scholarly journals In vitro: The Modulating Effect of Myricetin on the Atherosclerosis Related Processes in THP1 Macrophages

2021 ◽  
pp. 62-73
Author(s):  
Rehab F. Almassabi ◽  
Etimad A. Huwait ◽  
Sanaa J. Almowallad ◽  
Salma Y. Saddeek ◽  
Kalamegam Gauthaman
Keyword(s):  
Real Time ◽  
Cholesterol Efflux ◽  
Control Treatment ◽  
Intercellular Adhesion Molecule ◽  
Research Centre ◽  
Protective Effects ◽  
Qrt Pcr ◽  
Monocyte Migration ◽  
Ifn Γ

Aims: To assess the anti-atherosclerotic effects of Myricetin (pharmaceutical) in human THP-1 macrophages following IFN-γ or MCP-1 stimulation. Study Design: The protective effects of myricetin against atherosclerosis was evaluated using the humanTHP-1 macrophages and studying the following parameters namely, cell viability, cell proliferation, cell migration, inflammation related gene expression and cholesterol efflux in vitro. Place and Duration of Study: THP-1 cell line: Department of biochemistry (faculty of science), Cell Culture Unit, Experimental Biochemistry Unit (King Fahad Medical Research Centre), King Abdul Aziz University, between September 2019 and September 2020. Methodology: The THP-1 cell lines were differentiated into macrophages by incubation with PMA (160 nM) for 24 hours. The viability percentage was determined using Pierce LDH cytotoxicity assay kit, the percentage change in macrophages proliferation was evaluated by crystal violet dye, the RNA was extracted then the cDNA was synthesized and the quantitative real time polymerase chain reaction (qRT-PCR) was done for inflammation-related genes, ICAM-1 and MCP-1. The percentage of monocyte migration and cholesterol efflux were also calculated. Results: Cytotoxicity assays demonstrated no significant toxicity with myricetin at 25μM and 50 μM concentrations on THP-1 macrophages. The quantitative real-time RT-PCR (qRT-PCR) demonstrated a significant increase in interferon gamma (IFN-γ) mediated expression of both intercellular adhesion molecule (ICAM-1) and monocyte chemo-attractant protein-1 (MCP-1) by 2.1 and 7.1 fold respectively, compared to the control. Treatment with myricetin (25 μM and 50 μM) significantly inhibited the IFN-γ induced overexpression of ICAM-1 by 42.86% & 71.34% and MCP-1 by 53.52% & 87.32% respectively. Myricetin (25 μM) significantly reduced the migration of monocytes by 33.66% compared to MCP-1. The cholesterol efflux from THP-1 macrophages treated with myricetin was significantly increased by 47% and 57% in the absence and presence of IFN-γ, respectively compared to the control. Conclusion: Myricetin has anti-inflammatory effects and supports cholesterol efflux, which can help in prevention of atherosclerosis. Furthermore, myricetin did not exhibit any cytotoxic effects and therefore is a safe phytochemical which can complement conventional therapeutics.

scholarly journals Punicalagin Regulates Key Processes Associated with Atherosclerosis in THP-1 Cellular Model

2020 ◽  
Vol 13 (11) ◽  
pp. 372
Author(s):  
Sanaa Almowallad ◽  
Etimad Huwait ◽  
Rehab Al-Massabi ◽  
Salma Saddeek ◽  
Kalamegam Gauthaman ◽  
...  
Keyword(s):  
Cholesterol Efflux ◽  
Cell Model ◽  
In Vitro Cytotoxicity ◽  
Intercellular Adhesion Molecule ◽  
Potential Candidate ◽  
Monocyte Chemoattractant Protein 1 ◽  
Cytotoxicity Assays ◽  
Monocyte Migration ◽  
Ifn Γ

Atherosclerosis may lead to cardiovascular diseases (CVD), which are the primary cause of death globally. In addition to conventional therapeutics for CVD, use of nutraceuticals that prevents cholesterol deposition, reduce existing plaques and hence anti-atherosclerotic effects of nutraceuticals appeared to be promising. As such, in the present study we evaluated the beneficial effects of punicalagin, a phytochemical against an atherosclerotic cell model in vitro. Cytotoxicity assays were examined for 10 µM concentration of punicalagin on THP-1 macrophages. Real-time-polymerase chain reaction (RT-PCR) was used to analyze monocyte chemoattractant protein-1 (MCP-1) and Intercellular adhesion molecule (ICAM-1) expressions. Monocyte migration and cholesterol efflux assays were performed to investigate punicalagin’s further impact on the key steps of atherosclerosis. Cytotoxicity assays demonstrated no significant toxicity for punicalagin (10 µM) on THP-1 macrophages. Punicalagin inhibited the IFN-γ-induced overexpression of MCP-1 and ICAM-1 in macrophages by 10 fold and 3.49 fold, respectively, compared to the control. Punicalagin also reduced the MCP-1- mediated migration of monocytes by 28% compared to the control. Percentages of cellular cholesterol efflux were enhanced in presence or absence of IFN-γ by 88% and 84% compared to control with 58% and 62%, respectively. Punicalagin possesses anti-inflammatory and anti-atherosclerotic effects. Punicalagin also did not exhibit any cytotoxicity and therefore can be considered a safe and potential candidate for the treatment and prevention of atherosclerosis.

scholarly journals Antiatherogenic Effects of Quercetin in the THP-1 Macrophage Model In Vitro, With Insights Into Its Signaling Mechanisms Using In Silico Analysis

2021 ◽  
Vol 12 ◽  
Author(s):  
Etimad A. Huwait ◽  
Salma Y. Saddeek ◽  
Rehab F. Al-Massabi ◽  
Sanaa J. Almowallad ◽  
Peter Natesan Pushparaj ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Silico ◽  
Cholesterol Efflux ◽  
Antioxidant Properties ◽  
Intercellular Adhesion Molecule ◽  
Lipid Uptake ◽  
Gene Expression Assay ◽  
Monocyte Migration ◽  
Signaling Mechanisms

Background: Atherosclerosis (AS), a major risk factor for stroke and brain tissue destruction, is an inflammatory disease of the blood vessels, and the underlying pathology is inflammation mediated by various chemokines and cytokines. Quercetin, a natural flavonol, is reported to have both anti-inflammatory and antioxidant properties. As such, in the present study, we evaluated the antiatherogenic effects of quercetin in a human THP-1 cell line in vitro and also the signaling mechanisms using in silico analysis.Materials and Methods: THP-1 macrophages exposed to different concentrations of quercetin (5–100 μM for 24 h) were tested for cytotoxicity. Real-time gene expression assay for intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) was carried out following treatment with quercetin at 15 and 30 μM for 24 h either in the absence or presence of interferon (IFN-γ) for 3 h to induce inflammation. Monocyte migration and cholesterol efflux were also assessed.Results: Quercetin did not exert any cytotoxic effects on THP-1 cells at the various concentrations tested. The gene expression assay showed a significant decrease in ICAM-1 (by 3.05 and 2.70) and MCP-1 (by 22.71 and 27.03), respectively. Quercetin at 15 µM decreased THP-1 monocyte migration by 33% compared to the MCP-1-treated cells. It also increased cholesterol efflux significantly by1.64-fold and 1.60-fold either alone or in combination with IFN-γ, respectively. Ingenuity Pathway Analysis of the molecular interactions of quercetin identified canonical pathways directly related to lipid uptake and cholesterol efflux. Furthermore, CD36, SR-A, and LXR-α also demonstrated significant increases by 72.16-, 149.10-, and 29.68-fold, respectively.Conclusion: Our results from both in vitro and in silico studies identified that quercetin inhibited the THP-1 monocyte migration, MCP-1, and ICAM-1 and increased cholesterol efflux probably mediated via the LXR/RXR signaling pathway. Therefore, quercetin will help prevent cell infiltration in atherosclerotic plaques and reduce the risk of stroke or brain destruction.

Tu1989 Real Time In Vitro Monitoring of Inflammation Induced Motility Changes and Protective Effects of STW 5 in a Newly Designed Model

2012 ◽  
Vol 142 (5) ◽  
pp. S-895
Author(s):  
Andrei Sibaev ◽  
Birol Yuoce ◽  
Olaf Kelber ◽  
Dieter Weiser ◽  
Heba Abdel-Aziz ◽  
...  
Keyword(s):  
Real Time ◽  
Protective Effects

scholarly journals Poxvirus-encoded TNF receptor homolog dampens inflammation and protects from uncontrolled lung pathology during respiratory infection

2020 ◽  
Vol 117 (43) ◽  
pp. 26885-26894
Author(s):  
Zahrah Al Rumaih ◽  
Ma. Junaliah Tuazon Kels ◽  
Esther Ng ◽  
Pratikshya Pandey ◽  
Sergio M. Pontejo ◽  
...  
Keyword(s):  
Inflammatory Cytokine ◽  
Cytokine Production ◽  
Tumor Necrosis Factor Receptor ◽  
Binding Domain ◽  
Lung Pathology ◽  
Protective Effects ◽  
Inflammatory Cytokine Production ◽  
Reverse Signaling ◽  
Ifn Γ

Ectromelia virus (ECTV) causes mousepox, a surrogate mouse model for smallpox caused by variola virus in humans. Both orthopoxviruses encode tumor necrosis factor receptor (TNFR) homologs or viral TNFR (vTNFR). These homologs are termed cytokine response modifier (Crm) proteins, containing a TNF-binding domain and a chemokine-binding domain called smallpox virus-encoded chemokine receptor (SECRET) domain. ECTV encodes one vTNFR known as CrmD. Infection of ECTV-resistant C57BL/6 mice with a CrmD deletion mutant virus resulted in uniform mortality due to excessive TNF secretion and dysregulated inflammatory cytokine production. CrmD dampened pathology, leukocyte recruitment, and inflammatory cytokine production in lungs including TNF, IL-6, IL-10, and IFN-γ. Blockade of TNF, IL-6, or IL-10R function with monoclonal antibodies reduced lung pathology and provided 60 to 100% protection from otherwise lethal infection. IFN-γ caused lung pathology only when both the TNF-binding and SECRET domains were absent. Presence of the SECRET domain alone induced significantly higher levels of IL-1β, IL-6, and IL-10, likely overcoming any protective effects that might have been afforded by anti–IFN-γ treatment. The use of TNF-deficient mice and those that express only membrane-associated but not secreted TNF revealed that CrmD is critically dependent on host TNF for its function. In vitro, recombinant Crm proteins from different orthopoxviruses bound to membrane-associated TNF and dampened inflammatory gene expression through reverse signaling. CrmD does not affect virus replication; however, it provides the host advantage by enabling survival. Host survival would facilitate virus spread, which would also provide an advantage to the virus.

scholarly journals CD137 Agonist Induces Gastric Cancer Cell Apoptosis by Enhancing The Functions of CD8+ T Cells via NF-κB Signaling

2020 ◽  
Author(s):  
Ben-Shun Hu ◽  
Tian Tang ◽  
Tie-Long Wu ◽  
Ying-Yue Sheng ◽  
Yu-Zheng Xue
Keyword(s):  
Gastric Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
Real Time ◽  
Peripheral Blood ◽  
Cell Apoptosis ◽  
Nuclear Translocation ◽  
Granzyme B ◽  
Ifn Γ

Abstract Background: CD137 is identified as a target for tumor immunotherapy. However, the role of CD137 in gastric cancer (GC), especially in inducing GC cell apoptosis has not been studied yet. Methods: Foxp3+ and CD8+ T cells in GCs were investigated by immunohistochemistry (IHC). CD137 expression in GCs was detected by flow cytometry, IHC and immunofluorescence (IF). Peripheral blood mononuclear cells (PBMCs) and CD8+ T cells isolated from peripheral blood were stimulated with a CD137 agonist in vitro. CD8+ T cells proliferation and p65 expression were explored by flow cytometry. p65 nuclear translocation was analyzed by IF. IL-10, TGF-β, IFN-γ, Perforin and Granzyme B were detected by real-time quantitative PCR (real-time PCR). PBMCs and primary GC cells were cocultured and stimulated with the CD137 agonist in vitro. Apoptosis of the primary GC cells was detected by flow cytometry. Results: Our data demonstrated that GC tumors show characteristics of an immunosuppressive microenvironment. CD137 was predominantly expressed in CD8+ T cells in GCs and had a positive correlation with tumor cell differentiation. CD137 agonist promoted CD8+ T cells proliferation and increased the secretion of IFN-γ, Perforin and Granzyme B, which induced primary GC cell apoptosis. Mechanistically, this study found that CD137 agonist could induce NF-κB nuclear translocation in CD8+ T cells. Conclusion: Our results demonstrate that CD137 agonist can induce primary GC cell apoptosis by enhancing CD8+ T cells via activating NF-κB signaling.

scholarly journals Rehin Mitigates Chronic Kidney Injury by Decreasing the Release of Perforin and IFN-γ from Immune Cells Via Inhibiting the STING/TBK1/IRF3 Pathway Activation

2022 ◽  
Author(s):  
Jingyu Wang ◽  
Xin Huang ◽  
Yong Liao ◽  
Xintian Cai ◽  
Jing Xu ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Immune Cells ◽  
Medicinal Preparation ◽  
Kidney Injury ◽  
Normal Subjects ◽  
Inflammatory Factor ◽  
Mice Model ◽  
Qrt Pcr ◽  
Ifn Γ

Abstract Shenkang suppository (SKS), a Chinese medicinal preparation rich in various natural ingredients, has not been reported in any studies related to fibrosis. Our experiments validated the anti-fibrotic and anti-inflammatory effects of Rhein (Rh), which is a major component of SKS, and explored its potential immune mechanisms. Tissue and serum specimens from chronic kidney disease (CKD) patients and normal subjects were collected in 30 cases each, and the expression differences of perforin and IFN-γ were analyzed by ELISA. Further, the CKD mice model constructed with folic acid (FA) was used to validate these differences by WB and qRT-PCR to explore the potential nephroprotective mechanism of Rh. Besides, in vitro experiments were conducted to identify the release sources of perforin and IFN-γ. ELISA showed that perforin and IFN-γ were upregulated in CKD patients, and this phenomenon was also corroborated in CKD mice. WB and qRT-PCR data showed that Rh reversed perforin and IFN-γ upregulation, inflammatory factor recruitment, and extracellular matrix (ECM) protein upregulation. Results from in vitro experiments demonstrate that the upregulation of perforin and IFN-γ originates from the stress response of CD4+ T lymphocytes (CD4+ cells), CD8+ T lymphocytes (CD8+ cells) and natural killer cells (NK cells), which can be suppressed by Rh. More importantly, the activated STING/TBK1/IRF3 pathway in CKD was also inhibited by Rh. Our data suggest that Rh possesses anti-fibrotic and nephroprotective effects, which mechanistically are associated with decreased release of perforin and IFN-γ from immune cells, which may be achieved by suppressing the STING/TBK1/IRF3 pathway.

Gene Expression Profiling in Multiple Myeloma Cells Treated with the Novel Anti-Myeloma Agents Zoledronate and Fluvastatin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5078-5078
Author(s):  
Timothy J. Molloy ◽  
Baulch-Brown Cindy ◽  
Yi-Mo Deng ◽  
Andrew Spencer ◽  
David F. Ma
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Real Time ◽  
Real Time Pcr ◽  
Mevalonate Pathway ◽  
Annexin V ◽  
Quantitative Real Time Pcr ◽  
Myeloma Cells ◽  
Qrt Pcr

Abstract We have shown in vitro that multiple myeloma (MM) cells can be destroyed by treating them with the mevalonate pathway inhibitors zoledronate and fluvastatin. While the efficacy of these compounds singly and combination have been demonstrated, their exact modes of action remain largely unknown. The present study aimed to use microarray and quantitative real-time PCR (QRT-PCR) techniques to analyse gene expression in treated myeloma cells to identify novel genes and pathways involved in the anti-myeloma action of these compounds. The human MM cell line NCI-H929 was treated with zoledronate and fluvastatin singly and in combination, and RNA was extracted and used to interrogate oligonucleotide microarrays consisting of 19,000 features representing known and unknown genes. Quantitative real-time PCR was subsequently used to confirm the expression of several genes of interest. Flow cytometry with Annexin V FITC staining was used to detect apoptosis. It was observed that genes related to apoptosis (caspases and p53-related genes), cell cycle control (cyclins), GTPase signalling (Rabs), and growth and proliferation (growth factors) were particularly affected by zoledronate and fluvastatin, and some of these genetic effects were synergistic when a combination of zoledronate and fluvastatin was used. QRT-PCR confirmed the effects on the caspase- and p53-related apoptotic pathways, and these effects were correlated with increased apoptosis in the myeloma cells. The mevalonate pathway inhibitors fluvastatin and zoledronate are highly efficient at killing MM cells, and their effects appear to be synergistic. Our microarray and QT-PCR analyses demonstrated that the expression of specific groups of genes important to the survival and proliferation of myeloma cells are affected by these compounds. p53 and caspase-dependent pathways appear to be the key apoptotic cascades stimulated. Insights into the mechanisms of these novel therapeutics are important as they might help to define their roles in the treatment of multiple myeloma.

scholarly journals MicroRNA files in the prevention of intestinal ischemia/reperfusion injury by hydrogen rich saline

2020 ◽  
Vol 40 (1) ◽  
Author(s):  
Weifeng Yao ◽  
Xiaoyu Lin ◽  
Xue Han ◽  
Lanfen Zeng ◽  
Anshun Guo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mammalian Target Of Rapamycin ◽  
Mucosal Injury ◽  
Regulatory Subunit ◽  
Protective Effects ◽  
Intestinal Ischemia Reperfusion

Abstract Background: Hydrogen-rich saline (HRS) has been proven effective against ischemia/reperfusion (I/R) injury. However, knowledge on the underlying signaling events remain poor. Having recent highlight of microRNAs (miRNAs) in mediating intestinal I/R injury, we hypothesized that HRS may protect intestine against I/R injury by regulating miRNAs. Method: Mice were given intraperitoneal injection of saline or HRS once daily for five consecutive days before undergoing intestinal I/R that was induced by 60-min ischemia followed by 180-min reperfusion of superior mesenteric artery. The intestine was collected for histopathological assay, miRNA microarray profiling, Real-Time PCR, and Western blotting. Next, miR-199a-3p mimics or inhibitors were transfected into IEC-6 cells to explore the relationship between HRS treatment and miR-199a-3p. Results: I/R-induced mucosal injury and epithelial cells apoptosis were attenuated by HRS pretreatment. A total of 64 intestinal I/R-responsive miRNAs were altered significantly by HRS pretreatment, in which we validated four novel miRNAs with top significance by Real-Time PCR, namely miR-199a-3p, miR-296-5p, miR-5126, and miR-6538. Particularly, miR-199a-3p was drastically increased by I/R but reduced by HRS. Computational analysis predicts insulin-like growth factor (IGF)-1, mammalian target of rapamycin (mTOR), and phosphoinositide-3-kinase (PI3K) regulatory subunit 1 as targets of miR-199a-3p, suggesting involvement of the pro-survival pathway, IGF- 1/PI3K/Akt/mTOR. In in vitro experiment, HRS treatment reduced miR-199a-3p level, increase IGF-1, PI3K and mTOR mRNA expression, restore IEC-6 cells viability, and this protective effects were reversed under miR-199a-3p mimics treatment. Conclusion: Collectively, miR-199a-3p may serve a key role in the anti-apoptotic mechanism of HRS that contributes to its protection of the intestine against I/R injury.

Comprehensive Analysis of CD8 T Cell Immune Response Specific for Two Novel HLA-A*0201 Restriced CMV pp65 Peptides.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3928-3928
Author(s):  
Laura Bracci ◽  
David F. Stroncek ◽  
Stefanie Slezak ◽  
Giulio C. Spagnoli ◽  
Maurizio Provenzano
Keyword(s):  
Gene Expression ◽  
Hla Class I ◽  
Cd8 T Cell ◽  
Comprehensive Analysis ◽  
Protein Release ◽  
Cmv Infection ◽  
Qrt Pcr ◽  
Ifn Γ ◽  
In Vitro Induction

Abstract In immune compromised subjects such as patients undergoing bone marrow, organ transplantation or immunosuppressive therapies, Cytomegalovirus (CMV) infection is associated with significant morbidity until the individual’s immune system is completely reconstituted. One method of preventing CMV infection during immune suppression in transplant patients is represented by adoptive administration of CMV peptide-specific cytotoxic T lymphocytes (CTLs) from HLA-matched donors. Despite the strong immunogenicity demonstrated by specific HLA class I peptides, the peptide’s responsiveness varies among individuals. Therefore, it is important to identify additional epitopes for each HLA determinant of interest. In this study we report a comprehensive analysis of CD8 T cell-specific activity against two novel CMV pp65 HLA-A*0201 associated peptides in CMV-experienced HLA-A*0201 subjects. PBMCs from CMV seropositive HLA-A*0201 restricted healthy subjects were peptide-stimulated ex vivo and IFN-γ gene expression was analyzed by qrt-PCR. An IFN-γ ELISPOT assay was carried out on peptide-specific elicited CD8 T lymphocytes to confirm the qrt-PCR results. Tetrameric HLA/epitope complexes (tHLA) were used to track the levels of peptide-specific CD8 T cells responsiveness to cognate epitopes. In addition, in vitro peptide-specific expanded populations of CTLs were used in 51Cr release assay. Based on qrt-PCR results, four out of eight HLA-A*0201 peptides identified by computer algorithms were selected. Two are preaviously published peptides: pp65495–503 (NLVPMVATV) and pp65347–355 (ALFFFDIDL), while two are novel: pp65340–348 (RQYDPVAAL) and pp65310–318 (LMNGQQIFL). In spite the four peptides induced comparable mRNA IFN-γ transcript production, peptides pp65495–503, pp65340–348 and pp65310–318 induced a consistent and sustained IFN-γ protein release and specific killing, while the pp65347–355 failed to induce IFN-γ protein secretion and killing activity (if we exclude a positive IFN-γ protein release after 2-week in vitro induction in one of the donors tested). Comparative assays carried on the functional activity of the three peptides pp65495–503, pp65340–348 and pp65310–318 revealed no intrinsic differences in term of IFN-γ protein release and cytotoxic activity save for the CTL affinity to the HLA-A*0201/epitope complexes (pp65495–503 ~2.6% in nearly 100% of donors vs pp65340–348 ~0.67% or pp65310–318 ~0.77% in nearly one third of the donors after 2-week in vitro induction). The tHLA binding results could be possibly ascribed to differencies in the peptide avidity and stability for the HLA class I. Taken together, these results lead to the conclusion that different peptides can induce a variable levels of immune responses ranging between mere cytokine gene expression and effective cytotoxic activity. In addition, the two novel peptides selected here broaden the panel of potential reagents useful for adoptive immune therapy. Thus, in anticipation of a specific epitope-targeted immune intervention, the three HLA-A*0201 peptides described could be used in combination for adoptive transfer of epitope-specific T cells or epitope-specific vaccination.

Expression and cellular distribution of estrogen and progesterone receptors and the real-time proliferation of porcine cumulus cells

Zygote ◽  
2014 ◽  
Vol 23 (6) ◽  
pp. 836-845 ◽  
Author(s):  
Bartosz Kempisty ◽  
Agnieszka Ziółkowska ◽  
Sylwia Ciesiółka ◽  
Hanna Piotrowska ◽  
Paweł Antosik ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Progesterone Receptors ◽  
Cumulus Cells ◽  
Proliferation Index ◽  
Estrogen And Progesterone Receptors ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Cell Proliferation Index

SummaryAlthough the expression of estrogen and progesterone receptors within porcine ovary and cumulus–oocyte complexes (COCs) is well recognized, still little information is known regarding expression of the progesterone receptor (PGR), PGR membrane component 1 (PGRMC1) and of estrogen-related receptors (ERRγ and ERRβ/γ) in separated cumulus cells in relation to real-time proliferation. In this study, a model of oocytes-separated cumulus cells was used to analyze the cell proliferation index and the expression PGR, PGRMC1 and of ERRγ and ERRβ/γ during 96-h cultivation in vitro using real-time quantitative PCR (qRT-PCR) and confocal microscopic observation. We found that PGR protein expression was increased at 0 h, compared with PGR protein expression after 96 h of culture (P < 0.001). The expression of PGRMC1, ERRγ and ERRβ/γ was unchanged. After using qRT-PCR we did not found statistical differences in expression of PGR, PGRMC1, ERRγ and ERRβ/γ during 96 h of cumulus cells in vitro culture (IVC). We supposed that the differential expression of the PGR protein at 0 h and after 96 h is related to a time-dependent down-regulation, which may activate a negative feedback. The distribution of PGR, PGRMC1 proteins may be linked with the translocation of receptors to the cytoplasm after the membrane binding of respective agonists and intra-cytoplasmic signal transduction. Furthermore, cumulus cells analyzed at 0 h were characterized by decreased proliferation index, whereas those after 96 h of culture revealed a significant increase of proliferation index, which may be associated with differentiation/luteinization of these cells during real-time proliferation.

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