Aldosterone up-regulates voltage-gated potassium currents and NKCC1 protein membrane fractions

Abstract Na+–K+–2Cl− Cotransporter (NKCC1) is a protein that aids in the active transport of sodium, potassium, and chloride ions across cell membranes. It has been shown that long-term systemic treatment with aldosterone (ALD) can enhance NKCC1 protein expression and activity in the aging cochlea resulting in improved hearing. In the present work, we used a cell line with confirmed NKCC1 expression to demonstrate that in vitro application of ALD increased outward voltage-gated potassium currents significantly, and simultaneously upregulated whole lysate and membrane portion NKCC1 protein expression. These ALD-induced changes were blocked by applying the mineralocorticoid receptor antagonist eplerenone. However, application of the NKCC1 inhibitor bumetanide or the potassium channel antagonist Tetraethyl ammonium had no effect. In addition, NKKC1 mRNA levels remained stable, indicating that ALD modulates NKCC1 protein expression via the activation of mineralocorticoid receptors and post-transcriptional modifications. Further, in vitro electrophysiology experiments, with ALD in the presence of NKCC1, K+ channel and mineralocorticoid receptor inhibitors, revealed interactions between NKCC1 and outward K+ channels, mediated by a mineralocorticoid receptor-ALD complex. These results provide evidence of the therapeutic potential of ALD for the prevention/treatment of inner ear disorders such as age-related hearing loss.

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Age-related decline in the expression of GDF9 and BMP15 genes in follicle fluid and granulosa cells derived from poor ovarian responders

Journal of Ovarian Research ◽

10.1186/s13048-020-00757-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yan Gong ◽

Jesse Li-Ling ◽

Dongsheng Xiong ◽

Jiajing Wei ◽

Taiqing Zhong ◽

...

Keyword(s):

Granulosa Cells ◽

Enzyme Linked Immunosorbent Assay ◽

Mrna Levels ◽

Clinical Trial Registry ◽

Altered Expression ◽

Vitro Fertilization ◽

Age Related ◽

Tertiary Teaching ◽

Poor Ovarian Responders

Abstract Background Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) genes play important roles in folliculogenesis. Altered expression of the two have been found among patients with poor ovarian response (POR). In this prospective cohort study, we have determined the expression of the GDF9 and BMP15 genes in follicle fluid (FF) and granulosa cells (GCs) derived from poor ovarian responders grouped by age, and explored its correlation with the outcome of in vitro fertilization and embryo transfer (IVF-ET) treatment. Methods A total of 196 patients with POR were enrolled from a tertiary teaching hospital. The patients were diagnosed by the Bologna criteria and sub-divided into group A (< 35 year old), group B (35–40 year old), and group C (> 40 year old). A GnRH antagonist protocol was conducted for all patients, and FF and GCs were collected after oocyte retrieval. Expression of the GDF9 and BMP15 genes in the FF and GCs was determined with enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. Results Compared with group C, groups A and B had significantly more two pronuclei (2PN) oocytes and transplantable embryos, in addition with higher rates of implantation and clinical pregnancy (P < 0.05). The expression level of GDF9 and BMP15 genes in the FF and GCs differed significantly among the three groups (P < 0.05), showing a trend of decline along with age. The ratio of GDF9/BMP15 mRNA levels were similar among the three groups (P > 0.05). The relative levels of GDF9 and BMP15 proteins in GCs have correlated with the relative mRNA levels in GCs and protein concentrations in FF (P < 0.05). Conclusions For poor ovarian responders, in particular those over 40, the expression of GDF9 and BMP15 is declined along with increased age and in accompany with poorer oocyte quality and IVF outcome, whilst the ratio of GDF9/BMP15 mRNA levels remained relatively constant. Trial registration Chinese Clinical Trial Registry Center (ChiCTR1800016107). Registered on 11 May 2018.

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Effects of CREG1 on Age-Associated Metabolic Phenotypes and Renal Senescence in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms22031276 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1276

Author(s):

Michihiro Hashimoto ◽

Ayumi Goto ◽

Yuki Endo ◽

Masataka Sugimoto ◽

Jun Ueda ◽

...

Keyword(s):

Renal Dysfunction ◽

Body Weight Gain ◽

Mrna Levels ◽

Wild Type ◽

Metabolic Phenotypes ◽

Age Related ◽

Secretory Phenotype ◽

Filtering Function ◽

Renal Senescence

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a secreted glycoprotein that accelerates p16-dependent cellular senescence in vitro. We recently reported the ability of CREG1 to stimulate brown adipogenesis using adipocyte P2-CREG1-transgenic (Tg) mice; however, little is known about the effect of CREG1 on aging-associated phenotypes. In this study, we investigated the effects of CREG1 on age-related obesity and renal dysfunction in Tg mice. Increased brown fat formation was detected in aged Tg mice, in which age-associated metabolic phenotypes such as body weight gain and increases in blood glucose were improved compared with those in wild-type (WT) mice. Blood CREG1 levels increased significantly in WT mice with age, whereas the age-related increase was suppressed, and its levels were reduced, in the livers and kidneys of Tg mice relative to those in WT mice at 25 months. Intriguingly, the mRNA levels of Ink4a, Arf, and senescence-associated secretory phenotype (SASP)-related genes and p38MAPK activity were significantly lowered in the aged kidneys of Tg mice, in which the morphological abnormalities of glomeruli as well as filtering function seen in WT kidneys were alleviated. These results suggest the involvement of CREG1 in kidney aging and its potential as a target for improving age-related renal dysfunction.

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Sestrin 2 Attenuates Rat Hepatic Stellate Cell (HSC) Activation and Liver Fibrosis via an mTOR/AMPK-Dependent Mechanism

Cellular Physiology and Biochemistry ◽

10.1159/000495829 ◽

2018 ◽

Vol 51 (5) ◽

pp. 2111-2122 ◽

Cited By ~ 7

Author(s):

Yi-Bing Hu ◽

Xiao-Ting Ye ◽

Qing-Qing Zhou ◽

Rong-Quan Fu

Keyword(s):

Liver Fibrosis ◽

Protein Expression ◽

Hepatic Fibrosis ◽

Transforming Growth Factor ◽

Histopathological Examination ◽

Stellate Cell ◽

Smooth Muscle Actin ◽

Mrna Levels ◽

Alpha Smooth Muscle Actin

Background/Aims: Sestrin 2 is associated with the pathophysiology of several diseases. The aim of this study was to investigate the effects and potential mechanisms of Sestrin 2 in rat hepatic stellate cells (HSCs) during liver fibrogenesis. Methods: In this study, Sestrin 2 protein expression was detected in rat HSC-T6 cells challenged with transforming growth factor-β (TGF-β) and in mice treated with carbon tetrachloride (CCl4), a well-known model of hepatic fibrosis. Next, HSC-T6 cells and fibrotic mice were transfected with lentivirus. The mRNA expression levels of markers of liver fibrosis [alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1)] were analyzed by quantitative reverse transcription–polymerase chain reaction (RT-PCR). Cell death and proliferation were evaluated by the MTT assay, and biochemical markers of liver damage in serum [alanine transaminase (ALT) and aspartate transaminase (AST)] were also measured using a biochemical analyzer. Histopathological examination was used to evaluate the degree of liver fibrosis, and protein expression [phospho-adenosine monophosphate-activated protein kinase (p-AMPK), AMPK, phospho-mammalian target of rapamycin (p-mTOR), and mTOR] was determined by western blotting. Results: We found that Sestrin 2 was elevated in both the HSC-T6 cell and hepatic fibrosis models. In vitro, overexpression of Sestrin 2 attenuated the mRNA levels of α-SMA and Col1A1, suppressed α-SMA protein expression, and modulated HSC-T6 cell proliferation. In vivo, overexpression of Sestrin 2 reduced the ALT and AST levels as well as the α-SMA and Col1A1 protein expression in the CCl4 model of liver fibrosis. Moreover, the degree of liver fibrosis was ameliorated. Interestingly, overexpression of Sestrin 2 increased p-AMPK but decreased p-mTOR protein expression. Conclusion: Our findings indicate that Sestrin 2 may attenuate the activation of HSCs and ameliorate liver fibrosis, most likely via upregulation of AMPK phosphorylation and suppression of the mTOR signaling pathway.

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Garlic Extract: Inhibition of Biochemical and Biophysical Changes in Glycated HSA

Applied Sciences ◽

10.3390/app112211028 ◽

2021 ◽

Vol 11 (22) ◽

pp. 11028

Author(s):

Mohd W. A. Khan ◽

Ahmed A. Otaibi ◽

Arwa F. M. Alhumaid ◽

Abdulmohsen K. D. Alsukaibi ◽

Asma K. Alshamari ◽

...

Keyword(s):

Structural Changes ◽

Therapeutic Potential ◽

Inhibitory Effect ◽

Significant Inhibition ◽

Garlic Extract ◽

Diabetic Patients ◽

Age Related ◽

Health Complications ◽

Biophysical Changes

Glycation of various biomolecules contributes to structural changes and formation of several high molecular weight fluorescent and non-fluorescent, advanced glycation end products (AGEs). AGEs and glycation are involved in various health complications. Synthetic medicines, including metformin, have several adverse effects. Natural products and their derivatives are used in the treatment of various diseases due to their significant therapeutic qualities. Allium sativum (garlic) is used in traditional medicines because of its antioxidant, anti-inflammatory, and anti-diabetic properties. This study aimed to determine the anti-glycating and AGEs inhibitory activities of garlic. Biochemical and biophysical analyses were performed for in vitro incubated human serum albumin (HSA) with 0.05 M of glucose for 1, 5, and 10 weeks. Anti-glycating and AGEs inhibitory effect of garlic was investigated in glycated samples. Increased biochemical and biophysical changes were observed in glycated HSA incubated for 10 weeks (G-HSA-10W) as compared to native HSA (N-HSA) as well as glycated HSA incubated for 1 (G-HSA-1W) and 5 weeks (G-HSA-5W). Garlic extract with a concentration of ≥6.25 µg/mL exhibited significant inhibition in biophysical and biochemical changes of G-HSA-10W. Our findings demonstrated that garlic extract has the ability to inhibit biochemical and biophysical changes in HSA that occurred due to glycation. Thus, garlic extract can be used against glycation and AGE-related health complications linked with chronic diseases in diabetic patients due to its broad therapeutic potential.

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β-Caryophyllene Reduces the Inflammatory Phenotype of Periodontal Cells by Targeting CB2 Receptors

Biomedicines ◽

10.3390/biomedicines8060164 ◽

2020 ◽

Vol 8 (6) ◽

pp. 164 ◽

Cited By ~ 2

Author(s):

Giacomo Picciolo ◽

Giovanni Pallio ◽

Domenica Altavilla ◽

Mario Vaccaro ◽

Giacomo Oteri ◽

...

Keyword(s):

Oral Mucositis ◽

Cannabinoid Receptor ◽

Therapeutic Potential ◽

Preclinical Model ◽

Mrna Levels ◽

Innovative Strategies ◽

Inflammatory Phenotype ◽

Cb2 Receptors ◽

Vitro Model

Human gingival fibroblasts (GF) and human oral mucosa epithelial cells (EC) with an inflammatory phenotype represent a valuable experimental paradigm to explore the curative activity of agents to be used in oral mucositis. The role of cannabinoid receptor 2 (CB2) has not yet been investigated in oral mucositis. The aim of this study was to evaluate the therapeutic potential of β-Caryophyllene (BCP), a CB2 agonist, in an in vitro model of oral mucositis. GF and EC were stimulated with LPS (2 µg/mL) alone or in combination with BCP; a group of LPS challenged GF and EC were treated with BCP and AM630, a CB2 antagonist. LPS increased the inflammatory cytokines TNF-α, IL-1β, IL-6 and IL-17A whereas it decreased the anti-inflammatory cytokine IL-13. The upstream signals were identified in an augmented expression of NF-κB and STAT-3 and in reduced mRNA levels of PPARγ and PGC-1α. BCP blunted the LPS-induced inflammatory phenotype and this effect was reverted by the CB2 antagonist AM630. These results suggest that CB2 receptors are an interesting target to develop innovative strategies for oral mucositis and point out that BCP exerts a marked curative effect in a preclinical model of oral mucositis which deserves to be confirmed in a clinical setting.

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High-Frequency Near-Infrared Diode Laser Irradiation Attenuates IL-1β-Induced Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Human Primary Chondrocytes

Journal of Clinical Medicine ◽

10.3390/jcm9030881 ◽

2020 ◽

Vol 9 (3) ◽

pp. 881 ◽

Cited By ~ 1

Author(s):

Shuzo Sakata ◽

Ryo Kunimatsu ◽

Yuji Tsuka ◽

Ayaka Nakatani ◽

Tomoka Hiraki ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Protein Expression ◽

Laser Irradiation ◽

Diode Laser ◽

High Frequency ◽

Near Infrared ◽

Mrna Levels ◽

Induced Expression ◽

Tnf Α

High-frequency near-infrared diode laser provides a high-peak output, low-heat accumulation, and efficient biostimulation. Although these characteristics are considered suitable for osteoarthritis (OA) treatment, the effect of high-frequency near-infrared diode laser irradiation in in vitro or in vivo OA models has not yet been reported. Therefore, we aimed to assess the biological effects of high-frequency near-infrared diode laser irradiation on IL-1β-induced chondrocyte inflammation in an in vitro OA model. Normal Human Articular Chondrocyte-Knee (NHAC-Kn) cells were stimulated with human recombinant IL-1β and irradiated with a high-frequency near-infrared diode laser (910 nm, 4 or 8 J/cm2). The mRNA and protein expression of relevant inflammation- and cartilage destruction-related proteins was analyzed. Interleukin (IL) -1β treatment significantly increased the mRNA levels of IL-1β, IL-6, tumor necrosis factor (TNF) -α, matrix metalloproteinases (MMP) -1, MMP-3, and MMP-13. High-frequency near-infrared diode laser irradiation significantly reduced the IL-1β-induced expression of IL-1β, IL-6, TNF-α, MMP-1, and MMP-3. Similarly, high-frequency near-infrared diode laser irradiation decreased the IL-1β-induced increase in protein expression and secreted levels of MMP-1 and MMP-3. These results highlight the therapeutic potential of high-frequency near-infrared diode laser irradiation in OA.

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A Pilot Study on the Beneficial Effects of the Additional Selenium Supplementation to Methimazole for Treating Patients with Graves? disease

TURKISH JOURNAL OF MEDICAL SCIENCES ◽

10.3906/sag-1808-67 ◽

2019 ◽

Author(s):

BIN XU ◽

DI WU ◽

HONG YING ◽

YING ZHANG

Keyword(s):

Protein Expression ◽

Combination Group ◽

Thyroid Peroxidase ◽

Graves Disease ◽

Mrna Levels ◽

Thyroid Cells ◽

Beneficial Effects ◽

Thyroid Peroxidase Antibody ◽

Thyroglobulin Antibody

Backgroud/aim: The aim of this study was to assess the effect of a combination use of methimazole (MMI) and selenium (Se) in the treatment of Graves’ disease (GD). Materials and methods: A total of 103 newly-diagnosed hyperthyroidism patients were randomized to MMI and MMI+Se combination group. After treatment for six months, the levels of triiodothyronine (FT3), free thyroxine (FT4), thyrotrophin receptor antibody (TRAb), thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TGAb) were observed. Besides, an in vitro culture model of thyroid cells was established and the protein expression and mRNA levels of TRAb, TPOAb and TGAb were determined by western blot and RT-PCR. Results: A significant decrease in the levels of FT3, FT4, TRAb, TPOAb and TGAb were observed in both groups along with a marked increase in TSH levels. Furthermore, the in vitro experiments showed that the protein expression and mRNA levels of TRAb, TPOAb and TGAb decreased significantly. Also, compared to the MMI group, there was a greater improvement of these indices in the MMI+Se group. Conclusion: We suggest that the combined use of MMI and Se could improve the thyroid activity in patients which may provide effective therapy for the treatment of GD in clinical settings. Key words: Graves’ disease; methimazole; selenium; TRAb; TPOAb; TGAb

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Comparison of CXC Chemokines ENA-78 and Interleukin-8 Expression in Helicobacter pylori-Associated Gastritis

Infection and Immunity ◽

10.1128/iai.69.1.81-88.2001 ◽

2001 ◽

Vol 69 (1) ◽

pp. 81-88 ◽

Cited By ~ 29

Author(s):

Gabriele Rieder ◽

Wolfgang Einsiedl ◽

Rudolf A. Hatz ◽

Manfred Stolte ◽

Georg A. Enders ◽

...

Keyword(s):

Helicobacter Pylori ◽

Protein Expression ◽

Outer Membrane Proteins ◽

Water Soluble ◽

Interleukin 8 ◽

Gastric Epithelium ◽

Mrna Levels ◽

Rt Pcr ◽

H Pylori

ABSTRACT Colonization of the gastric mucosa with Helicobacter pylori is associated with a dense infiltration of granulocytes into the lamina propria in the active phase of gastritis. In this study, we investigated the involvement of epithelial cell-derived neutrophil-activating protein 78 (ENA-78) in development of H. pylori-associated gastritis. Antral biopsies from 27 patients with H. pylori-associated gastritis and 25 from H. pylori-negative individuals were first analyzed for ENA-78 and interleukin-8 (IL-8) mRNA by semiquantitative reverse transcription (RT)-PCR. In H. pylori-positive patients, significantly elevated levels were found for both chemokines (P < 0.05). Only IL-8 mRNA levels differed significantly (P< 0.05) in H. pylori-infected individuals who had serum antibodies for cytotoxin-associated protein CagA versus H. pylori-infected CagA-negative persons. Quantification of ENA-78 transcript levels by competitive RT-PCR yielded a significant 45-fold upregulation for ENA-78 transcripts in biopsies of H. pylori-positive versus H. pylori-negative patients (P < 0.05). In contrast to earlier findings with IL-8, the degree of ENA-78 mRNA upregulation was independent of the grade of activity of gastritis. Immunofluorescence studies on tissues of antral biopsies localized ENA-78 protein expression mainly to the gastric epithelium of H. pylori-positive patients, while control tissues were negative. Upregulation of ENA-78 and IL-8 mRNA and protein expression was also observed in an in vitro system using a gastric adenocarcinoma cell line. Only viable H. pyloriyielded a strong ENA-78 and IL-8 induction, while H. pyloriouter membrane proteins or water-soluble proteins had no significant effect. These data provide evidence for the importance of both IL-8 and ENA-78 in the development and perpetuation of H. pylori-associated gastritis.

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Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells

Biochemistry and Cell Biology ◽

10.1139/o09-060 ◽

2009 ◽

Vol 87 (6) ◽

pp. 933-942 ◽

Cited By ~ 34

Author(s):

Li Yang ◽

Hai-wei Zhang ◽

Rong Hu ◽

Yong Yang ◽

Qi Qi ◽

...

Keyword(s):

Cyclin D1 ◽

Cervical Carcinoma ◽

Hela Cells ◽

Therapeutic Potential ◽

P53 Gene ◽

Mrna Levels ◽

Human Cervical Carcinoma ◽

Phase Arrest

Wogonin, a naturally occurring flavonoid, has been shown to have tumor therapeutic potential both in vitro and in vivo. To better understand its anticancer mechanism, we examined the effect of wogonin on human cervical carcinoma HeLa cells. In this study, we observed that G1 phase arrest was involved in wogonin-induced growth inhibition in HeLa cells. Over a 24 h exposure of HeLa cells to 90 µmol·L–1 wogonin, the promoters of G1–S transition, including cyclin D1/Cdk4 and pRb, decreased within 12 h and E2F-1 depleted in the nucleus at the same time. As the G1 phase arrest developed, p53 and the Cdk inhibitor p21Cip1 elevated both at protein and mRNA levels. Furthermore, the up-regulation of p21Cip1 induced by wogonin was dramatically inhibited by siRNA-mediated p53 gene silencing. Collectively, our data suggested that wogonin induced G1 phase arrest in HeLa cells by modulating several key G1 regulatory proteins, such as Cdk4 and cyclin D1, as well as up-regulation of a p53-midiated p21Cip1 expression. This mechanism of wogonin may play an important role in the killing of cancerous cells and offer a potential mechanism for its anticancer action in vivo.

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Connexin 43 confers chemoresistance through activating PI3K

Oncogenesis ◽

10.1038/s41389-022-00378-7 ◽

2022 ◽

Vol 11 (1) ◽

Author(s):

Kevin J. Pridham ◽

Farah Shah ◽

Kasen R. Hutchings ◽

Kevin L. Sheng ◽

Sujuan Guo ◽

...

Keyword(s):

Poor Prognosis ◽

Connexin 43 ◽

Therapeutic Potential ◽

Mrna Levels ◽

Phosphatidylinositol 3 Kinase ◽

Junction Protein ◽

Gap Junction Protein ◽

Cx43 Mrna

AbstractCircumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.

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