The following reagents and equipment were used in the study: recombinant human epidermal growth factor (Shanghai Haohai Biotechnology Co., Ltd., National Medicine Standard S20010099), Cetuximab injection (Merck, Germany, product lot number 264637), fecal occult blood (OB) reagent (pilamid), Hole semi-quantitative detection method) (Zhuhai Bezo Biotechnology Co., Ltd., production batch number: B191001), Rabbit Anti EGFR (ab52894, Abcam, 1/2000), Rabbit Anti p-AKT (AF0016, Affnity, 1/1000), Rabbit Anti p-ERK1/2 (AF1015, Affnity, 1/1000), Mouse Monoclonal Anti-GAPDH (TA-08, Zhongshan Jinqiao, 1/2000), horseradish enzyme labeled goat anti-mouse IgG (H+L) (ZB-2305, Zhongshan Jinqiao, 1/2000), EGFR (AF6043 Affinity), Ki67 (ab15580 abcam), horseradish enzyme labeled goat anti-rabbit IgG (H+L) (ZB-2301, Zhongshan Jinqiao), hematoxylin staining solution (AR11800-1, BOSTER), eosin staining solution (AR11800-2, BOSTER), automatic microplate reader (WD-2102B, Beijing Liuyi Instrument Factory), ultra-high sensitivity chemiluminescence imaging system (Chemi DocTM XRS+, protein vertical electrophoresis instrument (DYY-6C, Beijing Liuyi Instrument Factory), Biorad Life Medical Products of Shanghai Co., Ltd.), optical microscope (CX41, OLYMPUS), microtome (BQ-318D, Bernard).

Twelve 8-week-old C57BL/6 male mice (about 25g) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (License number: SCXK (Xiang) 2019-0004). The mice were divided into four groups, each having 3 mice: a control group, a model group, model mice treated with EGF, and model mice treated with EGF in combination with EGFR monoclonal antibody. To develop the model, mice were injected with a small dose of 7.5mg/kg of AOM intraperitoneally, which was followed by 2% DSS in drinking water for 7 days, then switched to purified water for 14 days. This DSS feeding cycle was repeated 3 times. The mice developed symptoms such as diarrhea and bloody defecation from the first cycle. Mice in the EGF treatment group were given an EGF enema from the onset of symptoms. In contrast, the monoclonal antibody intervention group received an intraperitoneal injection of EGFR monoclonal antibody after EGF enema. Mice were weighed weekly and sacrificed 9 weeks after the symptoms appeared. The colon tissue was carefully excised. The intestinal cavity was emptied, and the colon was weighed and photographed.

Tissues were first fixed with 4% paraformaldehyde and rinsed with running water for several hours. The samples were then dehydrated sequentially in 70%, 80%, and 90% ethanol, followed by treatment with a 1:1 mixture of absolute alcohol and xylene for 15 minutes, xylene I for 15 minutes, and II for 15 minutes (until it became transparent). The samples were infiltrated with a 1:1 mixture of xylene and paraffin for 15 minutes. Later, samples were embedded in paraffin I and II for 50–60 minutes each. Later, tissue sections were cut, dewaxed, and hydrated. For staining, sections were immersed in hematoxylin solution for 3 minutes, differentiated with hydrochloric acid ethanol for 15 seconds, rinsed, and treated with bluing solution for 15 seconds. After rinsing with running water, sections were stained with eosin for 3 min, again rinsed with running water, dehydrated, cleared, and sealed with neutral resin. Prepared slides were inspected by microscopy.

Tissue sections were heated at 65 °C for 2 hours, then immersed in xylene twice for 10 minutes each. The sections were subsequently placed in 100% ethanol twice, 95% ethanol, 80% ethanol and purified water for 5 minutes each. The sections were then incubated with pepsin repair solution at 37°C for 30 minutes, then rinsed with PBS. The sections were treated with 3% hydrogen peroxide to block endogenous peroxidase. After washing the sections with PBS thrice for five minutes each, the sections were incubated with 5% BSA at 37 °C for 30 minutes, followed by primary antibody incubation overnight at 4 °C. The Sections were then incubated at room temperature for 45 minutes, then washed with PBS thrice for three minutes each. After washing, sections were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L 1:100 1:100)at 37 °C for 30 min, followed by rinsing thoroughly with PBS, developed with DAB for 5–10 min, and examined under a microscope. After rinsing with PBS or tap water for 1 minute, sections were counterstained with hematoxylin for 3 minutes, differentiated with hydrochloric acid and alcohol, and treated with blueing solution. The sections were then rinsed with tap water for 1 minute, dehydrated, cleared, mounted, and examined under a microscope.

Lysis buffer was used to lyse the cells at 4°C for 30 min. Samples were then centrifuged at 10,000 rpm/min for 10 minutes, and the supernatant was carefully collected as total protein. Protein concentration was determined using the BCA Kit. Proteins were then denatured and separated by electrophoresis for 1–2 hours, followed by wet transfer for 30–50 minutes. Membranes were incubated with primary antibodies overnight at 4°C. The next day, the membranes were washed thrice with 1X TBST and incubated with secondary antibody at room temperature for 1–2 hours. After washing with 1X TBS-T thrice, ECL exposure solution was used for chemiluminescence, which was detected under a BioRad ChemiDoc imaging system. Protein band intensity was measured through “Quantity One” software.

The Shapiro-Wilk test assessed the normality of data in each group. Data conforming to a normal distribution are expressed as mean ± standard deviation (mean ± SD). The Levene’s test was employed to evaluate the homogeneity of variance, with P > 0.05 indicating equal variances. When both normality and variance homogeneity assumptions were satisfied, comparisons among multiple groups were performed using one-way analysis of variance (one-way ANOVA), followed by Bonferroni post hoc tests. For data that met normality but exhibited unequal variances (heteroscedasticity), the Dunnett T3 test was used for post hoc comparisons among groups. For body weight comparison over time (throughout the experiment), the paired t-test was used. Statistical significance was considered at a P-value of < 0.05. All data are presented as mean ± standard error of the mean (SEM). GraphPad Prism software (Version 9.0) performed all the statistical analyses.

To evaluate the therapeutic potential of EGF enema and EGFR mAb injection against IBD, we developed an AOM/DSS-induced mouse model of IBD. By the second week, all non-control groups developed diarrhea and hematochezia, with significant initial weight loss observed in the model and EGF+EGFR Ab groups (P<0.05, compared to baseline) (Figure 1A). EGF enema with and without EGFR mAb injection was started at that point. Although all groups showed significant weight recovery from the second week by the end of the intervention (P<0.001, P<0.05, P<0.05, P<0.001, compared to the second week), mice treated with EGF or EGF+EGFR Ab exhibited no notable increase in body weight compared to the model group (Figure 1A). In addition, we also compared the changes in the weights of colon tissue among the four groups. Although the average weight of colon tissues in the model group was higher than that of the control group, it was not statistically significant. On the other hand, EGFR mAb treatment decreased the average weight of colon tissue compared to the EGF enema group, though it was also not significant (Figure 1B). Overall, the AOM/DSS model was successfully established; however, neither EGF monotherapy nor combination therapy with an EGFR mAb significantly affected body weight.

Figure 1.

The effect of EGFR mAb treatment combined with EGF enema in the IBD model in vivo. (A) Line-graph showing changes in body weight of mice (n=3 per group) in control, AOM/DSS model, model with EGF enema and model with EGF enema and EGFR mAb treatment. Treatment time and cycles are mentioned. (B) Bar-graph showing changes in weight of colon tissues (grams) among control, AOM/DSS model, model with EGF enema and model with EGF enema and EGFR mAb treatment. Data is presented as mean ± SEM. * indicates comparison to the initial body weight (Week 0), and # denotes comparison to the body weight at Week 2. ns; non-significant. ^*P<0.05, ^**P<0.01, ^***P<0.001, ^# P<0.05, ^##P<0.01, ^###P<0.001

Next, we performed H&E staining on colon tissues to assess histological changes across the experimental groups (Figure 2) and made the following observations. In the control group, the colon structure was intact and clearly defined. Goblet cells were abundant between the mucosal and intestinal gland epithelial cells. There were no signs of swelling, congestion, or structural disruption. The glands were well-developed and orderly, with a systematically arranged mucosal muscularis, indicating normal histological features (Figure 2A). In the AOM/DSS model group, significant pathological changes were observed. The intestinal mucosa showed congestion and edema, while the lumen was significantly narrowed. The glandular epithelial cells exhibited proliferation, and the glands were disorganized in their arrangement. A large infiltration of inflammatory cells was evident, with increased lamina propria cellular components. The muscle fibers of the muscularis mucosa were disrupted, and the gap between the muscularis and submucosa was widened. The submucosa contained large, loose connective tissue infiltrated with many inflammatory cells (Figure 2B). In the EGF enema group, partial alleviation of inflammation was observed, but pathological changes were still prominent. The mucosal muscular layer was thickened, and inflammatory cell infiltration persisted in the lamina propria. Detached epithelial cells were visible, having shed into the intestinal lumen, suggesting incomplete recovery from the AOM/DSS-induced damage (Figure 2C). The colon structure showed significant improvement in the EGF enema combined with EGFR mAb group, with histology returning close to normal. The lamina propria structure remained intact, and the infiltration of inflammatory cells was greatly reduced. No congestion or edema was observed in the intestinal mucosa; only a few epithelial cells detached from the mucosal surface (Figure 2D). These findings highlight the therapeutic potential of EGFR mAb treatment in reducing inflammation and restoring normal colon histology in the AOM/DSS-induced inflammatory carcinogenesis model.

Figure 2.

EGFR mAb treatment combined with EGF enema alleviates colonic inflammation in the IBD model in vivo. (A-D) Representative H&E staining images (100x) of colon tissue from control (A), AOM/DSS model (B), model with EGF enema (C) and model with EGF enema and EGFR mAb treatment (D)

Ki67 is a widely recognized marker of cellular proliferation, as it is expressed in actively dividing cells but absent in quiescent, non-dividing cells. Elevated levels of Ki67 indicate increased proliferation, which is often associated with inflammation, tissue repair, and carcinogenesis [16]. We performed IHC to evaluate the expression of Ki67 and EGFR across the experimental groups (Figure 3) and made the following observations. In the model group, Ki67 expression was significantly increased compared to the control group, reflecting enhanced epithelial cell proliferation (Figure 3A and B). Compared to the model group, the EGF group and the EGF+EGFR Ab group exhibited significantly reduced Ki67 protein expression levels (P<0.05, P<0.01); however, the combination of EGF and EGFR Ab did not further decrease Ki67 levels compared to the EGF group alone (Figure 3A and B). These findings demonstrate that both EGFR mAb and EGF enema treatments reduce the pathological upregulation of Ki67 and effectively inhibit proliferation.

Figure 3.

EGFR mAb treatment combined with EGF enema reduces proliferation in the IBD model in vivo. (A) IHC images (100x) showing changes in expression of Ki67 and EGFR among control, AOM/DSS model, model with EGF enema and model with EGF enema and EGFR mAb treatment. (B) Bar-graph showing quantification of staining in (A). Data is presented as mean ± SEM. ^*P<0.05, ^**P<0.01, ^***P<0.001

Next, we aimed to investigate whether EGFR mAb treatment combined with EGF enema impacts oncogenic signaling in the IBD model in vivo. Earlier studies have shown that elevated expression of phosphorylated AKT (p-AKT) and phosphorylated ERK (p-ERK) positively correlates with the degree of tumor tissue differentiation [17,18]. As expected, EGFR expression was significantly reduced in the model compared to control, aligning with the notion that EGFR expression is generally downregulated in inflammatory tissues in IBD [10,11]. However, EGF enema led to a significant increase in EGFR expression, suggesting the involvement of a compensatory mechanism where hyperactive EGFR signaling due to elevated EGF levels in the microenvironment further induces EGFR expression [9]. Notably, Compared to the model group, EGFR protein expression was downregulated in both the EGF-treated (enema) group and the EGF+EGFR Ab combination group (P<0.001, P<0.001); furthermore, the combination group exhibited a further decrease in EGFR levels compared to the EGF group alone (P<0.001)(Figure 4A and B). On the other hand, expression of oncogenic proteins, p-Akt and p-ERK1/2, was significantly elevated in the model group compared to the control group, indicating increased activation of oncogenic signaling pathways in response to AOM/DSS-induced inflammation (Figure 4A and B). Most importantly, both EGF enema with and without EGFR mAb treatment groups showed marked decreases in the expression of oncogenic proteins, p-Akt and p-ERK1/2, compared to the model group (P<0.001, P<0.01) (Figure 4A and B). These results suggest that combining EGFR mAb treatment with EGF enema effectively reduces oncogenic signaling pathways and keeps a check on hyperactive EGFR signaling by limiting EGFR expression, eventually mitigating inflammation-driven tumorigenesis.

Figure 4.

EGFR mAb treatment combined with EGF enema reduces oncogenic signaling in the IBD model in vivo. (A) Immunoblots showing changes in expression of p-AKT, p-ERK and EGFR among control, AOM/DSS model, model with EGF enema and model with EGF enema and EGFR mAb treatment. (B) Bar-graph showing quantification of changes in protein expression in (A). Data is presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001