scholarly journals Cryptotanshinone Regulates Androgen Synthesis through the ERK/c-Fos/CYP17 Pathway in Porcine Granulosa Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Danfeng Ye ◽  
Meifang Li ◽  
Yuehui Zhang ◽  
Xinhua Wang ◽  
Hua Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Mrna Expression ◽  
Molecular Mechanism ◽  
Granulosa Cells ◽  
Protein Level ◽  
Salvia Miltiorrhiza ◽  
Androgen Excess ◽  
Protein Levels ◽  
Androgen Synthesis ◽  
Porcine Granulosa Cells

The aim of the study is to investigate the molecular mechanism behind androgen reduction in porcine granulosa cells (pGCs) withSalvia miltiorrhizaBunge extract cryptotanshinone. PGCs were isolated from porcine ovaries and identified. Androgen excess model of the pGCs was induced with the MAPK inhibitor PD98059 and then treated with cryptotanshinone. The testosterone level was measured by radioimmunoassay in the culture media. The protein levels of P-ERK1/2, c-Fos, and CYP17 in the cells were measured by western blot. Cryptotanshinone decreased the concentration of testosterone and the protein level of CYP17 and increased the protein levels of P-ERK1/2 and c-Fos in the androgen excess mode. After the c-Fos gene was silenced by infection with c-Fos shRNA lentivirus, we measured the mRNA expression by quantitative RT-PCR and protein level by western blot of P-ERK1/2, c-Fos, and CYP17. This showed that the mRNA expression and protein level of P-ERK1/2 and c-Fos were significantly reduced in the shRNA–c-Fos group compared to the scrambled group, while those of CYP17 were significantly increased. So we concluded that cryptotanshinone can significantly reduce the androgen excess induced by PD98059 in pGCs. The possible molecular mechanism for this activity is regulating the ERK/c-Fos/CYP17 pathway.

MiR-335-5p Inhibits β-Amyloid (Aβ) Accumulation to Attenuate Cognitive Deficits Through Targeting c-jun-N-terminal Kinase 3 in Alzheimer’s Disease

2020 ◽  
Vol 17 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Dan Wang ◽  
Zhifu Fei ◽  
Song Luo ◽  
Hai Wang
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Transgenic Mice ◽  
Western Blot ◽  
Mrna Expression ◽  
Cognitive Deficits ◽  
Protein Levels ◽  
Amyloid Aβ ◽  
Β Amyloid ◽  
The Relationship

Objectives: Alzheimer's disease (AD), also known as senile dementia, is a common neurodegenerative disease characterized by progressive cognitive impairment and personality changes. Numerous evidences have suggested that microRNAs (miRNAs) are involved in the pathogenesis and development of AD. However, the exact role of miR-335-5p in the progression of AD is still not clearly clarified. Methods: The protein and mRNA levels were measured by western blot and RNA extraction and quantitative real-time PCR (qRT-PCR), respectively. The relationship between miR-335-5p and c-jun-N-terminal kinase 3 (JNK3) was confirmed by dual-luciferase reporter assay. SH-SY5Y cells were transfected with APP mutant gene to establish the in vitro AD cell model. Flow cytometry and western blot were performed to evaluate cell apoptosis. The APP/PS1 transgenic mice were used as an in vivo AD model. Morris water maze test was performed to assess the effect of miR- 335-5p on the cognitive deficits in APP/PS1 transgenic mice. Results: The JNK3 mRNA expression and protein levels of JNK3 and β-Amyloid (Aβ) were significantly up-regulated, and the mRNA expression of miR-335-5p was down-regulated in the brain tissues of AD patients. The expression levels of miR-335-5p and JNK3 were significantly inversely correlated. Further, the dual Luciferase assay verified the relationship between miR-335- 5p and JNK3. Overexpression of miR-335-5p significantly decreased the protein levels of JNK3 and Aβ and inhibited apoptosis in SH-SY5Y/APPswe cells, whereas the inhibition of miR-335-5p obtained the opposite results. Moreover, the overexpression of miR-335-5p remarkably improved the cognitive abilities of APP/PS1 mice. Conclusion: The results revealed that the increased JNK3 expression, negatively regulated by miR-335-5p, may be a potential mechanism that contributes to Aβ accumulation and AD progression, indicating a novel approach for AD treatment.

GATA6 Depletion Reduces Cyclic AMP-Stimulated IGF1 mRNA and Free Protein Levels in Luteinizing Porcine Granulosa Cells.

2010 ◽  
Vol 83 (Suppl_1) ◽  
pp. 634-634 ◽  
Author(s):  
Holly A. LaVoie ◽  
Jonathan B. Nguyen ◽  
Richard J. Kordus ◽  
Yvonne Y. Hui
Keyword(s):  
Cyclic Amp ◽  
Granulosa Cells ◽  
Free Protein ◽  
Protein Levels ◽  
Porcine Granulosa Cells

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses

2016 ◽  
Vol 28 (9) ◽  
pp. 1340 ◽  
Author(s):  
Dragos Scarlet ◽  
Ingrid Walter ◽  
Juraj Hlavaty ◽  
Christine Aurich
Keyword(s):  
Western Blot ◽  
Granulosa Cells ◽  
Hormone Receptors ◽  
Ovarian Function ◽  
Ovarian Tissue ◽  
Cumulus Cells ◽  
Protein Levels ◽  
Fshr Gene ◽  
Luteal Tissue ◽  
Immunofluorescence Labelling

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4 mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal’s age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.

scholarly journals Cardiac deletion of α-E-catenin leads to redused expression of the main component of desmosomes – γ-catenin

1970 ◽  
Vol 21 ◽  
pp. 293-296
Author(s):  
V. V. Balatskyi ◽  
L. L. Matsevych ◽  
O. O. Piven
Keyword(s):  
Heart Failure ◽  
Transgenic Mice ◽  
Western Blot ◽  
Real Time ◽  
Knockout Mice ◽  
Protein Level ◽  
Conditional Knockout ◽  
Protein Levels ◽  
Main Component ◽  
Real Time Qpcr

Aim. In our present work, we have addressed to the γ-catenin, known main component of desmosomes, expression in hearts with heterozygous and homozygous knockout of α-E-catenin gene. Methods. Alpha-E-catenin conditional knockout mice were bred with α-MHC-Cre transgenic mice. We analyze expression of γ-catenin with real time qPCR and Western blot. Results. Cardiac α-E-catenin deletion leads to downregulation of γ-catenin mRNA and protein levels only in homozygous mice, while we not observed any perturbation of γ-catenin expression in heterozygous mice. Conclusions. We have shown that homozygous knockout of α-E-catenin gene in embryonic heart occur reduction of the main component of desmosomes – γ-catenin mRNA and protein level of expression, which can lead to disruption of the desmosomes structure in adult myocardium. Keywords: α-E-catenin, heart failure, γ-catenin.

scholarly journals Is idiopathic hirsutism (IH) really idiopathic? mRNA expressions of skin steroidogenic enzymes in women with IH

2015 ◽  
Vol 173 (4) ◽  
pp. 447-454 ◽  
Author(s):  
Serpil Taheri ◽  
Gokmen Zararsiz ◽  
Sulbiye Karaburgu ◽  
Murat Borlu ◽  
Mahmut Tuncay Ozgun ◽  
...  
Keyword(s):  
Skin Biopsy ◽  
Mrna Expression ◽  
Androgen Excess ◽  
Functional Roles ◽  
Steroid Sulphatase ◽  
Healthy Women ◽  
Androgen Synthesis ◽  
Idiopathic Hirsutism ◽  
Skin Biopsies ◽  
Gene Mrna

ObjectiveHirsutism results from hyperandrogenemia and/or exaggerated androgen responsiveness. Among various causes of hirsutism, some patients do not exhibit androgen excess which is called idiopathic hirsutism (IH). The pathogenesis of IH could not so far be clearly established.DesignTo investigate the mRNA expression of aromatase enzyme and the other enzymes having functional roles in the steroidogenic pathway, in freshly obtained skin tissue from subumbilical skin and the arm of the patients with IH and healthy women.MethodsTwenty-one women with IH and 15 healthy women were included in the study. We aimed to determine mRNA expressions of genes associated with local androgen synthesis and metabolism (CYP11A1, STS, CYP19A1, SRD5A1, SRD5A2, HSD3B1, AR, COMT, ESR1, ESR2, HSD3B2, CYP17A1, SULT2A1, SULT1E1, HSD17B2, IL6, TGFB1, TNFA) from skin biopsy and blood samples of patients with IH and the data compared with healthy subjects.ResultsPatients with IH exhibit significantly lower interleukin 6 (IL6) mRNA expression and higher steroid sulphatase (STS) and hydroxysteroid (17beta) dehydrogenase 2 (HSD17B2), gene mRNA expression, respectively, in the subumbilical region skin biopsies. Similarly, patients with IH exhibit significantly lowerIL6mRNA expression and higherSTSandHSD17B2gene mRNA expression, respectively, in the arm skin compared to healthy women's subumbilical region.ConclusionsIn both arm and subumbilical skin biopsy of patients with IH, we observed an up-regulation ofHSD17B2andSTS, decreasedIL6mRNA expression, probably determining an increase in the local amount of active androgens, which could then be used as substrate for other androgen metabolic routes.

scholarly journals Canonical, Non-Canonical and Atypical Pathways of Nuclear Factor кb Activation in Preeclampsia

2020 ◽  
Vol 21 (15) ◽  
pp. 5574
Author(s):  
Agata Sakowicz ◽  
Michalina Bralewska ◽  
Tadeusz Pietrucha ◽  
Dominika E Habrowska-Górczyńska ◽  
Agnieszka W Piastowska-Ciesielska ◽  
...  
Keyword(s):  
Western Blot ◽  
Real Time ◽  
Protein Level ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Rt Pcr ◽  
Protein Levels ◽  
Activation Pathways ◽  
Expression And Activity

Although higher nuclear factor κB (NFκB) expression and activity is observed in preeclamptic placentas, its mechanism of activation is unknown. This is the first study to investigate whether the canonical, non-canonical, or atypical NFκB activation pathways may be responsible for the higher activation of NFκB observed in preeclamptic placentas. The study included 268 cases (130 preeclamptic women and 138 controls). We studied the expression of the genes coding for NFκB activators (NIK, IKKα, IKKβ, and CK2α) and inhibitors (IκBα and IκBβ) using RT-PCR in real time. The RT-PCR results were verified on the protein level using ELISA and Western blot. To determine the efficiency of the pathways, the ratios of activator(s) to one of the inhibitors (IκBα or IκBβ) were calculated for each studied pathway. The preeclamptic placentas demonstrated significantly lower IKKα and CK2α but higher IκBα and IκBβ protein levels. In addition, the calculated activator(s) to inhibitor (IκBα or IκBβ) ratios suggested that all studied pathways might be downregulated in preeclamptic placentas. Our results indicate that preeclamptic placentas may demonstrate mechanisms of NFκB activation other than the canonical, non-canonical, and atypical forms. In these mechanisms, inhibitors of NFκB may play a key role. These observations broaden the existing knowledge regarding the molecular background of preeclampsia development.

scholarly journals HIF1A-dependent increase in endothelin 2 levels in granulosa cells: role of hypoxia, LH/cAMP, and reactive oxygen species

Reproduction ◽  
2015 ◽  
Vol 149 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Ronit Yalu ◽  
Adepeju Esther Oyesiji ◽  
Iris Eisenberg ◽  
Tal Imbar ◽  
Rina Meidan
Keyword(s):  
Reactive Oxygen Species ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Time Window ◽  
Hypoxia Inducible Factor ◽  
Reactive Oxygen ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Protein Levels ◽  
Concomitant Reduction

Hypoxia-inducible factor 1 alpha (HIF1A) and endothelin 2 (EDN2) are transiently expressed during the same time window in the developing corpus luteum (CL). In this study, we sought to investigate the involvement of LH/cAMP, reactive oxygen species (ROS), and a hypoxia-mimetic compound (CoCl2) on HIF1A expression and how it affected EDN2 levels, using transformed human granulosa cells (thGCs) and primary bovine granulosa cells (GCs). CoCl2 elevated HIF1A protein levels in thGCs in a dose-dependent manner. Forskolin alone had no significant effect; however, forskolin and CoCl2 together further induced HIF1A protein and EDN2 mRNA expression in thGCs. Similarly, in primary GCs, LH with CoCl2 synergistically augmented HIF1A protein levels, which resulted in higher expression of EDN2 and another well-known hypoxia-inducible gene, VEGF (VEGFA). Importantly, LH alone elevated HIF1A mRNA but not its protein. The successful knockdown of HIF1A in thGCs using siRNA abolished hypoxia-induced EDN2 and also the additive effect of forskolin and CoCl2. We then examined the roles of ROS in thGCs: hydrogen peroxide (20 and 50 μM) elevated HIF1A protein as well as the expression of EDN2, implying that induction of HIF1A protein levels is sufficient to stimulate the expression of EDN2 (and VEGF) in normoxia. A broad-range ROS scavenger, butylated hydroxyanisole, inhibited CoCl2-induced HIF1A protein with a concomitant reduction in the mRNA expression of EDN2 and VEGF in thGCs. The results obtained in this study suggest that HIF1A, induced by various stimuli, is an essential mediator of EDN2 mRNA expression. The results may also explain the rise in the levels of HIF1A-dependent genes (EDN2 and VEGF) in the developing CL.

scholarly journals Dendrobium officinale Six nostrum promotes excretion of intestinal uric acid in hyperuricemia rat through inhibiting LPS/TLR4/NF-κB signaling pathway

2020 ◽  
Author(s):  
chen xue ◽  
ge hongzhang ◽  
lei shanshan ◽  
jiang zhetian ◽  
su jie ◽  
...  
Keyword(s):  
Uric Acid ◽  
Portal Vein ◽  
Western Blot ◽  
Protein Level ◽  
Dendrobium Officinale ◽  
Histopathological Changes ◽  
Intestinal Excretion ◽  
Protein Levels ◽  
Hepatic Portal Vein ◽  
Hepatic Portal

Abstract Background : To evaluate the effect of Dendrobium officinale Six nostrum (DOS) on promoting the intestinal excretion of uric acid (UA) in the rat model of hyperuricemia (HUA), and explored its possible mechanisms of action. Methods: In this study, HUA was induced in rat by administration of lipid emulsion (LE) for 6 weeks, meanwhile, the rat was orally administered with DOS for 6 weeks. Then the level of serum uric acid (SUA) and fecal uric acid (FUA) were measured by automatic biochemical analyzer. Lipopolysaccharide (LPS) in hepatic portal vein blood was detected by ELISA kit. The intestinal protein levels of ATP-binding cassette superfamily G member 2 (ABCG2) and glucose transporter 9 (GLUT9) were measured by Western blot assay. Toll-like receptor 4 (TLR4) and nuclear factor kappa-B (NF-κB) were determined by immunohistochemistry (IHC). Hematoxylin and eosin (H&E) staining was used to assess intestinal histological changes. Meanwhile, the main compositions of DOS were identified and determined by the High Performance Liquid Chromatography (HPLC). Results: According to HPLC analysis, acteoside and astilbin were identified as the main chemical components of DOS. Our results indicated that DOS significantly reduced SUA level and increased FUA level in the HUA rat induced by oral administration of LE for 6 weeks. IHC and Western blot showed that DOS could significantly increase protein level of ABCG2 and reduce protein level of GLUT9 in the intestine. It remarkably reduced the content of LPS in hepatic portal vein blood and decrease protein levels of TLR4 and NF-κB in the intestine. In addition, DOS could improve the histopathological changes of intestine through increasing the number of intestinal gland goblet cells, and recovering the complete and neatly-arranged structure of small intestinal epithelial cells. Conclusions: DOS has the effect of treating hyperuricemia, the molecular mechanism may be associated with up-regulating ABCG2 protein level, and down-regulating protein level of GLUT9 in the intestine to promote the intestinal excretion of UA, and then decreasing the SUA level. In addition, DOS can reduce the damage of inflammatory response to the intestine, improve the histopathological changes of intestine in HUA rat through inhibiting LPS/TLR4/NF-κB inflammatory pathway.

scholarly journals Production of VEGF receptor 1 and 2 mRNA and protein during endochondral bone repair is differential and healing phase specific

2010 ◽  
Vol 109 (6) ◽  
pp. 1930-1938 ◽  
Author(s):  
Marie K. Reumann ◽  
Turya Nair ◽  
Olga Strachna ◽  
Adele L. Boskey ◽  
Philipp Mayer-Kuckuk
Keyword(s):  
Mrna Expression ◽  
Protein Level ◽  
Bone Repair ◽  
Callus Formation ◽  
Detection Sensitivity ◽  
Vegf Receptor ◽  
Vegf Mrna ◽  
Endochondral Bone ◽  
Protein Levels ◽  
Healing Phase

Physiological disturbances, including temporary hypoxia, are expected to drive angiogenesis during bone repair. Evidence suggests that the angiogenic ligand vascular endothelial growth factor (VEGF)-A plays an important role in this process. We characterized the expression of two receptors that are essential for mediating VEGF signaling, VEGFR1/Flt-1 and VEGFR2/Flk-1/KDR, in a mouse rib fracture model. Their mRNA and protein levels were assessed in four healing phases, which were characterized histologically as hemorrhage formation on postfracture day (PFD) 1, inflammatory response on PFD 3, initiation of callus development on PFD 7, and the presence of a mature callus on PFD 14. Transcript was detected for VEGFR1 and VEGFR2, as well as VEGF. While mRNA expression of VEGFR1 was monophasic throughout all healing phases, VEGFR2 showed a biphasic profile with significantly increased mRNA expression during callus formation and maturation. Expression of VEGF mRNA was characterized by a more gradual increase during callus formation. The protein level for VEGFR1 was below detection sensitivity during the initial healing phase. It was then restored to a stable level, detectable through the subsequent healing phases. Hence, the VEGFR1 protein levels partially mirrored the transcript expression profile. In comparison, the protein level of VEGFR2 increased gradually during the healing phases and peaked at callus maturation. This correlated well with the transcriptional expression of VEGFR2. Intact bone from age-matched male mice had considerable protein levels of VEGFR1 and VEGF, but no detectable VEGFR2. Together, these findings uncovered expression signatures of the VEGF-VEGFR axis in endochondral bone repair.

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