scholarly journals The Effect and Mechanism of Celecoxib in Hypoxia-Induced Survivin Up-Regulation in HUVECs

2015 ◽  
Vol 37 (3) ◽  
pp. 991-1001 ◽  
Author(s):  
Ning-ning Liu ◽  
Ning Zhao ◽  
Na Cai
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Umbilical Vein ◽  
Survivin Expression ◽  
Mrna Levels ◽  
Cytoplasmic Staining ◽  
Hypoxic Conditions ◽  
Cox 2

Background/Aims: To investigate the roles of hypoxia-inducible factor 1α (HIF-1α), cyclooxygenase-2 (Cox-2) and its product, Prostaglandin E2 (PGE2), in the mechanisms underlying hypoxia-induced survivin expression in human umbilical vein endothelial cells (HUVECs) and to examine the effect of celecoxib, a selective Cox-2 inhibitor, on survivin expression. Methods: HUVECs were exposed to a normal (95% O2) or hypoxic (3% O2) environment for 24 hrs. We observed the localized expression of survivin, Cox-2 and HIF-1α in HUVECs using immunocytochemistry and detected the inhibitory effects of celecoxib on the growth of HUVECs using an MTT assay. mRNA and protein levels of Cox-2, HIF-1α and survivin were determined by real-time PCR and Western blot analysis under hypoxic conditions for 0, 6, 12, or 24 hrs. The time course changes of HIF-1α and survivin protein expression induced by cobalt chloride (CoCl2) were studied using Western blot analysis. We then treated HUVECs under hypoxia for 24 hrs with celecoxib (a Cox-2 selective inhibitor), genistein (a HIF-1α inhibitor) or exogenous PGE2 to further investigate the changes in hypoxia-induced survivin expression. Results: Following 24 hrs of hypoxic treatment, cells exhibited strongly positive survivin, HIF-1α and Cox-2 cytoplasmic staining. Celecoxib (65 μM) effectively inhibited cell proliferation under hypoxic conditions. The protein and mRNA levels of Cox-2, HIF-1α and survivin were increased under hypoxia. The patterns of HIF-1α and survivin expression induced by CoCl2 were similar to those induced by exposure to hypoxia. Genistein partially blocked survivin expression. Celecoxib reversed the hypoxia-induced survivin expression, whereas the addition of PGE2 partially restored this effect. Conclusions: Hypoxia-induced survivin expression in HUVECs may be mediated by dual interdependent mechanisms directly involving HIF-1α and indirectly involving the Cox-2/PGE2 pathways. Celecoxib may offset hypoxia-induced survivin expression.

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

2010 ◽  
Vol 299 (5) ◽  
pp. R1290-R1297 ◽  
Author(s):  
E. Zhao ◽  
Caleb L. Grey ◽  
Dapeng Zhang ◽  
Jan A. Mennigen ◽  
Ajoy Basak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Pcr Analysis ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

scholarly journals Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

scholarly journals Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on sodium/iodide symporter gene and protein expression

2002 ◽  
pp. 655-661 ◽  
Author(s):  
F Arturi ◽  
I Presta ◽  
D Scarpelli ◽  
JM Bidart ◽  
M Schlumberger ◽  
...  
Keyword(s):  
Western Blot ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Iodide Uptake ◽  
Protein Levels

BACKGROUND: Various clinical and experimental findings support the concept that human chorionic gonadotropin (hCG) can stimulate iodide uptake in thyroid cells. DESIGN: We investigated the molecular mechanisms underlying the effects of hCG on iodide uptake, and particularly its action on the expression of Na+/I- symporter (NIS) mRNA and protein. METHODS: Iodide uptake was analyzed in FTRL-5 cells by measuring (125)I concentrations in cells after a 30-min exposure to 0.1 microCi carrier-free Na (125)I in the presence or absence of hCG or, for control purposes, TSH. Expression of NIS mRNA and NIS protein synthesis were evaluated, respectively, with a semiquantitative 'multiplex' RT-PCR method and Western blot analysis. RESULTS: Iodide uptake was increased by hCG in a dose- and time-dependent manner: maximal effects were observed after 72 h of stimulation. The effect was cAMP dependent and paralleled that of TSH, although it lacked the early cycloheximide-independent component seen with TSH, and its peak effect was lower. Semiquantitative multiplex RT-PCR revealed that hCG produced a significant increase in NIS mRNA levels that was detectable after 4 h and peaked after 48 h. In contrast, in TSH-stimulated FRTL-5 cells, maximum NIS mRNA expression was observed after 24 h of stimulation. Western blot analysis demonstrated that hCG also caused a 2.5-fold increase over basal values in NIS protein levels, which was similar to that observed after TSH stimulation although the peak effects of the latter hormone were less marked and occurred earlier. CONCLUSION: Our data demonstrated that hCG stimulates iodide uptake in FRTL-5 cells by increasing NIS mRNA and protein levels. Thus, the functional status of the thyroid may be influenced by hCG-dependent changes in NIS expression occurring during pregnancy.

scholarly journals Identification of Matrine as a Novel Regulator of the CXCR4 Signaling Axis in Tumor Cells

2020 ◽  
Vol 21 (13) ◽  
pp. 4731 ◽  
Author(s):  
Young Yun Jung ◽  
Jae-Young Um ◽  
Acharan S. Narula ◽  
Ojas A. Namjoshi ◽  
Bruce E. Blough ◽  
...  
Keyword(s):  
Western Blot ◽  
Tumor Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Metastasis ◽  
Growth Factor Receptor ◽  
Boyden Chamber ◽  
Potential Candidate ◽  
Mrna Levels ◽  
Mobility Shift

Matrine, a quinolizidine alkaloid, is commonly employed for treating various viral and inflammatory disorders. Here, we have evaluated matrine for its activity on C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinases (MMP-9/2) expression, and its potential to affect tumor metastasis and invasion. The effects of matrine on CXCR4, MMP-9/2, and nuclear factor κB (NF-κB) activation in lung (A549), prostate (DU145), and pancreas (MIA PaCa-2) cells were investigated by diverse techniques. The expression level of CXCR4 and MMP-9/2 was analyzed by western blot analysis and reverse transcription polymerase chain reaction. NF-κB activation was also evaluated by western blot analysis, electrophoretic mobility shift assay as well as immunocytochemical experiments. Furthermore, we monitored cell invasion and metastasis activities by wound healing and Boyden chamber assays. We noted that matrine induced a down-regulation of CXCR4 and MMP-9/2 at both protein and mRNA levels. In addition, matrine negatively regulated human epidermal growth factor receptor 2 (HER2) and C-X-C Motif Chemokine Ligand 12 (CXCL12)-induced CXCR4 expression. Moreover, NF-κB suppression by matrine led to inhibition of metastatic potential of tumor cells. Our results suggest that matrine can block the cancer metastasis through the negative regulation of CXCR4 and MMP-9/2 and consequently it can be considered as a potential candidate for cancer therapy.

scholarly journals Expression of cyclooxygenase-2 in urinary bladder in rats with cyclophosphamide-induced cystitis

2007 ◽  
Vol 293 (2) ◽  
pp. R677-R685 ◽  
Author(s):  
Mary Beth Klinger ◽  
Abbey Dattilio ◽  
Margaret A. Vizzard
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Cell ◽  
Nerve Fibers ◽  
Cyclooxygenase 2 ◽  
Detrusor Smooth Muscle ◽  
Cox 2

These studies examined the expression of cyclooxygenase-2 (COX-2) expression in the urothelium and suburothelial space and detrusor from rats treated with cyclophosphamide (CYP) to induce acute (4 h), intermediate (48 h), or chronic (10-day) cystitis. Western blot analysis and immunohistochemistry were used to demonstrate COX-2 expression. In whole mount preparations of urinary bladder, nerve fibers in the suburothelial plexus, and inflammatory cell infiltrates were characterized for COX-2 expression after CYP-induced cystitis. COX-2 expression significantly ( P ≤ 0.01) increased in the urothelium + suburothelium and detrusor smooth muscle with acute, intermediate, and chronic (10-day) CYP-induced cystitis, but expression in urothelium + suburothelium was significantly greater. CYP-induced upregulation of COX-2 showed by immunostaining in the urothelium + suburothelium was similar to that observed with Western blot analysis and also demonstrated COX-2 inflammatory cell infiltrates (CD86+) and nerve fibers (PGP+) in the suburothelial plexus. Although COX-2 expression was significantly ( P ≤ 0.01) increased in detrusor smooth muscle, immunohistochemistry failed to demonstrate an obvious change in COX-2-immunoreactivity (IR) in detrusor muscle, but COX-2 inflammatory infiltrates were present throughout the detrusor. COX-2-IR nerve fibers exhibited increased density in the suburothelial plexus with acute or chronic CYP-induced cystitis. COX-2-IR macrophages (CD86+) were present throughout the urinary bladder with acute and chronic CYP-induced cystitis. These studies demonstrate cellular targets in the urinary bladder where COX-2 inhibitors may act.

Glucocorticoids upregulate taurocholate transport by ileal brush-border membrane

1997 ◽  
Vol 273 (1) ◽  
pp. G197-G203 ◽  
Author(s):  
M. J. Nowicki ◽  
B. L. Shneider ◽  
J. M. Paul ◽  
J. E. Heubi
Keyword(s):  
Steady State ◽  
Bile Acid ◽  
Western Blot ◽  
Brush Border ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Initial Velocity ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Mrna Levels

The regulation of the enterohepatic circulation of bile acids has not been fully elucidated. Substrate availability has been shown to have a regulatory role on the ileal uptake of taurocholate (TC) by a positive feedback mechanism. Other mechanisms are likely to be involved in regulating ileal bile acid uptake. The present study was designed to test the hypothesis that the ileal bile acid transporter (iBAT) is glucocorticoid sensitive and that changes in expression are mediated by changes in iBAT synthesis. Adult Sprague-Dawley rats (300–400 g) received intraperitoneal injections with either corticosterone (5 mg/ 100 g body weight) or an equivalent vehicle (control) daily for 3 days. On day 4, ileal brush-border membrane vesicles (BBMV) and hepatic basolateral membrane vesicles (BLMV) were prepared, and TC transport was performed using the rapid filtration technique. Initial velocity was measured at selected time points, and kinetics were calculated over a range of TC concentrations. Ileal RNA was isolated, and Northern analysis of steady-state iBAT mRNA levels was determined. Western blot analysis was performed to quantitate the level of the 48-kDa iBAT protein. The initial velocity of Na(+)-dependent TC uptake at 30 s by ileal BBMV was higher in treated animals (264.3 +/- 64.6 pmol/mg protein) compared with control animals (148.3 +/- 41.1 pmol/mg protein; P = 0.07). The maximal velocity of uptake (Vmax) was significantly higher in treated vs. control animals (1,091 +/- 62.7 vs. 689.1 +/- 55.0 pmol.min-1.mg protein-1, respectively; P = 0.002), whereas there was no significant difference in the Michaelis constant (Km) between the control and treated animals (43.3 +/- 7.2 vs. 35.3 +/- 8.7 microM, respectively; P = not significant). Steady-state iBAT mRNA levels were increased twofold in the treated vs. control groups. Western blot analysis showed that the abundance of the 48-kDa iBAT protein was eightfold higher in the treated animals compared with control. Kinetic analysis of hepatic Na(+)-dependent TC uptake revealed nearly identical Vmax and Km between the study and control animals. Therefore, we conclude that TC transport by ileal BBMV is upregulated by administration of glucocorticoids. The increase in BBMV transport Vmax corresponds to an increase in both iBAT transcript and protein.

scholarly journals Inhibiting Efferocytosis in Acute Myeloid Leukemia Decreases Checkpoint Blockade through Decreased CCL5/STAT6 Signaling and Increases Activation through NF-Kb

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1174-1174
Author(s):  
Kristen Allison ◽  
Michael R Verneris ◽  
Joselyn Cruz Cruz ◽  
Alisa Lee-Sherick
Keyword(s):  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Myeloid Leukemia ◽  
Advisory Board ◽  
M2 Macrophage ◽  
Mrna Levels ◽  
Potential Mechanism ◽  
Acute Myeloid

Abstract Efferocytosis is a tolerogenic wound-healing process carried out by macrophages including phagocytosis of cellular debris and production of T cell suppressive cytokines. MerTK, the prototypic efferocytic receptor, is expressed on macrophages in tissues that harbor high leukemic burden, or "leukemia-associated macrophages" (LAMs), and therefore efferocytosis is co-opted by leukemia for immune evasion. We previously reported that in acute myeloid leukemia (AML) models blocking efferocytosis through inhibition of MerTK led to increased serum [IL-18] and downregulation of checkpoint ligands PD-L1 and PD-L2 on LAMs, which was not attributable to decreased [INF-γ] given that INF-γ was actually increased with MerTK inhibition. To evaluate a potential mechanism of PD-L1/PD-L2 downregulation when efferocytosis is inhibited, we evaluated for IL-4, IL-10 and IL-6 - all previously associated with checkpoint ligand expression - in serum samples of C57Bl/6 mice inoculated with a syngeneic MLL-ENL AML, and treated with a MerTK efferocytosis inhibitor (MRX-2843, n=15) or vehicle (PBS, n=14). There was no appreciable [IL-4] or [IL-10] detected in either treatment group. There was decreased serum [IL-6] in some MRX-2843-treated samples (mean: 45.4pg/ml) compared to controls (mean: 379.1pg/ml, p=0.3), however nearly all treated and untreated samples were <40pg/ml. We then evaluated several additional serum cytokines from these mice. CCL5/RANTES, known to promote M2 macrophage polarization and modulate PD-L1/PD-L2, was significantly decreased in mice treated with MRX-2843 (12.92pg/ml) compared to vehicle (28.63pg/ml, p<0.001). To validate this as relevant signaling pathway in LAMs, we evaluated STAT6 phosphorylation by western blot analysis in bone marrow derived macrophages (BMDM) from C57Bl/6 mice co-cultured with AML cells and treated with MRX-2843 or vehicle. When MerTK was inhibited, STAT6 phosphorylation was consistently decreased compared to vehicle-treated BMDM. This CCL5/STAT6 axis alteration may be a potential mechanism of PD-L1/PD-L2 downregulation when efferocytosis is inhibited. We next sought to evaluate whether there was LAM autocrine cytokine signaling within tissues of high leukemic burden, which were not detected by serum cytokine analysis. BMDM co-cultured with AML cells, and treated with MRX-2843 or vehicle were harvested after 12 hours and RT-qPCR was performed. mRNA levels of IFN-β and IL-1β in BMDM treated with MRX-2843 were twice that of controls, suggesting classic macrophage activation. These BMDM did not amplify appreciable levels of IL-4, IL-10, or TGF-β mRNA. Given the increased IL-1β mRNA and previously reported increased IL-18 associated with MerTK inhibition in LAMs, we evaluated NF-κB activation and subsequent inflammasome assembly. By RT-qPCR we did not detect any consistent alteration in p105 (precursor of p50) or RelA (p65), however when BMDM co-cultured with AML cells and treated with MRX-2843 or vehicle, western blot analysis consistently demonstrated increased phosphorylation of p65 as well as increased NLRP3 when MerTK was inhibited. In conclusion, blocking efferocytosis through MerTK in LAMs decreased CCL5/STAT6 signaling, which likely contributes to decreased LAM PD-L1 and PD-L2 expression. Furthermore, MerTK inhibition led to activation of NF-κB p65, inflammasome assembly, and production of IL-1β and IL-18. Taken together, these data demonstrate a mechanism by which MerTK inhibition alters antigen presentation through decreased co-inhibition and augmented activating cytokine production. Given that MerTK inhibitors are currently in clinical trials for relapsed/refractory malignancies, these data are relevant, timely, and could provide additional justification for their use in acute leukemias. Disclosures Verneris: Fate Therapeutics: Consultancy; Novartis: Other: advisory board; jazz: Other: advisory board.

scholarly journals Quercetin Protects RIMVECs From Inflammatory Damage, Pyroptosis and Cell Migration by Inhibiting the TLR4/NF-κB/NLRP3 Pathway

2021 ◽  
Author(s):  
Huixin Zhang ◽  
Yeye Li ◽  
Zhongjie Liu
Keyword(s):  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Inflammatory Factors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Caspase 1 ◽  
Inflammatory Damage

Abstract Background: Intestinal mucosal microvascular endothelial cells (MEC) have multiple functions and play an important role in intestinal bowel diseases (IBD). Quercetin is a flavonoid found in many plants and fruits. It was reported that quercetin can treat several gastrointestinal cancers, but its effect on bacterial enteritis and pyroptosis-related diseases has been rarely studied. This article aims to explore the effect and mechanism of quercetin on inflammatory injury and pyroptosis of RIMVECs.Methods: The inflammatory damage and pyroptosis in RIMVECs were induced by LPS and ATP. Real-time quantitative polymerase chain reaction (RT-qPCR), western blot analysis, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence methods were used to detect TLR4/NF-κB/NLRP3 pathways, inflammatory factors (IL-1β and IL-18) and pyroptosis-related proteins (Caspase-1 and GSDMD). The expression and distribution of ZO-1 were detected by western blot analysis and immunofluorescence method. The late apoptosis and necrosis of cells were measured by cell flow cytometry. Results: The results showed that different concentrations (5, 10, 20μM) of quercetin not only significantly reduced the protein and mRNA levels of TLR4, NLRP3, Caspase-1 and GSDMD, but also down-regulated the protein expression, mRNA and secretion of IL-1β and IL-18. Quercetin also inhibited the phosphorylation of NF-κB p65 and the degradation of IκB. At the same time, quercetin increased the cell migration rate and the expression level of ZO-1, and reduced the number of late apoptotic cells (P<0.05). Conclusions: Our data indicated that Quercetin reduced the inflammatory response and pyroptosis induced by LPS/ATP through the TLR4/NF-κB/NLRP3 pathway, and protected the migration and tight junctions of RIMVECs.

Abstract 281: Resveratrol Enhances Cholesterol Efflux Through PPAR-γ--Dependent Signaling Pathways in Human Macrophages

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ofek Y Hai ◽  
Iryna Voloshyna ◽  
Michael J Littlefield ◽  
Steven Carsons ◽  
Allison B Reiss
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Gene Silencing ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cholesterol Efflux ◽  
Foam Cell Formation ◽  
Mrna Levels ◽  
Ppar Γ

Introduction: We previously reported that resveratrol, a plant derived polyphenol with numerous cardioprotective properties, improves cholesterol homeostasis by upregulating the expression of reverse cholesterol transport (RCT) proteins critical for preventing lipid overload and macrophage foam cell formation. However, the mechanism(s) by which resveratrol exerts this effect are unclear. The present study explores a possible mechanism for the atheroprotective actions of resveratrol on cholesterol metabolism. Hypothesis: We hypothesize that the anti-atherogenic effects of resveratrol are mediated through PPAR-γ dependent signaling pathways leading to upregulation of RCT proteins and enhanced cholesterol efflux. Methods: THP-1 macrophages, a pertinent model for atherosclerosis, were incubated for 20h with resveratrol (10μM) with/without the specific PPAR-γ antagonist GW6992 (1μM). Alternatively, cells were transfected with 100 nM of human PPAR-γ small interfering RNA (siRNA) for gene silencing prior to resveratrol treatment. Total RNA was isolated, converted to cDNA, and evaluated by QRT-PCR. Each reaction was done in triplicate. Western blot analysis was performed to confirm results of gene expression. Results: Resveratrol significantly increased expression of PPAR-γ, ABCA1, and 27-hydroxylase mRNA (mean±SEM, 136.2±8.5%, 168.3±9.2%, and 171.7±11.0% of control respectively, P<0.001). PPAR-γ gene silencing and PPAR-γ antagonist GW6992 effectively reduced PPAR-γ message by 90.4% and 54.7%, respectively (P<0.001). Both pharmacological blockade and gene knockdown of PPAR-γ nullified resveratrol effects on cholesterol efflux proteins. In cells treated with GW6992, mRNA levels of ABCA1 and 27-hydroxylase were decreased to 66.1±3.3% and 55.0±2.9% of control, respectively (P<0.001). Similarly, PPAR-γ silencing resulted in downregulation of ABCA1 and 27-hydroxylase expression to the level of control, which is significantly lower than in resveratrol treated cells (P<0.001). Data from gene expression studies were subsequently confirmed by Western blot analysis. Conclusions: We propose here a mechanistic model for the atheroprotective effects of resveratrol. Our data strongly suggests that resveratrol regulates cholesterol efflux and intracellular cholesterol processing via the PPAR-γ/LXR-α pathway.

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