CYP1A1 mRNA Levels Research Articles

Oral self-microemulsifying drug delivery system for honokiol's stress responses attenuation and anti-Cryptocaryon irritans efficacy enhancement in Trachinotus ovatus

In this study, an oral self-micro-emulsifying drug delivery system of honokiol (HN-SMEDDS) was developed to overcome honokiol's disadvantages, such as poor aqueous solubility and induced stress responses. The safeties of HN-SMEDDS and honokiol dimethyl sulfoxide aqueous solution (HN-DMSO) were evaluated by comparing their hemolytic activity and acute toxicity in pompano (Trachinotus ovatus). The one-h-ten-percent hemolytic concentration (EC10) of HN-SMEDDS on pompano erythrocytes was 1.206 mg/mL, 33.5 times that of HN-DMSO. And the minimum lethal doses (MLD) for HN-SMEDDS and HN-DMSO in pompano were 1000 mg/kg and 500 mg/kg, respectively. The 14-d relative percent survival (RPS) was calculated after orally administering substance HN-SMEDDS at various doses in pompano cryptocaryoniasis, revealing an optimal dose of 400 mg/kg. To assess efficacy, a comparison was carried out between the oral administration of HN-SMEDDS and HN-DMSO in treating pompano cryptocaryoniasis. This comparison was based on the 14-d RPS and the quantification of C. irritans trophonts in the right second gill (NCTG). In the challenged pompanos treated with HN-SMEDDS and HN-DMSO, the 14-d RPS and 7-d NCTG reached 68.89 ± 3.14% and 6.3 ± 2.1 individuals, and 53.33 ± 0.00% and 30.7 ± 2.1 individuals, respectively. Furthermore, stress-related markers, encompassing mRNA levels of cyp1a, hsp70, gst, and sod in the liver, spleen, and kidney, along with activities of ACP, CAT, SOD, GSH, and T-AOC in these organs and serum, were compared between the pompanos administered with HN-SMEDDS and HN-DMSO. The impact of HN-SMEDDS on the mRNA levels of cyp1a in the liver and kidney, hsp70 in all the analyzed tissues, gst in the liver and spleen, sod in the spleen, as well as the activities of ACP and CAT in liver, spleen, and serum, GSH content in spleen and kidney, SOD in the liver, kidney, and serum, and the T-AOC in liver, spleen, kidney, and serum, were significantly smaller than those of HN-DMSO. Compared to HN-DMSO, HN-SMEDDS could markedly reduce the stress responses of fish and enhance the efficacy of honokiol.

Bacterial endotoxin lipopolysaccharides regulate gene expression in human colon cancer cells

ObjectiveLipopolysaccharide (LPS) is a major cell wall component of gram-negative bacteria. Colon bacteria contribute to LPS which promotes colon cancer metastasis. The objective of this study was to survey the effect of LPS on cell viability and gene expression of 55 molecular targets in human colon cancer cells.ResultsLPS did not affect the viability of COLO 225 cells under the culture conditions but affected the expression of a number of genes important in inflammatory responses and cancer development. LPS increased TTP family, GLUT family and DGAT1 mRNA levels but decreased DGAT2a and DGAT2b expression in the human colon cancer cells. LPS also increased COX2, CXCL1, ELK1, ICAM1, TNFSF10 and ZFAND5 but decreased BCL2L1, CYP19A1 and E2F1 mRNA levels in the colon cancer cells. These data suggest that LPS has profound effects on gene expression in human colon cancer cells.

Effect of Growth Hormone Receptor Deficiency on Androgen-Associated Gene Expression of Hepatic Drug Metabolizing Enzymes and Drug Transporters in Pigs.

Growth hormone receptor (GHR)-deficient pigs were generated using the CRISPR/Cas9 system to investigate the involvement of GHR-mediated growth hormone (GH) signaling in androgen-associated gene expression of hepatic drug metabolizing enzymes (DMEs) and drug transporters. We initially confirmed that no wild-type GHR mRNA was present in GHR-/- (GHR-KO) pigs; in addition, as previously reported, those pigs exhibited decreases in body weight and serum insulin-like growth factor-1 concentration and an increase in serum GH concentration compared with the levels in GHR-/+ and GHR+/+ pigs with a wild-type GHR mRNA. The real-time RT-PCR results on the mRNA levels of hepatic DMEs and drug transporters in the GHR-KO pigs and the pigs with a wild-type GHR mRNA revealed that, among the examined hepatic DMEs, the mRNA levels of CYP1A2, CYP2A19, sulfotransferase (SULT) 1A1, and SULT2A1 were higher in GHR-KO pigs than in the pigs with a wild-type GHR mRNA, whereas the opposite trend was observed for the mRNA level of uridine 5'-diphospho-glucuronosyltransferase 1A6. No such significant differences in the mRNA levels of three hepatic drug transporters including multidrug resistance protein 1 were observed. In addition, the mRNA level of hepatic cut-like homeobox 2 (CUX2), which is expressed in an androgen-dependent manner and associated with the hepatic mRNA expression of several DMEs, was significantly decreased in GHR-KO pigs. The present findings strongly suggest that not only serum androgen but also GHR-mediated GH signaling contributes to the mRNA expression of several DMEs and CUX2, but not transporters, in the pig liver.

Scutellarin Mediates Cytochrome P450 3A4 and 2C19 Expression via Pregnane X Receptor and Constitutive Androstane Receptor.

Breviscapine is a flavonoid extracted from Erigeron breviscapus (Vant.) Hand.-Mazz., and mainly contains scutellarin. Nuclear receptors play important roles in regulating transporter and drug metabolic enzymes. To investigate the regulatory effects of scutellarin on CYP3A4 and 2C19 in HepG2 and Caco-2 cells based on nuclear receptors PXR and CAR. The proteins and mRNA levels of CYP3A4 and CYP2C19 treated with scutellarin were detected by Western Blot and RT-qPCR. Using assays of the dual-luciferase reporter, promoter sequences containing hPXR and hCAR protein recognition and binding regulatory elements CYP3A4 and CYP2C19 were inserted upstream of the reporter gene, and the expression vector and the reporter vector were cotransfected into HepG2 and Caco-2 cells. Scutellarin inhibited mRNA of CYP3A4 and PXR, and promoted mRNA expression of CYP2C19 and CAR in RT-qPCR results. Western-blot results showed scutellarin inhibited the expression of CYP3A4 and promoted the expression of CYP2C19. The dual-luciferase reporter genes showed that scutellarin enhanced the expression level of CYP2C19, and when its concentration was 40 and 80μmol/L, CYP3A4 was significantly increased. Scutellarin down-regulates CYP3A4 through PXR, and its mechanism may work by up-regulating CAR, binding to PXR to inhibit PXR-mediated expression of CYP3A4. Scutellarin up-regulates CYP2C19 through CAR.

Seasonal changes of vitamin D3 and ovarian steroidogenesis in the wild ground squirrels (Citellus dauricus Brandt)

There is mounting evidence that vitamin D3 regulates female reproductive function critically, while little is known about the function of seasonally variable vitamin D3 in regulating ovarian steroidogenesis. This study examined the seasonal expressions of vitamin D receptor (VDR), vitamin D metabolic molecules (CYP2R1, CYP27B1, and CYP24A1), and steroidogenic enzymes (P450scc, 3β-HSD, P450c17, and P450arom) in the ovaries of the wild ground squirrels (Citellus dauricus Brandt) during the different breeding seasons. VDR, CYP2R1, CYP27B1, and CYP24A1 were shown to be localized in different types of ovarian cells in the wild ground squirrels during the breeding and non-breeding seasons. Meanwhile, the mRNA levels of VDR, CYP2R1, CYP27B1, CYP11A1, HSD3B1, CYP17A1, and CYP19A1 in the ovaries were remarkably higher in the breeding season. Furthermore, RNA-seq data of ovaries revealed that 6036 genes were differentially expressed genes (DEGs); further analysis revealed that several DEGs known to be involved in ovarian steroidogenesis pathway and cellular response to vitamin D pathway were identified. In addition, during the breeding season, the concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone, and 17β-estradiol were greater in the serum of the wild female ground squirrels. This observation was positively correlated with seasonal changes in the concentration of 25(OH)D3, supporting the fact that the 25(OH)D3 content in the ovaries was significantly higher in the breeding season. These findings suggested that seasonal changes in vitamin D3 might regulate the ovarian steroidogenesis of the wild female ground squirrels.

Grape Polyphenols May Prevent High-Fat Diet-Induced Dampening of the Hypothalamic-Pituitary-Adrenal Axis in Male Mice.

Chronic high-fat diet (HFD) consumption causes obesity associated with retention of bile acids (BAs) that suppress important regulatory axes, such as the hypothalamic-pituitary-adrenal axis (HPAA). HFD impairs nutrient sensing and energy balance due to a dampening of the HPAA and reduced production and peripheral metabolism of corticosterone (CORT). We assessed whether proanthocyanidin-rich grape polyphenol (GP) extract can prevent HFD-induced energy imbalance and HPAA dysregulation. Male C57BL6/J mice were fed HFD or HFD supplemented with 0.5% w/w GPs (HFD-GP) for 17 weeks. GP supplementation reduced body weight gain and liver fat while increasing circadian rhythms of energy expenditure and HPAA-regulating hormones, CORT, leptin, and PYY. GP-induced improvements were accompanied by reduced mRNA levels of Il6, Il1b, and Tnfa in ileal or hepatic tissues and lower cecal abundance of Firmicutes, including known BA metabolizers. GP-supplemented mice had lower concentrations of circulating BAs, including hydrophobic and HPAA-inhibiting BAs, but higher cecal levels of taurine-conjugated BAs antagonistic to farnesoid X receptor (FXR). Compared with HFD-fed mice, GP-supplemented mice had increased mRNA levels of hepatic Cyp7a1 and Cyp27a1, suggesting reduced FXR activation and more BA synthesis. GP-supplemented mice also had reduced hepatic Abcc3 and ileal Ibabp and Ostβ, indicative of less BA transfer into enterocytes and circulation. Relative to HFD-fed mice, CORT and BA metabolizing enzymes (Akr1d1 and Srd5a1) were increased, and Hsd11b1 was decreased in GP supplemented mice. GPs may attenuate HFD-induced weight gain by improving hormonal control of the HPAA and inducing a BA profile with less cytotoxicity and HPAA inhibition, but greater FXR antagonism.

Impact of dietary soybean inclusion on the endocrine disruptive effect in female brood stock of Cyprinus carpio

Phytoestrogens, mostly found in legumes, mimic animal estrogens and induce either pro- or anti-estrogenic activity. A feeding trial for matured Cyprinus carpio females was carried out to assess the endocrine disrupting effect of phytoestrogens in SBM and to suggest a dietary inclusion of soybean meal for broodstock of carps. The fish were fed diets containing graded levels of soybean meal (SBM-0, 15, 20, 25, 30, 35, and 40%) for 60 days. To assess the endocrine disruption, sex hormone profiling, gene expression analysis of candidate genes, and histological examinations were conducted. Serum T3, serum vitellogenin, and mRNA levels of cyp19a1, cyp19b, and erβ were found to be higher in fish groups fed with diet containing low levels of SBM (SBM 15, 20, and 25), and significantly lower in high SBM diet (≥30%) fed groups. Serum concentrations of cholesterol, cortisol, and the mRNA level of 20βHSD all decreased as SBM increased. Increased levels of testosterone, estradiol, and the mRNA for the erα gene were observed in the diet when SBM was incorporated into the diet. In the treatment groups with ≥30% inclusion of SBM, the number of vitellogenic oocytes and the gonadosomatic index were significantly lower than the groups with low inclusion levels. Thus, soybean meal can be included in the diet of female carp broodstocks up to 25% without disrupting the reproductive function. However, an inclusion level beyond 25% could harm the endocrine axis, subsequently capable of impacting reproductive performance.

Maintenance of airway epithelial barrier integrity via the inhibition of AHR/EGFR activation ameliorates chronic obstructive pulmonary disease using effective-component combination

BackgroundAirway epithelial barrier dysfunction is highly related to the pathogenesis of chronic obstructive pulmonary disease (COPD). Effective-component combination (ECC) derived from Bufei Yishen formula (BYF) is an effective treatment regimen for patients with COPD and has previously been found to attenuate COPD and airway epithelial inflammation in rats. PurposeTo determine the mechanism underlying the protective effects of ECC-BYF against the disruption of the airway epithelial barrier in COPD. MethodsThe protective effects of ECC-BYF on the airway epithelial barrier were investigated in a rat COPD model. BEAS-2B epithelial cells were stimulated with cigarette smoke extract (CSE) to determine the direct effects of ECC-BYF on epithelial barrier function and aryl hydrocarbon receptor (AHR)/ epidermal growth factor receptor (EGFR) signaling. ResultsThe results revealed that ECC-BYF attenuated COPD in rats and maintained the airway epithelial barrier by upregulating the expression of apical junction proteins, including occludin (OCC), zonula occludens (ZO)-1, and E-cadherin (E-cad). In BEAS-2B cells, ECC-BYF decreased permeability, increased transepithelial electrical resistance, and prevented the decrease in OCC, ZO-1, and E-cad expression induced by CSE exposure. In addition, transcriptomics and network analysis revealed that the protective effects of ECC-BYF may be related to multiple signaling pathways, including ErbB, AHR, and PI3K–Akt–mTOR pathways. ECC-BYF treatment suppressed the protein levels of p-EGFR and p-ERK1/2 and mRNA levels of CYP1A1 in CSE-exposed BEAS-2B cells as well as the protein levels of p-EGFR, p-ERK1/2, and CYP1A1 in the lungs of rats with COPD. In BEAS-2B cells, the AHR agonist FICZ weakened the protective effect of ECC-BYF on the epithelial barrier by suppressing the increase in ZO-1 and OCC expression induced by ECC-BYF and preventing the inhibitory effects of ECC-BYF on EGFR phosphorylation. ConclusionsThis is the first study to demonstrate the protective effect of ECC-BYF on airway epithelial barrier function. The underlying mechanism may be associated with the suppression of the AHR/EGFR pathway to promote apical junction protein adhesion.

Comparison of two hepatocyte differentiation protocols in human umbilical cord mesenchymal stem cells: In vitro study

Human umbilical cord mesenchymal stromal cells (HUCMSCs) are an emerging source of cell therapy due to their self-renew and differentiation ability. They can differentiate into three germ layers, including the potential to generate hepatocytes. This study determined the transplantation efficiency and suitability of HUCMSCs-derived hepatocyte-like cells (HLCs) for their therapeutic application for liver diseases. This study aims to formulate ideal conditions to induce HUCMSCs into the hepatic lineage and investigate the efficiency of the differentiated HLCs based on their expression characteristics and capacity to integrate into the damaged liver of CCl4-challenged mice. Hepatocyte growth factor (HGF) and Activin A, Wnt3a were found to optimally promote the endodermal expansion of HUCMSCs, which showed phenomenal expression of hepatic markers upon differentiation in the presence of oncostatin M and dexamethasone. HUCMSCs expressed MSC-related surface markers and could undergo tri-lineage differentiations. Two hepatogenic differentiation protocols (differentiated hepatocyte protocol 1 [DHC1]: 32 days and DHC2: 15 days) were experimented with. The proliferation rate was faster in DHC2 than in DHC1 on day 7 of differentiation. The migration capability was the same in both DHC1 and DHC2. Hepatic markers like CK18, CK19, ALB, and AFP were upregulated. The mRNA levels of albumin, α1AT, αFP, CK18, TDO2, CYP3A4, CYP7A1, HNF4A, CEBPA, PPARA, and PAH were even higher in the HUCMSCs-derived HCLs than in the primary hepatocytes. Western blot confirmed HNF3B and CK18 protein expression in a step-wise manner differentiated from HUCMSCs. The metabolic function of differentiated hepatocytes was evident by increasing PAS staining and urea production. Pre-treating HUCMSCs with a hepatic differentiation medium containing HGF can drive their differentiation towards endodermal and hepatic lineages, enabling efficient integration into the damaged liver. This approach represents a potential alternative protocol for cell-based therapy that could enhance the integration potential of HUCMSC-derived HLCs.

Expression of CYP2B6 Enzyme in Human Liver Tissue of HIV and HCV Patients.

Background and Objectives: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections present significant public health challenges worldwide. The management of these infections is complicated by the need for antiviral and antiretroviral therapies, which are influenced by drug metabolism mediated by metabolic enzymes and transporters. This study focuses on the gene expression of CYP2B6, CYP3A4, and ABCB1 transporters in patients with HIV, HCV, and HIV/HCV co-infection, aiming to assess their potential association with the choice of therapy, patohistological and clinical parameters of liver damage such as the stage of liver fibrosis, serum levels of ALT and AST, as well as the grade of liver inflammation and other available biochemical parameters. Materials and Methods: The study included 54 patients who underwent liver biopsy, divided into HIV-infected, HCV-infected, and co-infected groups. The mRNA levels of CYP2B6, CYP3A4, and ABCB1 was quantified and compared between the groups, along with the analysis of liver fibrosis and inflammation levels. Results: The results indicated a significant increase in CYP2B6 mRNA levels in co-infected patients, a significant association with the presence of HIV infection with an increase in CYP3A4 mRNA levels. A trend towards downregulation of ABCB1 expression was observed in patients using lamivudine. Conclusions: This study provides insight into gene expression of CYP2B6 CYP3A4, and ABCB1 in HIV, HCV, and HIV/HCV co-infected patients. The absence of correlation with liver damage, inflammation, and specific treatment interventions emphasises the need for additional research to elucidate the complex interplay between gene expression, viral co-infection, liver pathology, and therapeutic responses in these particular patients population.

Liver-Specific Bmal1 Depletion Reverses the Beneficial Effects of Nobiletin on Liver Cholesterol Homeostasis in Mice Fed with High-Fat Diet.

Nobiletin (NOB), a naturally occurring small-molecule compound abundant in citrus peels, has displayed potential lipid-lowering and circadian-enhancing properties in preclinical studies. However, the requirement of specific clock genes for the beneficial effects of NOB is not well understood. In the current study, mice with a liver-specific deletion of the core clock component, Bmal1-Bmal1LKO-were fed a high-fat diet (HFD) ad libitum for eight weeks, while NOB (200 mg/kg) was administered by daily oral gavage from the fifth week and throughout the last four weeks. NOB decreased liver triglyceride (TG) alongside the decreasing mRNA levels of de novo lipogenesis (DNL) genes in both Bmal1flox/flox and Bmal1LKO mice. NOB increased serum very low-density lipoprotein (VLDL) levels in Bmal1LKO mice, which was consistent with higher liver Shp and lower Mttp mRNA expression levels, the key genes that facilitate VLDL assembly and secretion. NOB decreased liver and serum cholesterol levels in the Bmal1flox/flox mice, consistent with lower Hmgcr and higher Cyp7a1, Cyp8b1, Gata4 and Abcg5 mRNA levels in the liver. In contrast, in the Bmal1LKO mice, NOB increased Hmgcr mRNA levels and had no effect on the above-mentioned genes related to bile acid synthesis and cholesterol excretion, which might contribute to the elevation of liver and serum cholesterol levels in NOB-treated Bmal1LKO mice. NOB inhibited hepatic DNL and decreased liver TG levels in HFD-fed mice independently of liver Bmal1, whereas liver-specific Bmal1 depletion reversed the beneficial effects of NOB on liver cholesterol homeostasis. The complex interactions between NOB, the circadian clock and lipid metabolism in the liver warrant further research.

Cloning and Characterization of Cyp7a1 and Cyp27a1 Genes from the Non-Parasitic Japanese Lamprey Lethenteron reissneri.

Two cytochrome P450 genes homologous to human CYP7A1 and CYP27A1 were cloned from the non-parasitic Japanese lamprey Lethenteron reissneri. Lamprey cyp7a1 mRNA had varied expression levels among individuals: about four orders of magnitude differences in larval liver and nearly three orders of magnitude differences in male adult liver. Overexpressed Cyp7a1 protein tagged with green fluorescent protein (GFP) was localized to the endoplasmic reticulum. Lamprey cyp27a1 mRNA had relatively constant expression levels: within two orders of magnitude differences in larvae and adult liver and intestine. GFP-tagged Cyp27a1 protein was localized to mitochondria. The expression profiles of lamprey cyp7a1 and cyp27a1 genes and the cellular localizations of their products were in good agreement with their counterparts in mammals, where these two P450s catalyze initial hydroxylation reactions of cholesterol in classical and alternative pathways of bile acid synthesis, respectively. The cyp7a1 mRNA levels in adult male liver showed significant negative correlations to both body weight and total length of the animal, implying the involvement of the gene in the production of female-attractive pheromones in sexually matured male livers. The lamprey Cyp7a1 contains a long extension of 116 amino acids between helices D and E of the protein. Possible roles of this extension in regulating the enzymatic activity of lamprey Cyp7a1 are discussed.

Alterations in gonadotropin, prolactin, androgen and estrogen receptor and steroidogenesis-associated gene expression in gander testes in relation to the annual period

Physiological mechanisms of seasonal changes in testicular function in birds are not fully elucidated. The balance between androgens and estrogens and testis sensitivity for gonadotropin and gonadal steroids are still unclear. The aim of the study was to examine: (1) the changes in circulating and intra-testicular steroid hormone levels and their relationship; (2) the mRNA expression of testicular gonadotropin, prolactin (PRL), progesterone (P4), androgen, and estrogen receptors, and (3) key steroidogenesis processes-related genes with immunofluorescent localization of aromatase in gander testes during the annual period. Testes from ganders (n = 25) in the first reproduction season were obtained at five breeding stages, i.e., prebreeding (PrB), peak of reproduction (PR), postbreeding (PoB), nonbreeding (NB), and onset of reproduction (OR). Males were kept under breeding conditions. It was found that plasma P4 levels decreased at the PoB and NB stages, whereas intra-testicular P4 was the highest in the NB stage. Intra-testicular estradiol (E2) levels were higher at the PoB and NB stages than the other stages, whereas testosterone (T) levels showed a nearly opposite pattern. The plasma estradiol–to–testosterone ratios were higher at the PrB, PoB and NB stages compared to other stages. The transcript abundances for luteinizing hormone receptor (LHR), PRL receptor (PRLR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) also change in testicular tissue during the annual period. Moreover, StAR mRNA expression was upregulated at the PoB and NB stages, and CYP11A1 transcript level was the highest at the PoB stage. Stage-dependent changes in the CYP19A1 mRNA and aromatase protein levels with higher abundances of transcript at PoB and NB stages and protein at the NB stage were observed. Localization and immunofluorescent signal intensity for aromatase also differed in relation to the examined stages. It may be suggested that differential E2 levels, as well as aromatase expression and localization across annual stages are responsible for the seasonal activation/inactivation stages of testis spermatogenesis in domestic ganders. These data strongly suggest a role of aromatase in the control of gander steroidogenesis as changes in this enzyme level are associated with alternation in gonadal steroid hormones. In addition, joint action with others hormones, like PRL and LH, seems to be important in the final effect of seasonal reproduction potential.

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.

Characterization of ovarian follicles, serum steroid hormone concentration, and steroidogenic gene expression profiles in the developing ovarian follicles in White King pigeons

Paired pigeons only lay 2 eggs in a laying period, which is closely related to ovarian follicle development, but this process is not well understood. In this study, 60 pairs of 12-mo-old White King pigeons were selected and serum and follicles were collected at 4 stages of laying interval (LI), including the first (LI1), the third (LI3), the fifth (LI5), and the seventh day (LI7). Morphological results showed that paired pigeons normally had 2 preovulatory follicles and the second-largest follicle (F2) developed from LI3 and had been selected in LI5. Prehierarchical follicles were coupled and hierarchical, which was in accordance with its clutch size. The P4 concentration increased gradually from LI1 to LI5, reaching a maximum of 30.67 ng/mL in LI5 and decreasing to 27.83 ng/mL in LI7 (P < 0.05). The levels of T in LI1 and LI5 were higher than LI3 and LI7 (P < 0.05), although there was no significant difference in E2 in LI (P > 0.05), but it stayed at high levels. In the TCs of the largest follicle (F1), HSD3B1 mRNA and HSD17B1 mRNA levels peaked in LI7. The expression pattern of CYP17A1 and CYP19A1 was similar, increasing from LI3 to LI5 and then decreasing. In the TCs of F2, the expressions of HSD3B1 and CYP17A1 had no significant difference between LI5 and LI7 (P > 0.05), while the expression pattern of HSD17B1 and CYP19A1 was the opposite. In TCs of SF1, HSD3B1 mRNA level peaked in LI3 while CYP19A1 mRNA levels peaked in LI7. The expression of CYP17A1 had a minor change (P > 0.05) and the expression pattern of HSD17B1 was similar to F1. It was concluded that the morphological characteristics of follicles during the LI for the first time, including the number and diameter of small follicles (SFs) and hierarchical follicles in pigeon and the concentrations of steroid hormones and expressions of steroidogenic genes in TCs of different follicles could explain the growth and selection of 2 preovulatory follicles. This study facilitates further research into the regulation of ovulation and egg production in pigeons.

Ginsenoside Rb1 for overcoming cisplatin-insensitivity of A549/DDP cells in vitro and vivo through the dual-inhibition on two efflux pumps of ABCB1 and PTCH1

BackgroundThe multi-drug resistance is an inherent weakness in the chemotherapeutics of non-small cell lung cancer occurring frequently all over the world. Clinically, ginseng and Chinese medicinal prescriptions including ginseng usually used as anti-tumor adjuncts due to its characteristic of qi-invigorating, which could improve the curative effect of chemotherapy drugs and reduce their toxic side effects. Triterpenoid saponins are the crucial active ingredients in Panax ginseng, and Ginsenoside Rb1 is of the highest quantities. However, the research on the tumor drug-resistance reversal effect and mechanism of ginsenoside Rb1 is still not clear. PurposeThis study aimed to systematically estimate the reversal activity of Ginsenoside Rb1 on cisplatin-insensitivity of A549/DDP cells and to reveal its prospective molecular mechanism. MethodsMTT assay were conducted to evaluate the reversal activity on cisplatin-insensitivity of A549/DDP cells of Ginsenoside Rb1in vitro, and the behavior was also studied by establishing a subcutaneous transplanted tumor model of A549/DDP in BALB/c-nu mice. In addition, P-gp ATPase activity assay, cisplatin accumulation assay, Annexin V-FITC apoptosis assay, real-time qPCR analysis and western blotting analysis were used to clarify the potential mechanism. ResultsGinsenoside Rb1 could effectively reverse the cisplatin-resistance of A549/DDP in vitro and vivo. And after the co-treatment of Ginsenoside Rb1 plus cisplatin, the accumulation of cisplatin increased in A549/DDP cells, which was accompanied with the down-regulation of the mRNA and protein expression levels of ABCB1, SHH, PTCH1 and GLI2. Besides, the apoptosis-inducing ability of cisplatin improved by the relative regulation on the protein expression level of Bax and Bcl-2. Far more importantly, the changes of CYP3A4 mRNA and protein levels were not significant. ConclusionGinsenoside Rb1 could increase the concentration of intracellular cisplatin and improve the insensitivity for cisplatin on A549/DDP cells. Even better, there was perhaps no unpredictable CYP3A4-mediated pharmacokinetic interactions after the combination of Ginsenoside Rb1 plus cisplatin. Ginsenoside Rb1 was a probable reversal agent for the cisplatin-insensitivity of A549/DDP cells, with a bifunction of inhibiting the efflux of two drug pumps (P-gp and PTCH1) by targeting ABCB1 and Hedgehog (Hh) pathway. In general, this research laid the groundwork for the development of a new reversal agent for the cisplatin-insensitivity of NSCLC.

Colonic mechanism of serum NAD+ depletion induced by DEHP during pregnancy

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer in polyvinyl chloride products such as feed piping, packing bag, and medical consumable. Our previous studies have demonstrated that DEHP exposure reduced the concentration of nicotinamide adenine dinucleotide (NAD+) in pregnant mice serum, which cuts off the source of NAD+ to placenta and results fetal growth restriction. However, the mechanism of serum NAD+ depletion by DEHP remains elusive. This study investigated the intestinal mechanism of NAD+ shortage-induced by DEHP in pregnant mice. The transcriptome results implicated that the mRNA level of oxidative response genes Cyp1a1, Gsto2, Trpv1 and Trpv3 were upregulated in colon. These changes induced intestinal inflammation. Transmission Electron Microscopy results displayed that DEHP destroyed the tight junctions and cell polarity of colonic epithelial cells. These dysfunctions diminished the expression of NAD+ precursor transporters SLC12A8, SLC5A8, SLC7A5, and the NAD+ biosynthetic key enzymes NAMPT, NMNAT1–3, and TDO2 in colonic epithelial cells. Analysis of the gut microbiota showed that DEHP led to the dysbiosis of gut microbiota, reducing the relative abundance of Prevotella copri which possesses the VB3 biosynthetic pathway. Therefore, maternal DEHP exposure during pregnancy decreased the transportation of NAD+ precursors from enteric cavity to colonic epithelial cells, and inhibited the synthesis of NAD+ in colonic epithelial cells. Meanwhile, DEHP reduced the NAD+ precursors provided by gut microbiota. Eventually, serum NAD+ content was lowered. Taken together, our findings provide a new insight for understanding the intestinal mechanisms by which DEHP affects serum NAD+ levels.

MiR-27b-3p inhibits estrogen secretion of goose granulosa cells by targeting CYP1B1 through the AMPK signaling pathway

Although miR-27b-3p has been evidenced to regulate the proliferation, apoptosis, and differentiation of a variety of mammalian cell types, its actions and mechanisms on ovarian cell steroidogenesis remains largely unknown in both mammalian and avian species. In this study, we aimed to determine the expression profiles of miR-27b-3p in granulosa cell layers during goose ovarian follicle development and to reveal its actions on estrogen (E2) secretion of goose granulosa cells as well as the underlying regulatory mechanisms. It was observed that miR-27b-3p was ubiquitously expressed throughout follicle development but exhibited much higher levels in hierarchical- than in prehierarchical follicles. In cultured granulosa cells from the fourth through second largest preovulatory (F4-F2) follicles of goose, up- and downregulation of miR-27b-3p by using its mimic and inhibitor significantly decreased and increased E2 secretion, respectively. Meanwhile, the mRNA levels of STAR and CYP19A1 were significantly reduced while those of CYP11A1 and 3βHSD were elevated in the mimic-transfected granulosa cells. By comparison, downregulation of miR-27b-3p enhanced the mRNA levels of STAR but had no significant effects on those of CYP19A1, CYP11A1, and 3βHSD. Results from bioinformatic prediction and luciferase reporter assay demonstrated that CYP1B1 was a downstream target of miR-27b-3p. Although the siRNA-mediated downregulation of CYP1B1 did not significantly change E2 secretion by goose granulosa cells, it reduced the mRNA levels of STAR and CYP19A1 as well as those of LKB1 and AMPKα, which are involved in the AMPK signaling pathway. Taken together, these data suggest that miR-27b-3p plays an inhibitory role in E2 secretion by goose F4-F2 granulosa cells, at least in part, by targeting CYP1B1 through the AMPK signaling pathway.

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.

GLP-2 Improves Hepatic Inflammation and Fibrosis in Mdr2-/- Mice Via Activation of NR4a1/Nur77 in Hepatic Stellate Cells and Intestinal FXR Signaling

Glucagon-like peptide (GLP)-2 may exert antifibrotic effects on hepatic stellate cells (HSCs). Thus, we aimed to test whether application of the GLP-2 analogue teduglutide has hepatoprotective and antifibrotic effects in the Mdr2/Abcb4-/- mouse model of sclerosing cholangitis displaying hepatic inflammation and fibrosis. Mdr2-/- mice were injected daily for 4 weeks with teduglutide followed by gene expression profiling (bulk liver; isolated HSCs) and immunohistochemistry. Activated HSCs (LX2 cells) and immortalized human hepatocytes and human intestinal organoids were treated with GLP-2. mRNA profiling by reverse transcription polymerase chain reaction and electrophoretic mobility shift assay using cytosolic and nuclear protein extracts was performed. Hepatic inflammation, fibrosis, and reactive cholangiocyte phenotype were improved in GLP-2-treated Mdr2-/- mice. Primary HSCs isolated from Mdr2-/- mice and LX2 cells exposed to GLP-2 invitro displayed significantly increased mRNA expression levels of NR4a1/Nur77 (P < .05). Electrophoretic mobility shift assay revealed an increased nuclear NR4a1 binding after GLP-2 treatment in LX2 cells. Moreover, GLP-2 alleviated the Tgfβ-mediated reduction of NR4a1 nuclear binding activity. Invivo, GLP-2 treatment of Mdr2-/- mice resulted in increased intrahepatic levels of muricholic acids (accordingly Cyp2c70 mRNA expression was significantly increased), and in reduced mRNA levels of Cyp7a1 and FXR. Serum Fgf15 levels were increased in Mdr2-/- mice treated with GLP-2. Accordingly, GLP-2 treatment of human intestinal organoids activated their FXR-FGF19 signaling axis. GLP-2 treatment increased NR4a1/Nur77 activation in HSCs, subsequently attenuating their activation. GLP-2 promoted intestinal Fxr-Fgf15/19 signaling resulting in reduced Cyp7a1 and increased Cyp2c70 expression in the liver, contributing to hepatoprotective and antifibrotic effects of GLP-2 in the Mdr2-/- mouse model.