It is generally accepted that changes in intestinal microbiota are associated with human diseases, and the toxic effects of BaP have been widely characterized. 23 However, the effect of BaP on the intestinal tract is uncertain. This study observed that BaP exposure destroyed the gut microflora of mice, leading to the abnormal inflammatory responses and pyroptotic damage, and changes in intestinal function. Meanwhile, AHR which acts as the BaP receptor, plays a unique role in this process.
AHR is a crucial environmental regulator that influences the prevalence and severity of various immune and inflammatory diseases, making it a target for numerous disease therapies.24,25 The expression level of AHR gene is considered an important indicator of disease progession.26 When acting as a ligand, BaP has been found to elevate the mRNA expression of AHR and CYP1A1 genes. Through the intake of water and diet, BaP, obtained through water and diet, is metabolized into a key substance that triggers cancer development, leading to destruction of cellular macromolecules like DNA), resulting in gene mutations and diseases. BaP induces the metabolic activation of cytochrome P450 through the mechanism of BaP aromatic receptor (AHR), and ultimately becoming a DNA binding carcinogen.27 CYP1A1 and CYP1B1, two very important CYP450, are responsible for the metabolism of BaP in the liver, colon and ileum.28,29 Not surprisingly, our findings also demonstrated that BaP up-regulated the expression of AHR, CYP1A1 and CYP1B1 genes in mouse colon tissues (Fig. 1E).
The intestinal mucosal barrier is an important defense against intestinal foreign antigens and microorganisms, playing a vital role in maintaining the normal function of the body.30,31 Tight junction is a crucial component of the intestinal mucosal barrier, comprising various tight junction proteins. Among them, ZO-1, MUC2 and Occludin significantly impact intestinal mucosal permeability.32 Our study revealed a significant decrease in the mRNA expressions of ZO-1, MUC2 and Occludin following BaP treatment (Fig. 3). Treatment with CH223191, the activation of AHR was inhibited, the mRNA expressions of the ZO-1, MUC2 and Occludin increased, mitigating this effect. This indicates that BaP-induced colitis in mice may disrupt tight junction expression, causing intestinal mucosal barrier dysfunction by activating AHR. Meanwhile, the comparison of HE staining results across different treatment groups showed obvious inflammatory cell infiltration in BaP group. This suggested that in the colons of BaP-induced mice, abnormalities in intestinal mucosal barrier function results in increased intestinal permeability, allowing more inflammatory cytokines to enter the enterohepatic circulation. Inhibiting AHR activation during this process could alleviate this adverse effect of BaP (Fig. 2).
Pyroptosis is a type of proinflammatory programmed cell death.33 It is often associated with the secretion of IL-1β and IL-18.34,35 In fact, our observations revealed that BaP increased the mRNA expression of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) in the colon of mice. Additionally, BaP led to an upsurge in the expression of pyroptosis markers (NF-κB, NLRP3, caspase-1, and GSDMD) in colon tissues. Intervention with CH223191 could alleviate these changes. This suggested that BaP induced pyroptotic injury in colon tissue of mice, while CH223191 counteracted the promoting impact of BaP. Furthermore, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a high-affinity AHR agonist, triggers inflammation by enhancing the expression of proinflammatory cytokines through prolonged AHR activation.36 Therefore, it could be inferred that BaP induced pyroptotic injury of colon tissue by activating AHR.
In the process of evolution, there is a symbiotic relationship between gut microbiota and the intestinal environment. The intestinal tract provides nutrition and a reproductive environment for the gut microbiota. On the other hand, intestinal microflora can reduce intestinal permeability, increase the epithelial defense mechanism, assist in carbohydrate fermentation, and form mucosal barrier.37 The symbiotic relationship between gut microbiota and host is mutually beneficial. The host provides an important habitat and nutrients for the flora, and the gut microbiota supports the development of the metabolic system and the maturation of the intestinal immune system38 by providing beneficial nutrients, such as vitamins39 and short-chain fatty acids (SCFAs). Therefore, the interaction between gut microbiota and the intestinal immune system is crucial to maintaining mucosal homeostasis. However, when the balance of gut microbiota changes, it will lead to intestinal disease.40 In addition, the imbalance of gut microbiota is closely related to many diseases.
Studies have also revealed that patients with inflammatory bowel illness typically exhibit a decline in species richness, a reduction in many symbiotic and beneficial fecal bacteria, such as Bacteroidetes, and an increase or proliferation of Proteobacteria.41 We found that the gut microbiota changes caused by exposure to BaP in mice were mainly manifested at the phylum level. BaP increased the abundance of Firmicutes and Proteobacteria, but decreased the abundance of Bacteroidetes. An increase in Proteobacteria and a decrease in Bacteroidetes are commonly associated with PAHs.42,43 Lactobacillus and Helicobacter, intestinal probiotics, are reduced in colonic inflammatory environments.44 The abundance of Lactobacillus, Bacteroides and Oscillospira was markedly reduced by BaP. Bacteroides and Oscillospira are involved in carbohydrate fermentation of intestinal SCFAs. These reduced bacterial populations may lead to a decrease in SCFAs, which in turn disrupt the intestinal epithelial barrier and increase susceptibility to inflammation. As shown, the levels of acetate, propionate, and butyrate were dramatically lower in the BaP group than in the Control group. However, CH223191 treatment can alleviate the decrease of SCFAs content in the feces of BaP-exposed mice, thereby improving the BaP-induced changes in intestinal microbiota (Fig. 5A and B). It is tempting to speculate that the activation of AHR is important in the process of gut microbiota imbalance caused by BaP.
Recent studies have reported the role of AHR signaling in intestinal microbiota dysregulation caused by environmental pollutants. It found that dietary exposure to 2,3,7,8-tetrachlorodibenzofuran (TCDF), a dioxin-like pollutant, modifies the composition of gut bacterial community, triggers intestinal inflammation, and disturbs gut microbiota-host metabolic homeostasis in an AHR-dependent manner.45 CH223191 successfully eliminated the overall effect of dioxins on gut microbiota.46 This demonstrates that the close relation between AHR and the regulation of gut microbiota dynamics. It is inferred that BaP altered the gut microbiota by activating AHR, and CH223191 could effectively reverse this change.
It is well known that AHR can play a variety of functions, and a variety of experimental methods have been used to investigate the possible role of AHR ligands. In addition to these classic, synthetic AHR ligands, scientists have found many natural ligands in foods, such as flavonoids, stilbenes, carotenoids, and indoles. These natural compounds are less toxic but can still trigger reactions via the AHR pathway. Many studies have shown that activated AHR can reduce colitis in mice. AHR ligands, 6-Formylindolo[3,2-b] carbazole (FICZ) treatment in mice can reduce not only colitis induced by Dextran Sulfate Sodium Salt (DSS), but also colitis induced by trinitrobenzene sulfonic acid (TNBS).47 Obviously, this may conflict with our results, which reminds us that BaP is an immunotoxic and immunosuppressive ligand. Interestingly, we found that BaP and BaP + CH223191 groups could reduce the thickness of the footpad and the number of mesenteric lymph nodes and splenocytes in mice (Fig. 7), which was consistent with the characteristics of inhibiting delayed hypersensitivity. Abdulla et al. found that TCDD, an AHR ligand, inhibits the delayed-type hypersensitivity response and may modulate this effect by reversing the intestinal dysregulation induced in the inflammatory response.48 Therefore, we concluded that the immunomodulatory properties of BaP negate the beneficial effect of activating AHR, and thus contribute to the differences described above.
There is no doubt that BaP caused pyroptotic damage to colon tissue and a series of toxic reactions. The key element involved in this process is AHR. CH-223191 has been reported to significantly inhibit the toxicity of TCDD both in vitro and in vivo by blocking the binding of TCDD to AHR, as well as TCDD-mediated nuclear translocation and DNA binding activity of AHR. It has been suggested that modulating AHR signaling through dietary changes, smoking cessation, or using AHR antagonists may be a viable strategy for treating inflammatory bowel disease. In DSS-induced colitis, reducing AHR receptor expression has a protective effect, while the loss of AHR exacerbates the disease.49 This study also found that attenuation of the AHR signaling pathway will ameliorate colitis in a murine model exposed to BaP.