Tumor formation at ileocecal junction associated with interleukin-1ß upregulation in aryl hydrocarbon receptor-deficient mouse.

Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor. We previously reported spontaneous ileocecal tumorigenesis in AhR-deficient mice after the age of 10 weeks, which originated in the confined area between ileum and cecum. This study aimed to investigate the underlying mechanism that causes tumor development at this particular location. To observe mucosal architecture in detail, tissues of ileocecal region were stained with methylene blue. Gene expression profile in the ileocecal tissue was compared with cecum. Immunohistochemical analysis was performed with ileocecal tissues using antibodies against ileum-specific Reg3ß or cecum-specific Pitx2. In AhR+/+ mice and AhR+/- mice, that do not develop lesions, methylene blue staining revealed the gradually changing shape and arrangement of villi from ileum to cecum. It was also observed in AhR-deficient mice before developing lesions. Microarray-based analysis revealed abundant antimicrobial genes, such as Reg3, in the ileocecal tissue while FGFR2 and Pitx2 were specific to cecum. Immunohistochemical analysis of AhR-deficient mice indicated that lesions originated from the ileocecal junction, a boundary area between different epithelial types. Site-specific gene expression analysis revealed higher expression of IL-1ß at the ileocecal junction compared with the ileum or cecum of 9-11-week-old AhR-deficient mice. These findings indicate that AhR plays a vital function in the ileocecal junction. Regulating AhR activity can potentially manage the stability of ileocecal tissue possessing cancer-prone characteristics. This investigation contributes to understanding homeostasis in different epithelial transitional tissues, frequently associated with pathological states.

Induction of indoleamine 2,3-dioxygenase 1 expression in neurons of the central nervous system through inhibition of histone deacetylases blocks the progression of experimental autoimmune encephalomyelitis.

BACKGROUND: A wide array of histone deacetylase (HDAC) inhibitors and aryl hydrocarbon receptor (AHR) agonists commonly arrest experimental autoimmune encephalomyelitis (EAE). However, it is not known whether HDAC inhibition is linked to the AHR signaling pathway in EAE. METHODS: We investigated how the pan-HDAC inhibitor SB939 (pracinostat) exerted immunoregulatory action in the myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE mouse model by evaluating changes in of signal transducer and activator of transcription 3 (STAT3) acetylation and the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and AHR in inflamed spinal cords during EAE evolution. We proved the involvement of IDO1 and the AHR in SB939-mediated immunosuppression using Ido1-/- and Ahr-/- mice. RESULTS: Administration with SB939 halted EAE progression, which depended upon IDO1 expression in neurons of the central nervous system (CNS). Our in vitro and in vivo studies demonstrated that SB939 sustained the interleukin-6-induced acetylation of STAT3, resulting in the stable transcriptional activation of Ido1. The therapeutic effect of SB939 also required the AHR, which is expressed mainly in CD4+ T cells and macrophages in CNS disease lesions. Finally, SB939 was shown to markedly reduce the proliferation of CD4+ T cells in inflamed neuronal tissues but not in the spleen or draining lymph nodes. CONCLUSIONS: Overall, our results suggest that IDO1 tryptophan metabolites produced by neuronal cells may act on AHR in pathogenic CD4+ T cells in a paracrine fashion in the CNS and that the specific induction of IDO1 expression in neurons at disease-afflicted sites can be considered a therapeutic approach to block the progression of multiple sclerosis without affecting systemic immunity.

Microbiota-derived I3A protects the intestine against radiation injury by activating AhR/IL-10/Wnt signaling and enhancing the abundance of probiotics.

The intestine is prone to radiation damage in patients undergoing radiotherapy for pelvic tumors. However, there are currently no effective drugs available for the prevention or treatment of radiation-induced enteropathy (RIE). In this study, we aimed at investigating the impact of indole-3-carboxaldehyde (I3A) derived from the intestinal microbiota on RIE. Intestinal organoids were isolated and cultivated for screening radioprotective tryptophan metabolites. A RIE model was established using 13 Gy whole-abdominal irradiation in male C57BL/6J mice. After oral administration of I3A, its radioprotective ability was assessed through the observation of survival rates, clinical scores, and pathological analysis. Intestinal stem cell survival and changes in the intestinal barrier were observed through immunofluorescence and immunohistochemistry. Subsequently, the radioprotective mechanisms of I3A was investigated through 16S rRNA and transcriptome sequencing, respectively. Finally, human colon cancer cells and organoids were cultured to assess the influence of I3A on tumor radiotherapy. I3A exhibited the most potent radioprotective effect on intestinal organoids. Oral administration of I3A treatment significantly increased the survival rate in irradiated mice, improved clinical and histological scores, mitigated mucosal damage, enhanced the proliferation and differentiation of Lgr5+ intestinal stem cells, and maintained intestinal barrier integrity. Furthermore, I3A enhanced the abundance of probiotics, and activated the AhR/IL-10/Wnt signaling pathway to promote intestinal epithelial proliferation. As a crucial tryptophan metabolite, I3A promotes intestinal epithelial cell proliferation through the AhR/IL-10/Wnt signaling pathway and upregulates the abundance of probiotics to treat RIE. Microbiota-derived I3A demonstrates potential clinical application value for the treatment of RIE.

Biochemical and molecular biomarkers and their association with anthropogenic chemicals in wintering Manx shearwaters (Puffinus puffinus).

Anthropogenic pollution poses a threat to marine conservation by causing chronic toxic effects. Seabirds have contact throughout their lives with pollutants like plastic, metals, polychlorinated biphenyls (PCBs), and organochlorine pesticides such as hexachlorocyclohexanes (HCHs). We assessed 155 Manx shearwaters (Puffinus puffinus) stranded along the Brazilian coast, analyzing associations between organic pollutants, plastic ingestion, biomarkers (transcript levels of aryl hydrocarbon receptor, cytochrome P450-1A-5 [CYP1A5], UDP-glucuronosyl-transferase [UGT1], estrogen receptor alpha-1 [ESR1], and heat shock protein-70 genes) and enzymes activity (ethoxy-resorufin O-deethylase and glutathione S-transferase [GST]). Plastic debris was found in 29 % of the birds. The transcription of UGT1 and CYP1A5 was significantly associated with hexachlorobenzene (HCB) and PCBs levels. ESR1 was associated with HCB and Mirex, and GST was associated with Drins and Mirex. While organic pollutants affected shearwaters more than plastic ingestion, reducing plastic availability remains relevant as xenobiotics are also potentially adsorbed onto plastics.

(-)-Epicatechin gallate ameliorates cyprodinil-induced cardiac developmental defects through inhibiting aryl hydrocarbon receptor in zebrafish.

BACKGROUND: Cyprodinil is a widely used fungicide with broad-spectrum activity, but it has been associated with cardiac abnormalities. (-)-Epicatechin gallate (ECG), a natural polyphenolic compound, has been shown to possess protective properties in cardiac development. METHODS: In this study, we investigated whether ECG could mitigate cyprodinil-induced heart defects using zebrafish embryos as a model. Zebrafish embryos were exposed to cyprodinil with or without ECG. RESULTS: Our results demonstrated that ECG significantly improved the survival rate, embryo movement, and hatching delay induced by cyprodinil. Furthermore, ECG effectively ameliorated cyprodinil-induced cardiac developmental toxicity, including pericardial anomaly and impairment of cardiac function. Mechanistically, ECG attenuated the cyprodinil-induced alterations in mRNA expression related to cardiac development, such as amhc, vmhc, tbx5, and gata4, as well as calcium ion channels, such as ncx1h, atp2a2a, and cdh2. Additionally, ECG was found to inhibit the activity of the aryl hydrocarbon receptor (AhR) signaling pathways induced by cyprodinil. CONCLUSIONS: In conclusion, our findings provide evidence for the protective effects of ECG against cyprodinil-induced cardiac developmental toxicity, mediated through the inhibition of AhR activity. These findings contribute to a better understanding of the regulatory mechanisms and safe utilization of pesticide, such as cyprodinil.

In Silico and In Vitro multiple analysis approach for screening naturally derived ligands for red seabream aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a key ligand-dependent transcription factor that mediates the toxic effects of compounds such as dioxin. Recently, natural ligands of AHR, including flavonoids, have been attracting physiological and toxicological attention as they have been reported to regulate major biological functions such as inflammation and anti-cancer by reducing the toxic effects of dioxin. Additionally, it is known that natural AHR ligands can accumulate in wildlife tissues, such as fish. However, studies in fish have investigated only a few ligands in experimental fish species, and the AHR response of marine fish to natural AHR ligands of various other structures has not been thoroughly investigated. To explore various natural AHR ligands in marine fish, which make up the most fish, it is necessary to develop new screening methods that consider the specificity of marine fish. In this study, we investigated the response of natural ligands by constructing in vitro and in silico experimental systems using red seabream as a model species. We attempted to develop a new predictive model to screen potential ligands that can induce transcriptional activation of red seabream AHR1 and AHR2 (rsAHR1 and rsAHR2). This was achieved through multiple analyses using in silico/ in vitro data and Tox21 big data. First, we constructed an in vitro reporter gene assay of rsAHR1 and rsAHR2 and measured the response of 10 representatives natural AHR ligands in COS-7 cells. The results showed that FICZ, Genistein, Daidzein, I3C, DIM, Quercetin and Baicalin induced the transcriptional activity of rsAHR1 and rsAHR2, while Resveratrol and Retinol did not induce the transcriptional activity of rsAHR isoforms. Comparing the EC50 values of the respective compounds in rsAHR1 and rsAHR2, FICZ, Genistein, and Daidzein exhibited similar isoform responses, but I3C, Baicalin, DIM and Quercetin show the isoform-specific responses. These results suggest that natural AHR ligands have specific profiling and transcriptional activity for each rsAHR isoform. In silico analysis, we constructed homology models of the ligand binding domains (LBDs) of rsAHR1 and rsAHR2 and calculated the docking energies (U_dock values) of natural ligands with measured in vitro transcriptional activity and dioxins reported in previous studies. The results showed a significant correlation (R2=0.74(rsAHR1), R2=0.83(rsAHR2)) between docking energy and transcriptional activity (EC50) value, suggesting that the homology model of rsAHR1 and rsAHR2 can be utilized to predict the potential transactivation of ligands. To broaden the applicability of the homology model to diverse compound structures and validate the correlation with transcriptional activity, we conducted additional analyses utilizing Tox21 big data. We calculated the docking energy values for 1860 chemicals in both rsAHR1 and rsAHR2, which were tested for transcriptional activation in Tox21 data against human AHR. By comparing the U_dock energy values between 775 active compounds and 1085 inactive compounds, a significant difference (p<0.001) was observed between the U_dock energy values in the two groups, suggesting that the U_dock value can be applied to distinguish the activation of compounds. Furthermore, we observed a significant correlation (R2=0.45) between the AC50 of Tox21 database and U_dock values of human AHR model. In conclusion, we calculated equations to translate the results of an in silico prediction model for ligand screening of rsAHR1 and rsAHR2 transactivation. This ligand screening model can be a powerful tool to quantitatively estimate AHR transactivation of major marine agents to which red seabream may be exposed. The study introduces a new screening approach for potential natural AHR ligands in marine fish, based on homology model-docking energy values of rsAHR1 and rsAHR2, with implications for future agonist development and applications bridging in silico and in vitro data.

Association between estrogen receptor alpha and aryl hydrocarbon receptor gene polymorphisms in the prognosis of breast cancer in Egypt.

Breast cancer is the most malignant tumor among women in the world. Single nucleotide polymorphisms (SNPs) might better predict breast cancer prognosis. PvuII (T/C substitution), XbaI (A/G substitution), and aryl hydrocarbon (AhR) (G/A substitution) were evaluated as possible genetic prognostic factors for breast cancer. The aim of the current study was to assess the relation between PvuII (rs2234693), XbaI (rs9340799), and aryl hydrocarbon receptor gene polymorphisms AhR (rs2066853) in breast cancer prognosis. This was a case-control study that included 120 breast cancer patients classified into two groups. The first group included 60 patients with good prognostic factors, and the second group included 60 patients with poor prognostic factors. Blood samples were taken from all study participants to perform the genotyping assay. We found that positive genotypes of PvuII, XbaI, and AhR polymorphisms were strongly associated with better prognostic factors for breast cancer patients, while negative genotypes of PvuII and XbaI were more and significantly prevalent in poor prognostic breast cancer patients. We conclude that PvuII T/C, XbaI G/A, and AhR G/A alleles may be prognostic for breast cancer progression.

Role of aryl hydrocarbon receptors in infection and inflammation.

The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by various ligands, including pollutants, microorganisms, and metabolic substances. It is expressed extensively in pulmonary and intestinal epithelial cells, where it contributes to barrier defense. The expression of AhR is pivotal in regulating the inflammatory response to microorganisms. However, dysregulated AhR expression can result in endocrine disorders, leading to immunotoxicity and potentially promoting the development of carcinoma. This review focuses on the crucial role of the AhR in facilitating and limiting the proliferation of pathogens, specifically in relation to the host cell type and the species of etiological agents involved in microbial pathogen infections. The activation of AhR is enhanced through the IDO1-AhR-IDO1 positive feedback loop, which is manipulated by viruses. AhR primarily promotes the infection of SARS-CoV-2 by inducing the expression of angiotensin-converting enzyme 2 (ACE2) and the secretion of pro-inflammatory cytokines. AhR also plays a significant role in regulating various types of T-cells, including CD4+ T cells and CD8+ T cells, in the context of pulmonary infections. The AhR pathway plays a crucial role in regulating immune responses within the respiratory and intestinal barriers when they are invaded by viruses, bacteria, parasites, and fungi. Additionally, we propose that targeting the agonist and antagonist of AhR signaling pathways could serve as a promising therapeutic approach for combating pathogen infections, especially in light of the growing prevalence of drug resistance to multiple antibiotics.

Cell type-dependent response to benzo(a)pyrene exposure of human placental cell lines under normoxic, hypoxic, and pro-inflammatory conditions.

Benzo(a)pyrene (BaP) can be detected in the human placenta. However, little is known about the effects of BaP exposure on different placental cells under various conditions. In this study, we aimed to investigate the effects of BaP on mitochondrial function, pyrin domain-containing protein 3 (NLRP3) inflammasome, and apoptosis in three human trophoblast cell lines under normoxia, hypoxia, and inflammatory conditions. JEG-3, BeWo, and HTR-8/SVneo cell lines were exposed to BaP under normoxia, hypoxia, or inflammatory conditions for 24 h. After treatment, we evaluated cell viability, apoptosis, aryl hydrocarbon receptor (AhR) protein and cytochrome P450 (CYP) gene expression, mitochondrial function, including mitochondrial DNA copy number (mtDNAcn), mitochondrial membrane potential (ΔΨm), intracellular adenosine triphosphate (iATP), and extracellular ATP (eATP), nitric oxide (NO), NLPR3 inflammasome proteins, and interleukin (IL)-1ß. We found that BaP upregulated the expression of AhR or CYP genes to varying degrees in all three cell lines. Exposure to BaP alone increased ΔΨm in all cell lines but decreased NO in BeWo and HTR-8/SVneo, iATP in HTR-8/SVneo, and cell viability in JEG-3, without affecting apoptosis. Under hypoxic conditions, BaP did not increase the expression of AhR and CYP genes in JEG-3 cells but increased CYP gene expression in two others. Pro-inflammatory conditions did not affect the response of the 3 cell lines to BaP with respect to the expression of CYP genes and changes in the mitochondrial function and NLRP3 inflammasome proteins. In addition, in HTR-8/SVneo cells, BaP increased IL-1ß secretion in the presence of hypoxia and poly(I:C). In conclusion, our results showed that BaP affected mitochondrial function in trophoblast cell lines by increasing ΔΨm. This increased ΔΨm may have rescued the trophoblast cells from activation of the NLRP3 inflammasome and apoptosis after BaP treatment. We also observed that different human trophoblast cell lines had cell type-dependent responses to BaP exposure under normoxia, hypoxia, or pro-inflammatory conditions.

Indoleamine 2,3-dioxygenase (IDO1) - Can dendritic cells and monocytes expressing this moonlight enzyme change the phase of Parkinson's Disease?

Parkinson's Disease (PD) is the second most common neurodegenerative disease where central and peripheral immune dysfunctions have been pointed out as a critical component of susceptibility and progression of this disease. Dendritic cells (DCs) and monocytes are key players in promoting immune response regulation and can induce the enzyme indoleamine 2,3-dioxygenase 1 (IDO1) under pro-inflammatory environments. This enzyme with catalytic and signaling activity supports the axis IDO1-KYN-aryl hydrocarbon receptor (AhR), promoting disease-specific immunomodulatory effects. IDO1 is a rate-limiting enzyme of the kynurenine pathway (KP) that begins tryptophan (Trp) catabolism across this pathway. The immune functions of the pathway, which are extensively described in cancer, have been forgotten so far in neurodegenerative diseases, where a chronic inflammatory environment underlines the progression of the disease. Despite dysfunctions of KP have been described in PD, these are mainly associated with neurotoxic functions. With this review, we aim to focus on the immune properties of IDO1+DCs and IDO1+monocytes as a possible strategy to balance the pro-inflammatory profile described in PD. We also highlight the importance of exploring the role of dopaminergic therapeutics in IDO1 modulation to possibly optimize current PD therapeutic strategies.

Non-target analysis of crude oil photooxidation products at high latitudes and their biological effects.

With new oil and gas lease sales in high-latitude regions, there exists a need to better understand the chemical fate of spilled oil and its effects on biological life. To address this need, laboratory simulations of crude oil spills under sub-Arctic conditions were conducted using artificial seawater and exposure to solar irradiation to create Hydrocarbon Oxidation Products (HOPs). HOPs characterization and their biological effects were assessed using ultra high-performance liquid chromatography (UHPLC) with high resolution mass Orbitrap spectrometry and the aryl hydrocarbon receptor (AhR) chemically activated luciferase gene expression (CALUX) assay. Non-target UHPLC-Orbitrap mass spectrometry analysis identified 251 HOPs that were in greater abundance in light-exposed samples than dark controls. Oxidized polycyclic aromatic hydrocarbons were also detected, including phenanthrene quinone, anthraquinone, hydroxyanthraquinone, and 9-fluoreneone. The composition of HOPs were consistent with photo-products of alkylated two to four ring PAHs, primarily compounds between 1 and 3 aromatic rings and 1-3 oxygens. The HOP mixture formed during photochemical weathering of Cook Inlet crude oil induced greater AhR activity than parent petroleum products solubilized in dark controls, indicating that HOPs, as a complex mixture, may contribute to petroleum toxicity more than the parent petroleum compounds. These non-targeted approaches provide the most comprehensive analysis of hydrocarbon oxidation products to date, highlighting the diversity of the complex mixture resulting from the photooxidation of crude oil and the limitations of targeted analyses for adequately monitoring HOPs in the environment. Taken together, these data identify a critical "blind spot" in environmental monitoring and spill clean-up strategies as there is a diverse pool of HOPs that may negatively impact human and ecosystem health.

Exploring aryl hydrocarbon receptor expression and distribution in the tumor microenvironment, with a focus on immune cells, in various solid cancer types.

Introduction: Aryl hydrocarbon receptor (AhR) is a transcription factor that performs various functions upon ligand activation. Several studies have explored the role of AhR expression in tumor progression and immune surveillance. Nevertheless, investigations on the distribution of AhR expression, specifically in cancer or immune cells in the tumor microenvironment (TME), remain limited. Examining the AhR expression and distribution in the TME is crucial for gaining insights into the mechanism of action of AhR-targeting anticancer agents and their potential as biomarkers. Methods: Here, we used multiplexed immunohistochemistry (mIHC) and image cytometry to investigate the AhR expression and distribution in 513 patient samples, of which 292 are patients with one of five solid cancer types. Additionally, we analyzed the nuclear and cytosolic distribution of AhR expression. Results: Our findings reveal that AhR expression was primarily localized in cancer cells, followed by stromal T cells and macrophages. Furthermore, we observed a positive correlation between the nuclear and cytosolic expression of AhR, indicating that the expression of AhR as a biomarker is independent of its localization. Interestingly, the expression patterns of AhR were categorized into three clusters based on the cancer type, with high AhR expression levels being found in regulatory T cells (Tregs) in non-small cell lung cancer (NSCLC). Discussion: These findings are anticipated to serve as pivotal evidence for the design of clinical trials and the analysis of the anticancer mechanisms of AhR-targeting therapies.

A tryptophan-derived uremic metabolite/Ahr/Pdk4 axis governs skeletal muscle mitochondrial energetics in chronic kidney disease.

Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR), which has been shown to be activated in CKD. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared with controls with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD, and the magnitude of AHR activation was inversely correlated with mitochondrial respiration. In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was markedly impaired and strongly correlated with the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR substantially improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD + probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite/AHR/mitochondrial axis in skeletal muscle was verified using muscle-specific AHR knockdown in C57BL/6J mice harboring a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were present only when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significantly increased pyruvate dehydrogenase kinase 4 (Pdk4) expression and phosphorylation of pyruvate dehydrogenase enzyme. These findings establish a uremic metabolite/AHR/Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.

Benzo[a]pyrene exposure causes exonal switch resulting in reduced surface CD5 expression in an AHR-dependent manner.

The function of CD5 protein in T cells is well documented, but regulation of its surface-level expression has yet to be fully understood. However, variation in its surface expression is associated with various immunopathological conditions and haematological malignancies. Briefly, expression of an alternate exon E1B of a human endogenous retroviruses (HERV) origin directly downregulates the conventional transcript variant (E1A), as its expression leads to the retention of the resultant protein at the intracellular level (cCD5). A separate promoter governs the expression of E1B and may be influenced by different transcription factors. Hence, we performed in silico transcription factor binding site (TFBS) analysis of the 3 kb upstream region from TSS of exon E1B and found five putative DREs (Dioxin Response elements) with good similarity scores. Further, we observed the upregulation in E1B expression after the exposure of BaP (a dioxin) and the reduction of E1A expression and their respective protein, i.e. sCD5 and cCD5. The binding of AHR at the predicted DRE sites was confirmed by ChIP qPCR and AHR specific inhibitor and gene silencing studies suggested the involvement of AHR in exonal switch. This study indicates that the polycyclic aromatic hydrocarbon decreases the sCD5 expression by upregulating alternative exon expression, which may adversely affect the overall T cell functions.

Zhilining Formula alleviates DSS-induced colitis through suppressing inflammation and gut barrier dysfunction via the AHR/NF-κBp65 axis.

BACKGROUND: Repairing the intestinal mucosal barrier and reducing persistent inflammation is the key strategies for the treatment of ulcerative colitis (UC). Zhilining Formula (ZLN), composed of Andrographis herba (AH), Sophorae flavescentis radix (SFA), and Aucklandia radix (AR), is a well-tried formula for the clinical treatment of enteritis and dysentery in China, and its mechanism has not been clarified. PURPOSE: This study aims to investigate the effect of ZLN on UC and elucidate its underlying mechanism via metabolomics analysis and experimental verification. METHODS: The effect of ZLN on UC was evaluated in a 3.5 % dextran sulfate sodium (DSS)-induced mice model via the body weight, disease activity index (DAI), colon length, colonic histopathology, expression of inflammation factors, and intestinal barrier in mice. An UPLC-Q-TOF-MS/MS approach-based metabolomics analysis was performed to preliminary explore the mechanism of ZLN in colitis. Based on the results of metabolomics analysis, the expression of related protein or mRNA in AHR/NF-κBp65 axis was determined by qPCR and western blotting. Moreover, the potential interactions of active ingredients of ZLN with NF-κBp65 and AHR were investigated in vitro through using agonists and inhibitors of NF-κBp65 and AHR, respectively. RESULTS: ZLN alleviated body weight loss and colonic shortening in colitis mice, and down-regulated the DAI and histopathological score as well. ZLN also decreased the levels of inflammatory factors (MPO, IL-1ß, TNF-α and IL-18), protected goblet cell function and intestinal barrier in DSS-induced mice. Metabolomics results revealed that 36 metabolites that were significantly altered in mice after induction with DSS, which involved in 16 metabolic pathways, including biosynthesis of unsaturated fatty acid, phenylalanine metabolism, arachidonic acid (AA) metabolism, tryptophan (Trp) metabolism, retinol metabolism, and sphingolipid metabolism, etc. ZLN restored 26 different metabolites (DEMs) of them to normal-like levels, indicating ZLN regulated the AA metabolism and Trp-metabolism in UC mice, which hinted its potential pharmacological mechanism related to AHR/NF-κBp65 axis. We further confirmed that ZLN could restrain the activation of NF-κBp65 signaling pathway and then inhibit the expression of its mediated inflammatory cytokines, such as IL-1ß, TNF-α, COX-2 and IL17A. Moreover, ZLN increased nuclear translocation of AHR and IL22 expression, which is an important regulatory signal for intestinal mucosal barrier repaired. Finally, we elucidated in vitro that the active ingredients of ZLN exerted anti-colitis effects by activating AHR and simultaneously inhibiting NF-κBp65. CONCLUSION: ZLN relieved colitis by AHR/NF-κBp65 axis. This study highlighted the important role of AHR and NF-κBp65 in UC, and provided a theoretical basis for the application of ZLN.

The Role of Endocrine Disruption Chemical-Regulated Aryl Hydrocarbon Receptor Activity in the Pathogenesis of Pancreatic Diseases and Cancer.

The aryl hydrocarbon receptor (AHR) serves as a ligand-activated transcription factor crucial for regulating fundamental cellular and molecular processes, such as xenobiotic metabolism, immune responses, and cancer development. Notably, a spectrum of endocrine-disrupting chemicals (EDCs) act as agonists or antagonists of AHR, leading to the dysregulation of pivotal cellular and molecular processes and endocrine system disruption. Accumulating evidence suggests a correlation between EDC exposure and the onset of diverse pancreatic diseases, including diabetes, pancreatitis, and pancreatic cancer. Despite this association, the mechanistic role of AHR as a linchpin molecule in EDC exposure-related pathogenesis of pancreatic diseases and cancer remains unexplored. This review comprehensively examines the involvement of AHR in EDC exposure-mediated regulation of pancreatic pathogenesis, emphasizing AHR as a potential therapeutic target for the pathogenesis of pancreatic diseases and cancer.

The Antioxidant Drug Edaravone Binds to the Aryl Hydrocarbon Receptor (AHR) and Promotes the Downstream Signaling Pathway Activation.

A considerable effort has been spent in the past decades to develop targeted therapies for the treatment of demyelinating diseases, such as multiple sclerosis (MS). Among drugs with free radical scavenging activity and oligodendrocyte protecting effects, Edaravone (Radicava) has recently received increasing attention because of being able to enhance remyelination in experimental in vitro and in vivo disease models. While its beneficial effects are greatly supported by experimental evidence, there is a current paucity of information regarding its mechanism of action and main molecular targets. By using high-throughput RNA-seq and biochemical experiments in murine oligodendrocyte progenitors and SH-SY5Y neuroblastoma cells combined with molecular docking and molecular dynamics simulation, we here provide evidence that Edaravone triggers the activation of aryl hydrocarbon receptor (AHR) signaling by eliciting AHR nuclear translocation and the transcriptional-mediated induction of key cytoprotective gene expression. We also show that an Edaravone-dependent AHR signaling transduction occurs in the zebrafish experimental model, associated with a downstream upregulation of the NRF2 signaling pathway. We finally demonstrate that its rapid cytoprotective and antioxidant actions boost increased expression of the promyelinating Olig2 protein as well as of an Olig2:GFP transgene in vivo. We therefore shed light on a still undescribed potential mechanism of action for this drug, providing further support to its therapeutic potential in the context of debilitating demyelinating conditions.

Paeonol attenuates nonalcoholic steatohepatitis by regulating intestinal flora and AhR/NLRP3/Caspase-1 metabolic pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic steatohepatitis (NASH) is a common metabolic liver injury disease that is closely associated with obesity and metabolic disorders. Paeonol, an active ingredient found in Moutan Cortex, a traditional Chinese medicine which exhibits significant therapeutic effect on liver protection, has shown promising effects in treating liver diseases, particularly NASH. However, the specific intervention mechanism of paeonol on NASH is still unknown. AIM OF THE STUDY: Our objective is to elucidate the pharmacological mechanism of paeonol in intervening NASH at the in vivo level, focusing on the impact on intestinal flora, tryptophan-related targeted metabolome, and related Aryl hydrocarbon receptor (AhR) pathways. MATERIALS AND METHODS: Here, we explored the intervention effect of paeonol on NASH by utilizing the NASH mouse model. The Illumina highthroughput sequencing technology was preformed to determine the differences of gut microbiota of model and paeonol treatment group. The concentration of Indoleacetic acid is determined by ELISA. The intervention effect of NASH mouse and AhR/NLRP3/Caspase-1 metabolic pathway is analyzed by HE staining, oil red O staining, Immunohistochemistry, Immunofluorescence, Western blot and qRT-PCR assays. Fecal microbiota transplantation experiment also was performed to verify the intervention effect of paeonol on NASH by affecting gut microbiota. RESULTS: Firstly, we discovered that paeonol effectively reduced liver pathology and blood lipid levels in NASH mice, thereby intervening in the progression of NASH. Subsequently, through 16S meta-analysis, we identified that paeonol can effectively regulate the composition of intestinal flora in NASH mice, transforming it to resemble that of normal mice. Specifically, paeonol decreased the abundance of certain Gram-negative tryptophan-metabolizing bacteria. Moreover, we discovered that paeonol significantly increased the levels of metabolites Indoleacetic acid, subsequently enhancing the expression of AhR-related pathway proteins. This led to the inhibition of the NOD-like receptor protein 3 (NLRP3) inflammasome production and inflammation generation in NASH. Lastly, we verified the efficacy of paeonol in intervening NASH by conducting fecal microbiota transplantation experiments, which confirmed its role in promoting the AhR/NLRP3/cysteinyl aspartate specific proteinase (Caspase-1) pathway. CONCLUSIONS: Our findings suggest that paeonol can increase the production of Indoleacetic acid by regulating the gut flora, and promote the AhR/NLRP3/Caspase-1 metabolic pathway to intervene NASH.

Chlorocholine chloride exposure induced spermatogenic dysfunction via iron overload caused by AhR/PERK axis-dependent ferritinophagy activation.

Chlorocholine chloride (CCC) is a plant growth regulator used worldwide that is detectable in cereals, fruits and animal products. The health effects of CCC exposure have raised public concern. Our previous research showed that CCC exposure decreased testosterone synthesis in pubertal rats. However, little is known about whether and how pubertal CCC exposure impacts spermatogenesis. In this study, we used BALB/c mice and spermatogonia-derived GC-1 cells to examine CCC-induced spermatogenic dysfunction. In vivo, pubertal CCC exposure led to decreased testicular weight, decreased testicular germ cells and poor sperm quality. This effect worsened after cessation of CCC exposure for the next 30 days. RNA-seq and western blot analysis revealed that CCC induced aryl hydrocarbon receptor (AhR) signaling, endoplasmic reticulum stress (ERS) and ferritinophagy. Increased iron content and lipid peroxidation levels were also observed in CCC-treated testes. In vitro, it was identified that iron overload mediated by enhanced ferritinophagy occurred in CCC-treated GC-1 cells, which might be attributed to the PERK pathway in ERS. Further, for the first time, our study elucidated the involvement of AhR in CCC-induced iron overload, which aggravated testicular oxidative damage via lipid peroxidation. Considering the adverse impact of CCC exposure on rodents, supportive evidence from GC-1 cells, and the critical importance of spermatogenesis on male development, the effects of CCC on the male reproduction warrant increased attention.

Investigating the Aryl Hydrocarbon Receptor Agonist/Antagonist Conformational Switch Using Well-Tempered Metadynamics Simulations.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates biological signals to control various complicated cellular functions. It plays a crucial role in environmental sensing and xenobiotic metabolism. Dysregulation of AhR is associated with health concerns, including cancer and immune system disorders. Upon binding to AhR ligands, AhR, along with heat shock protein 90 and other partner proteins undergoes a transformation in the nucleus, heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), and mediates numerous biological functions by inducing the transcription of various AhR-responsive genes. In this manuscript, the 3-dimensional structure of the entire human AhR is obtained using an artificial intelligence tool, and molecular dynamics (MD) simulations are performed to study different structural conformations. These conformations provide insights into the protein's function and movement in response to ligand binding. Understanding the dynamic behavior of AhR will contribute to the development of targeted therapies for associated health conditions. Therefore, we employ well-tempered metadynamics (WTE-metaD) simulations to explore the conformational landscape of AhR and obtain a better understanding of its functional behavior. Our computational results are in excellent agreement with previous experimental findings, revealing the closed and open states of helix α1 in the basic helix-loop-helix (bHLH domain) in the cytoplasm at the atomic level. We also predict the inactive form of AhR and identify Arginine 42 as a key residue that regulates switching between closed and open conformations in existing AhR modulators.