CYP1A1 mRNA Expression Research Articles

Effects of Atorvastatin on Bile Acid Metabolism in High-fat Diet-fed ApoE -/- Mice.

Statins are considered as the cornerstone of the prevention and treatment of atherosclerotic cardiovascular disease, where pleiotropic effects are thought to contribute greatly in addition to the lipid-lowering effect. Bile acid metabolism has been gradually reported to be involved in the antihyperlipidemic and antiatherosclerotic effects of statins, but with inconsistent results and few studies carried out on animal models of atherosclerosis. The study aimed to examine the possible role of bile acid metabolism in the lipid-lowering and antiatherosclerotic effects of atorvastatin (ATO) in high-fat diet-fed ApoE -/- mice. The results showed that the levels of liver and faecal TC as well as ileal and faecal TBA were significantly increased in mice of the model group after 20 weeks of high-fat diet feeding compared with the control group, with significantly downregulated mRNA expression of liver LXR-α, CYP7A1, BSEP, and NTCP. ATO treatment further increased the levels of ileal and faecal TBA and faecal TC, but no obvious effect was observed on serum and liver TBA. In addition, ATO significantly reversed the mRNA levels of liver CYP7A1 and NTCP, and no obvious changes were observed in the expression of LXR-α and BSEP. Our study suggested that statins may enhance the synthesis of bile acids and facilitate the reabsorption of bile acids from the ileum via portal into the liver, possibly through the upregulation of the expression of CYP7A1 and NTCP. The results are helpful in enriching the theoretical basis for the clinical use of statins and have good translational value.

Nrf2-/- regulated lung DNA demethylation and CYP2E1 DNA methylation under PM2.5 exposure.

Cytochrome P450 (CYP450) can mediate fine particulate matter (PM2.5) exposure leading to lung injury. Nuclear factor E2-related factor 2 (Nrf2) can regulate CYP450 expression; however, the mechanism by which Nrf2-/- (KO) regulates CYP450 expression via methylation of its promoter after PM2.5 exposure remains unclear. Here, Nrf2-/- (KO) mice and wild-type (WT) were placed in a PM2.5 exposure chamber (PM) or a filtered air chamber (FA) for 12weeks using the real-ambient exposure system. The CYP2E1 expression trends were opposite between the WT and KO mice following PM2.5 exposure. After exposure to PM2.5, CYP2E1 mRNA and protein levels were increased in WT mice but decreased in KO mice, and CYP1A1 expression was increased after exposure to PM2.5 in both WT and KO mice. CYP2S1 expression decreased after exposure to PM2.5 in both the WT and KO groups. We studied the effect of PM2.5 exposure on CYP450 promoter methylation and global methylation levels in WT and KO mice. In WT and KO mice in the PM2.5 exposure chamber, among the methylation sites examined in the CYP2E1 promoter, the CpG2 methylation level showed an opposite trend with CYP2E1 mRNA expression. The same relationship was evident between CpG3 unit methylation in the CYP1A1 promoter and CYP1A1 mRNA expression, and between CpG1 unit methylation in the CYP2S1 promoter and CYP2S1 mRNA expression. This data suggests that methylation of these CpG units regulates the expression of the corresponding gene. After exposure to PM2.5, the expression of the DNA methylation markers ten-eleven translocation 3 (TET3) and 5-hydroxymethylcytosine (5hmC) was decreased in the WT group but significantly increased in the KO group. In summary, the changes in CYP2E1, CYP1A1, and CYP2S1 expression in the PM2.5 exposure chamber of WT and Nrf2-/- mice might be related to the specific methylation patterns in their promoter CpG units. After exposure to PM2.5, Nrf2 might regulate CYP2E1 expression by affecting CpG2 unit methylation and induce DNA demethylation via TET3 expression. Our study revealed the underlying mechanism for Nrf2 to regulate epigenetics after lung exposure to PM2.5.

Effects of four hormones on the mitigation of ovarian damage in tilapia (Oreochromis niloticus) after copper and cadmium exposure

Female tilapia of the Genetic Improvement of Farmed Tilapia (GIFT) strain were selected as an animal model to study the effects of four hormonal drugs in mitigating ovarian damage following exposure to copper and cadmium. After combined exposure to copper and cadmium in aqueous phase for 30 d, tilapia were randomly injected with oestradiol (E2), human chorionic gonadotropin (HCG), luteinizing hormone releasing hormone (LHRH), or coumestrol and raised in clear water for 7 d Ovarian samples were collected after combined exposure to heavy metals for 30 d and after recovery for 7 d Gonadosomatic index (GSI), copper and cadmium levels in the ovary, reproductive hormone levels in serum, and mRNA expression of key reproductive regulatory factors were determined. After 30 d of exposure to the combined copper and cadmium in aqueous phase, the Cd2+ content in tilapia ovarian tissue increased by 1,242.46% (p < 0.05), whereas the Cu2+ content, body weight, and GSI decreased by 68.48%, 34.46%, and 60.00% (p < 0.05), respectively. Additionally, E2 hormone levels in tilapia serum decreased by 17.55% (p < 0.05). After drug injection and recovery for 7 d, compared to the negative control group, the HCG group exhibited an increase of 39.57% (p < 0.05) in serum vitellogenin levels. Increases of 49.31%, 42.39%, and 45.91% (p < 0.05) in serum E2 levels were observed, and mRNA expression of 3β-HSD increased by 100.64%, 113.16%, and 81.53% (p < 0.05) in the HCG, LHRH, and E2 groups, respectively. The mRNA expression of CYP11A1 in tilapia ovaries increased by 282.26% and 255.08% (p < 0.05) and mRNA expression of 17β-HSD increased by 109.35% and 111.63% in the HCG and LHRH groups, respectively (p < 0.05). All four hormonal drugs, particularly HCG and LHRH, promoted the restoration of tilapia ovarian function to varying degrees after injury induced by combined exposure to copper and cadmium. This study presents the first hormonal treatment protocol for the mitigation of ovarian damage in fish exposed to combined aqueous phases of copper and cadmium as a strategy to prevent and treat fish ovarian damage induced by heavy metals.

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.

Mitoguardin2 Is Associated With Hyperandrogenism and Regulates Steroidogenesis in Human Ovarian Granulosa Cells.

Polycystic ovary syndrome (PCOS) is an endocrinopathy characterized by hyperandrogenism, anovulation, and polycystic ovaries, in which hyperandrogenism manifests by excess androgen and other steroid hormone abnormalities. Mitochondrial fusion is essential in steroidogenesis, while the role of mitochondrial fusion in granulosa cells of hyperandrogenic PCOS patients remains unclear. In this study, mRNA expression of mitochondrial fusion genes mitoguardin1, -2 (MIGA 1, -2) was significantly increased in granulosa cells of hyperandrogenic PCOS but not PCOS with normal androgen levels, their mRNA expression positively correlated with testosterone levels. Dihydrotestosterone (DHT) treatment in mice led to high expression of MIGA2 in granulosa cells of ovulating follicles. Testosterone or forskolin/ phorbol 12-myristate 13-acetate treatments increased expression of MIGA2 and the steroidogenic acute regulatory protein (StAR) in KGN cells. MIGA2 interacted with StAR and induced StAR localization on mitochondria. Furthermore, MIGA2 overexpression significantly increased cAMP-activated protein kinase A (PKA) and phosphorylation of AMP-activated protein kinase (pAMPK) at T172 but inhibited StAR protein expression. However, MIGA2 overexpression increased CYP11A1, HSD3B2, and CYP19A1 mRNA expression. As a result, MIGA2 overexpression decreased progesterone but increased estradiol synthesis. Besides the androgen receptor, testosterone or DHT might also regulate MIGA2 and pAMPK (T172) through LH/choriogonadotropin receptor-mediated PKA signaling. Taken together, these findings indicate that testosterone regulates MIGA2 via PKA/AMP-activated protein kinase signaling in ovarian granulosa cells. It is suggested mitochondrial fusion in ovarian granulosa cells is associated with hyperandrogenism and potentially leads to abnormal steroidogenesis in PCOS.

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.

Effects of asprosin on estradiol and progesterone secretion and proliferation of bovine granulosa cells

Asprosin is an adipokine synthesized by the white adipose tissue that regulates glucose homeostasis and that has been reported to affect bovine theca cell function and follicular growth, but its role on granulosa cell functions remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on granulosa cell steroidogenesis. Bovine granulosa cells from small ovarian follicles were cultured in vitro to investigate the effects of asprosin on cell proliferation, production of steroids, mRNA abundance of genes that encode steroidogenic enzymes and cell cycle regulators, and protein relative abundance of steroidogenic signaling pathways. Asprosin was shown to affect granulosa cell functions in a dose-dependent manner. In the presence of FSH, asprosin enhanced estradiol production and stimulated an increase in mRNA expression of FSHR and CYP19A1 in a dose-dependent manner. In the presence of IGF1, asprosin decreased estradiol production, increased progesterone production, altered PKA relative protein expression, and tended to alter the ratio of p-ERK1/2/total ERK1/2 protein expression in a dose-dependent manner. Furthermore, asprosin increased p-53 gene expression in basal culture conditions and with or without FSH and IGF1. Taken together, findings of this study show that asprosin is a regulator of granulosa cell functions and the effects of asprosin depend on dose and cell culture conditions.

Influence of ursodeoxycholic acid on blood glucose, insulin and GLP-1 in rats with liver fibrosis induced by bile duct ligation

BackgroundThe prevalence of impaired glucose tolerance and diabetes is much higher in people with cirrhosis than that in the general population. However, there are inadequate concrete guidelines for the management of diabetes in these patients, particularly in the early stage. Bile aids (BAs) have been found to exert hormone-like functions in the control of lipid and glucose metabolism. We studied the effect of ursodeoxycholic acid (UDCA) on glucose levels in rats with cirrhosis induced by bile duct ligation (BDL).MethodsSD rats were divided into three groups: sham operation (Group A); BDL (Group B), and UDCA plus BDL (Group C). After 4 weeks, oral glucose tolerance tests were performed. Serum biochemical parameters and the levels of glucose, insulin, and glucagon-like peptide 1 (GLP-1) were measured. Histopathology of the liver and islet was observed. The gene expression of cholesterol 7α-hydroylase (CYP7A1), microsomal oxysterol 7a-hydroxylase (CYP7B1) in the liver, and Takeda G-protein-coupled receptor-5 (TGR5) in the intestine was determined by real-time PCR.ResultsCompared with Group A, fasting glucose and 1-h and 2-h postprandial glucose levels increased slightly (all P > 0.05), 2-h postprandial insulin levels increased significantly (P < 0.05), 15 min postprandial GLP-1 levels decreased (P < 0.05) in Group B. Compared with Group B, fasting glucose and 1-h postprandial glucose levels decreased (all P < 0.05), 2-h postprandial insulin levels decreased (P < 0.01), and 15 min postprandial GLP-1 levels increased (P < 0.05) in Group C. After UDCA intervention, liver fibrosis induced by BDL was alleviated, and the islet areas were increased (P < 0.05). Compared with Group A, the mRNA expression of CYP7A1 and CYP7B1 in the liver increased, and the mRNA expression of TGR5 in the intestine decreased in Group B (all P < 0.05). Compared with Group B, the mRNA expression of CYP7A1 and CYP7B1 in the liver decreased, and TGR5 in the intestine increased in Group C (P < 0.05).ConclusionsAfter 4 weeks of BDL, the rats developed liver fibrosis and abnormal glucose metabolism. UDCA administration improved liver fibrosis, increased islet area, decreased glucose levels, inhibited genes in BA synthesis, enhanced TGR5 gene expression in the intestine, and further improved islet function.

Cytochrome P450 3A4 suppression by epimedium and active compound kaempferol leads to synergistic anti-inflammatory effect with corticosteroid.

Introduction: Cytochrome P450 (CYP) 3A4 is a major drug metabolizing enzyme for corticosteroids (CS). Epimedium has been used for asthma and variety of inflammatory conditions with or without CS. It is unknown whether epimedium has an effect on CYP 3A4 and how it interacts with CS. We sought to determine the effects of epimedium on CYP3A4 and whether it affects the anti-inflammatory function of CS and identify the active compound responsible for this effect. Methods: The effect of epimedium on CYP3A4 activity was evaluated using the Vivid CYP high-throughput screening kit. CYP3A4 mRNA expression was determined in human hepatocyte carcinoma (HepG2) cells with or without epimedium, dexamethasone, rifampin, and ketoconazole. TNF-α levels were determined following co-culture of epimedium with dexamethasone in a murine macrophage cell line (Raw 264.7). Active compound (s) derived from epimedium were tested on IL-8 and TNF-α production with or without corticosteroid, on CYP3A4 function and binding affinity. Results: Epimedium inhibited CYP3A4 activity in a dose-dependent manner. Dexamethasone enhanced the expression of CYP3A4 mRNA, while epimedium inhibited the expression of CYP3A4 mRNA and further suppressed dexamethasone enhancement of CYP3A4 mRNA expression in HepG2 cells (p < 0.05). Epimedium and dexamethasone synergistically suppressed TNF-α production by RAW cells (p < 0.001). Eleven epimedium compounds were screened by TCMSP. Among the compounds identified and tested only kaempferol significantly inhibited IL-8 production in a dose dependent manner without any cell cytotoxicity (p < 0.01). Kaempferol in combination with dexamethasone showed complete elimination of TNF-α production (p < 0.001). Furthermore, kaempferol showed a dose dependent inhibition of CYP3A4 activity. Computer docking analysis showed that kaempferol significantly inhibited the catalytic activity of CYP3A4 with a binding affinity of -44.73kJ/mol. Discussion: Inhibition of CYP3A4 function by epimedium and its active compound kaempferol leads to enhancement of CS anti-inflammatory effect.

Oxytocin enhances progesterone production with upregulation of BMP-15 activity by granulosa cells

To elucidate the reproductive role of oxytocin (OXT) in ovarian steroidogenesis and its functional interaction with bone morphogenetic proteins (BMPs), the effects of OXT on ovarian steroidogenesis were investigated by utilizing primary culture of rat granulosa cells and human granulosa KGN cells. Here we revealed that the OXT receptor was expressed in both rat and human granulosa cells and that OXT treatment significantly increased follicle-stimulating hormone (FSH)- and forskolin (FSK)-induced progesterone production, but not estradiol production, by rat and human granulosa cells, respectively. In accordance with the effects of OXT on progesterone production, OXT enhanced mRNA expression of CYP11A1 and HSD3B2 induced by FSK in human granulosa cells. Of note, OXT enhanced the phosphorylation of SMAD1/5/9 and the transcription of ID1 induced by BMP-15, but not those induced by BMP-6, in human granulosa cells. It was also revealed that OXT treatment upregulated the expression of BMPR2, a crucial type-II receptor of BMP-15, and enhanced the BMP-15-induced expression of inhibitory SMAD6 by human granulosa cells. Collectively, it was shown that OXT accelerates ovarian progesterone synthesis with upregulation of BMP-15 activity, leading to a fine-tuning of ovarian steroidogenesis (186 words).

Study on the hepatotoxicity and potential mechanism of gefitinib based on CYP450 in mice and AML12 cells.

Gefitinib is mainly used for the treatment of non-small-cell lung cancer. Hepatotoxicity is one of the main side effects of gefitinib, and seriously affects the treatment process of the disease. However, the hepatotoxicity mechanism of gefitinib remains unclear. The hepatotoxicity of different doses of gefitinib was investigated in mice and AML-12 cells, and the possible correlation of hepatotoxicity with CYP450 was analysed. The toxic effects of gefitinib were confirmed by the increased liver index, decreased body weight and survival rate, injured liver function and histopathology followed 16 days of oral administration. Gefitinib (400 mg/kg) upregulated the hepatic mRNA expression of CYP1A1 and downregulated the CYP2D9 and CYP2D10 in mice. Furthermore, we verified that gefitinib produced cytotoxicity on AML-12 cells in a dose and time-dependent manner, and confirmed that gefitinib (20 μM) induced cell apoptosis, upregulated mRNA expression of CYP1A1 and downregulated CYP2D9 and CYP2D10. Pearson correlation analysis also showed that the hepatotoxicity of gefitinib was positively correlated with CYP1A1 and negatively correlated with CYP2D9 and CYP2D10. Our results suggested that the hepatotoxicity gefitinib may be associated with CYP1A1, CYP2D9 and CYP2D10. These findings will contribute to a better understanding of the mechanism of gefitinib hepatotoxicity.

The β-Blocker Carvedilol Prevents Benzo(a)pyrene-Induced Lung Toxicity, Inflammation and Carcinogenesis.

The current study evaluated the effects of the β-blocker carvedilol on benzo(a)pyrene (B(a)P) and its active metabolite benzo(a)pyrene diol epoxide (BPDE)-induced lung toxicity, inflammation and carcinogenesis and explored the potential mechanisms. Carvedilol blocked the BPDE-induced malignant transformation of human bronchial epithelial cells BEAS-2B. In BEAS-2B cells, B(a)P strongly activated ELK-1, a transcription factor regulating serum response element (SRE) signaling, which was attenuated by carvedilol. Carvedilol also inhibited the B(a)P-induced AhR/xenobiotic responsive element (XRE) and mRNA expression of CYP1A1 and attenuated B(a)P-induced NF-κB activation. In a B(a)P-induced acute lung toxicity model in CD-1/IGS mice, pretreatment with carvedilol for 7 days before B(a)P exposure effectively inhibited the B(a)P-induced plasma levels of lactate dehydrogenase and malondialdehyde, inflammatory cell infiltration and histopathologic abnormalities in the lung, and upregulated the expression of GADD45α, caspase-3 and COX-2 in the lung. In a B(a)P-induced lung carcinogenesis model in A/J mice, carvedilol treatment for 20 weeks did not affect body weight but significantly attenuated tumor multiplicity and volume. These data reveal a previously unexplored role of carvedilol in preventing B(a)P-induced lung inflammation and carcinogenesis by inhibiting the cross-talk of the oncogenic transcription factors ELK-1, AhR and NF-κB.

Taraxasterol alleviates aflatoxin B1-induced liver damage in broiler chickens via regulation of oxidative stress, apoptosis and autophagy

Aflatoxin B1 (AFB1) is the most dangerous and abundant mycotoxin, which is toxic to almost all animals, and poultry is more sensitive to AFB1 toxicity. Ingesting AFB1-contaminated feed can cause significant liver damage and brings serious harm to poultry, which greatly restricts the development of the poultry industry. The present research was implemented to explore the intervention effect and its mechanism of taraxasterol on liver damage induced by AFB1 in broiler chickens. The liver damage model in broiler chickens was established by feeding 0.5 mg/kg AFB1 feed, and taraxasterol (25, 50 and 100 mg/kg BW, respectively) was given in the drinking water for 21 days. The growth performance, liver function, oxidative stress, apoptosis and autophagy were evaluated. The results showed that taraxasterol increased BW and reduced feed-to-gain ratio of broiler chickens induced by AFB1. Taraxasterol improved the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), total bilirubin (TBIL) and alkaline phosphatase (ALP), and attenuated hepatic histopathological changes induced by AFB1. Meantime, taraxasterol down-regulated cytochrome P450 (CYP450) enzyme system CYP1A1 and CYP2A6 mRNA expression, inhibited the overproduction of reactive oxygen species (ROS) and malondialdehyde (MDA), and enhanced the activities of antioxidant enzymes glutathione (GSH) and catalase (CAT) and the content of antioxidant superoxide dismutase (SOD) of the liver in broiler chickens induced by AFB1. Furthermore, taraxasterol up-regulated the mRNA and protein expression of hepatic nuclear factor E2 related factor 2 (Nrf2), heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and down-regulated the expression of hepatic kelch like ECH associated protein 1 (Keap1) induced by AFB1 in Keap1/Nrf2 signaling pathway. The ultrastructural observation and RT-qPCR results found that taraxasterol inhibited apoptosis of hepatocytes, up-regulated the expression of B-cell lymphoma-2 (Bcl-2) mRNA and down-regulated the expression of Bax and caspase3 mRNA. Further, taraxasterol restored the autophagy of hepatocytes and down-regulated the mRNA expression of phosphatidylinositol 3-kinase K (PI3K), protein kinase B (AKT) and mammalian target of rapamycin (mTOR) in AFB1-induced liver of broiler chickens. The above results indicate that taraxasterol alleviates liver damage induced by AFB1 in broiler chickens through regulation of Keap1/Nrf2 signaling pathway to exert its antioxidant effect, mitochondrial apoptosis pathway to improve anti-apoptotic ability and PI3K/AKT/mTOR pathway to restore autophagy.

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.

Metabolic Profiling and Investigation of the Modulatory Effect of Fagonia cretica L. Aerial Parts on Hepatic CYP3A4 and UGT2B7 Enzymes in Streptozotocin-Induced Diabetic Model.

Drug-metabolizing enzymes are either boosted or suppressed by diabetes mellitus. This research was designed to explore Fagonia cretica L. aerial parts' impact on CYP3A4 and UGT2B7 activity and their mRNA expression in diabetic rats. Fagonia cretica (F. cretica) dried powder was sequentially extracted with n-hexane, chloroform, ethyl acetate, methanol, and water. The methanol extract and aqueous fraction presented the most significant potential to decrease the concentration of alpha-hydroxyl midazolam, with 176.0 ± 0.85 mg/Kg and 182.9 ± 0.99 mg/Kg, respectively, compared to the streptozotocin (STZ)-induced diabetic group, reflecting the inhibition in CYP3A4 activity. The fold change in mRNA expression of CYP3A4 was decreased significantly by the methanol extract, and the aqueous fraction of F. cretica estimated by 0.15 ± 0.002 and 0.16 ± 0.001, respectively, compared with the diabetic group. Morphine metabolism was significantly increased in rats treated with F. cretica methanol extract and its aqueous fraction, displaying 93.4 ± 0.96 mg/Kg and 96.4 ± 1.27 mg/Kg, respectively, compared with the metabolism of morphine in the diabetic group, which highlights the induction of UGT2B7 activity. The fold change in mRNA expression of UGT2B7 was significantly increased by the methanol extract and the aqueous fraction, estimated at 8.14 ± 0.26 and 7.17 ± 0.23 respectively, compared to the diabetic group. Phytochemical analysis was performed using high-performance liquid chromatography (HPLC), where the methanol extract showed more flavonoids and phenolic compounds compared to the aqueous fraction of F. cretica. The obtained results were further consolidated by molecular docking studies, where quercetin showed the best fitting within the active pocket of CYP3A4, followed by gallic acid, displaying free binding energies (∆G) of -30.83 and -23.12 kcal/mol, respectively. Thus, F. cretica could serve as a complementary medicine with standard anti-diabetic therapy that can modulate the activity of the drug-metabolizing enzymes.

Effects of insulin-like growth factor-1 on the mRNA expression of estradiol receptors, steroidogenic enzymes, and steroid production in bovine follicles.

Insulin-like growth factor-1 (IGF-1) plays a crucial role in follicular growth and stimulates steroid hormone production in bovine follicles. Steroid hormones are synthesized through the actions of steroidogenic enzymes, specifically STAR, CYP11A1, HSD3B, and CYP19A1 in both theca cells (TCs) and granulosa cells (GCs), under the influence of gonadotropins. Particularly, estradiol 17β (E2) assumes a central role in follicular development and selection by activating estrogen receptors β (ESR2) in GCs. We assessed ESR2 mRNA expression in GCs of developing follicles and investigated the impact of IGF-1 on the mRNA expression of ESR2, CYP19A1, FSHR, and LHCGR, STAR, CYP11A1, and HSD17B in cultured GCs and TCs, respectively. Additionally, we assessed the influence of IGF-1 on androstenedione (A4), progesterone (P4), and testosterone (T) production in TCs. Small-sized follicles (< 6 mm) exhibited the highest levels of ESR2 mRNA expression, whereas medium-sized follicles (7-8 mm) displayed higher levels than large-sized follicles (≥ 9 mm) (P < 0.05). IGF-1 increased the mRNA expression of ESR2, CYP19A1, and FSHR in GCs of follicles of both sizes, except for FSHR mRNA in medium-sized follicles (P < 0.05). IGF-1 significantly elevated mRNA expression of LHCGR, STAR, CYP11A1, and CYP17B in TCs of small- and medium-sized follicles (P < 0.05). Moreover, IGF-1 augmented the production of A4 and P4 but had no impact on T production in TCs of small- and medium-sized follicles. Taken together, our findings indicate that IGF-1 upregulates steroidogenic enzymes and steroid hormone production, underscoring the crucial role of IGF-1 in follicle development and selection.

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.

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.

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.