Preliminary Study on Total Component Analysis and In Vitro Antitumor Activity of Eucalyptus Leaf Residues.

Eucalyptus globulus is widely introduced and cultivated in Yunnan province. Its foliage is mainly used to extract eucalyptus oil, but the by-product eucalyptus residue has not been fully utilized. Based on the above reasons, in this study, we sought to explore the comprehensive utilization potential of eucalyptus resources. The total composition of eucalyptus residue was analyzed by ultra performance liquid chromatography-time-of-flight mass spectrometry (UPLC-Q/TOF MS), and the active components and nutrient components of eucalyptus leaf residue were determined by chemical methods and liquid phase techniques. Meanwhile, the antitumor activity of triterpenoids in eucalyptus leaves was evaluated by tetramethylazazole blue colorimetric assay (MTT). The results of qualitative analysis indicated that 55 compounds were identified from eucalyptus residue, including 28 phloroglucinols, 17 terpenoids, 3 flavonoids, 5 fatty acids, 1 amino acid and 2 polyphenols. Among them, the pentacyclic triterpenoids, in eucalyptus residue, were mainly oleanane type and urthane type. The results of quantitative determination indicated that the content of triterpenoid compounds was 2.84% in eucalyptus residue, which could be enhanced to 82% by silicone separation. The antitumor activity results showed that triterpenoid compounds have moderate inhibitory effects on human breast cancer cell MDA-MB-231, gastric adenocarcinoma cell SGC-7901 and cervical cancer cell Hela. The half maximal inhibitory concentration (IC50) was 50.67, 43.12 and 42.65 µg/mL, respectively. In this study, the triterpenoids from eucalyptus leaf residues were analyzed to reveal that the triterpenoids from eucalyptus leaf have antitumor effects and have potential to be developed as antitumor drugs.

The time-dependent expression of FPR2 and ANXA1 in murine deep vein thrombosis model and its relation to thrombus age.

Thrombus age determination in fatal venous thromboembolism cases is an important task for forensic pathologists. In this study, we investigated the time-dependent expressions of formyl peptide receptor 2 (FPR2) and Annexin A1 (ANXA1) in a stasis-induced deep vein thrombosis (DVT) murine model, with the aim of obtaining useful information for thrombus age timing. A total of 75 ICR mice were randomly classified into thrombosis group and control group. In thrombosis group, a DVT model was established by ligating the inferior vena cava (IVC) of mice, and thrombosed IVCs were harvested at 1, 3, 5, 7, 10, 14, and 21 days after modeling. In control group, IVCs without thrombosis were taken as control samples. The expressions of FPR2 and ANXA1 during thrombosis were detected using immunohistochemistry and double immunofluorescence staining. Their protein and mRNA levels in the samples were determined by Western blotting and quantitative real-time PCR. The results reveal that FPR2 was predominantly expressed by intrathrombotic neutrophils and macrophages. ANXA1 expression in the thrombi was mainly distributed in neutrophils, endothelial cells of neovessels, and fibroblastic cells. After thrombosis, the expressions of FPR2 and ANXA1 were time-dependently up-regulated. The percentage of FPR2-positive cells and the level of FPR2 protein significantly elevated at 1, 3, 5 and 7 days after IVC ligation as compared to those at 10, 14 and 21 days after ligation (p < 0.05). Moreover, the mRNA level of FPR2 were significantly higher at 5 days than that at the other post-ligation intervals (p < 0.05). Besides, the levels of ANXA1 mRNA and protein peaked at 10 and 14 days after ligation, respectively. A significant increase in the mRNA level of ANXA1 was found at 10 and 14 days as compared with that at the other post-ligation intervals (p < 0.01). Our findings suggest that FPR2 and ANXA1 are promising as useful markers for age estimation of venous thrombi.

Time-Dependent Sequential Changes of IL-10 and TGF-ß1 in Mice with Deep Vein Thrombosis.

OBJECTIVES: To detect the expression changes of interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) during the development of deep vein thrombosis in mice, and to explore the application value of them in thrombus age estimation. METHODS: The mice in the experimental group were subjected to ligation of inferior vena cava. The mice were sacrificed by excessive anesthesia at 1 d, 3 d, 5 d, 7 d, 10 d, 14 d and 21 d after ligation, respectively. The inferior vena cava segment with thrombosis was extracted below the ligation point. The mice in the control group were not ligated, and the inferior vena cava segment at the same position as the experimental group was extracted. The expression changes of IL-10 and TGF-ß1 were detected by immunohistochemistry (IHC), Western blotting and real-time qPCR. RESULTS: IHC results revealed that IL-10 was mainly expressed in monocytes in thrombosis and TGF-ß1 was mainly expressed in monocytes and fibroblast-like cells in thrombosis. Western blotting and real-time qPCR showed that the relative expression levels of IL-10 and TGF-ß1 in each experimental group were higher than those in the control group. The mRNA and protein levels of IL-10 reached the peak at 7 d and 10 d after ligation, respectively. The mRNA expression level at 7 d after ligation was 4.72±0.15 times that of the control group, and the protein expression level at 10 d after ligation was 7.15±0.28 times that of the control group. The mRNA and protein levels of TGF-ß1 reached the peak at 10 d and 14 d after ligation, respectively. The mRNA expression level at 10 d after ligation was 2.58±0.14 times that of the control group, and the protein expression level at 14 d after ligation was 4.34±0.19 times that of the control group. CONCLUSIONS: The expressions of IL-10 and TGF-ß1 during the evolution of deep vein thrombosis present time-dependent sequential changes, and the expression levels of IL-10 and TGF-ß1 can provide a reference basis for thrombus age estimation.

Acute cold stress induces intestinal injury via CIRP-TLR4-IRE1 signaling pathway in pre-starter broilers.

BACKGROUND: Cold stress is a common environmental stress in broiler chicks. Cold-inducible RNA-binding protein (CIRP) is a conserved cold shock protein that can regulate inflammatory response through Toll-like receptor 4 (TLR4). The mechanism that how CIRP involves in the regulation of cold stress in broilers remains unclear. METHODS AND RESULTS: In this study, 360 7-day-old healthy male SZ901 chicks were selected and randomly allocated to four groups, and then subjected to acute cold exposure at the ambient temperature of 12 ± 1 °C for 0 h, 4 h, 8 h, and 12 h, respectively. After cold exposure, abdominall skin temperature, gene expression of CIRP-TLR4-IRE1 signaling pathway in ileum mucosa, and small intestinal structure were measured. The results showed that cold exposure decreased abdominall skin temperature, upregulated the gene expression of endoplasmic reticulum stress (ERS) markers IRE1, inflammatory factors IL-1ß, IL-6, IL-10, TNF-α, and tight junction proteins ZO-1 and Occludin in ileum of chicks compared with the control group with no (0 h) cold exposure. Compared with the control group, a long time cold exposure upregulated the gene expression of CIRP, TLR4, GRP78, NF-κB in ileum mucosa, and decreased the villus height and V/C of small intestine. CONCLUSIONS: The above results suggest that acute cold stress induces endoplasmic reticulum stress via upregulating the gene expression of CIRP-TLR4-IRE1 signaling pathway, and results in the structural damage of chick intestine.

Mechanisms of in-hospital acute ischemic stroke and their relevance to prognosis: A retrospective analysis.

OBJECTIVES: In-hospital stroke (IHS) is common and has a poor prognosis. Limited data were about the mechanisms of IHS, posing a challenge in taking measures to prevent stroke during hospitalization. This study aims to investigate the mechanisms of IHS and their relevance to prognosis. MATERIALS AND METHODS: Patients with in-hospital acute ischemic stroke at Peking Union Medical College Hospital from June 2012 to April 2022 were consecutively enrolled. The Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification of stroke and detailed mechanisms were evaluated by two experienced neurologists. Functional outcome at discharge was evaluated. RESULTS: A total of 204 IHS patients were included, with a median age of 64 (IQR 52-72) and 61.8% male. The most common mechanism was embolism (57.8%), followed by hypoperfusion (42.2%), hypercoagulation (36.3%), small vessel mechanism (19.1%), discontinuation of antithrombotic drugs (13.2%), and iatrogenic injury (9.8%). Iatrogenic injury (P = 0.001), hypoperfusion (P = 0.006), embolism (P = 0.03), and discontinuation of antithrombotic drugs (P = 0.004) were more common in perioperative stroke compared to non-perioperative stroke. Median NIHSS improvement (2 vs 1, P = 0.002) and median mRS improvement (1 vs 0.5, P = 0.02) at discharge were higher in perioperative patients. Advanced age and higher NIHSS at onset were significantly associated with a poorer prognosis, whereas embolism mechanism was associated with a better prognosis. CONCLUSIONS: The etiologies and mechanisms of IHS are complex. Perioperative and non-perioperative IHS have different mechanisms and prognostic features. Determining the causes and mechanisms of IHS will help to identify the population at risk and prevent stroke appropriately during hospitalization.

Comparative assessment on ammonia nitrogen adsorption onto a saline soil-groundwater environment: distribution, multi-factor interaction, and optimization using response surface methodology and artificial neural network.

The adsorption of soil can reduce the leaching of NH4+-N from the external environment into groundwater. The adsorption of NH4+-N is affected by many factors. It is critical to use statistical model to quantitatively describe the effects of interaction between two or more factors on the system response. In this study, HJ-Biplot was used to analyze the correlation characteristics of soil water, salt, and nitrogen, and the response surface methodology and artificial neural network were used to statistically visualize the interaction between factors, including concentration, total dissolved solids (TDS), temperature, and pH. The results showed that the study soil was a typical saline soil, with maximum soil NH4+-N content of 85.45 mg/kg. For the adsorption experiments of NH4+-N on saline soils, the effects of factors on the adsorption capacity were assessed using the RSM model. The RSM model was coupled with an ANN to predict the adsorption of NH4+-N by saline soils. The NH4+-N concentration and water pH were both significant at a linear level (p < 0.0001). The interaction between NH4+-N concentration and pH was also more significant (p < 0.01). Under optimal conditions (concentration: 800 mg/L; temperature: 24 °C; TDS: 637 mg/L; pH: 7.83), the NH4+-N adsorption capacity was 1650.2 ug/g, which was in general agreement with the calculated values from the Box-Behnken and RSM model. In addition, a statistical error criterion for the model showed that the RSM-ANN model had greater predictive ability than RSM model.

Analysis of 5-Year-old children's oral health service utilization and influencing factors in Guizhou Province, China (2019-2020).

BACKGROUND: This study aimed to investigate the utilization patterns and factors related to oral health care for 5-year-old preschoolers based on Andersen's Behavioural Model in Guizhou Province, Western China. METHOD: A cross-sectional study of 4,862 5-year-old preschoolers in 66 kindergartens was conducted in 2019 and 2020. A basic oral examination and a survey of parents and grandparents were conducted to gather data on oral health services. The results were analysed using chi-square tests and logistic regression analysis. RESULT: The utilization rate of oral health services for children in Guizhou province was 20.5%. The dmft was 4.43, and the rate of caries was 72.2%. The average cost of a dental visit was higher in rural areas and higher for girls. Logistic regression analysis revealed that dmft ≥ 6 teeth, a history of toothache, starting toothbrushing at age ≤ 3 years and limited parental knowledge were the primary factors impacting dental visits. CONCLUSION: Needs factors such as severe oral conditions and pain in children are the main reasons for the utilization of these services. This study underscores the urgency to actively promote the importance of oral health and expand insurance coverage for oral health services.

Health risk assessment of groundwater nitrogen pollution in Songnen Plain.

Access to safe drinking water is one of the fundamental human rights and an important part of healthy living. This study considered various land use methods, used geostatistical analysis, and triangular random model to explore nitrogen pollution and estimate its potential risk to human health for local populations in Songnen Plain of Northeast China and recognize parameter uncertainties. Nitrate concentrations in groundwater ranged from 0.01 to 523.45 mg/L, more than 72.35% of the samples exceeded Grade III threshold (20 mg/L of N) as per China's standard, and nitrate nitrogen content is greater than 20 mg/L accounted for around 60% of the research area, mainly distributed in the eastern and central high plain area. The nitrate-nitrogen content of groundwater in the town land was significantly higher than that of agricultural land, and the ammonia nitrogen content was conversely. The townland's risk value was two times that of agricultural land, considering different land use methods would avoid overestimating or underestimating regional risk value. Non-carcinogenic risks (HI) of two land use were above the safety level (i.e., HI > 1), suggesting that groundwater nitrate would have significant health effects on the age groups, and further threaten children. There was a wide range of fluctuations in the uncertainty of nitrogen concentration and model evaluation parameters; triangular random model was more sensitive to data changes, which could reduce the uncertainty. The contribution rate of nitrate-nitrogen concentration to risk was above 90%, which explained the need for random sampling to improve the evaluation results reliability. The findings in this paper will provide new insight for solving uncertainties in water safety management.

Copper-Catalyzed Highly Selective Protoboration of CF3 -Containing 1,3-Dienes.

The copper-catalyzed highly selective protoboration of CF3 -containing conjugated diene with proton source and B2 Pin2 has been developed. This chemistry could suppress the well-known defluorination and provide borated reagents with an intact CF3 -group. Further studies indicated that the functional group tolerance of this chemistry is very well, and the products could be used as versatile precursors for different types of transformations. Importantly, using chiral diphosphine ligand, we have developed the first example for using such starting material to synthesis allylic boron-reagents which bearing a CF3 -containing chiral center. Notably, the reaction mechanism was intensively studied by DFT calculations, which could reveal the reason that defluorination was inhibited.

Mechanistic insights into the effects of SREBP1c on hepatic stellate cell and liver fibrosis.

Sterol regulatory element-binding protein 1c (SREBP1c) plays key roles in maintenance of hepatic stellate cell (HSC) quiescence. The present researches investigated the mechanisms underlying the effects of SREBP1c on HSCs and liver fibrogenesis by HSC-targeted overexpression of the active SREBP1c using adenovirus in vitro and in vivo. Results demonstrated that SREBP1c exerted inhibitory effects on TAA-induced liver fibrosis. SREBP1c down-regulated TGFß1 level in liver, reduced the receptors for TGFß1 and PDGFß, and interrupted the signalling pathways of Smad3 and Akt1/2/3 but not ERK1/2 in HSCs. SREBP1c also led to the decreases in the protein levels of the bromodomain-containing chromatin-modifying factor bromodomain protein 4, methionine adenosyltransferase 2B (MAT2B) and TIMP1 in HSCs. In vivo activated HSCs did not express cyclin D1 and cyclin E1 but SREBP1c down-regulated both cyclins in vitro. SREBP1c elevated PPARγ and MMP1 protein levels in the model of liver fibrosis. The effect of SREBP1c on MAT2B expression was associated with its binding to MAT2B1 promoter. Taken together, the mechanisms underlying the effects of SREBP1c on HSC activation and liver fibrosis were involved in its influences on TGFß1 level, the receptors for TGFß1 and PDGFß and their downstream signalling, and the molecules for epigenetic regulation of genes.

Toxicity of Two Different Size Ceria Nanoparticles to Mice After Repeated Intranasal Instillation.

Ceria nanoparticles (nanoceria) have been used as a fuel-borne catalyst, and are emitted in the particulate phase in the diesel exhaust. To evaluate the potential health risks of airborne nanoceria, the pulmonary and systemic consequences of nanoceria to ICR mice were studied under repeated transnasal instillation. Two different size nanoceria (7-nm and 25-nm) were chose, representing the nanoceria mostly used as fuel additives and the combustion products of the very nanoceria, respectively. As the first target organ of nanoceria, the lungs suffered serious damages manifested in the results of their biochemical and pathological examinations. The results demonstrated the penetration of nanoceria through air-blood barrier after the lung burden. Nanoceria were further transferred to the secondary target organs, mainly the liver and spleen. Nanoceria could also arrive at the central neural system via olfactory nerve pathway. The systemic accumulation of nanoceria finally triggered lipid peroxidation in multiple organs. Generally, the smaller nanoceria induced more severe pulmonary damage but similar systemic toxicity when compared with the larger one. These findings imply that using nanoceria as diesel fuel catalyst may cause health concerns. Additional studies are warranted to investigate the pulmonary and systemic health consequences of nanoceria additive to diesel fuel.

Colorimetric and Ratiometric Chemosensor for Visual Detection of Gaseous Phosgene Based on Anthracene Carboxyimide Membrane.

In this work, we reported an anthracene carboxyimide-based chemosensor (AC-Phos) for colorimetric and ratiometric fluorescence detection of highly toxic phosgene, which displayed rapid response (<5 min) toward phosgene with a high selectivity and a low detection limit (2.3 nM). Furthermore, a facile testing membrane with a polystyrene immobilizing chemosensor has been fabricated for real-time visualizing of gaseous phosgene.

Magnetic solid-phase extraction of benzoylurea insecticides by Fe3 O4 nanoparticles decorated with a hyper-cross-linked porous organic polymer.

A magnetic polytriphenylamine porous organic polymer was prepared through simple self-polycondensation of triphenylamine followed by coprecipitation with Fe2+ and Fe3+ . It was applied as a magnetic adsorbent for the extraction of six benzoylurea insecticides from tomato, cucumber, and watermelon samples before their high-performance liquid chromatography and mass spectral detection. Under the optimized experimental conditions, the established method gave a low limit of detection ranging from 0.05 to 0.1 ng/g and a good linear response ranging from 0.2 to 40 ng/g with coefficients of determination >0.99. The method recoveries for spiked analytes at the concentrations of 3 and 15 ng/g in real samples were in the range of 87.7-106.7% with the relative standard deviations <6.4%. The results indicated that it had a good adsorption capability toward the target analytes due to the π-stacking and hydrogen bonding interactions. The polymer material showed great potential in the efficient extraction of organic compounds from real samples with complex matrixes.

Sex-determining region Y-box 9 acts downstream of NADPH oxidase to influence the effect of leptin on PPARγ1 expression in hepatic stellate cells.

Leptin, an adipocyte-derived hormone, promotes liver fibrogenesis and inhibits the expression of peroxisome-proliferator activated receptor γ (PPARγ), a key transcription factor in inhibition of hepatic stellate cell (HSC) activation, in HSCs. This research aimed to further investigate the mechanisms underlying leptin regulation of PPARγ1 in HSCs in vivo and in vitro. Results demonstrated that sex-determining region Y-box 9 (Sox9) could bind to a site around -2275 within leptin response region of PPARγ1 promoter and inhibited PPARγ1 expression. Sox9 upregulated the expressions of α1(I)collagen and alpha-smooth muscle actin in HSCs. Leptin stimulated Sox9 expression and Sox9 binding to PPARγ1 promoter. The signaling pathways of NADPH oxidase, ß-catenin, and delta-like homolog1 (DLK1) mediated leptin upregulation of Sox9 expression. Moreover, there existed crosstalk between NADPH oxidase pathway and ß-catenin or DLK1 signaling pathway. Human liver specimens of cirrhosis were shown to be of a large number of the positive HSCs for p47phox (playing a central role in NADPH oxidase activity), 4-hydroxynonenal (a lipid peroxidation product), Sox9, and α-smooth muscle actin whereas PPARγ-positive HSCs were rarely detected. These results might deepen understanding of the molecular mechanisms for leptin inhibition of PPARγ1 expression in HSCs and for the liver fibrosis associated with leptin.

Simultaneous inhibition of NMDA and mGlu1/5 receptors by levo-corydalmine in rat spinal cord attenuates bone cancer pain.

Bone cancer pain is a challenge for its not completely clarified mechanism and broad clinical morbidity. Therefore, novel and more effective drugs are urgent needed for improvement of patients' quality of life. Glutamate receptors have been associated with the development of the central sensitization of chronic pain. Inhibition of N-methyl-d-aspartate (NMDA) and metabotropic glutamate (mGlu) receptors can effectively attenuate bone cancer pain, respectively. Herein, our results indicated that levo-Corydalmine (l-CDL), a compound from Corydalis yanhusuo W.T. Wang, which has been used in traditional Chinese medicine for pain relief could effectively attenuate bone cancer pain induced by tibia bone cavity tumor cell implantation (TCI) through simultaneously inhibiting the NMDA and mGlu1/5 receptors in rat spinal cord without notable side effects. Both intragastric and intrathecal administration of l-CDL significantly alleviated the mechanical hypersensitivity induced by TCI in rats, and the analgesic effect of l-CDL could be reversed by intrathecal administration of NMDA receptor agonist NMDA and mGlu1/5 receptor agonist DHPG but not AMPA receptor agonist AMPA. l-CDL could also selectively suppress NMDA and DHPG induced rapid rise in Ca2+ oscillations in primary cultures neurons of spinal cord in vitro. The antinociception of l-CDL were partially mediated by the reduced phosphorylation of PKC γ and ERK1/2 in spinal cord of TCI rats in a NMDA and mGlu1/5 dependent manner. In conclusion, these results suggested that l-CDL attenuates TCI induced bone cancer pain through simultaneously inhibiting the NMDA and mGlu1/5 receptors and the downstream PKC γ, ERK1/2 signaling pathways in the spinal cord.

Magnetic spherical carbon as an efficient adsorbent for the magnetic extraction of phthalate esters from lake water and milk samples.

Magnetic spherical carbon was synthesized by a facile hydrothermal carbonization procedure with biomass glucose as the carbon precursor and nanoclusters iron colloid as magnetic precursor. The textures of the as-prepared magnetic spherical carbon were characterized by nitrogen adsorption-desorption isotherms, X-ray diffraction, transmission electron microscopy, scanning electron microscopy and vibration sample magnetometry. Results indicated that the magnetic spherical carbon possessed high surface area as well as strong magnetism, which endows the material with good adsorption capability and easy separation properties. To assess its absorption performance, the magnetic spherical carbon was employed as adsorbent for the extraction and preconcentration of phthalate esters from lake water and milk samples before high-performance liquid chromatographic analysis. Some key parameters that could influence the enrichment efficiency were investigated. Under the optimum conditions, a good linearity was achieved with the linear correlation coefficients higher than 0.9973. The limits of detection (S/N = 3) were 0.05-0.08 ng/mL for lake water and 0.1-0.2 ng/mL for milk samples. The recoveries of the analytes for the method were in the range 80.1-112.6%.

Phytic acid induced three-dimensional graphene for the enrichment of phthalate esters from bottled water and sports beverage samples.

A three-dimensional graphene was synthesized through a hydrothermal reaction of graphene oxide with phytic acid. The microstructure and morphology of the phytic acid induced three-dimensional graphene were investigated by nitrogen adsorption-desorption isotherms, scanning electron microscopy, and transmission electron microscopy. With a large surface area and three-dimensional structure, the graphene was used as the solid-phase extraction adsorbent for the extraction of phthalate esters from bottled water and sports beverage samples before high-performance liquid chromatographic analysis. The results indicated that the graphene was efficient for the solid-phase extraction of phthalate esters. The limits of detection (S/N = 3) of the method for the analytes were 0.02-0.03 ng/mL for the water samples and 0.03-0.15 ng/mL for the sports beverage sample. The limits of quantitation (S/N = 9) for the analytes were 0.06-0.09 ng/mL for water samples and 0.09-0.45 ng/mL for sports beverage sample. The calibration curves for the phthalate esters by the method had a good linearity from 0.1 to 80.0 ng/mL with correlation coefficients larger than 0.9997. The recoveries of the analytes for the method fell in the range of 86.7-116.2% with the relative standard deviations between 1.5 and 6.8%.

Effective targeting of colorectal cancer cells using TORC1/2 kinase inhibitors in vitro and in vivo.

AIM: We investigated the effects of TORC1/2 kinase inhibitors on colorectal cancer (CRC) cell lines. MATERIALS & METHODS: Using selective TORC1/2 inhibitors, rapamycin and PP242, we assessed their effect on the growth of CRC cells in vitro and tumor growth in vivo. RESULTS: Rapamycin and PP242 inhibit proliferation and induce apoptosis of CRC cells. They also enhance proapoptotic effect of conventional chemo drug doxorubicin in CRC cells in vitro. When combined with doxorubicin, rapamycin and PP242 almost completely inhibit tumor growth in vivo. Rapamycin and PP242 inhibit phosphorylation of Akt, ribosomal S6 kinase, 4EBP1 and mTOR. CONCLUSION: Our study suggests rapamycin and PP242 may be a useful therapeutic agent and inhibiting mTOR signaling pathway represents a new targeted therapy for CRC.

A role for retinoids in human oocyte fertilization: regulation of connexin 43 by retinoic acid in cumulus granulosa cells.

Retinoids are essential for ovarian steroid production and oocyte maturation in mammals. Oocyte competency is known to positively correlate with efficient gap junction intercellular communication (GJIC) among granulosa cells in the cumulus-oocyte complex. Connexin 43 (C x 43) is the main subunit of gap junction channels in human cumulus granulosa cells (CGC) and is regulated by all-trans retinoic acid (ATRA) in other hormone responsive cell types. The objectives of this study were to quantify retinoid levels in human CGC obtained during IVF oocyte retrievals, to investigate the potential relationship between CGC ATRA levels and successful oocyte fertilization, and to determine the effects of ATRA on C x 43 protein expression in CGC. Results showed that CGC cultures actively metabolize retinol to produce ATRA. Grouped according to fertilization rate tertiles, mean ATRA levels were 2-fold higher in pooled CGC from women in the highest versus the lowest tertile (P < 0.05). ATRA induced a rapid dephosphorylation of C x 43 in CGC and granulosa cell line (KGN) cultures resulting in a >2-fold increase in the expression of the functional non-phosphorylated (P0) species (P < 0.02). Similar enhancement of P0 by ATRA was shown in CGC and KGN cultures co-treated with LH or hCG which, by themselves, enhanced the protein levels of C x 43 without altering its phosphorylation profile. Correspondingly, the combination of ATRA+hCG treatment of KGN caused a significant increase in GJIC compared with single agent treatments (P < 0.025) and a doubling of GJIC from that seen in untreated cells (P < 0.01). These findings indicate that CGC are a primary site of retinoid uptake and ATRA biosynthesis. Regulation of C x 43 by ATRA may serve an important role in folliculogenesis, development of oocyte competency, and successful fertilization by increasing GJIC in CGC.

Estrogen increases Nrf2 activity through activation of the PI3K pathway in MCF-7 breast cancer cells.

The actions of the transcription factor Nuclear factor erythroid 2-related factor (Nrf2) in breast cancer have been shown to include both pro-oncogenic and anti-oncogenic activities which is influenced, at least in part, by the hormonal environment. However, direct regulation of Nrf2 by steroid hormones (estrogen and progesterone) has received only scant attention. Nrf2 is known to be regulated by its cytosolic binding protein, Kelch-like ECH-associated protein 1 (Keap1), and by a Keap1-independent mechanism involving a series of phosphorylation steps mediated by phosphatidylinositol 3-kinase (PI3K) and glycogen synthase kinase 3 beta (GSK3ß). Here, we report that estrogen (E2) increases Nrf2 activity in MCF7 breast cancer cells through activation of the PI3K/GSK3ß pathway. Utilizing antioxidant response element (ARE)-containing luciferase reporter constructs as read-outs for Nrf2 activity, our data indicated that E2 increased ARE activity >14-fold and enhanced the action of the Nrf2 activators, tertiary butylhydroquinone (tBHQ) and sulforaphane (Sul) 4 to 9 fold compared with cells treated with tBHQ or Sul as single agents. This activity was shown to be an estrogen receptor-mediated phenomenon and was antagonized by progesterone. In addition to its action on the reporter constructs, mRNA and protein levels of heme oxygenase 1, an endogenous target gene of Nrf2, was markedly upregulated by E2 both alone and in combination with tBHQ. Importantly, E2-induced Nrf2 activation was completely suppressed by the PI3K inhibitors LY294002 and Wortmannin while the GSK3ß inhibitor CT99021 upregulated Nrf2 activity. Confirmation that E2 was, at least partly, acting through the PI3K/GSK3ß pathway was indicated by our finding that E2 increased the phosphorylation status of both GSK3ß and Akt, a well-characterized downstream target of PI3K. Together, these results demonstrate a novel mechanism by which E2 can regulate Nrf2 activity in estrogen receptor-positive breast cancer cells and suggest that patients׳ hormonal status through this activity may play a significant role in some therapeutic outcomes.