Aryl hydrocarbon receptor dynamics in esophageal squamous cell carcinoma:From immune modulation to therapeutic opportunities

Esophageal squamous cell carcinoma(ESCC)is a substantial global health burden.Immune escape mechanisms are important in ESCC progression,enabling cancer cells to escape the surveillance of the host immune system.One key player in this process is the Aryl Hydrocarbon Receptor(AhR),which influences multiple cellular processes,including proliferation,differentiation,metabolism,and immune regulation.Dysregulated AhR signaling participates in ESCC development by stimulating carcinogenesis,epithelial-mesenchymal transition,and immune escape.Targeting AhR signaling is a potential therapeutic approach for ESCC,with AhR ligands showing efficacy in preclinical studies.Additionally,modification of AhR ligands and combination therapies present new opportunities for therapeutic intervention.This review aims to address the knowledge gap related to the role of AhR signaling in ESCC pathogenesis and immune escape.

Aryl hydrocarbon receptor nuclear translocator 2 as a prognostic biomarker and immunotherapeutic indicator for clear cell renal cell carcinoma

Background:In many cancer types,aryl hydrocarbon receptor nuclear translocator 2(ARNT2)has been found to be associated with tumor cell proliferation and prognosis.However,the role of ARNT2 in clear cell renal cell carcinoma(ccRCC)has not been completely elucidated.In this study,the potential role of ARNT2 in ccRCC development was characterized.Methods:A pan-cancer dataset(TCGA-TARGET-GTEx)was accessed from UCSC Xena Data Browser.ARNT2 expression in normal and tumor samples was compared.Univariate Cox regression was performed to evaluate the prognostic value of ARNT2.Single sample gene set enrichment analysis(ssGSEA)was used to estimate the enrichment of functional pathways and gene signatures.CIBERSORT and ESTIMATE methods evaluated the immune infiltration.The ARNT2 expression was determined in ccRCC tissue and cell lines using RT-qPCR and Western blot.Results:ARNT2 expression was significantly dysregulated in 23 out of 30 cancer types.Pan-cancer data revealed a strong correlation between ARNT2 expression and immune modulators,immune cell infiltration,and genomic alternations.In ccRCC patients,the low-ARNT2 expression group had higher immune infiltration,CD8 T cells,and programmed cell death ligand 1 expression,as well as higher enrichment score of immunotherapeutic predictors than those in the high-ARNT2 expression group.Low-ARNT2 expression group was more responsive to immunotherapy.Moreover,low ARNT2 expression was observed in ccRCC tissue and cell lines.Conclusions:Dysregulated ARNT2 expression is involved in cancer development and the modulation of the immune microenvironment.ARNT2 can be potentially used as a prognostic indicator and an immunotherapeutic indicator for ccRCC.

Constitutive aryl hydrocarbon receptor facilitates the regenerative potential of mouse bone marrow mesenchymal stromal cells

BACKGROUND Bone marrow mesenchymal stromal cells(BMSCs)are the commonly used seed cells in tissue engineering.Aryl hydrocarbon receptor(AhR)is a transcription factor involved in various cellular processes.However,the function of constitutive AhR in BMSCs remains unclear.AIM To investigate the role of AhR in the osteogenic and macrophage-modulating potential of mouse BMSCs(mBMSCs)and the underlying mechanism.METHODS Immunochemistry and immunofluorescent staining were used to observe the expression of AhR in mouse bone marrow tissue and mBMSCs.The overexpression or knockdown of AhR was achieved by lentivirus-mediated plasmid.The osteogenic potential was observed by alkaline phosphatase and alizarin red staining.The mRNA and protein levels of osteogenic markers were detected by quantitative polymerase chain reaction(qPCR)and western blot.After coculture with different mBMSCs,the cluster of differentiation(CD)86 and CD206 expressions levels in RAW 264.7 cells were analyzed by flow cytometry.To explore the underlying molecular mechanism,the interaction of AhR with signal transducer and activator of transcription 3(STAT3)was observed by co-immunoprecipitation and phosphorylation of STAT3 was detected by western blot.RESULTS AhR expressions in mouse bone marrow tissue and isolated mBMSCs were detected.AhR overexpression enhanced the osteogenic potential of mBMSCs while AhR knockdown suppressed it.The ratio of CD86+RAW 264.7 cells cocultured with AhR-overexpressed mBMSCs was reduced and that of CD206+cells was increased.AhR directly interacted with STAT3.AhR overexpression increased the phosphorylation of STAT3.After inhibition of STAT3 via stattic,the promotive effects of AhR overexpression on the osteogenic differentiation and macrophage-modulating were partially counteracted.CONCLUSION AhR plays a beneficial role in the regenerative potential of mBMSCs partially by increasing phosphorylation of STAT3.

Potential therapeutic significance of increased expression of aryl hydrocarbon receptor in human gastric cancer

AIM： To determine the functional significance of aryl hydrocarbon receptor （AhR） in gastric carcinogenesis, and to explore the possible role of AhR in gastric cancer （GC） treatment. METHODS： RT-PCR, real-time PCR, and Western blotting were performed to detect AhR expression in 39 GC tissues and five GC cell lines. AhR protein was detected by immunohistochemistry （IHC） in 290 samples： 30 chronic superficial gastritis （CSG）, 30 chronic atrophic gastritis （CAG）, 30 intestinal metapiasia （IN）, 30 atypical hyperplasia （AH）, and 70 GC. The AhR agonist tetrachlorodibenzo-para-dioxin （TCDD） was used to treat AGS cells. MTr assay and flow cytometric analysis were performed to measure the viability, cell cycle and apoptosis of AGS cells.RESULTS： AhR expression was significantly increased in GC tissues and GC cell lines. IHC results indicated that the levels of AhR expression gradually increased, with the lowest levels in CSG, followed by CAG, IM, AH and GC. AhR expression and nuclear translocation were significantly higher in GC than in precancerous tissues. TCDD inhibited proliferation of AGS cells via induction of growth arrest at the G1-S phase. CONCLUSION： AhR plays an important role in gastric carcinogenesis. AhR may be a potential therapeutic target for GC treatment.

Expression and role of aryl hydrocarbon receptor in Aspergillus fumigatus keratitis

●AIM:To observe the expression and role of aryl hydrocarbon receptor(Ah R)in the immune response of mouse cornea infected with Aspergillus fumigatus(A.fumigatus).●METHODS:Murine models of A.fumigatus keratitis were established by scraping the central epithelium of mouse cornea,daubing A.fumigatus on the cornea and covering with a contact lens.The mice were randomly divided into the control group and the A.fumigatus-infected(A.F.)group for 1,3 and 5 d respectively,which corneas were daily monitored by a slit lamp microscope and the clinical scores were also recorded timely after infection.In this study,immunofluorescence staining was used to detect the expression and localization of Ah R in mouse corneas,and the m RNA and protein of Ah R were detected by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.In addition,mouse peritoneal macrophages were stimulated by A.fumigatus with or without the pretreatment of Ah R antagonist CH223191 and Ah R agonist FICZ,and the tumor necrosis factor alpha(TNF-α),inducible nitric oxide synthase(i NOS),interleukin-10(IL-10)and Arg-1 m RNA were detected by RT-PCR.●RESULTS:According to the results of the slit light photography,it was clearly indicated that the corneal inflammation were the most severe and the clinical score became the highest as well on the 3 rd day after the infection of A.fumigatus.Contrasted with the control group,the expression of Ah R in the corneal epithelial cells infected with A.fumigatus was significantly increased detected by immunofluorescence staining.Ah R mainly expressed in the nucleus and cytoplasm of corneal epithelial cells.Consistent with the transcriptional level of Ah R m RNA,the expression level of Ah R protein reached the peak on the 3 rd day after infection which was detected by Western blot.Furthermore,RT-PCR showed that CH223191 up-regulated the expression of TNF-αand i NOS and down-regulated the expression of IL-10 and Arg-1 in peritoneal macrophages;inversely,FICZ reduced the expression of TNF-αand i NOS while elevated the expression of IL-10 and Arg-1.●CONCLUSION:Ah R is involved in the pathogenesis of A.fumigatus keratitis and induced immune protection in anti-A.fumigatus immune response by inhibiting M1 and increasing M2 phenotype macrophage-related inflammatory factors.

The oral commensal Streptococcus mitis activates the aryl hydrocarbon receptor in human oral epithelial cells

Streptococcus mitis （S. mitis） is a pioneer commensal bacterial species colonizing many of the surfaces of the oral cavity in healthy individuals. Yet, not much information is available regarding its interaction with the host. We used examination of its transcriptional regulation in oral keratinocytes to elucidate some of its potential roles in the oral cavity. Transcription factor analysis of oral keratinocytes predicted S. mitis.mediated activation of aryl hydrocarbon receptor （AhR）, Activation and functionality of AhR was confirmed through nuclear translocation determined by immunofluorescence microscopy and real-time polymerase chain reaction with reverse transcription analysis of CYPIA1, the hallmark gene for AhR activation. Addition of Streptococcus mutans or Streptococcus gordonfi did not induce CYPIA1 transcription in the keratinocyte cultures. Introduction of an AhR-specific inhibitor revealed that S. mitis-mediated transcription of CXCL2 and CXCL8 was regulated by AhR. Elevated levels of pmstaglandin E2 （enzyme-linked immunosorbent assay） in supernatants from S. mitis-treated oral epithelial cells were also attenuated by inhibition of AhR activity. The observed AhR-regulated activities point to a contribution of S. mitis in the regulation of inflammatory responses and thereby to wound healing in the oral cavity. The concept that the oral commensal microbiota can induce AhR activation is important, also in view of the role that AhR has in modulation of T-cell differentiation and as an anti-inflammatory factor in macrophaees.

Activation of Aryl Hydrocarbon Receptor Prolongs Survival of Fully Mismatched Cardiac Allograft

Recent data suggest that activation of aryl hydrocarbon receptor （AhR） by its high-affinity ligand 2,3,7,8-tetrachlorodihenzo-p-dioxin （TCDD） results in expansion of regulatory T （Treg） cells and suppresses the development of autoimmune and allergic diseases in several models. Treg cells have been increasingly documented to suppress allograft rejection and even to establish stable long-term graft acceptance. However, the involvement of TCDD in the regulation of solid organ transplantation rejec- tion is largely unknown. Here, we examined whether activation of AhR with TCDD altered cardiac al- lograft rejection in an allogeneic heart transplant model. Recipient C57BL/6 （H-2b） mice were adminis- trated with a single intraperitoneal injection of TCDD, and the murine cardiac transplant models from BALB/c （H-2d） to C57BL/6 （H-2b） were built 24 h later. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of TCDD on T-cell proliferation was assessed by mixed lymphocyte reaction （MLR）. Histological and immunohistochemical analyses were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry and enzyme-linked immunosorbent assay （ELISA）. Activation of AhR remarkably pro- longed the survival of cardiac allografts to more than 20 days. In vitro, TCDD ugregulated the fre- quency of CD4＋CD25＋Foxp3＋ Treg cells and suppressed the proliferation of T lymphocytes. In vivo, the prolonged survival time was associated with increased number of Treg cells in allografls and spleens Furthermore, the secretion of interferon-3, （IFN-3,） and interleukin-17 （IL-17） was reduced to less than 50% of that of the PBS treatment control group by TCDD treatment, whereas IL-10 was elevated to 10-fold of that of the PBS treatment control group. Collectively, our data indicate that activation of AhR with a single dose of TCDD significantly prolonged the survival of fully allogeneic cardiac grafts, and the mechanism underlying this effect might be involved in the induction of Treg cells.

补肾益精法调控AhR通路治疗硬皮病的机制:基于网络药理学及分子对接

目的通过网络药理学方法、分子对接方法分析芳香烃受体(AhR)信号通路在补肾益精法治疗硬皮病中的作用及其机制。方法通过TCMSP等数据库及LC-MS分析结果获取补肾益精法中药复方的有效活性成分及其靶点基因、AhR信号通路基因和硬皮病疾病基因,构建补肾益精法活性成分-AhR信号通路-硬皮病交集靶点网络图;使用STRING数据库构建蛋白互作网络;进行GO、KEGG富集分析;最后通过分子对接验证有效活性成分与AhR结合能力。结果阿魏酸、槲皮素、山奈酚、异阿魏酸、木犀草素在补肾益精法靶向AhR信号通路治疗硬皮病中发挥关键作用;其机制可能与调控参与硬皮病发病机制的IL-6、CTNNB1、HSP90AA1、TNF、PTGS2等AhR信号通路分子有关;分子对接显示阿魏酸、山奈酚、槲皮素、异阿魏酸、木犀草素等活性成分与AhR结合能均低于-6.0 kcal/mol,具有较好结合活性。结论补肾益精法治疗硬皮病的机制可能与阿魏酸、槲皮素、山奈酚、异阿魏酸、木犀草素等多种关键活性成分,靶向AhR信号通路,调控IL-6、CTNNB1、HSP90AA1、TNF、PTGS2等核心靶点相关。

Gene-environment interactions in male reproductive health： special reference to the aryl hydrocarbon receptor signaling pathway

Over the last few decades, there have been numerous reports of adverse effects on the reproductive health of wildlife and laboratory animals caused by exposure to endocrine disrupting chemicals （EDCs）. The increasing trends in human male reproductive disorders and the mounting evidence for causative environmental factors have therefore sparked growing interest in the health threat posed to humans by EDCs, which are substances in our food, environment and consumer items that interfere with hormone action, biosynthesis or metabolism, resulting in disrupted tissue homeostasis or reproductive function. The mechanisms of EDCs involve a wide array of actions and pathways. Examples include the estrogenic, androgenic, thyroid and retinoid pathways, in which the EDCs may act directly as agonists or antagonists, or indirectly via other nuclear receptors. Dioxins and dioxin-like EDCs exert their biological and toxicological actions through activation of the aryl hydrocarbon-receptor, which besides inducing transcription of detoxifying enzymes also regulates transcriptional activity of other nuclear receptors. There is increasing evidence that genetic predispositions may modify the susceptibility to adverse effects of toxic chemicals. In this review, potential consequences of hereditary predisposition and EDCs are discussed, with a special focus on the currently available publications on interactions between dioxin and androgen signaling.

The Aryl Hydrocarbon Receptor: A Target for Breast Cancer Therapy

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates a battery of genes in response to exposure to a broad class of environmental poly aromatic hydrocarbons (PAH). AhR is historically characterized for its role in mediating the toxicity and adaptive responses to these chemicals, however mounting evidence has established a role for it in ligand-independent physiological processes and pathological conditions, including cancer. The AhR is overexpressed and constitutively activated in advanced breast cancer cases and was shown to drive the progression of breast cancer. In this article we will review the current state of knowledge on the possible role of AhR in breast cancer and how it will be exploited in targeting AhR for breast cancer therapy.

Aryl Hydrocarbon Receptor is Involved in the Proinflammatory Cytokine Response to Cadmium

Objective To investigate involvement of the aryl hydrocarbon receptor(Ah R)in the immunomodulatory effects of cadmium(Cd).Methods The effect of Cd on Ah R activation(CYP1 A1 and CYP1 B1 m RNA expression)was examined in lung leukocytes of Cd-exposed rats(5 and 50 mg/L,30 d orally)and by in vitro leukocyte exposure.The involvement of Ah R signaling in the effects of Cd on the interleukin(IL)-1β,IL-6,and tumor necrosis factor(TNF)lung leukocyte response was investigated in vitro using the receptor antagonist CH-223191.Results Cd increased CYP1 B1(in vivo and in vitro)and CYP1 A1(in vitro)m RNA,indicating Ah R involvement in the action of Cd.In response to Cd,lung leukocytes increased IL-6 and decreased TNF at the gene expression and protein levels,but decreased IL-1βproduction due to reduced NLRP3.The Ah R antagonist CH-223191 abrogated the observed effects of Cd on the cytokine response.The absence of Ah R reactivity and cytokine response to Cd of leukocytes from the lungs of a rat strain that is less sensitive to Cd toxicity coincided with a high Ah R repressor m RNA level.Conclusion Ah R signaling is involved in the lung leukocyte proinflammatory cytokine response to Cd.The relevance of the Ah R to the cytokine response to Cd provides new insight into the mechanisms of Cd immunotoxicity.

Blocking the Aryl Hydrocarbon Receptor Alleviates Myocardial Ischemia/Reperfusion Injury in Rats

Objective Restoring the blood perfusion of ischemic heart tissues is the main treatment for myocardial ischemia.However,the accompanying myocardial ischemia reperfusion injury(IRI)would aggravate myocardial damage.Previous studies have confirmed that aryl hydrocarbon receptor(AhR)is closely correlated to kidney and intestinal IRI.The present study aimed to explore the relationship between AhR and myocardial IRI.Methods An oxygen glucose deprivation/reoxygenation(OGD/R)model of H9c2 cells and an ischemia/reperfusion(I/R)model of Sprague-Dawley rat myocardium were established.OGD/R cells and myocardial IRI rats were treated with different concentrations of the AhR antagonist CH-223191 or agonist 6-formylindolo[3,2-b]carbazole(FICZ).Under the conditions of normoxia and hypoxia/reoxygenation,the activity of cardiomyocytes,lactate dehydrogenase(LDH)and cell reactive oxygen species(ROS)were detected.In rats,myocardial pathological damage and markers of myocardial injury were detected.Results According to the results of the cell viability,LDH and ROS tests in vitro,both CH-223191 and FICZ showed no myocardial protection under OGD/R conditions.However,the histological staining and analysis of myocardial injury marker LDH in vitro revealed that CH-223191 could significantly reduce the myocardial IRI.Conclusion AhR exhibited a different effect on myocardial IRI in vitro and in vivo.In vivo,CH-223191 could significantly alleviate the myocardial IRI,suggesting that inhibition of AhR may play a role in myocardial protection,and AhR may serve as a potential treatment target for myocardial IRI.

Aryl hydrocarbon receptor as a new therapeutic target for cancer and immune disorders

The aryl hydrocarbon receptor （AhR） was discovered more than three decades ago, and initially was char-acterized as a transcription factor with a role in xe-nobiotic metabolism. However, based on subsequent observations that AhR remains active under physiologi-cal conditions, exhibits constitutive expression during development, and has a high degree of conservation among species, it was hypothesized that AhR is re-sponsible for functions in addition to its role in detoxif-cation. Correspondingly, recent studies have elucidated novel physiological roles for this ligand-dependent transcription factor that link it to several pathways associated with disease development. In this review, studies are presented that support a role for AhR in cell proliferation, apoptosis, and immune homeostasis, thereby highlighting the therapeutic potential of this receptor for cancer and immune disorders.

AhR对急性胰腺炎大鼠肠道微环境及屏障功能的影响

目的探讨芳香烃受体(aryl hydrocarbon receptor,AhR)对急性胰腺炎(acute pancreatitis,AP)大鼠肠道微环境、屏障功能及核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf-2)通路的影响。方法通过胆胰管注射5%牛磺胆酸钠的方法建立AP大鼠模型,将50只大鼠随机分组:AP组、NC组(尾部注射5μl慢病毒空白载体)、AhR过表达组(尾部注射5μl过表达AhR慢病毒载体)、AhR抑制剂组(腹腔注射10μg 2-甲基-2H-吡唑-3-羧酸)、AhR过表达+ML385组(在AhR过表达组基础上,腹腔注射20 mg/kg ML385),另取10只大鼠为假手术组。心脏采血,检测血清中脂肪酶、淀粉酶水平以及SOD、MDA、CRP水平;分离胰腺组织,观察胰腺组织损伤程度及AhR、Nrf-2、HO-1蛋白表达水平;分离回肠组织,观察肠黏膜损伤情况并进行评分。结果与假手术组相比,模型组大鼠回肠、胰腺病理评分、CRP、MDA含量、脂肪酶、淀粉酶水平均升高,SOD含量、Nrf-2、HO-1、AhR表达显著降低(P<0.05);与模型组、NC组相比,AhR过表达组大鼠回肠、胰腺病理评分、CRP、MDA含量、脂肪酶、淀粉酶水平均降低,SOD含量、Nrf-2、HO-1、AhR蛋白表达水平显著增加(P<0.05);与模型组相比,AhR抑制剂组大鼠回肠、胰腺病理评分、CRP、MDA含量、脂肪酶、淀粉酶水平均升高,SOD含量、Nrf-2、HO-1、AhR蛋白表达水平显著降低(P<0.05);ML385逆转了AhR过表达对AP大鼠的保护作用(P<0.05)。结论增强AhR表达可以改善AP大鼠肠道微环境,恢复肠道功能,缓解大鼠胰腺和肠黏膜损伤,可能与激活Nrf-2通路有关。

AhR、25-OHD和DOCK8在小儿特应性皮炎中的表达及相关性研究

目的分析芳香烃受体(AhR)、25-羟维生素D(25-OHD)和胞质分裂蛋白8(DOCK8)在小儿特应性皮炎(AD)中的表达及相关性。方法选取2019年8月—2020年8月本院收治的92例AD患儿作为AD组,80例系统性红斑狼疮(SLE)患儿作为SLE组,以同期在本院健康体检的90例健康儿童作为健康对照组。测定3组研究对象AhR、25-OHD和DOCK8的mRNA表达情况。采用Logistic回归分析影响AD发生的影响因素,采用Pearson相关性分析法分析AhR、25-OHD和DOCK8的相关性,受试者工作特征曲线(ROC曲线)分析影响因素对AD发生的预测价值。结果与健康对照组比较,SLE组及AD组AhR、DOCK8的mRNA表达升高,25-OHD的mRNA表达降低,差异有统计学意义(P<0.05);与SLE组比较,AD组AhR、DOCK8的mRNA表达升高,25-OHD的mRNA表达降低,差异有统计学意义(P<0.05)。随着疾病进展,AD患儿AhR、DOCK8的mRNA表达升高,25-OHD的mRNA表达降低。与轻度AD患儿比较,中度、重度AD患儿AhR、DOCK8的mRNA表达升高,25-OHD的mRNA表达降低(P<0.05)。与中度比较,重度AD患儿AhR、DOCK8的mRNA表达升高,25-OHD的mRNA表达降低(P<0.05)。以小儿AD作为因变量(否=1,是=0),以AhR、25-OHD、DOCK8作为自变量纳入方程,建立Logistic回归模型,结果显示,高AhR[优势比(OR)=13.283,95%可信区间(95%CI):4.371~40.365]和高DOCK8(OR=3402.792,95%CI:100.332~115406.258)为小儿AD发生的独立危险因素,高25-OHD(OR=0.679,95%CI:0.561~0.822)为小儿AD发生的保护因素(均P<0.05)。Pearson相关性分析结果显示,AhR与25-OHD之间呈负相关(r=-0.230,P=0.003);AhR与DOCK8之间呈正相关(r=0.410,P=0.001);25-OHD与DOCK8之间呈负相关(r=-0.455,P=0.001)。与AhR(AUC=0.897)、25-OHD(AUC=0.766)和DOCK8(AUC=0.918)单项预测比较,3项联合(AUC=0.975,当截断值为0.431时,其敏感度为98.24%,特异度为89.13%)预测小儿AD发生的价值较高(P<0.05)。结论AhR在小儿AD中表达升高,DOCK8、25-OHD在小儿AD中表达降低,且与患儿疾病严重程度相关,三者线性相关,临床可根据其表达高低早期诊断此病。

Aryl hydrocarbon receptor:Linking environment to aging process in elderly patients with asthma

Aging is a significant risk factor for various diseases,including asthma,and it often leads to poorer clinical outcomes,particularly in elderly individuals.It is recognized that age-related diseases are due to a time-dependent accumulation of cellular damage,resulting in a progressive decline in cellular and physiological functions and an increased susceptibility to chronic diseases.The effects of aging affect not only the elderly but also those of younger ages,posing significant challenges to global healthcare.Thus,understanding the molecular mechanisms associated with aging in different diseases is essential.One intriguing factor is the aryl hydrocarbon receptor(AhR),which serves as a cytoplasmic receptor and ligand-activated transcription factor and has been linked to the aging process.Here,we review the literature on several major hallmarks of aging,including mitochondrial dysfunction,cellular senescence,autophagy,mitophagy,epigenetic alterations,and microbiome disturbances.Moreover,we provide an overview of the impact of AhR on these hallmarks by mediating responses to environmental exposures,particularly in relation to the immune system.Furthermore,we explore how aging hallmarks affect clinical characteristics,inflammatory features,exacerbations,and the treatment of asthma.It is suggested that AhR signaling may potentially play a role in regulating asthma phenotypes in elderly populations as part of the aging process.

Predicting Chemical Immunotoxicity through Data-Driven QSAR Modeling of Aryl Hydrocarbon Receptor Agonism and Related Toxicity Mechanisms

Computational modeling has emerged as a time-saving and cost-effective alternative to traditional animal testing for assessing chemicals for their potential hazards.However,few computational modeling studies for immunotoxicity were reported,with few models available for predicting toxicants due to the lack of training data and the complex mechanisms of immunotoxicity.In this study,we employed a data-driven quantitative structure–activity relationship(QSAR)modeling workflow to extensively enlarge the limited training data by revealing multiple targets involved in immunotoxicity.To this end,a probe data set of 6,341 chemicals was obtained from a high-throughput screening(HTS)assay testing for the activation of the aryl hydrocarbon receptor(AhR)signaling pathway,a key event leading to immunotoxicity.Searching this probe data set against PubChem yielded 3,183 assays with testing results for varying proportions of these 6,341 compounds.100 assays were selected to develop QSAR models based on their correlations to AhR agonism.Twelve individual QSAR models were built for each assay using combinations of four machine-learning algorithms and three molecular fingerprints.5-fold cross-validation of the resulting models showed good predictivity(average CCR=0.73).A total of 20 assays were further selected based on QSAR model performance,and their resulting QSAR models showed good predictivity of potential immunotoxicants from external chemicals.This study provides a computational modeling strategy that can utilize large public toxicity data sets for modeling immunotoxicity and other toxicity endpoints,which have limited training data and complicated toxicity mechanisms.

Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tetrachlorodibenzo-p dioxin (TCDD) to generate TCDD:AhR:DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

基于NF-κB/AHR通路探讨电针“足三里”和“内关”对肠易激综合征大鼠内脏高敏感的调节机制

目的 观察电针“足三里”和“内关”穴对肠易激综合征(irritable bowel syndrome, IBS)大鼠内脏高敏感的调节机制。方法 将40只新生SD大鼠随机分为模型制备组(n=32)和空白组(n=8),采用“母婴分离结合醋酸灌肠”复制内脏高敏感大鼠模型,将模型复制成功的内脏高敏感大鼠随机分为模型组、假刺激组、电针组,每组8只。假刺激组大鼠仅电刺激非经非穴部位,空白组及模型组大鼠仅抓取,不做其他干预;电针组大鼠给予电针同侧足三里和内关穴,每次30 min,隔日1次,共4周。观察各组大鼠一般情况,体质量,粪便性状评分,内脏痛阈值,血清白细胞介素-17A(interleukin-17A,IL-17A)水平,结肠组织中芳香烃受体(aryl hydrocarbon receptor, AHR)、细胞色素P1(cytochrome P1,CYP1)、核因子-κB(nuclear factor-κB,NF-κB) P65蛋白表达水平。结果 与空白组比较,模型组大鼠粪便性状评分显著增加(P<0.05),体质量显著下降(P<0.05),内脏痛阈值显著降低(P<0.05),血清IL-17A水平显著升高(P<0.05),结肠组织AhR、CYP1、NF-κB P65蛋白表达水平均显著降低(P<0.05);与模型组比较,电针组大鼠粪便性状评分显著减少(P<0.05),体质量显著增加(P<0.05),内脏痛阈值显著增加(P<0.05),血清IL-17A水平显著降低(P<0.05),结肠组织AHR、CYP1、NF-κB P65蛋白表达水平显著上升(P<0.05)。结论 电针“足三里”和“内关”穴能降低IBS大鼠的内脏高敏感,其作用机制可能通过激活NF-κB/AHR通路,上调AHR、CYP1、NF-κB P65蛋白的表达而实现的。

BaP通过AhR和Nrf2信号通路对HepG2细胞中GSTP1的影响

目的探究苯并芘(benzo[a]pyrene,BaP)通过芳香烃受体(aryl hydrocarbon receptor,AhR)和核因子E2相关因子(subcellular localization of nuclear factor E2-related factor 2,Nrf2)信号通路对HepG2细胞中谷胱甘肽-S-转移酶P1(glutathione s-transferase P1,GSTP1)的影响。方法将体外培养HepG2细胞分为Control组、BaP(10μmol/L)组、BaP(10μmol/L)+AhR抑制剂(1μmol/L)组和BaP(10μmol/L)+Nrf2抑制剂(1μmol/L)组。BaP组给予10μmol/L BaP培养24 h,抑制剂组给予1μmol/L抑制剂0.5 h后,加入10μmol/L BaP培养24 h,采用Western Blot和qPCR实验方法检测各组AhR、Nrf2、细胞色素P450s(cytochrome P450s,CYPs)、血红素加氧酶-1(heme oxygenase-1,HO-1)及GSTP1基因和蛋白的表达。结果与Control组相比,BaP组中AhR、Nrf2、GSTP1、CYP1A1及HO-1的mRNA和蛋白表达均升高(P<0.05);与BaP组相比,BaP+AhR抑制剂组中AhR、GSTP1和CYP1A1的mRNA和蛋白表达均降低(P<0.05),BaP+Nrf2抑制剂组中Nrf2、GSTP1和HO-1的表达均降低(P<0.05);与BaP+AhR抑制剂组相比,BaP+Nrf2抑制剂组GSTP1 mRNA和蛋白的表达降低,差异有统计学意义(P<0.05)。结论BaP通过AhR和Nrf2两条信号通路增加GSTP1的表达,以Nrf2信号通路为主导影响GSTP1的表达。