CYP1A1 Protein Research Articles

Effects of clarithromycin on the pharmacokinetics of tacrolimus and expression of CYP3A4 and P-glycoprotein in rats.

The objective of this study was to investigate the effect of clarithromycin on the pharmacokinetics of tacrolimus in rats and better understand its mechanism. In the control group (n = 6), rats received a single oral dose of 1mg tacrolimus on day 6. In the experimental group (n = 6), rats received 0.25 g of clarithromycin daily for five consecutive days and then a single oral dose of 1mg tacrolimus on day 6. Orbital venous blood (250 μL) was collected at 0, 0.25, 0.50, 0.75, 1, 2, 4, 8, 12, and 24 h before and after tacrolimus administration. Blood drug concentrations were detected via mass spectrometry. Small intestine and liver tissue samples were collected after rats were euthanized via dislocation, and CYP3A4 and P-glycoprotein (P-gp) protein expression was determined using western blotting. Clarithromycin increased the blood tacrolimus concentration and affected its pharmacokinetic properties in rats. Compared with those in the control group, the AUC0-24 , AUC0-∞ , AUMC(0-t) , and AUMC(0-∞) of tacrolimus in the experimental group were significantly increased, whereas the CLz/F was significantly lower (P < 0.01). Simultaneously, clarithromycin significantly inhibited CYP3A4 and P-gp expression in the liver and intestine. Protein expression of CYP3A4 and P-gp in the liver and the intestinal tract was significantly downregulated in the intervention group compared with that in the control group. Clarithromycin significantly inhibited the protein expression of CYP3A4 and P-gp in the liver and intestine, thereby increasing the mean blood concentration and significantly increasing the AUC of tacrolimus.

Comparison of moderate-intensity continuous training and high-intensity interval training effects on the Ido1-KYN-Ahr axis in the heart tissue of rats with occlusion of the left anterior descending artery

Myocardial infarction (MI) affects many molecular pathways in heart cells, including the Ido1-KYN-Ahr axis. This pathway has recently been introduced as a valuable therapeutic target in infarction. We examined the effects of moderate-intensity continuous training (MICT) and high-intensity interval training (HIIT) on the axis in the heart tissue of male Wistar rats with occluded left anterior descending (OLAD). Thirty rats (age 10–12 weeks, mean weight 275 ± 25 g) were divided into five groups with 6 animals: Control (Ct) group, MICT group, rats with OLAD as MI group, rats with OLAD treated with MICT (MIMCT group) and rats with OLAD treated with HIIT (MIHIIT group). Rats performed the training protocols for 8 weeks, 5 days a week. HIIT included 7 sets of 4 min running with an intensity of 85–90% VO2max and 3 min of recovery activation between sets. MICT included continuous running at the same distance as HIIT with an intensity of 50–60% VO2max for 50 min. The expressions of Ahr, Cyp1a1, and Ido1 were assayed by real-time PCR. Malondialdehyde (MDA) and Kynurenine levels, and AHR, CYP1A1, and IDO1 proteins were detected using ELISA. Data were analyzed using the ANOVA and MANOVA tests. Compared to the CT group, MI caused an increase in all studied factors, but only statistically significant (P < 0.05) for MDA and IDO1. With a greater effect of HIIT, both protocols significantly lowered the proteins expressions in the MIHIIT and MIMCT groups, compared with the MI group (P < 0.001). In healthy rats, only AHR protein significantly decreased in the MICT group compared to the Ct group (P < 0.05). HIIT and MICT protocols significantly reduced the gene and protein expression of Cyp1a1 (P < 0.05) and Ido1 (P < 0.01), and HIIT had a greater effect. In conclusion, both protocols were effective at reducing the levels of Ido1-Kyn-Ahr axis components and oxidative stress in the infarcted heart tissue and HIIT had a higher significant effect.

Liver microsomal cytochrome P450 3A-dependent drug oxidation activities in individual dogs

Drug oxidations are mediated mainly by cytochromes P450 (P450s or CYPs). CYP3As are an important P450 subfamily and include liver-specific CYP3A12 and intestine-specific CYP3A98 in dogs. Individual differences in drug oxidation activities were investigated, including correlations with immunoreactive CYP3A protein intensities and CYP3A mRNA expression levels in livers. Pooled and individual dog liver microsomes showed activities towards nifedipine, midazolam, alprazolam, and estradiol, but the levels of catalytic activities varied approximately twofold among the individual dogs. One dog harboured a CYP1A2 variant causing protein deletion but showed higher activities than the other dogs towards nifedipine oxidation, midazolam 1′-hydroxylation, alprazolam 4-hydroxylation, estradiol 16α-hydroxylation activities, and caffeine C 8-hydroxylation; the latter is used as a reference reaction for CYP1A. In individual dog liver microsomes, the intensities of the immunochemical bands with anti-human CYP3A4 and anti-rat CYP3A2 antibodies along with CYP3A12 and CYP3A26 mRNA expression levels showed good correlations (p < 0.05) with nifedipine oxidation, midazolam 1′- and 4-hydroxylation, alprazolam 1′- and 4-hydroxylation, and estradiol 16α-hydroxylation activities. These results suggest that the oxidation activities of dog liver microsomes towards nifedipine and other typical CYP3A-catalyzed drugs exhibit approximately twofold individual differences and were predominantly mediated by liver-specific CYP3A12 in the dogs.

Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes

Novel cytochrome P450 3A5 (CYP3A5) cDNA in tree shrews (which are non-rodent primate-like species) and pig CYP3A227 cDNA were identified, along with known pig CYP3A22, CYP3A29, and CYP3A46 cDNAs. All five cDNAs contained open reading frames encoding a polypeptide of 503 amino acids that shared high sequence identity (72–78 %) with human CYP3A4 and were more closely related to human CYP3As than rat CYP3As by phylogenetic analysis. CYP3A5 was the only CYP3A in the tree shrew genome, but pig CYP3A genes formed a CYP3A gene cluster in the genomic region corresponding to that of human CYP3A genes. Tree shrew CYP3A5 mRNA was predominantly expressed in liver and small intestine, among the tissues analyzed, whereas pig CYP3A227 mRNA was most abundantly expressed in jejunum, followed by liver. Metabolic assays established that tree shrew CYP3A5 and pig CYP3A proteins heterologously expressed in Escherichia coli metabolized typical human CYP3A4 substrates nifedipine and midazolam. These results suggest that novel tree shrew CYP3A5 and pig CYP3A227 were functional enzymes able to metabolize human CYP3A4 substrates in liver and small intestine, similar to human CYP3A4, although pig CYP3A227 mRNA was minimally expressed in all tissues analyzed.

A multi-targeted investigation of Deepwater Horizon crude oil exposure impacts on the marine teleost stress axis

The toxicity of the polycyclic aromatic hydrocarbons (PAHs) in Deepwater Horizon (DWH) oil is well-established, but a knowledge gap exists regarding how this combination of PAHs affects the vertebrate stress axis. We hypothesized that (1) marine vertebrates exposed to DWH PAHs experience stress axis impairment, and co-exposure to an additional chronic stressor may exacerbate these effects, (2) serotonin (5-hydroxytryptamine; 5-HT) may act as a secondary cortisol secretagogue in DWH PAH-exposed fish to compensate for impairment, and (3) the mechanism of stress axis impairment may involve downregulation of cyclic adenosine monophosphate (cAMP; as proxy for melanocortin 2 receptor (MC2R) functionality), total cholesterol, and/or mRNA expression of CYP1A and steroidogenic proteins StAR, P450scc, and 11β-h at the level of the kidney. We found that in vivo plasma cortisol and plasma adrenocorticotropic hormone (ACTH) concentrations in Gulf toadfish exposed to an environmentally relevant DWH PAH concentration (ΣPAH50= 4.6 ± 1.6 μg/L) for 7 days were not significantly different from controls, whether fish were chronically stressed or not. However, the rate of cortisol secretion by isolated kidneys after acute stimulation with ACTH was significantly lower in PAH-exposed toadfish compared to clean seawater (SW) controls. 5-HT does not appear to be acting as a secondary cortisol secretagogue, rather, PAH-exposed + stressed toadfish exhibited significantly lower plasma 5-HT concentrations than clean SW + stressed fish as well as a reduced sensitivity to 5-HT at the level of the kidney. There was a tendency for kidney cAMP concentrations to be lower in PAH-exposed fish (p = 0.069); however, mRNA expression of steroidogenic proteins between control and PAH-exposed toadfish were not significantly different and a significant elevation in total cholesterol concentration in PAH-exposed toadfish compared to controls was measured. Future work is needed to establish whether the slower cortisol secretion rate by isolated kidneys of PAH-exposed fish is detrimental, to determine the potential role of other secretagogues in compensating for the impaired kidney interrenal cell function, and to determine whether there is a reduction in MC2R mRNA expression or an impairment in the function of steroidogenic proteins.

15,16-dihydrotanshinone I in Danshen ethanol extract aggravated cholestasis by inhibiting Cyp3a11 mediated bile acids hydroxylation

Our previous study found that high-dose Tanshinones Capsule (TC) aggravated cholestasis in mice. To explore its underlying mechanism, main tanshinones components (15,16-dihydrotanshinone I (DTI), cryptotanshinone (CTS) and tanshinone IIA (TSA)) form TC were studied separately. Bile acids (BAs) that were primarily metabolized by hydroxylation were identified, and then the inhibitory effect of each tanshinones on their hydroxylation were evaluated. The anti-cholestasis effect of each tanshinones were studied in mice, the hepatic concentrations of BAs and tanshinones were measured and analyzed as well. The effect of tanshinones on Cyp3a11 protein expression was investigated. DTI exhibited inhibitory effect on the hydroxylation of lithocholic acid (LCA), taurolithocholic acid (TLCA) and taurochenodeoxycholic acid (TCDCA), their IC50 values were 0.81, 0.36 and 1.29 μM, respectively. The hydroxylation of LCA, TLCA and TCDCA were mediated by Cyp3a11. Low-dose DTI, CTS and TSA ameliorated cholestatic liver injury in mice, while high-dose DTI didn’t exhibit anti-cholestatic effect. The hepatic BAs profiles indicated that hydroxylation of BAs was inhibited in high-dose DTI group. DTI and TSA up-regulated the protein expression of Cyp3a11. As the hepatic concentration of DTI increased, the inhibitory effect at enzymatic activity level overwhelmed its up-regulation effect at protein level, thus resulted in worsening of cholestasis.

Functionalized Sulfur-Containing Heterocyclic Analogs Induce Sub-G1 Arrest and Apoptotic Cell Death of Laryngeal Carcinoma In Vitro

In this study, we speculate that the hydroxyl-containing benzo[b]thiophene analogs, 1-(3-hydroxybenzo[b]thiophen-2-yl) ethanone (BP) and 1-(3-hydroxybenzo[b]thiophen-2-yl) propan-1-one hydrate (EP), might possess antiproliferative activity against cancer cells. Hydroxyl-containing BP and EP show selectivity towards laryngeal cancer cells (HEp2), with IC50 values of 27.02 ± 1.23 and 35.26 ± 2.15 µM, respectively. The hydroxyl group present in the third position is responsible for the anticancer activity and is completely abrogated when the hydroxyl group is masked. BP and EP enhance the antioxidant enzyme activity and reduce the ROS production, which are correlated with the antiproliferative effect in HEp-2 cells. An increase in the BAX/BCL-2 ratio occurs during the BP and EP treatment and activates the caspase cascade, resulting in apoptosis stimulation. It also arrests the cells in the Sub-G1 phase, indicating the induction of apoptosis. The molecular docking and simulation studies predicted a strong interaction between BP and the CYP1A2 protein, which could aid in combinational therapy by enhancing the bioavailability of the drugs. BP and EP possess an antioxidant property with low antiproliferative effects (~5.18 µg/mL and ~7.8 µg/mL) as a standalone drug, therefore, they can be combined with other drugs for effective chemotherapy that might trigger the effect of pro-oxidant drug on healthy cells.

Psoralen induces liver injury and affects hepatic bile acids metabolism in female and male C57BL/6J mice.

Psoralen is a major component of Fructus Psoraleae that could induce liver injury. In this study, C57BL/6J mice were administered with psoralen at doses of 80 mg/kg for 3, 7 and 14 days. Blood and liver samples were collected for serum biochemistry and histopathology examinations, respectively. Psoralen led to liver injury with significantly increased liver weight and liver coefficient and up regulated serum ALT, AST and TG but down regulated serum TC and TP. The expression of bile acid-associated transporters and enzymes was detected by western blot, and the results showed that psoralen significantly down-regulates the expressions of CYP7A1, CYP27A1, BSEP and OSTα protein while up-regulates the expressions of HMGCR and FASN, resulting in the obstacles of bile acid efflux in the liver. The contents of 24 kinds of bile acids in the liver were measured by LC-MS/MS, and the results showed that psoralen led to the accumulation of unconjugated bile acids in the liver, such as ALCA and CA, which were more severe in male mice than female mice. It was indicated that psoralen may disrupt the balance of bile acid metabolism by inhibiting the expression of the efflux transporter, which then leads to liver damage.

Exposure to acrylamide inhibits testosterone production in mice testes and Leydig cells by activating ERK1/2 phosphorylation.

Acrylamide (ACR) is formed during the cooking of starchy foods at high temperatures. Accumulating evidence has shown that ACR has toxic effects, but the mechanism of its potential reproductive toxicity remains unclear. In this study, we observed that ACR caused weight loss in mice. There was no significant difference in the weight of testis and epididymis between the low/medium-dose ACR group and the control group. And the number of epididymal sperms, testicular Leydig cells, serum testosterone level, testicular steroidogenic genes and enzymes, including cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1), were decreased in the medium/high-dose ACR group. Additional cell experiments showed that the apoptosis rate and the level of reactive oxygen species (ROS) were increased, and testosterone levels and CYP17A1 protein expression were reduced in Leydig cells with treated ACR. Furthermore, the phosphorylation levels of extracellular signal-regulated kinases (ERK1/2) increased significantly; however, there was no significant difference in the levels of serine-threonine protein kinase (AKT) phosphorylation in the testis of mice and Leydig cells treated with ACR. These results suggest that ACR exposure leads to the damage of testicular structure and function and a decline in testosterone synthesis in Leydig cells and mouse testis, which may be related to the activated phosphorylation of ERK1/2.

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

CYP3A4 and CYP3A5 Expression is Regulated by CYP3A4*1G in CRISPR/Cas9-Edited HepG2 Cells.

Functional CYP3A4*1G (G>A, rs2242480) in cytochrome P450 3A4 (CYP3A4) regulates the drug-metabolizing enzyme CYP3A4 expression. The objective of this study was to investigate whether CYP3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5 in gene-edited human HepG2 cells. CYP3A4*1G GG and AA genotype HepG2 cells were established using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) single nucleotide polymorphism technology and homology-directed repair in the CYP3A4*1G GA HepG2 cell line. In CYP3A4*1G GG, GA, and AA HepG2 cells, CYP3A4*1G regulated expression of CYP3A4 and CYP3A5 mRNA and protein in an allele-dependent manner. Of note, significantly decreased expression level of CYP3A4 and CYP3A5 was observed in CYP3A4*1G AA HepG2 cells. Moreover, the results after RIF treatment showed that CYP3A4*1G decreased the induction level of CYP3A4 and CYP3A5 mRNA expression in CYP3A4*1G AA HepG2 cells. At the same time, CYP3A4*1G decreased CYP3A4 enzyme activity and tacrolimus metabolism, especially in CYP3A4*1G GA HepG2 cells. In summary, we successfully constructed CYP3A4*1G GG and AA homozygous HepG2 cell models and found that CYP3A4*1G regulates both basal and RIF-induced expression and enzyme activity of CYP3A4 and CYP3A5 in CRISPR/Cas9 CYP3A4*1G HepG2 cells. SIGNIFICANCE STATEMENT: Cytochrome P450 (CYP) 3A4*1G regulates both basal and rifampicin (RIF)-induced expression and enzyme activity of CYP3A4 and CYP3A5. This study successfully established CYP3A4*1G (G>A, rs2242480), GG, and AA HepG2 cell models using CRISPR/Cas9, thus providing a powerful tool for studying the mechanism by which CYP3A4*1G regulates the basal and RIF-induced expression of CYP3A4 and CYP3A5.

Sulfathiazole treats type 2 diabetes by restoring metabolism through activating CYP19A1

Globally, diabetes mellitus has been a major epidemic bringing metabolic and endocrine disorders. Currently, 1 in 11 adults suffers from diabetes mellitus, among the patients >90% contract type 2 diabetes mellitus (T2DM). Therefore, it is urgent to develop new drugs that effectively prevent and treat type 2 diabetes through new targets. With high-throughput screening, we found that sulfathiazole decreased the blood glucose and improved glucose metabolism in T2DM mice. Notably, we discovered that sulfathiazole treated T2DM by activating CYP19A1 protein to synthesize estrogen. Collectively, sulfathiazole along with CYP19A1 target bring new promise for the better therapy of T2DM.

Effects of maternal exposure to procymidone on hepatic metabolism in the offspring of mice.

As an effective fungicide widely used in agricultural production, the excessive procymidone (PRO) residue has been detected in the environment and food. Our previous study demonstrated that PRO could destroy the intestinal barrier in mice and has a joint toxic effect. To explore the cross-generational impact of maternal exposure, 10-week-old C57BL/6 female mice were orally administrated to 10 and 100 mg/kg body weight/day of PRO during pregnancy and lactation. The offspring obtained nutrients from the maternal through the placenta and breast milk, and PRO residues were detected in the liver, intestine, and feces of F1 generation. Fecal examination found that the residual PRO had been completely metabolized when the offspring mice grew to 35 days. The drug residue of F1 generation male mice was higher than that of female mice. We attributed this result to the difference in cytochrome P450 (CYP450) enzyme expression between male and female mice. The transcriptional levels of CYP1A1, CYP1A2, CYP2D9, and CYP3A4, and CYP450 protein expression levels, were higher in female mice. Furthermore, targeted MS of plasma revealed abnormal amino acid levels. In addition, PRO-induced hepatic metabolite changes in F0 and F1-7w mice. KEGG pathway analysis further showed that PRO jointly changed the amino acid biosynthesis pathway of the maternal and offspring. In summary, these results indicated that maternal exposure to PRO during a special period would interfere with self metabolism, and offspring will also have metabolic disorders.

Control of Arterial Hypertension by the AhR Blocker CH-223191: A Chronopharmacological Study in Chronic Intermittent Hypoxia Conditions.

Chronic intermittent hypoxia (CIH) is a major contributor to the development of hypertension (HTN) in obstructive sleep apnea (OSA). OSA subjects frequently display a non-dipping pattern of blood pressure (BP) and resistant HTN. After discovering that AHR-CYP1A1 axis is a druggable target in CIH-HTN, we hypothesized that CH-223191 could control BP in both active and inactive periods of the animals, recovering the BP dipping profile in CIH conditions.We evaluated the chronopharmacology of the antihypertensive efficacy of the AhR blocker CH-223191 in CIH conditions (21% to 5% of O2, 5.6cycles/h, 10.5h/day, in inactive period of Wistar rats). BP was measured by radiotelemetry, at 8am (active phase) and at 6pm (inactive phase) of the animals. The circadian variation of AhR activation in the kidney in normoxia was also assessed, measuring the CYP1A1 (hallmark of AhR activation) protein levels.Despite drug administration before starting the inactive period of the animals, CH-223191 was not able to decrease BP during the inactive phase, in CIH conditions, therefore not reverting the non-dipping profile. These results suggest that a higher dose or different time of administration of CH-223191 might be needed for an antihypertensive effect throughout the 24-h cycle.

Focal adhesion alterations in G0-positive melanoma cells.

Melanoma is a highly heterogeneous malignant tumor that exhibits various forms of drug resistance. Recently, reversal transition of cancer cells to the G0 phase of the cell cycle under the influence of therapeutic drugs has been identified as an event associated with tumor dissemination. In the present study, we investigated the ability of chemotherapeutic agent dacarbazine to induce a transition of melanoma cells to the G0 phase as a mechanism of chemoresistance. We used the flow cytometry to analyze cell distribution within cell cycle phases after dacarbazine treatment as well as to identifyG0 -positive cells population. Transcriptome profiling was provided to determine genes associated with dacarbazine resistance. We evaluated the activity of β-galactosidase in cells treated with dacarbazine by substrate hydrolysis. Cell adhesion strength was measured by centrifugal assay application with subsequent staining of adhesive cells with Ki-67 monoclonal antibodies. Ability of melanoma cells to metabolize dacarbazine was determined by expressional analysis of CYP1A1, CYP1A2, CYP2E1 followed by CYP1A1 protein level evaluation by the ELISA method. The present study determined that dacarbazine treatment of melanoma cells could induce an increase in the percentage of cells in G0 phase without alterations of β-galactosidase positive cells which corresponded to the fraction of the senescent cells. Transcriptomic profiling of cells under dacarbazine induction of G0 -positive cells percentage revealed that 'VEGFA-VEGFR2 signaling pathway' and 'Cell cycle' signaling were mostly enriched by dysregulated genes. 'Focal adhesion' signaling was also found to be triggered by dacarbazine. In melanoma cells treated with dacarbazine, an increase in G0 -positive cells among adherent cells was found. Dacarbazine induces the alteration in a percentage of melanoma cells residing in G0 phase of a cell cycle. The altered adhesive phenotype of cancer cells under transition in the G0 phase may refer to a specific intercellular communication pattern of quiescent/senescent cancer cells.

Usnea improves high-fat diet- and vitamin D3-induced atherosclerosis in rats by remodeling intestinal flora homeostasis.

Background: Usnea has various pharmacological properties, including anti-inflammatory, antitumor, antioxidant, antiviral, and cardiovasculoprotective effects. Aim of the study: To investigate the potential mechanisms underlying the anti-atherosclerosis (AS) activity of Usnea ethanol extract (UEE) via the regulation of intestinal flora. Materials and Methods: The chemical composition of UEE was determined using ultra-performance liquid chromatography with quadrupole exactive orbitrap mass spectrometry (UPLC-Q-EOMS). Thirty-six male Sprague-Dawley rats were divided into six groups. A high-fat diet and intraperitoneal vitamin D3 injections were used to establish a rat model of AS. After 4weeks of treatment with UEE, hematoxylin-eosin staining was performed to evaluate the pathomorphology of the aorta, liver, and colon. The composition and diversity of the rat intestinal flora were determined using high-throughput 16S rRNA sequencing. Enzyme-linked immunosorbent assays were used to measure the levels of plasma trimethylamine oxide (TMAO), serum bile acid (BA), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). The protein expression of cholesterol 7α-hydroxylase (CYP7A1) and flavin monooxygenase 3 (FMO3) in the liver and zonula occludens-1 (ZO-1) and occludin in colon tissue was detected via western blotting. Results: Forty-four compounds were identified in UEE. In the rat model of AS, UEE significantly prevented calcium deposition; decreased the serum levels of TC, TG, LDL-C, LPS, TNF-α, and IL-6; and increased the serum level of HDL-C. Additionally, all UEE dosages decreased the relative abundance of Verrucomicrobiota while increased that of Bacteroidetes. FMO3 protein expression and TMAO levels decreased, whereas CYP7A1 protein expression and BA levels increased. The absorption of intestinal-derived LPS was minimized. Furthermore, the protein expression of ZO-1 and occludin was upregulated. Conclusion: UEE ameliorated AS. The underlying mechanism was the reversal of imbalances in the intestinal flora by Usnea, thereby inhibiting calcium deposition, abnormal lipid metabolism, and inflammatory response.

Formation of CYP3A-specific metabolites of ibrutinib in vitro is correlated with hepatic CYP3A activity and 4β-hydroxycholesterol/cholesterol ratio.

Ibrutinib is an orally administered Bruton's tyrosine kinase inhibitor approved for the treatment of B-cell malignancies, including chronic lymphocytic leukemia. Ibrutinib is metabolized primarily via oxidation by cytochrome P450 (CYP) 3A4/5 to M37 (the primary active metabolite), M34, and M25. The objectives of this study were to assess the relationship between formation of the major CYP3A-specific ibrutinib metabolites in vitro and hepatic CYP3A activity and protein abundance, and to evaluate the utility of the endogenous CYP3A biomarker, plasma 4β-hydroxycholesterol (4β-HC) to cholesterol ratio, to predict ibrutinib metabolite formation in individual cadaveric donors with matching hepatocytes. Ibrutinib (5μM) was incubated with single-donor human liver microsomes (n=20) and primary human hepatocytes (n=15), and metabolites (M37, M34, and M25) were measured by liquid chromatography-tandem mass spectrometry analysis. CYP3A4/5 protein concentrations were measured by quantitative targeted absolute proteomics, and CYP3A activity was measured by midazolam 1'-hydroxylation. Ibrutinib metabolite formation positively correlated with midazolam 1'-hydroxylation in human liver microsomes and hepatocytes. Plasma 4β-HC and cholesterol concentrations were measured in plasma samples obtained at the time of liver harvest from the same 15 donors with matching hepatocytes. Midazolam 1'-hydroxylation in hepatocytes correlated with plasma 4β-HC/cholesterol ratio. When an infant donor (1 year old) was excluded based on previous ontogeny studies, M37 and M25 formation correlated with plasma 4β-HC/cholesterol ratio in the remaining 14 donors (Spearman correlation coefficients [r] 0.62 and 0.67, respectively). Collectively, these data indicate a positive association among formation of CYP3A-specific ibrutinib metabolites in human hepatocytes, hepatic CYP3A activity, and plasma 4β-HC/cholesterol ratio in the same non-infant donors.

337 Multi-level dysregulation of the AHR/CYP1A1 axis in psoriasis

The aryl hydrocarbon receptor (AHR) is an environmentally-regulated transcription factor with homeostatic and anti-inflammatory roles in skin. AHR activation leads to expression of the cytochrome P4501 (CYP1) enzyme family, which provides critical negative feedback by degrading AHR ligands. We have previously shown that excessive CYP1A1 activity impairs AHR anti-inflammatory activity in psoriasis-like skin inflammation in mice, while suppression of aberrant enzymatic activity restores AHR beneficial effect. Moreover, CYP1A1 activity is significantly enhanced in blood cells of psoriasis patients, suggesting that dysregulation of CYP1A1 activity may play a role in psoriasis. Here, we performed a multi-modal investigation of the AHR/CYP1A1 axis in the blood and skin of psoriasis patients and healthy controls, assessing availability of AHR ligands and precursors by mass spectrometry, AHR and CYP1A1 mRNA by qPCR, and CYP1A1 enzymatic activity by EROD assay. Levels of the ligand precursor tryptophan were significantly (p≤0.001) decreased in the serum of psoriasis patients (n=20) versus healthy (n=20). Expression of AHR mRNA was significantly (p≤0.001) decreased in both psoriasis blood and psoriasis lesional (PsL) skin, while CYP1A1 mRNA did not differ in blood but was significantly (p≤0.05) decreased in PsL skin. In vitro culture of skin epidermal sheets with an AHR agonist significantly (p≤0.05) upregulated CYP1A1 mRNA in healthy and psoriasis skin, but, surprisingly, CYP1A enzymatic activity was not increased by AHR ligation in PsL. We are currently studying AHR and CYP1A1 protein expression in the skin at single-cell level by imaging mass cytometry to gain further insights into the complex regulation of the pathway. Taken together, our data show that there is a multi-level dysregulation of the AHR/CYP1A1 axis in psoriasis and suggest a failure of multiple pathway checkpoints that could be targeted to restore AHR beneficial effects in skin inflammation.

Gegen Qinlian decoction activates AhR/IL-22 to repair intestinal barrier by modulating gut microbiota-related tryptophan metabolism in ulcerative colitis mice

Ethnopharmacological relevanceGegen Qinlian decoction (GQD) is a traditional Chinese medicine derived from Treatise on febrile diseases and is clinically used for the treatment of acute ulcerative colitis (UC). However, the potential mechanism of GQD treatment for UC remains elusive. Aim of studyIn this study, we aimed to explore the involvement of gut microbiota-related tryptophan metabolism in mediating protective effects of GQD against intestinal barrier damage. Materials and methodsMice with colitis were treated with 3% dextran sulfate sodium (DSS) for 6 days. The therapeutic effects of GQD in UC mice were examined based on body weight, disease activity index (DAI), organ index, length and pathological changes in the colon. The distribution of fluorescein isothiocyanate dextran (FITC-dextran) in the intestinal tract was observed using small animal imaging, while concentration of FITC-dextran in serum was detected using a fluorescein microplate analyser. Bacterial infiltration in colon tissues was observed by fluorescence in situ hybridisation (FISH), and the bacterial load in mesenteric lymph nodes (MLNs) was further examined through bacterial culture. Subsequently, colonic goblet cells were detected using Alcian blue staining. The tight junctions of the colonic epithelium were observed using transmission electron microscopy, and the expression of tight junction proteins was detected by immunofluorescence (IF) and western blot. In addition, flow cytometry was used to analyse the proportion of interleukin-22-positive (IL-22+) ILC3 cells in lamina propria lymphocytes, and the content of IL-22 in colon homogenates was determined using an ELISA kit. In addition, targeted tryptophan metabolomics was used to detect the concentration of indole derivatives produced by tryptophan metabolism in faeces, and 16S rDNA was used to investigate the composition and abundance of gut microbiota-related tryptophan metabolism. ResultsAdministration of GQD significantly alleviated the pathological symptoms, including weight loss, increased DAI score, changes in organ index, colon shortening, and colon pathological injury in UC mice. In addition, GQD reduced the diffusion of FITC-dextran in the intestinal tract, the content of FITC-dextran in serum, and bacterial infiltration in MLNs and colon tissues. Additionally, GQD significantly increased the number of colonic goblet cells, repaired the structure of epithelial tight junctions and increased the expression of tight junction proteins. Furthermore, GQD significantly increased the proportion of IL-22+ ILC3 in the lamina propria, the expression of CYP1A1 protein in colon tissue, and the level of IL-22 in colon homogenates. However, the above protective effects of GQD were inhibited by co-administration of GQD and aryl hydrocarbon receptor (AhR) antagonist. Additionally, GQD restored the content of indole derivatives generated by tryptophan metabolism, regulated the diversity of the gut microbiota, and significantly increased the abundance of genes related to tryptophan metabolism. ConclusionOur results confirmed that GQD repaired the damaged intestinal barrier in UC mice by regulating gut microbiota-related tryptophan metabolism and restoring the generation of indole derivatives to activate AhR-mediated IL-22 production.

Alizarin as a New Activator of the Aryl Hydrocarbon Receptor Signaling Pathway

Alizarin (1,2-dihydroxyanthraquinone) is a natural red dye extracted from the roots of Rubia cordifolia L. (family Rubiaceae). Alizarin has been used as a biological red stain for calcium. The aryl hydrocarbon receptor (AhR) has critical roles in multiple physiological pathways. This study aimed to determine whether alizarin is an unreported ligand of AhR. In the present study, we investigated the effects on cytochrome P450 (CYP) 1A1 mRNA, protein expression, AhR nuclear translocation, aryl hydrocarbon response element (AHRE) reporter activity, and AhR-specific antagonist following alizarin treatment of cells of the human hepatoma cell line, HepG2, and murine hepatoma cell line, Hepa-1c1c7. Alizarin induced CYP1A1 mRNA and protein expression in HepG2 and Hep-1c1c7 cells. Such induction was not present in C4 (B13NBii1) cells, which are AhR signal deficient, C12 (B15ECiii2) cells, which reduce AhR protein levels. The alizarin-induced responses were blocked by CH-223191, which is an AhR antagonist. Alizarin, the same as with the AhR ligand, induced the nuclear localization of AhR, as well as stimulated the transcriptional activity of AHRE. The results of this study suggest that alizarin is an AhR agonist.