The regulation of retinoid X receptor-mediated oxidative stress pathway in rat pulmonary ischemia/reperfusion injury / 生理学报

The aim of this study was to investigate the regulatory role of retinoid X receptor (RXR)-mediated oxidative stress pathway in rat pulmonary ischemia/reperfusion injury (PIRI) and the underlying mechanism. Seventy-seven male Sprague-Dawley (SD) rats were randomly divided into 7 groups (n = 11): control group, sham group, sham+9-cis-retinoid acid (9-cRA, RXR agonist) group, sham+HX531 (RXR inhibitor) group, ischemia/reperfusion (I/R) group, I/R+9-cRA group, and I/R+HX531 group. The unilateral lung I/R model was established by obstruction of left lung hilus for 30 min and reperfusion for 180 min in vivo. The rats in I/R+9-cRA and I/R+HX531 groups were given intraperitoneal injection of 9-cRA and HX531 before thoracotomy. After reperfusion, the left lung tissue was taken to evaluate the lung tissue injury, and the oxidative stress-related indexes of the lung tissue were detected by the corresponding kits. The lung tissue morphology and the ultrastructure of the alveolar epithelial cells were observed by HE staining and transmission electron microscope, respectively. The protein expression of RXR in lung tissue was observed by immunofluorescence labeling method, and the expression level of nuclear factor E2-related factor (Nrf2) protein was detected by Western blot. The results showed that, compared with the sham group, the I/R group exhibited obviously injured lung tissue, decreased SOD activity, increased MDA content and MPO activity, and down-regulated expression level of Nrf2 protein. Compared with the I/R group, the I/R+9-cRA group showed alleviated lung tissue injury, increased activity of SOD, decreased MDA content and MPO activity, and up-regulated expression levels of RXR and Nrf2 protein. The above-mentioned improvement effects of 9-cRA were reversed by HX531 treatment. These results suggest that RXR activation can effectively protect the lung tissue against I/R injury, and the mechanism may involve the activation of Nrf2 signaling pathway, the enhancement of antioxidant level and the reduction of oxidative stress response.

Effects and related mechanism of retinoid X receptor agonist bexarotene on atherosclerosis progression in diabetic apoE(-/-) mice / 中华心血管病杂志

<p><b>OBJECTIVE</b>To explore the effect of retinoid X receptor (RXR) agonist bexarotene on atherosclerosis and the potential mechanism in streptozotocin (STZ) induced diabetic apolipoprotein E knockout (apoE(-/-)) mice.</p><p><b>METHODS</b>Eight C57BL/6 mice served as control, 46 apoE(-/-) mice were randomized into 4 groups: apoE(-/-) group (n = 10), STZ+apoE(-/-) group (n = 12), STZ+apoE(-/-)+Bex 10 (10 mg×kg⁻¹×d⁻¹)group (n = 12), STZ+ apoE(-/-)+Bex 30 (30 mg×kg⁻¹×d⁻¹) group (n = 12). Diabetic apoE(-/-) animal model was established by intraperitoneal injection of STZ. Blood glucose was determined by glucose oxidase method. Patch area in thoracic aorta was measured by HE staining. Western blotting was used to determine the RXR and gp91(phox) protein level in thoracic aorta. Reactive oxygen species (ROS) level in blood and thoracic aorta homogenates was detected by Fenton and Griess method.</p><p><b>RESULTS</b>(1) Patch areas of thoracic aorta were larger in apoE(-/-) group than in C57BL/6 group [(38.40 ± 8.95)µm² vs. (0.10 ± 0.01) µm², P < 0.01], further increased in STZ+apoE(-/-) group [(94.06 ± 8.04)µm², P < 0.05 vs. apoE(-/-) group] and significantly reduced in STZ+apoE(-/-)+Bex 10 group [(78.72 ± 4.62)µm², P < 0.05 vs. STZ+apoE(-/-) group] and further educed in STZ+apoE(-/-)+Bex 30 group [(46.13 ± 7.56)µm², P < 0.05 vs. STZ+apoE(-/-)+Bex 10 group]. (2) Blood glucose level, TG, TC, LDL-C, thoracic aorta gp91(phox) protein level and ROS level in blood and thoracic aorta homogenates were significantly higher in STZ+apoE(-/-) group than in apoE(-/-) group (all P < 0.05). Blood glucose level and TG, TC, LDL-C levels were similar between STZ+apoE(-/-)+Bex10 and STZ+apoE(-/-) group. Thoracic aorta gp91(phox) protein level and ROS level in blood and thoracic aorta homogenates were lower in STZ+apoE(-/-)+Bex 10 group than in STZ+apoE(-/-) group (P < 0.05). Blood glucose level, TG, TC, LDL-C levels, gp91(phox) expression in thoracic aorta, ROS level in blood and thoracic in STZ+apoE(-/-)+Bex 30 group were lower than in STZ+apoE(-/-) group (all P < 0.05).</p><p><b>CONCLUSION</b>Bexarotene treatment could attenuate arteriosclerosis progression in STZ induced diabetic apoE(-/-) mice, the underlying mechanism might be related to suppressing oxidative stress and decreasing blood glucose level and improving lipids metabolism.</p>

Effect of Thyroid Hormone to the Expression of ATP-binding Cassette Transporter ABCG5 / 대한갑상선학회지

BACKGROUND AND OBJECTIVES: Excretion of bile acid and free cholesterol of bile was important to maintain cholesterol homeostasis. ATP-binding cassette transporter G5 (ABCG5) and G8 (ABCG8) promoted biliary cholesterol excretion. In previous study, hepatic secretion of cholesterol and ABCG5/G8 expression are strongly stimulated in hypophysectomized rats during treatment with thyroid hormone. In this study, we aimed to evaluate the effect of thyroid hormone to expression of ABCG5 and G8 in mouse liver. MATERIALS AND METHODS: We administered thyroid hormone (T3) to C57BL/6 mice and then RNA and protein was isolated from liver. We isolated primary hepatocyte and administered T3 to evaluate in vitro effect. HepG2 cells were cotransfected with either a control plasmid or expression plasmids for human thyroid hormone receptor (hTR)beta/human retinoid X receptor (hRXR)alpha or human liver X receptor (hLXR)alpha in combination with reporter plasmids TK-LXRE3-LUC with or without T3. RESULTS: Serum total cholesterol was decreased after 5 days of T3 treatment. Expression of ABCG5/8 mRNA and ABCG5 protein was increased after T3 treatment. In primary hepatocytes, T3 also increased ABCG5/8 mRNA expression. LXRalpha mRNA was not increased by T3. However, when we cotransfected liver X receptor response element (LXRE) construct and TRbeta/RXRalpha with T3, the activity of LXRE containing construct was markedly increased. CONCLUSION: We confirmed that thyroid hormone increased expression of ABCG5/8. This result suggested that thyroid hormone played an important role in decreasing serum cholesterol through bile excretion.

Effect of retinoid X receptor activation on oxidized low-density lipoprotein induced cell differentiation of murine macrophage cell line into dendritic like cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effect and related mechanism of retinoid X receptor (RXR) activation on oxidized low-density lipoprotein (ox-LDL) induced differentiation of macrophage into dendritic cell.</p><p><b>METHODS</b>RAW264.7 murine macrophage cell line was cultured with ox-LDL for 48 h in the absence and presence of RXR activator 9-cisRA or SR11237. Cell morphology was observed by phase contrast microscope and cell surface markers involved in dendritic cell immune maturation and activation was analyzed by FACS. Cellular reactive oxygen species production was detected by CM-H2DCFDA fluorescent probe.</p><p><b>RESULTS</b>ox-LDL-treated RAW264.7 murine macrophage cell line differentiated into dendritic like cells after 48 h and cell surface markers CD40, CD86, CD83, MHC Class II and CD1d were upregulated. These changes could be attenuated by cotreatment with 9-cisRA or SR11237. Upregulated cell surface markers CD40, CD86, CD83, MHC Class II and CD1d by ox-LDL were decreased about 47%, 43%, 48%, 32% and 17% respectively by 9-cisRA and 38%, 38%, 46%, 36% and 32% respectively by SR11237. The effect of 9-cisRA and SR11237 was dose dependent. Cellular reactive oxygen species were significantly increased in ox-LDL-treated RAW264.7 cells (MFI 38.24 +/- 4.20 vs. 4.46 +/- 0.39, P < 0.05) and which was significantly reduced by 9-cisRA (10(-7) mol/L) and SR11237 (10(-6) mol/L) to 12.60 +/- 1.52 and 17.89 +/- 1.91 respectively (all P < 0.05).</p><p><b>CONCLUSION</b>RXR activation partly inhibits the differentiation of ox-LDL induced macrophage into dendritic cell by reducing oxidative stress injury.</p>

Combinational Treatment with Retinoic Acid Derivatives in Non-small Cell Lung Carcinoma In Vitro

The growth inhibitory effects of four retinoic acid (RA) derivatives, 9-cis RA, 13-cis RA, N-(4-hydroxyphenyl) retinamide (4-HPR), and all-trans retinoic acid (ATRA) were compared. In addition, the effects of various combinations of these four agents were examined on non-small cell lung carcinoma (NSCLC) cell-lines, and on the expressions of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) on these cells. At the clinically achievable concentration of 1 micrometer, only 4-HPR inhibited the growths of H1299 and H460 cells-lines. However, retinoic acid receptor beta(RAR beta) expression was up-regulated on H460 and H1299 cells treated with 1 micrometer of ATRA, 13-cis RA, or 9-cis RA. All NSCLC cell lines showed growth inhibition when exposed sequentially to 1 micrometer ATRA and 0.1 micrometer 4-HPR. In particular, sequential treatment with 1 micrometer ATRA or 13-cis RA and 4-HPR markedly inhibited H1703 cell growth; these cells exhibited no basal RAR beta expression and were refractory to 4-HPR. However, in NSCLC cell lines that expressed RAR beta, the expressional levels of RAR beta were up-regulated by ATRA alone and by sequential treatment with ATRA and 4-HPR. 4-HPR was found to be the most active of the four agents in terms of NSCLC growth-inhibition. Moreover, sequential treatments with ATRA or 13-cis RA followed by 4-HPR were found to have synergistic growth-inhibitory effects and to regulate RAR expression.

Reverse Cholesterol Transport in Cultured Gallbladder Epithelial Cells / 대한소화기학회지

Bile is the major route of cholesterol excretion from the body. It is concentrated in the gallbladder, and often results in supersaturation of cholesterol. The high levels of cholesterol in gallbladder bile has clinical implications with respect to cholesterol gallstone formation and cholesterolosis of the gallbladder wall. Gallbladder epithelial cells (GBEC) are exposed to high cholesterol concentrations on their apical surfaces. Therefore, GBEC are uniquely positioned to play an important role in modulating biliary cholesterol concentrations. Recently, it has been documented that the key-transporter for polarized cholesterol and phospholipid efflux in GBEC is ATP-binding cassette transporter A1 (ABCA1) and Liver X receptor (LXR) and retinoid X receptor (RXR) in the nucleus of GBEC have a role that regulates ABCA1 expression. In addition, GBEC synthesize apolipoprotein A-I and E as cholesterol acceptors. These results indicate that GBEC has a perfect system for reverse cholesterol transport. We introduce the roles and mechanisms of ABCA1, scavenger receptor class B-I, LXR and RXR related to reverse cholesterol transport in GBEC with a review of our study experience and related literature.

Thyroid Hormone Receptor Dynamics in Thyroid Hormone Response Elements of the Human Type 1 Iodothyronine Deiodinase Gene / 대한내분비학회지

BACKGROUND: Type 1 iodothyronine deiodinase (D1), the product of the hdio1 gene, is involved in thyroid hormone activation by the deiodination of thyroxine (T4) to form 3, 5, 3'-triiodothyronine (T3). Recent studies have identified two thyroid hormone response elements (TREs) in the 5 flanking region of the hdio1 gene. TRE1, proximal to TRE in the hdio1 gene, consists of a direct repeat of thyroid hormone receptor (TR) binding octamers with 10 bp separating the two TR binding sites. The upstream TRE, TRE2, is a classical direct repeat of retinoid X receptor (RXR)/TR binding half-sites with a 4-bp separation. There are few studies clarifying the TR dynamics in the TRE of a specific gene with or without the exposure of activated thyroid hormone. We evaluated TR binding patterns in the proximal and distal TREs of the hdio1 gene before and after T3 stimulation. METHODS: We employed chromatin immunoprecipitation (ChIP) technique to investigate the TR-TRE interaction before and after T3 stimulation in human hepatocellular carcinoma HepG2 cell line.Following cross-linking and sonication of the cells, immunoprecipitation was performed overnight at 4degrees C with TR 1, TR 1 and TR 2 antibodies. We analyzed the binding patterns and amounts of TR 1, TR 1 and TR 2 to TRE1 and TRE2 before and after 12 hours stimulation with 100 nM T3 by using conventional and quantitative real-time polymerase chain reactions (RQ-PCR). Reverse transcriptional PCR (RT-PCR) and Western blot with TR 1, TR 1 and TR 2 antibodies were performed to measure the levels of hdio1 mRNA and TR 1, TR 1 and TR 2 proteins before and after 12 hours exposure to 100 nM T3. RESULTS: In TRE1, TR 1 binding was significantly decreased after 12 hours stimulation with 100nM T3 (3.74-->1.97, delta=-47.3%, p3.01, delta=-71.1%, p 2.93, delta=-76.7%, p 9.84, delta=+7.3%). Total TR bindings in TRE2 were significantly decreased after 12 hours stimulation with 100 nM T3 (32.14 --> 15.78, delta=-50.9%, p<0.05). The TR bindings to TRE1 and TRE2 were not significantly different by the amounts of TR antibodies used during ChIP assays. The levels of hdio1 mRNA were significantly increased, 2.03 times, after 12 hours exposure to 100nM T3 (p<0.001). Western blot showed no significant change of the level of each TR isoform protein before and after 12 hours exposure to 100 nM T3. CONCLUSION: Our results demonstrate the dynamics of TR 1 at proximal TRE (TRE1) and the switching phenomenon of TR isoforms at distal TRE (TRE2) of the hdio1 gene after T3 stimulation. Further investigation, however, is needed to clarify the mechanisms of these observations.

Effect of retinoid kappa receptor alpha (RXRalpha) transfection on the proliferation and phenotype of rat hepatic stellate cells in vitro / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the effect of retinoid kappa receptor alpha (RXRalpha) transfection plus treatment with the RXRalpha ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF) activated hepatic stellate cells (HSCs).</p><p><b>METHODS</b>PDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1- human RXRalpha, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (alpha-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis.</p><p><b>RESULTS</b>Transfection of the RXRalpha gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRalpha protein for at least 168 hours. Cell proliferation and expressions of alpha- smooth muscle actin (alpha-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs.</p><p><b>CONCLUSION</b>Transfection with the RXRalpha gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.</p>

Retinoic Acid Enhances Drug-Induced Cell Death in Anticancer Drug-Resistant Cell Lines / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Retinoids (RA), a group of vitamin A derivatives, is known to be important for regulation of normal cellular growth and differentiation. RA treatment of various cancers resulted in cell growth inhibition and apoptosis. Therefore, the chemotherapeutic and chemopreventative activities of various types of tumor have been examined. Biological actions of RA are mediated through nuclear receptors, including the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In this study, we examined the effect of all-trans-retinoic acid (atRA) as an anticancer drug-sensitiser in cancer cell lines and in its drug- resistant cancer cell lines MATERIALS AND METGODS: Cells were maintained by RPMI 1640 medium containing 10% fetal bovine serum. Cells were treated with 1 micro M atRA for 48 h, then with the desired concentration of anticancer drug for 24 h. Cell viability was measured spectrophotometrically at 540 nm using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. Western blot analyses were performed with the desired antibodies. RESULTS: We investigated if pre-treatment with atRA enhanced the drug-sensitivity of various cancer cell lines to either 5-fluorouracil, adriamycin, or cisplatin. 5-FU (SNU638-F2) and CDDP-resistant cell (SNU638-Cis) lines, from a Korean gastric cancer cell line (SNU638) and the ADR-resistant cells (AD600) was established from a colon cancer cell line (SW620). Treatment of each cell line, with 1 micro M atRA, prior to drug exposure resulted in enhanced cell death in these cell lines. Furthermore, the effect of atRA on growth inhibition, in each drug-resistant cell line, was more obvious than in their parent cell lines. Increased activity of Transglutaminase II (TgaseII) and cleavage of Poly (ADP-ribose) polymerase (PARP) were also observed (western blot analysis CONCLUSION: Based on our data, we suggest that atRA enhances anticancer drug-induced cell death and reverses the drug-sensitivity of the drug-resistant cancer cell lines.

Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

Retinoic acids (RA) play a key role in myeloid differentiation through their agonistic nuclear receptors (RAR alpha/RXR) to modulate the expression of target genes. In acute promyelocytic leukemia (APL) cells with rearrangement of retinoic acid receptor a (RAR alpha) (including: PML-RAR alpha, PLZF-RAR alpha, NPM-RAR alpha, NuMA- RAR alpha or STAT5b-RAR alpha) as a result of chromosomal translocations, the RA signal pathway is disrupted and myeloid differentiation is arrested at the promyelocytic stage. Pharmacologic dosage of all-trans retinoic acid (ATRA) directly modulates PML-RAR alpha and its interaction with the nuclear receptor co-repressor complex, which restores the wild-type RAR alpha/RXR regulatory pathway and induces the transcriptional expression of downstream genes. Analysing gene expression profiles in APL cells before and after ATRA treatment represents a useful approach to identify genes whose functions are involved in this new cancer treatment. A chronologically well coordinated modulation of ATRA-regulated genes has thus been revealed which seems to constitute a balanced functional network underlying decreased cellular proliferation, initiation and progression of maturation, and maintenance of cell survival before terminal differentiation.

Growth Regulation of Ovarian Cancer Cells through the Inactivation of AP-1 by Retinoid Derivatives / 대한암학회지

PURPOSE: The growth regulatory effect of retinoid derivatives could be mediated by the transcriptional inactivation of AP-1 oncogenic transcription factor. By using ovarian cancer cell lines we were to investigate the cross-regulation mechanism between retinoids and AP-1. MATERIALS AND METHODS: Cell proliferation assays were performed in 4 ovarian cancer cells (A2774, PA-1, OVCAR-3, SKOV-3) by increasing the concentrations of all-trans retinoic acid (ATRA), 9-cis retinoic acid (9RA), 13-cis RA (13RA), 4-hydroxyphenyl retinamide (4-HPR). Transient transfection and CAT ELISA were done to determine the selective activity of each retinoid on the RAR (alpha, beta, gamma), RXR (alpha, beta, gamma). and the negative activity on AP-1 (c-Jun). RESULTS: Antiproliferative effect of 4-HPR (IC50; 0.7~2.7 micrometer) was more potent than those of other retinoid derivatives (IC50; 2.7~9.0 micrometer). To assess the anticancer mechanism, we examined the effect of 4-HPR on the transriptional activity of retinoic acid receptors (RAR/RXR) and of c-jun. Contrary to other retinoid derivatives that are active for RAR and RXR with some different levels, 4-HPR showed weak activity only for RARgamma. However, 4-HPR exerted the strongest suppression on AP-1 (c-Jun) activity. CONCLUSION: Based on our results showing much 4-HPR's potent antiproliferative activity coupled with the most effectively inhibiting activity on AP-1 and minimum activity on RA receptor (selective for RARgamma) than other retinoid derivatives, we suggest that 4-HPR may be a novel, and very effective anticancer drugs for ovarian cancer.