Metabolites-Based Network Pharmacology to Preliminarily Verify In Vitro Anti-Inflammatory Effect of Ardisiacrispin B.

Ardisiae Crenatae Radix is an ethnic medicinal herb with good anti-inflammatory activity. Ardisiacrispin B is one of the main components in Ardisiae Crenatae Radix extract, with a content of up to 16.27%, and it may be one of the pharmacological components through which Ardisiae Crenatae Radix exerts anti-inflammatory activity. At present, reports on ardisiacrispin B mainly focus on anti-tumor effects, and there have been no reports on anti-inflammatory activities. As a triterpenoid saponin, due to its large molecular weight and complex structure, the composition of substances that function in the body may include other forms after metabolism, in addition to compounds with original structures. Exploring the anti-inflammatory effects on the prototypes and metabolites of the compound may provide a more comprehensive response to the characteristics of ardisiacrispin B's anti-inflammatory action. In this study, ardisiacrispin B was analyzed for metabolites to explore its metabolic processes in vivo. Subsequently, the anti-inflammatory effects of the prototypes and metabolites were further analyzed through network pharmacology, with the expectation of discovering the signaling metabolic pathways through which they may act. Finally, the anti-inflammatory effects of ardisiacrispin B in vitro and the effects on key signaling pathways at the protein level were explored. The results of this study showed that the isolated compounds were confirmed to be ardisiacrispin B. After the metabolite analysis, a total of 26 metabolites were analyzed, and the metabolism process in rats mainly involves oxidation, dehydration, glucuronide conjugation, and others. Speculation as to the anti-inflammatory molecular mechanisms of the prototypes and metabolites of ardisiacrispin B revealed that it may exert its anti-inflammatory effects mainly by affecting the PI3K-AKT pathway. Further anti-inflammatory mechanisms demonstrated that ardisiacrispin B had a good anti-inflammatory effect on LPS-induced RAW264.7 cells and a strong inhibitory effect on NO, TNF-α, and IL-1ß release in cells. Furthermore, it had significant inhibitory effects on the expression of PI3K, P-PI3K, AKT, and P-AKT. This study supplements the gaps in the knowledge on the in vivo metabolic process of ardisiacrispin B and explores its anti-inflammatory mechanism, providing an experimental basis for the development and utilization of pentacyclic triterpenoids.

Ptbp1 knockdown failed to induce astrocytes to neurons in vivo.

The conversion of non-neuronal cells to neurons is a promising potential strategy for the treatment of neurodegenerative diseases. Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons. However, some groups have disputed the interpretation of the data underlying the reported neuron conversion events. These controversies surrounding neuron conversion may be due to differences in the astrocyte fate-mapping systems. Here, we suppressed Ptbp1 using Cas13X and labelled astrocytes with an HA tag fused to Cas13X (Cas13X-NLS-HA). We found no astrocyte-to-neuron conversion in the mouse striatum via the HA-tagged labelling system compared with the GFAP-driven tdTomato labelling system (AAV-GFAP::tdTomato-WPRE) used in previous studies. Our findings indicate that Cas13X-mediated Ptbp1 knockdown failed to induce neuron conversion in vivo.

The pathogenesis and therapeutic strategies of heat stroke-induced myocardial injury.

Heat stroke (HS) is a febrile illness characterized by an elevation in the core body temperature to over 40°C, accompanied by central nervous system impairment and subsequent multi-organ dysfunction syndrome. In recent years, the mortality rate from HS has been increasing as ambient temperatures continue to rise each year. The cardiovascular system plays an important role in the pathogenesis process of HS, as it functions as one of the key system for thermoregulation and its stability is associated with the severity of HS. Systemic inflammatory response and endothelial cell damage constitute pivotal attributes of HS, other factors such as ferroptosis, disturbances in myocardial metabolism and heat shock protein dysregulation are also involved in the damage to myocardial tissue in HS. In this review, a comprehensively detailed description of the pathogenesis of HS-induced myocardial injury is provided. The current treatment strategies and the promising therapeutic targets for HS are also discussed.

Parametric MRI Detects Aristolochic Acid Induced Acute Kidney Injury.

Exposure to aristolochic acid (AA) is of increased concern due to carcinogenic and nephrotoxic effects, and incidence of aristolochic acid nephropathy (AAN) is increasing. This study characterizes renal alterations during the acute phase of AAN using parametric magnetic resonance imaging (MRI). An AAN and a control group of male Wistar rats received administration of aristolochic acid I (AAI) and polyethylene glycol (PEG), respectively, for six days. Both groups underwent MRI before and 2, 4 and 6 days after AAI or PEG administration. T2 relaxation times and apparent diffusion coefficients (ADCs) were determined for four renal layers. Serum creatinine levels (sCr) and blood urea nitrogen (BUN) were measured. Tubular injury scores (TIS) were evaluated based on histologic findings. Increased T2 values were detected since day 2 in the AAN group, but decreased ADCs and increased sCr levels and BUN were not detected until day 4. Significant linear correlations were observed between T2 of the cortex and the outer stripe of outer medulla and TIS. Our results demonstrate that parametric MRI facilitates early detection of renal injury induced by AAI in a rat model. T2 mapping may be a valuable tool for assessing kidney injury during the acute phase of AAN.

Design and synthesis of novel diosgenin-triazole hybrids targeting inflammation as potential neuroprotective agents.

Alzheimer's disease is a progressive neurodegenerative disease, and its incidence is expected to increase as the global population ages. Recent studies provide increasing evidence that inflammation plays a key role in the pathogenesis and progression of AD. Diosgenin, an active ingredient in Dioscorea nipponica Makino, is a promising bioactive lead compound in the treatment of Alzheimer's disease, which exhibited anti-inflammatory activity. To search for more efficient anti-Alzheimer agents, a series of novel diosgenin-triazolyl hybrids were designed, synthesized, and their neuroprotective effects against oxygen-glucose deprivation-induced neurotoxicity and LPS-induced NO production were evaluated. Most of these new hybrids displayed better activities than DIO. In particular, the promising compound L6 not only demonstrated an excellent neuroprotective effect but also showed the best anti-inflammatory activity. The structure-activity relationship study illustrated that the introduction of benzyl or phenyl triazole did improve the activity, and the introduction of benzyl triazole was better than that of phenyl triazole. The results we obtained showed that the diosgenin skeleton could be a promising structural template for the development of new anti-Alzheimer drug candidates, and compound L6 has the potential to be an important lead compound for further research.

Design, synthesis, and biological evaluation of diosgenin-indole derivatives as dual-functional agents for the treatment of Alzheimer's disease.

The complex pathogenesis of Alzheimer's disease (AD) has become a major obstacle in its treatment. An effective approach is to develop multifunctional agents that simultaneously target multiple pathological processes. Here, a series of diosgenin-indole compounds were designed, synthesized and evaluated for their neuroprotective effects against H2O2 (hydrogen peroxide), 6-OHDA (6-hydroxydopamine) and Aß (beta amyloid) damages. Preliminary structure-activities relationship revealed that the introduction of indole fragment and electron-donating group at C-5 on ring indole could be beneficial for neuroprotective activities. Results indicated that compound 5b was the most promising candidate against cellular damage induced by H2O2 (52.9 ± 1.9%), 6-OHDA (38.4 ± 2.4%) and Aß1-42 (54.4 ± 2.7%). Molecular docking study suggested the affinity for 5b bound to Aß1-42 was -40.59 kcal/mol, which revealed the strong binding affinity of 5b to Aß1-42. The predicted values of brain/blood partition coefficient (-0.733) and polar surface area (85.118 Å2) indicated the favorable abilities of BBB permeation and absorption of 5b. In addition, 5b significantly decreased ROS (reactive oxygen species) production induced by H2O2. In the following in vivo experiment, 5b obviously attenuated memory and learning impairments of Aß-injected mice. In summary, compound 5b could be considered as a promising dual-functional neuroprotective agent against AD.

Twin drug design, synthesis and evaluation of diosgenin derivatives as multitargeted agents for the treatment of vascular dementia.

A novel series of multitargeted molecules were designed and synthesized by combining the pharmacological role of cholinesterase inhibitor and antioxidant of steroid as potential ligands for the treatment of Vascular Dementia (VD). The oxygen-glucose deprivation (OGD) model was used to evaluate these molecules, among which the most potent compound ML5 showed the highest activity. Firstly, ML5 showed appropriate inhibition of cholinesterases (ChEs) at orally 15 mg/kg in vivo. The further test revealed that ML5 promoted the nuclear translocation of Nrf2. Furthermore, ML5 has significant neuroprotective effect in vivo model of bilateral common carotid artery occlusion (BCCAO), significantly increasing the expression of Nrf2 protein in the cerebral cortex. In the molecular docking research, we predicted the ML5 combined with hAChE and Keap1. Finally, compound ML5 displayed normal oral absorption and it was nontoxic at 500 mg/kg, po, dose. We can draw the conclusion that ML5 could be considered as a new potential compound for VD treatment.

Physiological system analysis of the kidney by high-temporal-resolution T2∗ monitoring of an oxygenation step response.

PURPOSE: Examine the feasibility of characterizing the regulation of renal oxygenation using high-temporal-resolution monitoring of the T2∗ response to a step-like oxygenation stimulus. METHODS: For T2∗ mapping, multi-echo gradient-echo imaging was used (temporal resolution = 9 seconds). A step-like renal oxygenation challenge was applied involving sequential exposure to hyperoxia (100% O2 ), hypoxia (10% O2 + 90% N2 ), and hyperoxia (100% O2 ). In vivo experiments were performed in healthy rats (N = 10) and in rats with bilateral ischemia-reperfusion injury (N = 4). To assess the step response of renal oxygenation, a second-order exponential model was used (model parameters: amplitude [A], time delay [Δt], damping constant [D], and period of the oscillation [T]) for renal cortex, outer stripe of the outer medulla, inner stripe of the outer medulla, and inner medulla. RESULTS: The second-order exponential model permitted us to model the exponential T2∗ recovery and the superimposed T2∗ oscillation following renal oxygenation stimulus. The in vivo experiments revealed a difference in Douter medulla between healthy controls (D < 1, indicating oscillatory recovery) and ischemia-reperfusion injury (D > 1, reflecting aperiodic recovery). The increase in Douter medulla by a factor of 3.7 (outer stripe of the outer medulla) and 10.0 (inner stripe of the outer medulla) suggests that this parameter might be rather sensitive to (patho)physiological oxygenation changes. CONCLUSION: This study demonstrates the feasibility of monitoring the dynamic oxygenation response of renal tissues to a step-like oxygenation challenge using high-temporal-resolution T2∗ mapping. Our results suggest that the implemented system analysis approach may help to unlock questions regarding regulation of renal oxygenation, with the ultimate goal of providing imaging means for diagnostics and therapy of renal diseases.

Design, synthesis and biological evaluation of novel osthole-based derivatives as potential neuroprotective agents.

A total of 26 compounds based on osthole skeleton were designed, synthesized. Their cytoprotective abilities of antioxidation, anti-inflammation and Aß42(Amyloid ß-protein 42)-induced neurotoxicity were evaluated by MTT assays. Mechanism of the action of selected compounds were investigated by molecular docking. AlogP, logS and blood-brain barrier (BBB) permeability of all these compounds were simulated by admetSAR. Most of the compounds showed better antioxidative and anti-inflammatory activities compared with osthole, especially OST7 and OST17. The compound OST7 showed relative high activity in neuroprotection against H2O2 (45.7 ± 5.5%), oxygen glucose deprivation (64.6 ± 4.8%) and Aß42 (61.4 ± 5.2%) at a low concentration of 10 µM. EC50 of selected compounds were measured in both H2O2 and OGD induced cytotoxicity models. Moreover, NO inhibiting ability of OST17(50.4 ± 7.1%) already surpassed the positive drug indomethacin. The structure activity relationship study indicated that introduction of piperazine group, tetrahydropyrrole group and aromatic amine group might be beneficial for enhancement of osthole neuroprotective properties. Molecular docking explained that the reason OST7 exhibited relatively stronger neuroprotection against Aß because of the greater area of interactions between molecule and target protein. OST7 and OST17 both provided novel methods to investigate osthole as anti-AD drugs.

Non-Gaussian Diffusion Models and T1 rho Quantification in the Assessment of Hepatic Sinusoidal Obstruction Syndrome in Rats.

BACKGROUND: Non-Gaussian diffusion models and T1 rho quantification may reflect the changes in tissue heterogeneity in hepatic sinusoidal obstruction syndrome (SOS). PURPOSE: To investigate the feasibility of diffusion kurtosis imaging (DKI), stretched exponential model (SEM), and T1 rho quantification in detecting and staging SOS in a monocrotaline (MCT)-induced rat model. STUDY TYPE: Animal study. POPULATION: Thirty male Sprague-Dawley rats gavaged with MCT to induce hepatic SOS and six male rats without any intervention. FIELD STRENGTH/SEQUENCE: 3.0T, DWI with five b-values (0-2000 s/mm2 ) and T1 rho with five spin lock times (1-60 msec). ASSESSMENT: MRI was performed 1 day before and 1, 3, 5, 7, and 10 days after MCT administration. The corrected apparent diffusion coefficient (Dapp ), kurtosis coefficient (Kapp ), distributed diffusion coefficient (DDC), and intravoxel water molecular diffusion heterogeneity (α) were calculated from the corresponding non-Gaussian diffusion model. The T1 rho value was calculated using a monoexponential model. Specimens obtained from the six timepoints were categorized into normal liver (n = 6), early-stage (n = 16), and late-stage (n = 14) SOS in accordance with the pathological score. STATISTICAL TESTS: Parametric statistical methods and receiver operating characteristic (ROC) curves were employed to determine diagnostic accuracy. RESULTS: The Dapp , Kapp , DDC, α, and T1 rho values were correlated with pathological score with r values of -0.821, 0.726, -0.828, -0.739, and 0.714 (all P < 0.001), respectively. DKI (combined Dapp and Kapp ) and SEM (combined DDC and α) were better than T1 rho for staging SOS. The areas under the ROC curve of DKI, SEM, and T1 rho for differentiating normal liver and early-stage SOS were 0.97, 1.00, and 0.79, whereas those of DKI, SEM, and T1 rho for differentiating early-stage and late-stage SOS were 1.00, 0.97, and 0.92, respectively. DATA CONCLUSION: DKI, SEM, and T1 rho may be helpful in staging SOS. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2 J. Magn. Reson. Imaging 2020;52:1110-1121.

Design, synthesis and evaluation of diosgenin carbamate derivatives as multitarget anti-Alzheimer's disease agents.

In order to produce an effective and multi-targeted clinical drug that could prevent progressive neurodegeneration, a series of diosgenin carbamate derivatives were designed, synthesized and tested for their anti-inflammatory, antioxidant and anti-Aß activities. The results demonstrated that compound M15 was the most promising derivative against inflammatory (NO inhibition 22.7 ± 2.2%,10 µM) and cellular damage induced by H2O2 (SH-SY5Y cell protection = 75.3 ± 3.4%, 10 µM) or Aß (astrocytes protection = 70.2 ± 6.5%, 10 µM). Molecular docking studies revealed the strong binding affinity of M15 to the active site of nNOS, Aß42 and pro-inflammatory proteins. Western blot demonstrated that M15 decreased IL-1ß, IL-6 and TNF-α level, which may contribute to its anti-inflammatory effects. In addition, M15 maintained mitochondrial function as well as cell viability through reducing H2O2-induced ROS production. The results indicated that oral administration of M15 attenuated memory deficits and played a neuroprotective effect on subcutaneous (s.c.) D-gal aging mice. In summary, M15 could be considered as a potential multifunctional neuroprotective agent due to the effects of anti-inflammatory, antioxidant and anti-Aß activities.

Synthesis and biological evaluation of 3-carbamate smilagenin derivatives as potential neuroprotective agents.

Studies indicated that smilagenin, isolated from Anemarrhena asphodeloides Bunge, could improve cognitive impairment and exhibit neuroprotective activity. On the basis of the structure of smilagenin, a series of derivatives were synthesized and evaluated for their neuroprotective effects of H2O2-induced, oxygen glucose deprivation-induced neurotoxicity in SH-SY5Y cells and LPS-induced NO production in RAW264.7 cells. Structure activity relationship of derivatives revealed that benzyl-substituted piperazine formate derivatives showed the potent neuroprotective activity such as A12. These findings may provide new insights for the development of neuroprotective agents against Alzheimer's disease.

[Evaluation of renal oxygenation in rats with acute aristolochic acid nephropathy using blood oxygenation level-dependent magnetic resonance imaging].

OBJECTIVE: To evaluate the changes in renal oxygenation in rats with acute aristolochic acid nephropathy using blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) at 7.0T. METHODS: Wistar rats were randomly divided into AAN group (n=18) and control group (n=6) for intraperitoneal injections of AAI at 40 mg/kg and PEG400, respectively, on a daily basis for 6 consecutive days. All the control rats and 6 rats from AAN group underwent BOLD MRI scan before and at 2, 4, and 6 days after the initial injection for measuring renal cortical and medullary R2* values. At each of the 4 time points, 3 rats in AAN group were sacrificed for histological evaluation; the control rats were examined at 6 days after the initial injection. RESULTS: The cortical and medullary R2* values of the rats in AAN group on days 4 and 6 were significantly higher than those in the control group (P < 0.05). In AAN group, the cortical R2* values showed no obvious changes on day 2 as compared with the baseline values, but increased significantly on day 4 (P < 0.05) and day 6 (P < 0.01); the medullary R2* values increased progressively and were significantly higher than the baseline values on day 4 (P < 0.01) and day 6 (P < 0.01). In the control group, no significant changes were detected in either cortical or medullary R2* values throughout the experiment. CONCLUSIONS: BOLD MRI allows non-invasive measurement of renal oxygenation levels in rats with AAN. The increase of renal cortical and medullary R2* values, and particularly the latter, indicates a lowered renal oxygenation level, which provides potentially useful information for clinical decisions.

Design, synthesis and biological evaluation of 3-piperazinecarboxylate sarsasapogenin derivatives as potential multifunctional anti-Alzheimer agents.

A series of multifunctional 3-piperazinecarboxylate sarsasapogenin derivatives were designed and synthesized against Alzheimer's disease (AD). The protection against H2O2-triggered oxidative stress in PC12 cells, and inhibition on LPS-induced NO production in RAW264.7 cell lines in vitro by these derivatives were firstly evaluated. Most of the compounds showed better antioxidant and antiinflammatory activities compared with sarsasapogenin, especially AA34 and AA36. Structure-activity relationships revealed that benzyl group, electron-donating group and intramolecular hydrogen bond might be beneficial to enhancing their neuroprotective activities. Moreover, Aß42 was the optimum predicted target based on the high 3D molecular similarity between compound AA36 and caprospinol. In the following experiments, AA36 significantly protected PC12 cells from Aß-induced damage and improved learning and memory impairments in Aß-injected mice. Thus AA36 is regarded as a potent anti-AD agent and N-substituted piperazinecarboxylate can be served as a promising structural unit for anti-AD drug design.

[Differential diagnosis of hepatocellular carcinoma and hepatic hemangiomas based on radiomic features of gadoxetate disodium-enhanced magnetic resonance imaging].

OBJECTIVE: To evaluate the feasibility of using radiomic features for differential diagnosis of hepatocellular carcinoma (HCC) and hepatic cavernous hemangioma (HHE). METHODS: Gadoxetate disodium-enhanced magnetic resonance imaging data were collected from a total of 135 HCC and HHE lesions. The radiomic texture features of each lesion were extracted on the hepatobiliary phase images, and the performance of each feature was assessed in differentiation and classification of HCC and HHE. In multivariate analysis, the performance of 3 feature selection algorithms (namely minimum redundancy-maximum relevance, mRmR; neighborhood component analysis, NCA; and sequence forward selection, SFS) was compared. The optimal feature subset was determined according to the optimal feature selection algorithm and used for testing the 3 classifier algorithms (namely the support vector machine, RBF-SVM; linear discriminant analysis, LDA; and logistic regression). All the tests were repeated 5 times with 10-fold cross validation experiments. RESULTS: More than 50% of the radiomic features exhibited strong distinguishing ability, among which gray level co-occurrence matrix feature S (3, -3) SumEntrp showed a good classification performance with an AUC of 0.72 (P<0.01), a sensitivity of 0.83 and a specificity of 0.57. For the multivariate analysis, 15 features were selected based on the SFS algorithm, which produced better results than the other two algorithms. Testing of these 15 selected features for their average cross-validation performance with RBF-SVM classifier yielded a test accuracy of 0.82∓0.09, an AUC of 0.86∓0.12, a sensitivity of 0.88∓0.11, and a specificity of 0.76∓0.18. CONCLUSION: The radiomic features based on gadoxetate disodium-enhanced magnetic resonance images allow efficient differential diagnosis of HCC and HHE, and can potentially provide important assistance in clinical diagnosis of the two diseases.

Cell-based and in silico evidence against quercetin and structurally-related flavonols as activators of vitamin D receptor.

It has been reported that quercetin is an activator of rat vitamin D receptor (rVDR). However, the conclusion was based on experiments performed without all the appropriate control groups, raising the possibility of a false-positive finding. Furthermore, distinct differences exist in the chemical structures of quercetin and 1α,25-dihydroxyvitamin D3, which is a prototypic agonist of VDR. Therefore, we investigated systematically whether quercetin and other flavonols are agonists of rVDR, mouse VDR (mVDR), or human VDR (hVDR). Quercetin, 3-hydroxyflavone, galangin, datiscetin, kaempferol, morin, isorhamnetin, tamarixetin, myricetin, and syringetin did not activate rVDR, mVDR, or hVDR in HEK-293 and HepG2 cells transfected with the corresponding receptor expression plasmid and either the secreted phosphoprotein 1 (Spp1) or cytochrome P450 24A1 (CYP24A1) reporter plasmid, when compared to the respective empty vector control group transfected with one or the other reporter plasmid and treated with one of the flavonols. Control analysis indicated that lithocholic acid and 1α,25-dihydroxyvitamin D3, but not rifampicin, activated rVDR, mVDR, and hVDR. As shown in transfected HEK293 and HepG2 cells, the flavonols did not influence hVDR ligand binding domain transactivation, steroid receptor coactivator-1 recruitment, or hVDR target gene expression (transient receptor potential cation channel 6 and CYP24A1) in hVDR-expressing Caco-2 or LS180 cells. The cumulative data from the cell-based experiments were corroborated by results obtained from molecular docking analysis. In conclusion, quercetin, 3-hydroxyflavone, galangin, datiscetin, kaempferol, morin, isorhamnetin, tamarixetin, myricetin, and syringetin are not agonists of rVDR, mVDR, or hVDR, as judged by cell-based and in silico evidence.

Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli.

Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to pilus assembly. Type IV pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence pilus assembly and retraction, but their precise functions are not well defined. The Type IV pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for pilus assembly, and CofB can be detected in the pilus fraction. We solved the 2.0 Å crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate pilus assembly. We propose a model for CofB-initiated pilus assembly with implications for understanding filament growth in more complex Type IV pilus systems as well as the related Type II secretion system.

Retinoic acid biosynthesis is impaired in human and murine endometriosis.

Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1(-/-) mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1(-/-) mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an "endometriosis phenotype" that enables cells to implant and grow at ectopic sites.

Evaluation of Ginkgo biloba extract as an activator of human glucocorticoid receptor.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba, which is one of the most frequently used herbal medicines, is commonly used in the management of several conditions, including memory impairment. Previously, it was reported to decrease the expression of peripheral benzodiazepine receptor and the biosynthesis of glucocorticoids, thereby regulating glucocorticoid levels. However, it is not known whether Ginkgo biloba extract regulates the function of the glucocorticoid receptor. AIM OF THE STUDY: We determined whether Ginkgo biloba extract and several of its chemical constituents affect the activity of human glucocorticoid receptor (hGR). MATERIALS AND METHODS: A hGR-dependent reporter gene assay was conducted in HepG2 human hepatocellular carcinoma cells and hGR target gene expression assays were performed in primary cultures of human hepatocytes. RESULTS: Multiple lots and concentrations of the extract and several of its chemical constituents (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide) did not increase hGR activity, as assessed by a cell-based luciferase reporter gene assay. The extract did not influence the expression of hGR target genes, including tyrosine aminotransferase (hTAT), constitutive androstane receptor (hCAR), or pregnane X receptor (hPXR), in primary cultures of human hepatocytes. Moreover, hGR antagonism by mifepristone (also known as RU486) did not attenuate the extent of induction of hCAR- and hPXR-regulated target genes CYP2B6 and CYP3A4 by Ginkgo biloba extract. CONCLUSION: Ginkgo biloba extract, ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, and bilobalide are not activators of hGR. Furthermore, the extract does not influence the hGR-hCAR or the hGR-hPXR signaling pathway in primary cultures of human hepatocytes.

Selective agonism of human pregnane X receptor by individual ginkgolides.

Ginkgolide A, ginkgolide B, ginkgolide C, and ginkgolide J are structurally related terpene trilactones present in Ginkgo biloba extract. Pregnane X receptor (PXR), glucocorticoid receptor (GR), and constitutive androstane receptor (CAR) regulate the expression of genes involved in diverse biological functions. In the present study, we investigated the effects of individual ginkgolides as single chemical entities on the function of human PXR (hPXR), human GR (hGR), and human CAR (hCAR). In cell-based reporter gene assays, none of the ginkgolides activated hGR or hCAR (wild-type and its SV23, SV24, and SV25 splice variants). Concentration-response experiments showed that ginkgolide A and ginkgolide B activated hPXR and rat PXR to a greater extent than ginkgolide C, whereas ginkgolide J had no effect. As determined by a time-resolved fluorescence resonance energy transfer competitive binding assay, ginkgolide A and ginkgolide B, but not ginkgolide C or ginkgolide J, were shown to bind to the ligand-binding domain of hPXR, consistent with molecular docking data. Compared with tetraethyl 2-(3,5-di-tert-butyl-4-hydroxyphenyl)ethenyl-1,1-bisphosphonate (SR12813) (a known agonist of hPXR), ginkgolide A and ginkgolide B were considerably less potent in binding to hPXR. These two ginkgolides recruited steroid receptor coactivator-1 to hPXR and increased hPXR target gene (CYP3A4) expression, as assessed by a mammalian two-hybrid assay and real-time polymerase chain reaction, respectively. In conclusion, the individual ginkgolides regulate the function of nuclear receptors in a receptor-selective and chemical-dependent manner. This study identifies ginkgolide A and ginkgolide B as naturally occurring agonists of hPXR and provides mechanistic insight into the structure-activity relationship in ligand activation of hPXR.