CYP1A1 mRNA Research Articles

The effects of irisin and leptin on steroidogenic enzyme gene expression in human granulosa cells: In vitro studies

Reproduction and energy metabolism are closely related, and fertility can be directly affected by either obesity or malnutrition. In this study, we investigated the in vitro effects of irisin and leptin, two hormones primarily involved in energy metabolism, on the expression of genes encoding key steroidogenic enzymes in primary cultures of human granulosa cells. Granulosa cells were purified from follicular fluid samples obtained during in vitro fertilization (IVF) procedure, cultured, and treated with irisin (125-2000 ng/ml) or leptin (25–400 ng/ml) for 1–3 days. mRNA expression levels of cytochrome P450 enzymes [CYP11A1, CYP19A1, CYP21A2], hydroxy-delta-5-steroid dehydrogenase, 3 beta and steroid delta-isomerase 1 (HSD3B1), and hydroxysteroid 17-beta dehydrogenase 3 (HSD17B3) were measured using qRT-PCR analysis. Irisin significantly upregulated CYP19A1 mRNA levels, while leptin upregulated CYP19A1 and HSD3B1 mRNA levels. These preliminary results show that irisin and leptin may directly affect the expression of the genes important for ovarian steroidogenesis and female reproduction.

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.

Effects of Mangifera indica leaves improves blood lipids profile and biochemical indices in high-fat diet-induced hyperlipidaemia rats

Dyslipidemia a chronic, metabolic syndrome characterized by elevated lipid profiles together with lipid peroxidation in individuals with atherosclerotic cardiovascular disease (CVD). Functional ingredients such as high phenolic content and potent antioxidant activity obtained from agricultural waste by-products or waste are of great interest. However, the hypolipidemic effects of the waste mango (Mangifera indica L.) leaves (MLE) have not been investigated. Here, the specific lipid-lowering and potential hepatoprotective mechanisms by which the gavage administration of MLE affects lipid metabolism and liver steatosis in rats fed on a high-fat diet (HFD) were evaluated. In rats treated with high level of MLE, a persistent suppressive effect on liver weight and body weight gain was discovered after 28-day intervention. Furthermore, body lipid index and reduced inflammatory reaction and liver function parameters in HFD control rats were markedly ameliorated by supplementation with high doses of MLE. In addition, histological and histomorphometric analyses here demonstrated that fat accumulation changed in HFD supplemented hyperlipidaemia rats, but normalized in MLE treatment groups. Further real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses were carried out to determine the mRNA and protein abundance of PPARα receptors and CYP7A1 in liver tissues of rats. These results indicate that MLE supplement have the promising lipid lowering effects in HFD-induced hyperlipidaemia rats based on positive ameliorations in the serum lipid profile and liver function parameters.

Acute Cholesterol-Lowering Effect of Exendin-4 in Ldlr-/- and C57BL/6J Mice.

We previously reported that glucagon-like peptide-1 receptor agonists (GLP-1RAs) reduced serum low-density lipoprotein cholesterol (LDL-C) levels in patients with type 2 diabetes mellitus receiving statins, which increased LDL receptor (LDLR) expression. Nevertheless, it remains unclear how much LDLR expression contributes to the LDL-C-lowering effect of GLP-1RAs. We examined the effect of a GLP-1RA, namely, exendin-4, on serum LDL-C levels and its mechanism in Ldlr-/- and C57BL/6J mice. Ten-week-old Ldlr-/- and C57BL/6J mice received exendin-4 or saline for 5 days, and serum lipid profiles and hepatic lipid levels were examined. Cholesterol metabolism-related gene expression and protein levels in the liver and ileum and the fecal bile acid (BA) composition were also examined. Exendin-4 treatment significantly decreased serum very-low-density lipoprotein cholesterol (VLDL-C) and LDL-C levels and mature hepatic SREBP2 levels and increased hepatic Insig1/2 mRNA expression in both mouse strains. In Ldlr-/- mice, exendin-4 treatment also significantly decreased hepatic cholesterol levels and fecal BA excretion, decreased hepatic Cyp7a1 mRNA expression, and increased small intestinal Fgf15 mRNA expression. In C57BL/6J mice, exendin-4 treatment significantly decreased small intestinal NPC1L1 levels. Our findings demonstrate that exendin-4 treatment decreased serum VLDL-C and LDL-C levels in a manner that was independent of LDLR. Exendin-4 treatment might decrease serum cholesterol levels by lowering hepatic SREBP2 levels and cholesterol absorption in Ldlr-/- and C57BL/6J mice. Exendin-4 treatment might decrease cholesterol absorption by different mechanisms in Ldlr-/- and C57BL/6J mice.

Farnesoid X receptor is inhibited after ileum transposition in diabetic rats: its hypoglycemic effect

Background: Aim to investigate bile acid profile changes and the Farnesoid X receptor (FXR) status after ileotransposition (IT), and reveal its possible hypoglycemic mechanism. Methods: Twenty male diabetic rats were randomly assigned into the IT group and the sham IT (SH) group. Bile acid profiles were measured using an ultra-performance liquid chromatography-tandem mass spectrometry. Glucose metabolism was monitored after oral administration of FXR inhibitor and agonist. And the expression of key FXR target genes were measured. Results: The levels of β-muricholic acid (P = 0.047), tauro-α-muricholic acid and tauro-β-muricholic acid (P < 0.001) in plasma in the IT group were higher than those in the SH group, and the levels of taurocholic acid (P = 0.049) and turoursodeoxycholic acid (P = 0.030) were lower than those in the SH group. After inhibition of intestinal FXR, the glucose metabolism in the SH group was improved. When FXR agonist was given, the blood glucose level was increased in both groups. After sacrifice, the levels of glycoursodeoxycholic acid, tauro-α-muricholic acid and tauro-β-muricholic acid in liver and ileum tissues were higher than those in the SH group (P < 0.05), the level of α- muricholic acid (P < 0.001) in liver tissues were lower than that in the SH group. Moreover, the expression of CYP7A1 mRNA (P < 0.001) and FGF15 mRNA (P = 0.001) in the IT group was significantly higher, and the expression of PEPCK mRNA (P = 0.004), SREPB1c mRNA (P = 0.005) and SRB1 mRNA (P = 0.001) were significantly lower than that in the SH group. Conclusions: We demonstrate a remarkable heterogeneity of BA profiles after IT, FXR activation might has a detrimental effect on glucose metabolism.

Effect of maternal dietary 25OHD3 on the gonadal development of offspring mice

Context Studies have widely reported that a vitamin D3 metabolite 25OHD3 improves the growth performance of animals and offspring, but few studies have focused on offspring sex hormone secretion and gonadal development in the offspring. Aims This study focused on the effects of gestational feeding with 25OHD3 on the growth, serum calcium (Ca2+) and phosphorus (Pi) concentrations, and ovarian and testicular development in offspring. Methods From the beginning of gestation until the end of labour, maternal mice were gavaged daily with 25 ug of 25OHD3. We weighed offspring on Days 1, 10, and 21 postnatally and collected offspring serum and gonadal samples on Day 21. Key results The results showed that 25OHD3 significantly increased maternal serum Ca2+ and Pi concentrations, and increased the growth rate and serum Ca2+ and Pi concentrations of offspring, significantly increased the serum concentrations of luteinising hormone (LH) and follicle-stimulating hormone (FSH), and accelerated the follicle development in the female offspring. Male offspring showed significantly increased LH, FSH, and testosterone serum concentrations, and 3β-HSD and 17β-HSD mRNA expressions, and a significantly decreased CYP17A1 mRNA expression. Conclusions In conclusion, the present findings indicated that maternal feeding with 25OHD3 during pregnancy positively affects offspring hormone secretion and gonadal development. Implications These insights have shed light on the potential significance of maternal nutrition in ensuring healthier animal populations and offer valuable considerations for animal husbandry practices.

Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes

Tree shrews (Tupaia belangeri) are a non-rodent primate-like species sometimes used for biomedical research involving hepatitis virus infections and toxicology. Genome analysis has indicated similarities between tree shrews and humans in the numbers of cytochromes P450 (P450 or CYP), which constitute a family of important drug-metabolizing enzymes; however, P450s have not been fully investigated in tree shrews. In this study, we identified CYP1A1, CYP1A2, CYP1B1, and CYP1D1 cDNAs from tree shrew liver and compared their characteristics with dog, pig, and human CYP1As. The deduced amino acid sequences of tree shrew CYP1s were highly identical (82–87 %) to human CYP1s. In tree shrews, CYP1A1 and CYP1A2 mRNAs were preferentially expressed in liver, whereas CYP1D1 mRNA was preferentially expressed in kidney and lung. In contrast, CYP1B1 mRNA was expressed in various tissues, with the most abundant expression in spleen. Among the tree shrew CYP1 mRNAs, CYP1A2 mRNA was most abundant in liver, and CYP1B1 mRNA was most abundant in kidney, small intestine, and lung. All tree shrew CYP1 proteins heterologously expressed in Escherichia coli catalyzed caffeine and estradiol in a similar manner to tree shrew liver microsomes and human, dog, and pig CYP1 proteins. These results suggest that tree shrew CYP1A1, CYP1A2, CYP1B1, and CYP1D1 genes, different form human pseudogene CYP1D1P, are expressed in liver, small intestine, lung, and/or kidney and encode functional drug-metabolizing enzymes important in toxicology.

1716. In Vitro Drug-Drug Interaction Evaluation of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor

Abstract Background Epetraborole (EBO) is a boron-containing oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. EBO is being developed for the treatment of Mycobacterium avium complex lung disease patients and will be used in combination with other drugs. Therefore, EBO and its major circulating metabolite M3 were evaluated in a comprehensive drug-drug interactions (DDI) risk assessment. Methods Stability of EBO was evaluated in human liver microsomes, hepatocytes and recombinant CYP enzymes. The inhibitory potential of EBO (0.03-100 μM) and M3 (1-1000 μM) on cytochrome P450 (CYP) activities was assessed using human hepatic microsomes. The CYP induction potential of EBO (25-200 μM), M3 was evaluated and compared to prototypical inducers in human hepatocytes (three donors) and mRNA. Fold increase in mRNA expression was utilized to investigate the CYP induction potential of EBO (0.3-100 µM) and M3 (1-250 µM). Stably transfected cell lines that expressed individual transporters were used to determine whether EBO or M3 were substrates or inhibitors for these proteins. Results In vitro studies with microsomes, hepatocytes and recombinant cytochrome P450 (CYP) enzymes indicated that EBO was a poor substrate for major CYP enzymes; drug interactions with epetraborole as victim are considered unlikely. Neither EBO nor its major metabolite M3 was a potent reversible or time-dependent inhibitor of major CYP enzymes. Half maximal inhibitory concentration (IC50) values for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 were &amp;gt;100 µM. EBO was not an inducer of CYP1A2 mRNA in human hepatocytes from three donors, while it was a weak inducer of CYP2B6 and CYP3A4. No induction of mRNA was observed in human hepatocytes at concentrations relevant to planned clinical doses. EBO is unlikely to be a substrate for P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, MATE1 and MATE2K. EBO was an in vitro substrate for OCT2, a transporter involved in active renal secretion. At clinically relevant concentrations, neither EBO nor M3 inhibited major human efflux or uptake transporters. Conclusion At clinically relevant concentrations of EBO and its major circulating metabolite M3, there is a low risk of victim or perpetrator DDI. Disclosures Afshin Shafiee, PhD, AN2 Therapeutics: Stocks/Bonds Bradley K. Wong, PhD, AN2 Therapeutics: Advisor/Consultant Sanjay Chanda, PhD, AN2 Therapeutics: Stocks/Bonds.

Disrupted placental vitamin D metabolism and calcium signaling in gestational diabetes and pre-eclampsia patients.

Gestational diabetes (GDM) and pre-eclampsia (PE) represents the unrecognized risk factors for reduced bone content in neonates. The present study is planned to explore the components of vitamin D metabolism and calcium transport in placenta of GDM and PE cases and its effect on the neonatal bone mass determination using bone densitometry system. We have collected serum and placenta tissues from GDM (n = 20), PE (n = 20), and healthy pregnancies (n = 20). In the present study, we found mRNA expression of oxidative stress markers, vitamin D metabolic components and calcium channels, calcium channel binding proteins, plasma membrane calcium ATPase, ATP synthase and Ca2+ release genes; Ryanodine receptors genes were assessed by quantitative real-time PCR (qRT-PCR) in placental tissue of GDM, PE, and healthy pregnancies. We observed high level of oxidative stress in both GDM and PE placenta compared to normal pregnancies. CYP2R1 and VDR mRNA expression was significantly downregulated and upregulation of CYP27B1 and CYP24A1 in GDM and PE compared with healthy cases. Similarly, calcium transporters were downregulated in GDM and PE placental tissues. In addition, CYP24A1, VDR, CaBP28K, TRPV5 and PMCA3 mRNA expression were correlated with BMC of neonates. Oxidative stress is probably relevant to disrupted vitamin D homeostasis and calcium transport in the placenta of GDM and PE cases. The altered regulatory mechanism of CYP24A1 and VDR could indicates more pronounced serum 25(OH)D reduction. Additionally, reduced BMC in the neonates of these cases might be as consequences of modified CYP24A1, VDR, CaBP28K, TRPV5 and PMCA3 mRNA expression.

Crosstalk between the aryl hydrocarbon receptor (AhR) and the peroxisome proliferator-activated receptor gamma (PPARγ) as a key factor in the metabolism of silver nanoparticles in neuroblastoma (SH-SY5Y) cells in vitro

The potential usefulness of silver nanoparticles (AgNPs) in anticancer therapy has been postulated for many years. However, little is known to date about the exact impact of such NPs on intracellular detoxication pathways. Therefore, the aim of this study was to determine the impact of AgNPs on the AhR-PPARγ-CYP1A1 pathway in neuroblastoma (SH-SY5Y) cells. The obtained results showed a decrease in the metabolic activity of the SH-SY5Y cells at the 50 and 100 μg/mL concentrations with an increase in caspase-3 activity. An increase in the intercellular ROS production was observed at the 1 and 10 μg/mL concentrations. The co-treatment of the AgNP-treated cells with the AhR and PPARγ inhibitors abolished the effect of the tested AgNPs in the SH-SY5Y cells. In turn, the CYP1A1 activity assay showed a decrease in this parameter in the AgNP-treated cells. Moreover, the gene expression analysis demonstrated that AgNPs were able to increase the AhR and CYP1A1 mRNA expression and decrease the PPARγ gene expression after the 6-h treatment. In turn, an increase in the AhR and PPARγ protein expression was observed after 24 h. Summarizing, the study shows for the first time that AgNPs with a 5-nm diameter size are able to exert a cytotoxic effect on SH-SH5Y cells in a ROS-dependent manner affect the AhR-PPARγ-CYP1A1 pathway inter alia by inhibiting the activity of CYP1A1. This is important due to given present research approaches using such NPs as enhancer agents in the modern PPARγ inhibitor-based anticancer therapy.

Quantitative Cytochrome P450 3A4 Induction Risk Assessment Using Human Hepatocytes Complemented with Pregnane X Receptor-Activating Profiles.

Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.

Indole acetylated high-amylose maize starch: Synthesis, characterization and application for amelioration of colitis

Tryptophan metabolites such as indole-3-acetic acid (IAA) are critical for gut health, through their binding to the aryl hydrocarbon receptor (AhR), and may be useful for treatment of gastrointestinal diseases. Delivery of IAA to the colon is necessary, and one strategy is use of esterified starches which get digested in the colon by gut microbes. High amylose maize starch (HAMS) resists digestion in the upper gastrointestinal tract and is fermented by gut microbiota to release short-chain fatty acids (SCFAs), which are also beneficial to intestinal homeostasis. IAA esterified to HAMS (HAMSIAA) was synthesized with different degrees of substitution (DSs) by controlling the ratio of IAA vs HAMS. Successful incorporation of indole acetyl group was verified by NMR and FTIR spectra. XRD revealed that the crystalline type of HAMSIAA changed from B to V-type. SEM showed the destroyed surface of the starch granules. HAMSIAA with DS ~ 0.3 effectively increased IAA in the colon, to levels unachievable by oral IAA delivery. HAMSIAA increased pathways downstream of AhR activation, including CYP1A1 mRNA expression and IL-22 protein levels, and greatly improved DSS-induced colitis. HAMSIAA could serve as an ideal means for colon-targeted delivery of IAA and a promising nutraceutical for amelioration of inflammatory conditions.

Abstract A017: Lung cancer chemoprevention with the β-blocker carvedilol

Abstract Chronic exposure to carcinogens that are present in tobacco smoke is a causative factor for lung carcinogenesis. Although smoking cessation successfully reduced the prevalence of cigarette use in the US, lung cancer risk for current and former smokers remains high, for whom no effective chemopreventive agent currently exists. In the present study, carvedilol, an FDA-approved β-blocker was examined on lung carcinogenesis induced by the tobacco carcinogen benzo(a)pyrene [B(a)P] in vitro and in vivo. In non-tumorigenic human bronchial epithelial cell culture BEAS-2B, carvedilol inhibited benzo(a)pyrene diol epoxide (BPDE)-induced malignant transformation at non-toxic concentrations in a dose-dependent manner. Although β-adrenergic receptors (β-ARs) are expressed in BEAS-2B cells, the anticancer activity of carvedilol is independent of β-blockade since the non-β-blocking R-carvedilol enantiomer also blocked transformation while the β-blockers atenolol (β1-AR selective blocker), ICI-118,551 (β2-AR selective blocker), and propranolol (nonselective blocker) had no effect. Carvedilol’s anti-transformation activity is possibly mediated by aryl hydrocarbon receptor signaling because carvedilol, R- and S-carvedilol all inhibited B(a)P-activated xenobiotic responsive element (XRE) promoter and CYP1A1 mRNA expression. In a B(a)P-induced acute lung toxicity model in CD-1 mice, pretreatment with carvedilol, R- or S-carvedilol (20 mg/kg/day) for 7 days significantly attenuated increased plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration, histopathologic changes, and overexpression of COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol at 3.2 and 20 mg/kg significantly attenuated tumor multiplicity and burden, to a similar degree as the EGFR inhibitor gefitinib. Our study reveals a previously unexplored role for the FDA approved cardiovascular drug carvedilol in the prevention of tobacco carcinogen-associated lung cancer. Citation Format: Ayaz Shahid, Mengbing Chen, Carol Lin, Bradley T. Andresen, Cyrus Parsa, Robert Orlando, Ying Huang. Lung cancer chemoprevention with the β-blocker carvedilol [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A017.

Investigation of doxorubicin combined with ciprofloxacin-induced cardiotoxicity: from molecular mechanism to fundamental heart function.

This study was designed to investigate the impacts of Doxo alone and in combination with Cipro on the hepatic and cardiac CYP1A2, CYP2J3, and CYP3A1 mRNA levels. We also aimed to analyze the cardiac function by perfusing isolated rat hearts. Rats were given Doxo and/or Cipro in chronic (3-week) and acute (single-day) dosing schedules. Cardiac CYP2J3, CYP3A1, and CYP1A2 gene expression levels were measured by quantitative reverse transcription PCR. Cardiac functions of the isolated hearts were evaluated by using the Langendorff technique. Doxo alone (2.5mg/kg) and Doxo + Cipro (2.5mg + 20mg/kg) significantly decreased hepatic CYP1A2 expression compared to saline, whereas Doxo (2.5mg/kg) and Doxo + Cipro (2.5mg + 20mg/kg) showed significantly higher cardiac CYP1A2 expression in comparison to control. In the liver tissue, Doxo (2.5mg/kg) and Doxo + Cipro (2.5 + 20mg/kg) decreased the CYP2J3 expression than the control group. The Doxo (2.5mg/kg) and Doxo + Cipro (2.5 + 20mg/kg)-treated group had significantly higher cardiac CYP2J3 expression compared to control. Doxo (2.5mg/kg; cumulative dose 15mg/kg) and Doxo + Cipro (2.5 + 20mg/kg) showed significantly higher cardiac CYP3A1 expressions than the control. Rate-pressure product (HR × LVDP)/1000) showed an overall decrease in cardiac functions of Doxo (2.5mg/kg) and Doxo + Cipro (2.5 + 20mg/kg)-treated group. We found considerable effects in chronic protocol; Doxo alone high dose and plus Cipro decreased hepatic CYP1A2 and CYP2J3 mRNA. On the other hand, these treatment groups exhibited an increase in the cardiac CYP1A2, CYP2J3, and CYP3A1 expression and likewise deteriorated the overall hemodynamic parameters.

Cooperative effects of oocytes and estrogen on the forkhead box L2 expression in mural granulosa cells in mice

Forkhead box L2 (FOXL2) plays a critical role in the development and function of mammalian ovaries. In fact, the causative effects of FOXL2 misregulations have been identified in many ovarian diseases, such as primary ovarian insufficiency and granulosa cell tumor; however, the mechanism by which FOXL2 expression is regulated is not well studied. Here, we showed that FOXL2 expression in ovarian mural granulosa cells (MGCs) requires stimulation by both oocyte-derived signals and estrogen in mice. In the absence of oocytes or estrogen, expression of FOXL2 and its transcriptional targets, Cyp19a1 and Fst mRNA, in MGCs were significantly decreased. Moreover, expression levels of Sox9 mRNA, but not SOX9 protein, were significantly increased in the FOXL2-reduced MGCs. FOXL2 expression in MGCs was maintained with either oocytes or recombinant proteins of oocyte-derived paracrine factors, BMP15 and GDF9, together with estrogen, and this oocyte effect was abrogated with an ALK5 inhibitor, SB431542. In addition, the FOXL2 level was significantly decreased in MGCs isolated from Bmp15−/− /Gdf9+/− mice. Therefore, oocyte, probably with estrogen, plays a critical role in the regulation of FOXL2 expression in mural granulosa cells in mice.

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.

The physiological blood concentration of phenylalanine-proline can ameliorate cholesterol metabolism in HepG2 cells.

We have previously reported that the dipeptide Phe-Pro affects lipid metabolism in vivo and in vitro, but very little is known regarding the mechanism of action of Phe-Pro after it is absorbed by the intestines via PepT1. In this study, we administered a single oral dose of Phe-Pro to rats and quantified its concentration in the portal plasma using LC-TOF/MS analysis. Additionally, the physiological blood concentration of Phe-Pro was added to the lipid accumulation model of HepG2 cells to decrease intracellular cholesterol and increase the expression of CYP7A1 and PPARα mRNA levels. Moreover, we analyzed the binding of PPARα and Phe-Pro using AlphaFold2. We found that Phe-Pro is a ligand for PPARα. To the best of our knowledge, this is the first study that shows Phe-Pro to be present in the portal plasma. We found for the first time that Phe-Pro ameliorated cholesterol metabolism in HepG2 cells.

Induction mechanism of cigarette smoke components (CSCs) on dyslipidemia and hepatic steatosis in rats

ObjectiveThe purpose of this study was to explore the effect of cigarette smoke component (CSC) exposure on serum lipid levels in rats and the underlying molecular mechanism.MethodsMale SPF-grade SD rats were randomly divided into a control group and a CSC exposure group, with the CSC group being exposed to CSC for 6 weeks. RT–PCR and Western blotting methods were used to detect lipid metabolism gene expression in rats, and 16S RNA gene sequencing was used to detect the gut microbiota in the rat cecum. Rat serum exosomes were prepared and identified, and the interaction of exosomal miR-291a-3p and miR-126a-5p with AMPK and CYP7A1 was detected by a dual luciferase reporter gene assay (DLRG).ResultsSerum indicators, including cholesterol levels and trimethylamine oxide (TMAO) content, were significantly affected in the CSC exposure group compared with the control group (P < 0.05), and the expression levels of adenylate-activated protein kinase (AMPK), acetyl-coenzyme A carboxylase (ACC) and HMG-CoA reductase (HMG-CoAR) genes were significantly increased (P < 0.05) in the liver, while the expression level of cholesterol 7α-hydroxylase (CYP7A1) was markedly decreased (P < 0.01). 16S rRNA gene sequencing of the gut microbiota in the rat cecum showed that the abundance of Firmicutes in the CSC group increased significantly at the phylum level, while the abundances of Bacteroidota and Spirochaetota were reduced significantly (P < 0.01). The relative abundance of Romboutsia, Turicibacter, and Clostridium sensu stricto increased significantly (P < 0.01), and the relative abundance of Prevotella, Muribaculaceae_norank, Lachnospiraceae NK4A136 group, Roseburia, Treponema, and Ruminococcus significantly decreased (P < 0.01) at the genus level. In addition, the exosome miR-291a-3p and miR-126a-5p levels were markedly regulated by CSC exposure (P < 0.01). The interactions of miR-291a-3p and miR-126a-5p with AMPK and CYP7A1 mRNA were also validated by the DLRG method.ConclusionsIn summary, the rat dyslipidemia induced by CSC exposure may be related to the interference of gut microbiota structure and interaction of miRNAs from serum exosomes with target mRNAs, which further regulated AMPK-ACC/CYP7A1 signaling in rats.

Abstract 12525: Synergistic Effects of Pitavastatin and Pemafibrate on Hepatic Steatosis and Inflammation in Ldlr +/- Mice Fed Fat Diet

Introduction: The combination therapy of statin, ezetimibe and PCSK9 inhibitors enables us to reduce serum LDL-C and risk of atherosclerotic diseases. However, there remain patients who cause cardiovascular diseases. Recently, it has been reported that nonalcoholic fatty liver disease (NAFLD) might be a background for cardiovascular diseases. However, there are few reports on the combination therapy of statin and pemafibrate on hepatic steatosis and inflammation. Hypothesis: The aim of this study is to examine the impact of statin and/or pemafibrate on hepatic lipid contents and inflammatory gene expression in Ldlr +/- mice. Methods: Eight to ten weeks old male Ldlr +/- mice were used. Mice were fed high fat diet (HFD) ± 0.01% pitavastatin ± 0.00003% pemafibrate for four weeks. Serum lipids and liver histology/ lipid contents/ mRNA expression in four groups were examined. Results: Pemafibrate reduced serum triglycerides, particularly chylomicron and VLDL fractions, and increased HDL-C as previously reported. Hepatic triglyceride was unchanged, and very importantly, hepatic cholesterol and free-cholesterol were decreased with pitavastatin + pemafibrate compared to pitavastatin (pitavastatin vs pitavastatin + pemafibrate: cholesterol; 13.5±4.7 vs 9.4±1.9 mg/g of liver, P&lt;0.01, free-cholesterol; 10.4±4.8 vs 6.9±1.4 mg/g of liver, P&lt;0.01). The addition of pemafibrate resulted in a 46% increase in Cyp7a1 mRNA expression, suggesting that cholesterol in liver was reduced due to enhanced cholesterol excretion. Tnfa mRNA in liver was reduced by 62% and F4/80 staining of the liver showed decreased macrophage infiltration. Conclusions: Adding pemafibrate to pitavastatin did not alter hepatic triglyceride, however decreased cholesterol and free-cholesterol and improved inflammatory gene responses in liver, suggesting that the synergistic effect of pitavastatin and pemafibrate may be beneficial in NAFLD related atherosclerotic patients.

Altered Serum Bile Acid Profile Associated with Chronic Allograft Dysfunction in Kidney Transplant Recipients.

BACKGROUND Chronic allograft dysfunction (CAD) is the leading cause of graft loss among kidney transplant recipients (KTRs). Bile acids (BAs) play an important role in regulating inflammatory process, which is the major contributor to the development of CAD. The aim of this study was to evaluate the association between BAs metabolic dysregulation and CAD in KTRs. MATERIAL AND METHODS Fifteen serum BA species were determined in 43 healthy controls (HCs) and 131 KTRs by UPLC-MS/MS. KTRs were grouped into stable renal function (STA) and CAD1 and CAD2 groups based on eGFR levels. Circulating CYP7A1, CYP7B1, CYP27A1, and SLCO2B1 mRNA levels were determined by RT-PCR. RESULTS Total BA concentrations were comparable among the 4 groups. However, KTRs showed significantly different BAs profiling compared to HCs. KTRs with severe CAD (CAD2) had significantly lower unconjugated BAs and secondary BAs (SBAs) compared to the other 3 groups. KTRs had significantly lower SBAs/primary BAs (PBAs) ratios than HCs, which were comparable among the 3 KTR groups. Conjugated/unconjugated BAs ratios increased significantly with the deterioration of allograft function, which was further confirmed by correlation analysis. Differential correlation network analysis revealed that perturbations in intraclass and interclass BA coregulation existed during CAD progression. Moreover, relative gene expressions of CYP7B1 and CYP27A1 were positively correlated with eGFR. CONCLUSIONS BA species profiling, but not total BA concentrations, was significantly altered in KTRs with CAD. The shifts from unconjugated BAs toward conjugated BAs, SBAs toward PBAs, and distinct pairwise BAs coregulation patterns were the main characteristics of KTRs with CAD.