Role of aryl hydrocarbon receptor in down-regulation of Clara cell secretory protein expression during endotoxin-induced lung injury in rats / 中华麻醉学杂志

Objective:To evaluate the role of aryl hydrocarbon receptor (AhR) in the down-regulation of Clara cell secretory protein (CCSP) expression during endotoxin-induced lung injury in rats.Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 200-250 g, were divided into 4 groups ( n=6 each) using a random number table method: normal control group (group C), acute lung injury (ALI) group, ALI+ AhR antagonist group, and ALI+ vehicle group. Lipopolysaccharide(LPS) 1 mg/kg was intratracheally instilled to develop the model of lung injury, while the equal volume of normal saline was given instead in group C. At 2 h before LPS injection, AhR antagonist 6, 2′, 4′-trimethoxyflavone solution 5 mg/kg (diluted to 1 ml in dimethyl sulfoxide solution) was intraperitoneally injected in ALI+ AhR antagonist group, while dimethyl sulfoxide solution 1 ml was given in ALI+ vehicle group. The rats were sacrificed under anesthesia at 48 h after LPS administration. The left lung was lavaged and the broncho-alveolar lavage fluid (BALF) was collected for determination of the concentrations of CCSP by enzyme-linked immunosorbent assay, and the expression of CCSP in the bronchial epithelium in right lung tissues was determined by immunohistochemistry. Results:Compared with group C, the expression of CCSP in the bronchial epithelium was significantly down-regulated, and the concentrations of CCSP in BALF were decreased in the other three groups ( P<0.05 or 0.01). Compared with ALI group and ALI+ vehicle group, the histopathological injury was significantly reduced, the expression of CCSP in the bronchial epithelium was up-regulated, and the concentrations of CCSP in BALF were increased in ALI+ AhR antagonist group ( P<0.01). Conclusions:AhR partially mediates the down-regulation of CCSP expression during endotoxin-induced lung injury in rats.

The mechanism of microRNA-124-3p in regulating proliferation and invasion of gastric cancer cell by aryl hydrocarbon receptor-activated β-catenin pathway / 中华消化杂志

Objective:To explore the value of microRNA(miR)-124-3p and its target gene aryl hydrocarbon receptor (AHR) in the diagnosis and prognostic evaluation of gastric cancer, and the related molecular mechanisms in regulating proliferation and invasion of gastric cancer cell.Methods:The clinical and prognostic characteristics of patients with gastric adenocarcinoma expressing miR-124-3p were obtained from The Cancer Genome Atlas database and Genotype-Tissue Expression database. The correlation between miR-124-3p expression level and pathological stage, TNM stage, overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) in patients with gastric adenocarcinoma were studied by bioinformatics analysis. The interaction sites between miR-124-3p and AHR mRNA were predicted by Target Scan 7.1 online tool. The target binding sites of miR-124-3p in AHR mRNA were verified by subcutaneous tumorigenesis experiment in mice, immunohistochemistry, dual luciferase assay, quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Nine male Balb/c nude mice, aged 4 to 6 weeks with weight of (18.43±0.29) g were injected with miR-124-3p simulant (miR-124-3p group), negative control simulant (negative control group) and 0.9% sodium chloride solution (sodium chloride control group) through the tail vein. Gastric cancer cell lines (MKN-45, AGS) were transfected with RNA simulants (including miR-124-3p simulant, negative control simulant and 0.9% sodium chloride solution). The expression of AHR and Catenin β 1 gene ( CTNNB1) at mRNA level, the expression of AHR and β-catenin at protein level in 3 mice groups and the effects of miR-124-3p transfection on the proliferation and invasion of transfected gastric cancer cells were analyzed. Pearson correlation analysis and Holm-Sidak corrected multiple t test were used for statistical analysis. Results:Low expression of miR-124-3p was positively correlated with severe pathological stages and TNM stages in patients with gastric adenocarcinoma ( R2=0.83 and 0.86, P=0.031 and 0.023). High expression of miR-124-3p was positively correlated with OS, DSS and PFI ( R2=1.00, 0.99 and 0.99, P=0.029, 0.044 and 0.049). The results of subcutaneous tumorigenesis experiment in mice demonstrated that the number of apoptotic cells in the tumor of miR-124-3p group was more than that of negative control group and sodium chloride control group ((43.33±1.86)/high power field (HPF) vs. (20.00±1.73)/HPF and (18.67±1.76)/HPF), and the differences were statistically significant ( t=8.55 and 8.33, P=0.013 and 0.014). The results of immunohistochemistry showed that the optical density of AHR protein in mice tumor tissue of miR-124-3p group was lower than that of negative control group and sodium chloride control group (0.081±0.008 vs. 0.276±0.019 and 0.273±0.018), and the differences were statistically significant ( t=9.06 and 7.51, P=0.012 and 0.017). The results of dual luciferase assay indicated that the fluorescence intensity in wild-type AHR MKN-45 cells transfected with miR-124-3p simulant was lower than that of negative control group (0.293±0.020 vs. 1.000±0.032), and the difference was statistically significant ( t=18.56, P<0.001). The results of RT-qPCR demonstrated that the mRNA levels of AHR and CTNNB1 in MKN-45 cells transfected with miR-124-3p simulant were both lower than those in untreated MKN-45 cells (0.51±0.09 vs. 1.02±0.02, 0.46±0.03 vs. 1.03±0.01), and the differences were statistically significant ( t=4.51 and 16.60, P=0.046 and 0.004). The results of Western blotting experiments showed that the relative protein expression levels of AHR and β-catenin of MKN-45 cells transfected with miR-124-3p simulant were lower than those of transfected with 0.9% sodium chloride solution and negative control simulant (3 332.94±81.25 vs. 9 041.60±439.79 and 8 276.54±562.52, 2 725.79±167.57 vs. 9 701.94±410.02 and 8 081.66±275.84), and the differences were statistically significant ( t=15.49, 7.91, 17.35 and 19.42, P=0.004, 0.016, 0.003 and 0.003). Conclusions:MiR-124-3p is correlated with diagnosis and prognosis of gastric cancer. MiR-124-3p induces apoptosis of gastric cancer cells in vitro and vivo by negatively regulating AHR expression at mRNA and protein level, thereby down-regulating the expression of CTNNB1 mRNA and β-catenin pathway-related protein. Therefore, miR-124-3p may become a potential diagnostic and prognostic marker of gastric cancer.

Role of AhR and ARNT in recurrent spontaneous abortion / 中国医师杂志

Objective To explore the relationship between aryl hydrocar-bon receptor (AhR),aryl hydrocarbon receptor nuclear translocator (ARNT),estradiol (E2),estrogen receptor (ER) and recurrent spontaneous abortion (RSA) through observing the level of serum AhR,ARNT,E2,and AhR,ARNT,ER in decidua and chorionic tissues of the patients with recurrent spontaneous abortion.Methods 64 cases of RSA patients who induced abortion at the Shanxi Dayi hospital from May 2015 to September2017 were chosen as RSA group,and 30 cases of healthy abortion women of the same period who had born full-term normal fetus were choosen as the normal group.The serum,villi and decidua of each case were collected during abortion.The level of AhR,ARNT,ER of both groupswere detected by enzyme-linked immunoscrbent assay (ELISA) method.The Serum E2 lever were detected by the method of chemical luminescence.The data of all the patients were analyzed.Results (1) The level of ARNT in peripheral blood of RSA group was significantly higher than that of normal group (P ＜ 0.05),and the levels of AhR and E2 in peripheral blood were not statistically significant between the two groups (P ＞ 0.05).The levels of AhR and ARNT in villi and decidua were significantly higher in RSA group than in normal group (P ＜ 0.05),while the expression of ER in villi and decidua was significantly lower in RSA group than in normal group (P ＜ 0.05).(2) There was no significant difference in the ratio of AhR/ARNT between the two groups (P ＞ 0.05);the ratio of AhR/ER and ARNT/ER in the villi and decidua of the RSA group was higher than that of the normal group (P ＜ 0.05).Conclusions Overexpression of AhR and ARNT and low expression of ER in villi and decidua tissues may be related to the occurrence of RSA.

Advance of the role of Aromatic hydrocarbon receptor in immunoregulation related to tryptophan metabolism / 国际外科学杂志

Aromatic hydrocarbon receptor (AhR),an intracellular receptor,contains multiple ligand binding sites.Various ligands of AhR are divided into exogenous and endogenous ligands according to the origination.Different ligands bind and activate AhR,regulating the transcription of downstream target genes,especially CYP1 from Cypcytochrome P450 gene family,which plays an essential role in different pathological problems,as well as in the normal development and function of organism.Kynurenine(Kyn),a key metabolic product of tryptophan (Trp),metabolic products of which are major types of endogenous ligands of AhR,has an impact on adaptive immune by manipulating the polarization and activity of immunocytes.Kyn has been focus for its supressive effect in anti-tumor immunity and raised concern recently for its intriguing role in allograft immunoregulation.Research advances in the role of AhR in immunoregulation related to tryptophan metabolism will be illustrated in this review in detail.

Aryl hydrocarbon receptor overexpression in miniaturized follicles in female pattern hair loss

Abstract The etiopathogenesis of female pattern hair loss is still poorly understood. In addition to genetic and hormonal elements, environmental factors could be involved. The aryl hydrocarbon receptor is expressed in keratinocytes and can be activated by environmental pollutants leading to alterations in the cell cycle, inflammation, and apoptosis. Here we demonstrate the overexpression of nuclear aryl hydrocarbon receptors in miniaturized hair follicles in female pattern hair loss.

Regulation of CYP1A1 and Inflammatory Cytokine by NCOA7 Isoform 4 in Response to Dioxin Induced Airway Inflammation / 결핵및호흡기질환

BACKGROUND: Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, binds to a wide variety of synthetic and naturally occurring compounds. AhR is involved in the regulation of inflammatory response during acute and chronic respiratory diseases. We investigated whether nuclear receptor coactivator 7 (NCOA7) could regulate transcriptional levels of AhR target genes and inflammatory cytokines in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated human bronchial epithelial cells. This study was based on our previous study that NCOA7 was differentially expressed between normal and chronic obstructive pulmonary disease lung tissues. METHODS: BEAS-2B and A549 cells grown under serum-free conditions were treated with or without TCDD (0.15 nM and 6.5 nM) for 24 hours after transfection of pCMV-NCOA7 isoform 4. Expression levels of cytochrome P4501A1 (CYP1A1), IL-6, and IL-8 were measured by quantitative real-time polymerase chain reaction. RESULTS: The transcriptional activities of CYP1A1 and inflammatory cytokines were strongly induced by TCDD treatment in both BEAS-2B and A549 cell lines. The NCOA7 isoform 4 oppositely regulated the transcriptional activities of CYP1A1 and inflammatory cytokines between BEAS-2B and A549 cell lines. CONCLUSION: Our results suggest that NCOA7 could act as a regulator in the TCDD-AhR signaling pathway with dual roles in normal and abnormal physiological conditions.

Dynamic expression of interleukin-22 in grafts after allograft corneal transplantation and its relationship with graft rejection in rats / 中华实验眼科杂志

Background The rejection following keratoplasty still is a leading cause of corneal transplantation failure.Studies showed that the interleukin-22 (IL-22) ,one of the effector molecules of T helper cell 17 (Th17) participated on the rejection after heart,liver and bone marrow transplantation.However,the effect of IL-22 on corneal graft rejection is not well understood.Objective This study was to investigate the expression of IL-22 mRNA in the corneal grafts and the role of IL-22 in the immune rejection after corneal transplantation in rats.Methods Seventy-two Wistar rats were randomized into autologous keratoplasty group,allograft keratoplasty group and anti-rejection group,and other 4 normal Wistar rats served as normal control group.Autologous keratoplasty was operated on the Wistar rats of the autologous keratoplasty group,and allograft keratoplasty were carried out with the 24 SD rats as donors and 48 Wistar rats as recipients.Tobramycin and dexamethasone eye drops were topically administrated after autologous keratoplasty for 2 weeks in the anti-rejection group.The experimental eyes were examined by slit lamp microscope after surgery and graft survival was evaluated based on the rejection scoring criteria of Larkin.Intergroup accumulated survival rates of grafts were compared using Kaplan-Meier analysis.Histopathological examination of grafts was carried out in 5 and 14 days after operation respectively,and the related expression levels of IL-22 mRNA and aryl hydrocar-bon receptor (AhR) mRNA were carried out by real-time fluorescence quantitative PCR.The feeding and use of the experimental animals followed the Guangdong provincial regulations on the management of experimental animals.The experimental design was approved by the ethics committee of Southern Medical University.Results The median survival time of grafts in the allograft keratoplasty group was 10 days,and that in the anti-rejection group was 17 days,showing a significant survival extention in the anti-rejection group (x2=16.442,P =0.000).Significant differences were found among the 4 groups in the related expression levels of IL-22 mRNA in both 5 days and 14 days after surgery (postoperative 5 days : F=2.44,P =0.00;postoperative 14 days: F=267.92, P =0.00), and the related expression levels of IL-22 mRNA were remarkably higher in the allograft keratoplasty group than those in the anti-rejection group at different time points (postoperative 5 days :9.70±0.35 vs.0.46±0.21;postoperative 14 days : 23.12 ± 1.89 vs.3.14±0.94) (both at P＜0.05).The related expression levels of AhR mRNA in the grafts were considerably different among the 4 groups (postoperative 5 days : F =395.73, P =0.00;postoperative 14 days : F =942.37, P =0.00) , and the expression levels were significantly elevated in the allograft keratoplasty group compared with the anti-rejection group at various time points (postoperative 5 days:2.52±0.32 vs.1.89±0.10;postoperative 14 days:7.20±0.25 vs.2.60±0.17) (both at P＜0.05).Conclusions The expression level of IL-22 RNA up-regulates in the grafts with immuno-rejection.Topical administration of tobramycin and dexamethasone eye drops inhibits the rejection after keratoplasty.AhR plays a regulative role to the expression of IL-22 in rats after keratoplasty.

Relationship of polymorphisms of AhR - 1661G/A with GSTP1 - 313A/G and susceptibility to endometriosis / 中华妇产科杂志

Objective To investigate the association of polymorphisms of arylhydrocarbon receptor (AhR) - 1661G/A with glutathione S-transferase pi ( GSTP1 ) - 313A/G and the susceptibility to endometriosis in southern Han Chinese.Methods Total of 432 endometriosis patients undergoing laparoscopic or laparotomy surgery matched with 493 patients with fallopian tube ligation,tubal recanalization,laparoscopic hydrotubation,benign ovarian tumor and teratoma surgeries without endometriosis as control group were enrolled in this study.The single nucleotide polymorphism (SNP) of AhR -1661G/A and GSTP1 -313A/G were detected by using a fluorescent quantitative PCR-based high resolution melting (HRM).Results The numbers of combined genotypes AhR - 1661G/A and GSTP1 -313A/G were 120 patients with AG + AA,64 patients with AG + AG,8 patients with AG + GG,109 patients with GG +AA,84 patients with GG + AG,4 patients with GG + GG,31 patients with AA + AA,10 patients with AA + AG,1 patient with AA + GG at endometriosis group and 131 patients with AG + AA,68 patients with AG + AG,6 patients with AG + GG,157 patients with GG + AA,66 patients with GG + AG,4 patients with GG + GG,35 patients with AA + AA,20 patients with AA + AG,3 patients with AA + GG at endometriosis group.There was no statistically different frequencies of genotypes between endometriosis group and control group (x2 = 12.558,P = 0.128 ).Compared with genotype GG + AA,the risk of endometriosis with genotype GG + AG was increased 1.833 time (95％CI:1.233-2.274).Conclusion The combined genotype GG + AG [ from AhR - 1661G/A (GG) and GSTP1 - 313A/G (AG) ] might be related with susceptibility to endometriosis.

Signaling pathway for 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-Naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.