Carbonyl Reductase 1 Research Articles

Overexpression of carbonyl reductase 1 in ovarian cancer cells suppresses proliferation and activates the eIF2 signaling pathway.

High expression of carbonyl reductase 1 (CBR1) protein in ovarian cancer cells inhibits tumor growth and metastasis. However, the underlying mechanism is unknown. To investigate the mechanism by which CBR1 suppresses tumor growth, the present study generated ovarian cancer cells that constitutively overexpress human CBR1 (hCBR1) protein. Ovarian cancer cell lines (OVCAR-3 and SK-OV-3) were transfected with a plasmid encoding hCBR1, followed by selection with G418 to isolate hCBR1-overexpressing lines. The proliferation rates of hCBR1-overexpressing cells were then compared with those of negative control and wild-type cells. Overexpression of hCBR1 led to significant inhibition of proliferation (P<0.05). Subsequently, to investigate changes in intracellular signaling pathways, cellular proteins were extracted and subjected to proteome analysis using liquid chromatography followed by mass spectrometry. There was an inverse correlation between CBR1 protein expression and cell proliferation. In addition, Ingenuity Pathway Analysis of hCBR1-overexpressing cell lines was performed, which revealed changes in the expression of proteins involved in signaling pathways related to growth regulation. Of these, the eukaryotic translation initiation factor 2 (eIF2) signaling pathway was upregulated most prominently. Thus, alterations in multiple tumor-related signaling pathways, including eIF2 signaling, may lead to growth suppression. Taken together, the present data may lead to the development of new drugs that target CBR1 and related signaling pathways, thereby improving outcomes for patients with ovarian cancer.

Based on mRNA Sequencing Techniques to Explore the Molecular Mechanism of Buzhong Yiqi Decoction for Autoimmune Thyroiditis.

Autoimmune diseases (AD) account for a high percentage of the population. One of the most prevalent is autoimmune thyroiditis (AIT). However, the therapeutic effects of Buzhong Yiqi (BZYQ) decoction on AIT have not been studied yet. The majority of the present study was conducted on NOD.H-2h4 mice in an attempt to ascertain the therapeutic effects of BZYQ decoction on AIT. The 0.05% sodium iodide water (NaI)-induced AIT mice model was established. A total of nine NOD.H-2h4 mice were randomly divided into three groups: the normal group provided with regular water, the model group drinking freely 0.05% NaI, and the treatment group treated with BZYQ decoction (9.56 g/kg) after NaI supplementation (NaI + BZYQ). BZYQ decoction was administered orally once daily for eight weeks. The thyroid histopathology test was used to measure the severity of lymphocytic infiltration. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of anti-thyroglobulin antibody (TgAb), interleukin (IL)-1β, IL-6, and IL-17. The Illumina HiSeq X sequencing platform was utilized to analyze the thyroid tissue by mRNA expression profiles. Bioinformatics analysis was used to investigate the biological function of the differentially expressed mRNAs. In addition, the expression of Carbonyl Reductase 1 (CBR1), 6-Pyruvoyltetrahydropterin Synthase (PTS), Major Histocompatibility Complex, Class II (H2-EB1), Interleukin 23 Subunit Alpha (IL-23A), Interleukin 6 Receptor (IL-6RA), and Janus Kinase 1 (JAK1) was measured by quantitative real-time PCR (qRT-PCR). The treatment group exhibited significantly lower rates of thyroiditis and lymphocyte infiltration compared to the model group. Serum levels of TgAb, IL-1β, IL-6, and IL-17 were significantly higher in the model group, but they fell dramatically after BZYQ decoction administration. According to our results, 495 genes showed differential expression in the model group compared to the control group. Six hundred twenty-five genes were significantly deregulated in the treatment group compared to the model group. Bioinformatic analysis showed that most mRNAs were associated with immune-inflammatory responses and were involved in multiple signaling pathways, including folate biosynthesis and the Th17 cell differentiation pathway. CBR1, PTS, H2-EB1, IL- 23A, IL-6RA and JAK1 mRNA participated in folate biosynthesis and the Th17 cell differentiation pathway. The qRT-PCR analysis confirmed that the above mRNAs were regulated in the model group compared to the treatment group Conclusion: The results of this investigation have revealed novel insights into the molecular mechanism of action of BZYQ decoction against AIT. The mechanism may be partially attributed to the regulation of mRNA expression and pathways.

The cardioprotective properties of Persicaria maculosa and Citrus sinensis extracts against doxorubicin-induced cardiotoxicity in mice.

This study assessed the cardioprotective properties of Persicaria maculosa (PME) and Citrus sinensis (CME) hydro-methanolic extracts, besides Citrus sinensis aqueous extract (CWE) against doxorubicin (DOX)-induced cardiotoxicity. The extracts were characterized. Mice were divided into eight groups: control (saline), DOX, protected (injected with 200 mg/kg of PME, CWE or CME for 21 days, orally, and DOX), and extracts (PME, CWE or CME administration, orally, for 21 days). DOX was injected (5 mg/kg, ip) on days 8, 13 and 18 of the experiment. Cardiac tumor necrosis factor-alpha (TNF-α), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and carbonyl reductase 1 (CBR1) expression levels, besides superoxide dismutase, catalase, malondialdehyde, nitric oxide and total protein levels were evaluated. Serum lactate dehydrogenase, creatine phosphokinase cardiac isoenzyme, aspartate transaminase, cholesterol, triglycerides and creatinine levels, as well as the cardiac tissues were examined. Comparing with the control, DOX considerably (p<0.01) up-regulated TNF-α expression, malondialdehyde, nitric oxide, cardiac enzymes, lipids and creatinine levels, while it significantly (p<0.01) down-regulated Nrf2 and CBR1. Additionally, DOX interfered with antioxidant enzymes' activities (p<0.01). Conversely, protected groups showed a significant (p<0.01) amelioration of DOX-induced cardiotoxic effects. The current study provides a new understanding of P. maculosa and C. sinensis cardioprotective mechanisms. The extracts' cardioprotective effects may be due to their antioxidant activities, ability to maintain the redox homeostasis through regulation of important antioxidant genes and primary antioxidant enzymes, and capability to recover inflammatory cytokines and lipids levels. Noteworthy, the tested extracts showed no toxic changes on the normal mice.

In Silico and In Vitro Assessment of Carbonyl Reductase 1 Inhibition Using ASP9521-A Potent Aldo-Keto Reductase 1C3 Inhibitor with the Potential to Support Anticancer Therapy Using Anthracycline Antibiotics.

Anthracycline antibiotics (ANT) are among the most widely used anticancer drugs. Unfortunately, their use is limited due to the development of drug resistance and cardiotoxicity. ANT metabolism, performed mainly by two enzymes-aldo-keto reductase 1C3 (AKR1C3) and carbonyl reductase 1 (CBR1)-is one of the proposed mechanisms generated by the described effects. In this study, we evaluated the CBR1 inhibitory properties of ASP9521, a compound already known as potent AKR1C3 inhibitor. First, we assessed the possibility of ASP9521 binding to the CBR1 catalytic site using molecular docking and molecular dynamics. The research revealed a potential binding mode of ASP9521. Moderate inhibitory activity against CBR1 was observed in studies with recombinant enzymes. Finally, we examined whether ASP9521 can improve the cytotoxic activity of daunorubicin against human lung carcinoma cell line A549 and assessed the cardioprotective properties of ASP9521 in a rat cardiomyocytes model (H9c2) against doxorubicin- and daunorubicin-induced toxicity. The addition of ASP9521 ameliorated the cytotoxic activity of daunorubicin and protected rat cardiomyocytes from the cytotoxic effect of both applied drugs. Considering the favorable bioavailability and safety profile of ASP9521, the obtained results encourage further research. Inhibition of both AKR1C3 and CBR1 may be a promising method of overcoming ANT resistance and cardiotoxicity.

CBR1 decreases protein carbonyl levels via the ROS/Akt/CREB pathway to extend lifespan in the cotton bollworm, Helicoverpa armigera.

Reactive oxygen species (ROS) are considered a major cause of ageing and ageing-related diseases through protein carbonylation. Little is known about the molecular mechanisms that confer protection against ROS. Here, we observed that, compared with nondiapause-destined pupae, high protein carbonyl levels are present in the brains of diapause-destined pupae, which is a 'non-ageing' phase in the moth Helicoverpa armigera. Protein carbonyl levels respond to ROS and decrease metabolic activity to induce diapause in order to extend lifespan. However, protein carbonylation in the brains of diapause-destined pupae still occurs at a physiological level compared to young adult brains. We find that ROS activate Akt, and Akt then phosphorylates the transcription factor CREB to facilitate its nuclear import. CREB binds to the promoter of carbonyl reductase 1 (CBR1) and regulates its expression. High CBR1 levels reduce protein carbonyl levels to maintain physiological levels. This is the first report showing that the moth brain can naturally control protein carbonyl levels through a distinct ROS-Akt-CREB-CBR1 pathway to extend lifespan.

Development of transgenic mice overexpressing mouse carbonyl reductase 1.

Carbonyl reductase 1 (CBR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with broad substrate specificity. CBR1 catalyzes the reduction of numerous carbonyl compounds, including quinones, prostaglandins, menadione, and multiple xenobiotics, while also participating in various cellular processes, such as carcinogenesis, apoptosis, signal transduction, and drug resistance. In this study, we aimed to generate transgenic mice overexpressing mouse Cbr1 (mCbr1), characterize the mCbr1 expression in different organs, and identify changes in protein expression patterns. To facilitate a deeper understanding of the functions of CBR1, we generated transgenic mice overexpressing CBR1 throughout the body. These transgenic mice overexpress 3xFLAG-tagged mCbr1 (3xFLAG-mCbr1) under the CAG promoter. Two lines of transgenic mice were generated, one with 3xFLAG-mCbr1 expression in multiple tissues, and the other, with specific expression of 3xFLAG-mCbr1 in the heart. Pathway and network analysis using transgenic mouse hearts identified 73 proteins with levels of expression correlating with mCbr1 overexpression. The expression of voltage-gated anion channels, which may be directly related to calcium ion-related myocardial contraction, was also upregulated. mCbr1 transgenic mice may be useful for further in vivo analyses of the molecular mechanisms regulated by Cbr1; such analyses will provide a better understanding of its effects on carcinogenesis and cardiotoxicity of certain cancer drugs.

Quantitative Evaluation of the Contribution of Each Aldo-Keto Reductase and Short-Chain Dehydrogenase/Reductase Isoform to Reduction Reactions of Compounds Containing a Ketone Group in the Human Liver.

Enzymes of the aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase superfamilies are involved in the reduction of compounds containing a ketone group. In most cases, multiple isoforms appear to be involved in the reduction of a compound, and the enzyme(s) that are responsible for the reaction in the human liver have not been elucidated. The purpose of this study was to quantitatively evaluate the contribution of each isoform to reduction reactions in the human liver. Recombinant cytosolic isoforms were constructed, i.e., AKR1C1, AKR1C2, AKR1C3, AKR1C4, and carbonyl reductase 1 (CBR1), and a microsomal isoform, 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1), and their contributions to the reduction of 10 compounds were examined by extrapolating the relative expression of each reductase protein in human liver preparations to recombinant systems quantified by liquid chromatography-mass spectrometry. The reductase activities for acetohexamide, doxorubicin, haloperidol, loxoprofen, naloxone, oxcarbazepine, and pentoxifylline were predominantly catalyzed by cytosolic isoforms, and the sum of the contributions of individual cytosolic reductases was almost 100%. Interestingly, AKR1C3 showed the highest contribution to acetohexamide and loxoprofen reduction, although previous studies have revealed that CBR1 mainly metabolizes them. The reductase activities of bupropion, ketoprofen, and tolperisone were catalyzed by microsomal isoform(s), and the contributions of HSD11B1 were calculated to be 41%, 32%, and 104%, respectively. To our knowledge, this is the first study to quantitatively evaluate the contribution of each reductase to the reduction of drugs in the human liver. SIGNIFICANCE STATEMENT: To our knowledge, this is the first study to determine the contribution of aldo-keto reductase (AKR)-1C1, AKR1C2, AKR1C3, AKR1C4, carbonyl reductase 1, and 11β-hydroxysteroid dehydrogenase type 1 to drug reductions in the human liver by utilizing the relative expression factor approach. This study found that AKR1C3 contributes to the reduction of compounds at higher-than-expected rates.

LncRNA MYMLR promotes pituitary adenoma development by upregulating carbonyl reductase 1 via sponging miR-197-3p.

Long noncoding RNAs (lncRNAs) have been demonstrated to participate in various biological processes and play key roles in tumorigenesis and metastasis. Pituitary adenoma (PA) is one of the most common malignancies in central nervous system. Recently, multiple lncRNAs have been identified to regulate PA initiation, progression and metastasis. we aimed to elucidate the expression pattern and function of lncRNA MYMLR in PA development. The expression of lncRNA MYMLR in PA tissues and cells was examined by real-time quantitative PCR. Knockdown of MYMLR expression was achieved by using shRNA. The function of MYMLR and regulatory network were analyzed using CCK-8 assay, wound-healing assay, migration assay and Annexin V/PI staining. Xenograft tumor model was used to explore the function of MYMLR in vivo . Bioinformatics analysis and luciferase reporter assay were conducted to investigate the interaction between MYMLR and its regulatory network. LncRNA MYMLR was highly expressed in PA tissues compared with that in normal tissues. Knockdown of MYMLR suppressed cell proliferation, migration and invasion, while promoting PA cell apoptosis. Mechanistically, MYMLR functioned as a competing endogenous RNA (ceRNA) sponging microRNA miR-197-3p. Furthermore, miR-197-3p exerted its tumor inhibitory role via negatively regulating carbonyl reductase 1 (CBR1). Overexpression of CBR1 antagonized the inhibitory effect of lncRNA MYMLR knockdown or miR-197-3p overexpression. In addition, xenograft tumor model revealed that knockdown of lncRNA MYMLR suppressed PA tumor development in vivo via regulating CBR1. Our findings suggest a regulatory network of lncRNA MYMLR/miR-197-3p/CBR1, which benefits the understanding of PA development and provides a promising lncRNA-direct therapeutic strategy against PA.

MOLECULAR DOCKING STUDY, ANTIOXIDANT ACTIVITY, PROXIMATE CONTENT, AND TOTAL PHENOL OF Lemna perpusilla Torr. GROWN IN SUMEDANG, WEST JAVA, INDONESIA

Water lentils or duckweeds (Lemnoideae, Araceae family) grow abundantly in the tropical aquatic environments of West Java, Indonesia. There are several types of Lemna plants, however, in this study, we explored Lemna perpusilla Torr. grown in Ciparanje Pond, Sumedang, West Java, Indonesia for its potential to be utilized as a fish feed supplement. The antioxidant activity of this small-sized plant is believed to belong to the phenolic compounds or other active secondary metabolites contained in the plant. Methods used were molecular docking study, the proximate analysis, antioxidant activity using DPPH reagent, and determination of the total phenolic compounds of the plant. The molecular docking simulation was performed between zeaxanthin, lutein, carotenoid, and stigmasterol with the NADPH-dependent human carbonyl reductase-1 (hCBR-1), an enzyme that works by protecting cells against cellular damage resulting from oxidative stress. Phytochemical screening showed that our Lemna positively contains alkaloids, flavonoids or carotenoids, polyphenols, tannins, triterpenoids, and steroids. Molecular docking simulation revealed that stigmasterol, lutein, zeaxanthin, and carotenoid in this plant could interact with important residues in the catalytic site of hCBR-1. The proximate analysis indicated a high content of crude protein and fat. The antioxidant activity conveyed a strong activity which is parallel with its total phenolic compounds content. It can be concluded that Lemna perpusilla Torr. grown in Ciparanje Pond, Sumedang, West Java, Indonesia is the potential to be utilized as a fish feed supplement.

Effects of orexin A on PTGS2, PTGES, CBR1 and PGFS mRNA transcript abundances and prostaglandin E2 and F2α concentrations in culture medium of pig uterine explants collected during early gestation and the estrogenic cycle

In this study, aims were to evaluate orexin A (OXA) effects on mRNA abundance of important enzymes involved in prostaglandin production, such as cyclooxygenase 2 (PTGS2), microsomal PGE2 synthase-1 (PTGES), PGF2α synthase (PGFS) and carbonyl reductase 1 (CBR1), as well as prostaglandin E2 (PGE2) and F2α (PGF2α) culture medium concentrations for endometrial and myometrial explants. Tissues were collected from gilts during specific phases of the estrogenic cycle or early gestational period. There were greater concentrations of PGE2 with OXA treatments of endometrial tissues collected on days 12–13 and 27–28, as well as PGF2α on days 10–11 of the gestational period. The PGF2α concentrations were less in tissues collected on days 27–28 of the gestational period. The OXA treatments resulted in lesser concentrations of PGE2 from myometrial tissues collected on days 10–11 and greater PGF2α on days 10–11 of the gestational period and 10–11 of the estrogenic cycle. Effects of OXA may occur due to actions at PTGS2, PTGES, PGFS and CBR1 genes because mRNA abundances for proteins encoded by these genes were affected by OXA. Results indicate there is an OXA effect on mRNA abundances and prostaglandin culture medium concentrations of uterine tissue collected at different stages of the gestational period or estrogenic cycle using different doses of OXA. It, therefore, is concluded OXA may affect de novo synthesis and secretion of PGE2 and PGF2α in the uterus of pigs.

Induction of carbonyl reductase 1 (CR1) gene expression in Daphnia magna by TNT, but not its key metabolites 2-ADNT and 4-ADNT

2,4,6-trinitrotoluene (TNT) is a known source of reactive oxygen species (ROS), which cause oxidative stress in aquatic ecosystems. Carbonyl reductases (CRs) are one of several possible defense mechanisms induced against ROS products, especially those that result in the ‘so-called’ carbonyl stress. Daphnia magna, a freshwater organism living in stagnant freshwater bodies, expresses four copies of the CR gene (Dma_CR1, Dma_CR2, Dma_CR3 and Dma_CR4). In this study, induction of all four copies of Dma_CR by 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT), was investigated. Reverse transcription polymerase chain reaction (RT-PCR) analysis of treated daphnids revealed up-regulation of Dma_CR1 alone in response to TNT, but not 2-ADNT and 4-ADNT (which are key metabolites of TNT). This concentration- and time-dependent up-regulation in mRNA-expression was observed both in the presence and absence of light, in the same magnitude. Moreover, significant change in mRNA-expression could be observed 8 h after treatment with TNT. In the presence of TNT, the antioxidant N-acetylcysteine (NAc) could not reverse TNT-induced up-regulation of Dma_CR1 mRNA-expression. On the other hand, withdrawal of TNT from the culture medium caused a significant reduction in the TNT-induced mRNA-expression of Dma_CR1 within 24 h. These findings highlight the potential of Dma_CR1 as a biomarker for biomonitoring of TNT levels in freshwater bodies.

Chrysin induces autophagy-dependent ferroptosis to increase chemosensitivity to gemcitabine by targeting CBR1 in pancreatic cancer cells

Recent studies have verified that inducing reactive oxygen species (ROS) is one of the gemcitabine anti-tumor mechanisms of action. Human carbonyl reductase 1 (CBR1) plays an important role in protecting cells against oxidative damage. However, it is unclear whether CBR1 is involved in pancreatic cancer (PC) progression and resistance to gemcitabine. Based on the GEPIA database, we analyzed tumor tissue samples from PC patients using immunohistochemistry (IHC) and revealed that CBR1 was highly expressed in PC tissues and that this was significantly correlated with the clinicopathological features of PC. Genetic inhibition of CBR1 suppressed PC cell proliferation by regulating ROS generation. Furthermore, gemcitabine upregulated CBR1 expression, which could limit the anti-tumor activity of gemcitabine, and attenuation of CBR1 enhanced gemcitabine sensitivity in vitro and in vivo. Additionally, we report that chrysin directly binds to CBR1, which inhibited its enzymatic activity both at the molecular and cellular levels. Inhibition of CBR1 by chrysin increased cellular ROS levels and led to ROS-dependent autophagy, which resulted in the degradation of ferritin heavy polypeptide 1 (FTH1) and an increase in the intracellular free iron level that participates in ferroptosis in PC cells. Finally, our results showed that chrysin enhanced PC sensitivity to gemcitabine by inducing ferroptotic death in vitro and in vivo. Collectively, these findings indicate that CBR1 is a potential therapeutic target for PC treatment. In addition, we elucidated a novel mechanism underlying the anti-tumor effects of chrysin.

Loxoprofen enhances intestinal barrier function via generation of its active metabolite by carbonyl reductase 1 in differentiated Caco-2 cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide as antipyretic analgesics and agents for rheumatoid arthritis and osteoarthritis, but known to cause damage to the gastrointestinal mucosae as their serious adverse effects. Few studies showed the impairment of intestinal epithelial barrier function (EBF) by high concentrations (0.5–1 mM) of NSAIDs, but the underlying mechanism is not fully understood. This study is aimed at clarifying effects at a low concentration (50 μM) of three NSAIDs, loxoprofen (Lox), ibuprofen and indomethacin, on intestinal EBF using human intestinal epithelial-like Caco-2 cells. Among those NSAIDs, Lox increased the transepithelial electric resistance (TER) value, decreased the paracellular Lucifer yellow CH (LYCH) permeability, and upregulated claudin (CLDN)-1, −3 and −5, indicating that low doses of Lox enhanced EBF through increasing expression of CLDNs. Lox is known to be metabolized to a pharmacologically active metabolite, (2S,1′R,2′S)-loxoprofen alcohol (Lox-RS), by carbonyl reductase 1 (CBR1), which is highly expressed in human intestine. CBR1 was expressed in the Caco-2 cells, and the pretreatment with a CBR1 inhibitor suppressed both the Lox-evoked CLDN upregulation and EBF enhancement. In addition, the treatment of the cells with Lox-RS resulted in higher TER value and lower LYCH permeability than those with Lox. Thus, Lox-RS synthesized by CBR1 may greatly contribute to the improving efficacy of Lox on the barrier function. Since EBF is decreased in inflammatory bowel disease, we finally examined the effect of Lox on EBF using the Caco-2/THP-1 co-culture system, which is used as an in vitro inflammatory bowel disease model. Lox significantly recovered EBF which was impaired by inflammatory cytokines secreted from THP-1 macrophages. These in vitro observations suggest that Lox enhances intestinal EBF, for which the metabolism of Lox to Lox-RS by CBR1 has an important role.

Maternal, umbilical arterial metabolic levels and placental Nrf2/CBR1 expression in pregnancies with and without 25-hydroxyvitamin D deficiency

Background The aim of this case–control study was to document maternal, umbilical arterial metabolic levels and correlations in pregnancies with and without 25-hydroxyvitamin D [25(OH)D] deficiency, while, also investigating the expression of nuclear factor erythroid 2 related factor 2 (Nrf2) and carbonyl reductase 1 (CBR1) in the placenta. Methods One hundred participants, 50 deficient for 25(OH)D and 50 normal, were recruited from among hospitalized single-term pregnant women who had elected for cesarean section. Umbilical arterial and placental samples were collected during cesarean section. Metabolic levels were assessed for the 25(OH)D deficiency and control groups’ maternal, umbilical arterial samples. Nrf2 and CBR1 expression levels were investigated in the placentas of 12 pregnant women with 25(OH)D deficiency and 12 controls. Results Compared with the control participants, the 25(OH)D deficient women had significantly higher triglyceride (TG) levels (3.80 ± 2.11 vs. 2.93 ± 1.16 mmol/L, 3.64 ± 1.84 vs. 2.81 ± 1.16 mmol/L, p < .01, .001); lower high density lipoprotein cholesterol (HDL-C) levels (1.54 ± 0.32 vs. 1.82 ± 0.63 mmol/L, 1.41 ± 0.72 vs. 2.44 ± 1.68 mmol/L, p < .001, .01) in both material blood and the umbilical artery. In addition, Nrf2 and CBR1 expression levels were lower in the maternal 25(OH)D deficient placenta. Conclusion 25(OH)D deficient pregnant women have higher TG levels and lower HDL-C levels in both material blood and the umbilical artery. TG level is negatively correlated with 25(OH)D in both the maternal serum and infant umbilical artery. 25(OH)D deficiency also lowers placental expression of Nrf2 and CBR1. Supplemental data for this article is available online at here.

Diallyl disulfide prevents cigarette smoke-induced emphysema in mice

IntroductionCigarette smoke (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and pulmonary emphysema. The use of antioxidants has emerged as a potential therapeutic strategy to treat airway inflammation and lung diseases. In the current study, we investigated the potential therapeutic impact of diallyl disulfide (Dads) treatment in a murine model of CS-induced emphysema. MethodsC57BL/6 mice were exposed to CS for 60 consecutive days and treated with vehicle or Dads (30, 60 or 90 mg/kg) by oral gavage for the last 30 days, three times/week. The control group was sham-smoked and received vehicle treatment. All mice were euthanized 24 h after day 60; bronchoalveolar lavage (BAL) was performed and lungs were processed for further experimentation. Histological (HE stained sections, assessment of mean linear intercept (Lm)), biochemical (nitrite, superoxide dismutase (SOD), glutathione transferase (GST), and malondialdehyde (MDA) equivalents), and molecular biology (metalloproteinase (MMP) 12, SOD2, carbonyl reductase 1 (CBR1), nitrotyrosine (PNK), 4-hydroxynonenal (4-HNE), and CYP2E1) analyses were performed. ResultsTreatment with Dads dose-dependently reduced CS-induced leukocyte infiltration into the airways (based on BAL fluid counts) and improved lung histology (indicated by a reduction of Lm). Furthermore, CS exposure dramatically reduced the activity of the antioxidant enzymes SOD and GST in lung tissue and increased nitrite and MDA levels in BAL; these effects were all effectively counteracted by Dads treatment. Western blot analysis further confirmed the antioxidant potential of Dads, showing that treatment prevented the CS-induced decrease in SOD2 expression and increase in lung damage markers, such as CBR1, PNK, and 4-HNE. Furthermore, increased MMP12 (an important hallmark of CS-induced emphysema) and CYP2E1 lung protein levels were significantly reduced in mice receiving Dads treatment. ConclusionOur findings demonstrate that treatment with Dads is effective in preventing multiple pathological features of CS-induced emphysema in an in vivo mouse model. In addition, we have identified several proteins/enzymes, including 4-HNE, CBR1, and CYP2E1, that are modifiable by Dads and could represent specific therapeutic targets for the treatment of COPD and emphysema.

Influence of Clerodendrum volubile leaf extract on doxorubicin-induced toxicity and inhibition of carbonyl reductase mediated metabolism.

Doxorubicin (DOX) is a commonly used chemotherapeutic drug. However, its non-target organ toxicities pose a serious problem. This study is to assess the protective role of Clerodendrum volubile leaf extract (CVE) against DOX-induced toxicities in rats. In addition, the inhibitory activities of three phytochemical compounds (Rutin, Gallic acid and Rosmarinic acid) from CVE against Carbonyl reductase 1 (CBR1) were examined. Rats were randomly divided into 5 groups: (a) Control group rats were given 0.9% NaCl as vehicle, (b) DOX group: A single dose of DOX (25mg/kg; i.p.) was administered and rats were sacrificed 4days after DOX injection, while groups (c-e) CVE-treated DOX rat groups were given 125, 250 and 500mg/kg body weight of extracts orally for 12 consecutive days; 8days before, and 4days after the DOX administration. Computational techniques were used to determine the inhibitory activities of the compounds against CBR1. DOX intoxication caused a significant increase (p<0.05) in serum marker enzymes: ALT, AST, ALP, LDH, CK activities. The levels of liver and heart tissues antioxidant parameters: GPx, SOD, CAT, and GSH were significantly (p<0.05) decreased in DOX-intoxicated rats with concomitant elevation of malondialdehyde levels. Pretreatment with CVE reversed the above trends. From the structural analysis, Rutin and RSA exhibited the highest binding free energies against CBR1, and also exhibited structural stability when bound with CBR1. Our study indicates the protective effect of CVE when used in combination with doxorubicin thus improving its chemotherapeutic application via inhibition of CBR-mediated metabolism.

Carbonyl Reductase 1 Attenuates Ischemic Brain Injury by Reducing Oxidative Stress and Neuroinflammation.

Oxidative stress and neuroinflammatory response after the ischemic injury are important pathophysiologic mechanisms that cause brain tissue loss and neurological deficit. This study aims to observe the expression and role of carbonyl reductase 1 (CBR1), an NADPH-dependent oxidoreductase with specificity for carbonyl compounds such as 4-hydroxynonenal (4-HNE), in the brain after ischemic injury and to investigate the influence of CBR1 on ischemia-induced neuroinflammation. CBR1 expresses in the neurons, astrocyte, and microglia in the normal brain. The expression of CBR1 decreased in the ischemic regions following cerebral ischemia, and also reduced in primary neurons after OGD (oxygen-glucose deprivation); however, the expression of CBR1 significantly increased in microglia in the ischemic penumbra. Furthermore, TAT-CBR1 fusion protein played neuroprotective effects in reducing the infarct volume and improving neurological outcomes after ischemic injury. Mechanistically, CBR1 decreased the levels of 4-HNE in the brain after stroke; it also modulated microglial polarization toward the M2 phenotype, which was well-known to confer neuroprotection after ischemic injury. Our results demonstrate that CBR1 provides neuroprotection against ischemic injury by reducing oxidative stress and neuroinflammation, making a promising agent for cerebral ischemia treatment.

Carbonyl Reductase 1 Overexpression in Adipose Amplifies Local Glucocorticoid Action and Impairs Glucose Tolerance in Lean Mice

Glucocorticoids play a critical role in metabolic homeostasis. Chronic or excessive activation of the glucocorticoid receptor (GR) in adipose tissue contributes to metabolic disorders such as glucose intolerance and insulin resistance. Steroid-metabolising enzymes in adipose, such as 11β-HSD1 or 5α-reductase, modulate the activation of GR by converting primary glucocorticoids into more or less potent ligands. Carbonyl reductase 1 (CBR1) is a novel regulator of glucocorticoid metabolism, converting corticosterone/cortisol to 20β-dihydrocorticosterone/cortisol (20β-DHB/F); a metabolite which retains GR activity. CBR1 is abundant in adipose tissue and increased in obese adipose of mice and humans1 and increased Cbr1 expression is associated with increased fasting glucose1. We hypothesised that increased Cbr1/20β-DHB in obese adipose contributes to excessive GR activation and worsens glucose tolerance. We generated a novel murine model of adipose-specific Cbr1 over-expression (R26-Cbr1Adpq) by crossing conditional knock-in mice with Adiponectin-Cre mice. CBR1 protein and activity were doubled in subcutaneous adipose tissue of male and female R26-Cbr1Adpq mice compared with floxed controls; corresponding to a two-fold increase 20β-DHB (1.6 vs. 4.2ng/g adipose; P=0.0003; n=5-7/group). There were no differences in plasma 20β-DHB or corticosterone. Bodyweight, lean or fat mass, did not differ between male or female R26-Cbr1Adpq mice and floxed controls. Lean male R26-Cbr1Adpq mice had higher fasting glucose (9.5±0.3 vs. 8.4±0.3mmol/L; P=0.04) and worsened glucose tolerance (AUC 1819±66 vs. 1392±14; P=0.03). Female R26-Cbr1Adpq mice also had a worsened glucose tolerance but fasting glucose was not altered with genotype. There were no differences in fasting insulin or non-esterified fatty acid between genotypes in either sex. Expression of GR-induced genes Pnpla2, Gilz and Per1, were increased in adipose of R26-Cbr1Adpq mice. Following high-fat diet induced obesity, no differences in bodyweight, lean or fat mass, with genotype were observed in male and female mice, and genotype differences in fasting glucose and glucose tolerance were abolished. In conclusion, adipose-specific over-expression of Cbr1 in lean male and female mice led to increased levels of 20β-DHB in adipose but not plasma, and both sexes having worsened glucose tolerance. The influence of adipose CBR1/20β-DHB on glucose tolerance was not associated with altered fat mass or bodyweight and was attenuated by high-fat diet-induced obesity. These metabolic consequences of Cbr1 manipulation require careful consideration given the wide variation in CBR1 expression in the human population, the presence of inhibitors and enhancers in many foodstuffs and the proposed use of inhibitors as an adjunct for cancer treatment regimens. Reference: Morgan et al., Scientific Reports. 2017; 7.

Environmental and Genetic Factors in the Pathogenesis of COPD in the Road-Working Population.

Background Chronic obstructive pulmonary disease (COPD) is a typical heterogeneous condition caused by environmental and genetic risk factors. Objectives We investigated extrinsic (environmental) and intrinsic (genetic) factors contributing to the development of COPD in a nonsmoker road-working population in Northeast China. Method The target population was divided into a COPD group and an exposed control group. Another healthy nonroad working nonsmoker control group was also included for environmental factor comparison. Peripheral blood was collected and analyzed using inductively coupled plasma mass spectrometry for inorganic elements of PM2.5, and microarray, rt-PCR, and Multiplex ELISA for genetic factors. Results Forty-three COPD road workers, thirty-nine non-COPD road workers, and 52 age and gender-matched healthy nonroad workers were enrolled. There were significantly higher levels in all 24 inorganic elements in the COPD group compared with the healthy control group except potassium and manganese, while the majority of inorganic elements were similar between the COPD group and the exposed control group except in aluminum and cobalt. There were 39 genes showing significant differences between the COPD group and the exposed control group. Collagen, type XV, alpha 1 (COL15A1), Meis homeobox 1 (MEIS1), carbonyl reductase 3 (CBR3), and amine oxidase, copper containing 3 (AOC3) were confirmed by rt-PCR to be differentially expressed. Their correlations with blood cytokines were also evaluated. Conclusions Aluminum might contribute to the development of COPD in the road-working population. CBR3 and AOC3 seem expressed in different patterns than previously reported, evidenced by their correlation with proinflammatory and anti-inflammatory cytokines.

Carbonyl reduction pathway in hepatic in vitro metabolism of anthracyclines: Impact of structure on biotransformation rate

Carbonyl reduction biotransformation pathway of anthracyclines (doxorubicin, daunorubicin) is a significant process, associated with drug metabolism and elimination. However, it also plays a pivotal role in anthracyclines-induced cardiotoxicity and cancer resistance. Herein, carbonyl reduction of eight anthracyclines, at in vivo relevant concentrations (20 μM), was studied in human liver cytosol, to describe the relationship between their structure and metabolism. Significant differences of intrinsic clearance between anthracyclines, ranging from 0,62–74,9 μL/min/mg were found and associated with data from in silico analyses, considering their binding in active sites of the main anthracyclines-reducing enzymes: carbonyl reductase 1 (CBR1) and aldo-keto reductase 1C3 (AKR1C3). Partial atomic charges of carbonyl oxygen atom were also determined and considered as a factor associated with reaction rate. Structural features, including presence or absence of side-chain hydroxy group, a configuration of sugar chain hydroxy group, and tetracyclic rings substitution, affecting anthracyclines susceptibility for carbonyl reduction were identified.