Ecdysone Elicits Chronic Renal Impairment via Mineralocorticoid-Like Pathogenic Activities

Background/Aims: Ecdysteroids are steroidal insect molting hormones that also exist in herbs. Ecdysteroid-containing adaptogens have been popularly used to improve well-being and by bodybuilders for muscle growth. However, the use of ecdysone in mammals is also associated with kidney growth and enlargement, indications of disturbed kidney homeostasis. The underlying pathogenic mechanism remains to be clarified. Methods: Virtual screening tools were employed to identify compounds that are homologous to ecdysone and to predict putative ecdysone-interacting proteins. The kidney effect of ecdysone was examined in vitro and in vivo and compared with that of aldosterone. Cellular apoptosis was estimated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Cell motility was assessed by scratch-wound cell migration assay. Blood urea nitrogen was measured to evaluate renal function. Western immunblot analysis was employed to determine the expression profile of interested proteins. Results: Computational molecular structure analysis revealed that ecdysone is highly homologous to aldosterone. Moreover, virtual screening based on compound-protein interaction profiles identified the Mineralocorticoid Receptor (MR) to potentially interact with ecdysone. Accordingly, to assess potential biological functions of ecdysone in mammals, ecdysone was applied to mineralocorticoid-sensitive inner medullar collecting duct cells. Ecdysone induced mesenchymal accumulation of extracellular matrix and epithelial dedifferentiation characterized by de novo expression of α-smooth muscle actin. In addition, ecdysone elicited cellular apoptosis and retarded cell motility, akin to the effect of aldosterone. In vivo, daily treatment of mice with ecdysone increased cell apoptosis in the kidney, impaired renal function and elicited early signs of renal fibrogenesis, marked by deposition of collagen and fibronectin in tubulointerstitium, reminiscent of the action of aldosterone. The MR signaling pathway is likely responsible for the cellular and pathobiological effects of ecdysone, as evidenced by strong ecdysone-induced MR nuclear translocation in renal tubular cells both in vitro and in vivo, while blockade of MR by concomitant spironolactone treatment largely abolished the detrimental effects of ecdysone. Conclusion: Our findings suggest that ecdysone induces mineralocorticoid-dependent activities that impair renal function and elicit renal injury.

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Virtual Screening for Potential Phytobioactives as Therapeutic Leads to Inhibit NQO1 for Selective Anticancer Therapy

Molecules ◽

10.3390/molecules26226863 ◽

2021 ◽

Vol 26 (22) ◽

pp. 6863

Author(s):

Bhargav Shreevatsa ◽

Chandan Dharmashekara ◽

Vikas Halasumane Swamy ◽

Meghana V. Gowda ◽

Raghu Ram Achar ◽

...

Keyword(s):

Virtual Screening ◽

Binding Site ◽

Anticancer Agents ◽

Substrate Binding ◽

Tumor Burden ◽

Screening Tools ◽

Tumor Promoter ◽

Substrate Binding Site

NAD(P)H:quinone acceptor oxidoreductase-1 (NQO1) is a ubiquitous flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory two-electron reductions of quinones, quinonimines, nitroaromatics, and azo dyes. NQO1 is a multifunctional antioxidant enzyme whose expression and deletion are linked to reduced and increased oxidative stress susceptibilities. NQO1 acts as both a tumor suppressor and tumor promoter; thus, the inhibition of NQO1 results in less tumor burden. In addition, the high expression of NQO1 is associated with a shorter survival time of cancer patients. Inhibiting NQO1 also enables certain anticancer agents to evade the detoxification process. In this study, a series of phytobioactives were screened based on their chemical classes such as coumarins, flavonoids, and triterpenoids for their action on NQO1. The in silico evaluations were conducted using PyRx virtual screening tools, where the flavone compound, Orientin showed a better binding affinity score of −8.18 when compared with standard inhibitor Dicumarol with favorable ADME properties. An MD simulation study found that the Orientin binding to NQO1 away from the substrate-binding site induces a potential conformational change in the substrate-binding site, thereby inhibiting substrate accessibility towards the FAD-binding domain. Furthermore, with this computational approach we are offering a scope for validation of the new therapeutic components for their in vitro and in vivo efficacy against NQO1.

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In vitro and in vivo assessment of the protective effect of sufentanil in acute lung injury

Journal of International Medical Research ◽

10.1177/0300060520986351 ◽

2021 ◽

Vol 49 (2) ◽

pp. 030006052098635

Author(s):

Qi Gao ◽

Ningqing Chang ◽

Donglian Liu

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Protective Effect ◽

High Mobility ◽

Terminal Deoxynucleotidyl Transferase ◽

Inflammatory Factors ◽

Luciferase Reporter ◽

Hmgb1 Protein

Objectives To investigate the mechanisms underlying the protective effect of sufentanil against acute lung injury (ALI). Material and Methods Rats were administered lipopolysaccharide (LPS) by endotracheal instillation to establish a model of ALI. LPS was used to stimulate BEAS-2B cells. The targets and promoter activities of IκB were assessed using a luciferase reporter assay. Apoptosis of BEAS-2B cells was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Results Sufentanil treatment markedly reduced pathological changes in lung tissue, pulmonary edema and secretion of inflammatory factors associated with ALI in vivo and in vitro. In addition, sufentanil suppressed apoptosis induced by LPS and activated NF-κB both in vivo and in vitro. Furthermore, upregulation of high mobility group box protein 1 (HMGB1) protein levels and downregulation of miR-129-5p levels were observed in vivo and in vitro following sufentanil treatment. miR-129-5p targeted the 3ʹ untranslated region and its inhibition decreased promoter activities of IκB-α. miR-129-5p inhibition significantly weakened the protective effect of sufentanil on LPS-treated BEAS-2B cells. Conclusion Sufentanil regulated the miR-129-5p/HMGB1 axis to enhance IκB-α expression, suggesting that sufentanil represents a candidate drug for ALI protection and providing avenues for clinical treatment.

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A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

10.1101/2020.07.27.223008 ◽

2020 ◽

Author(s):

Yueyang Wang ◽

Alan Y. Hsu ◽

Eric M. Walton ◽

Ramizah Syahirah ◽

Tianqi Wang ◽

...

Keyword(s):

Cell Motility ◽

Neutrophil Migration ◽

Transgenic Fish ◽

Subcellular Location ◽

Vector System ◽

Specific Gene ◽

Tissue Specific ◽

Biological Studies

AbstractTissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

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Ivermectin induces autophagy-mediated cell death through the AKT/mTOR signaling pathway in glioma cells

Bioscience Reports ◽

10.1042/bsr20192489 ◽

2019 ◽

Vol 39 (12) ◽

Cited By ~ 5

Author(s):

Jingjing Liu ◽

Hongsheng Liang ◽

Chen Chen ◽

Xiaoxing Wang ◽

Faling Qu ◽

...

Keyword(s):

Cell Death ◽

Molecular Mechanisms ◽

Anticancer Agent ◽

Glioma Cells ◽

Mtor Signaling ◽

Terminal Deoxynucleotidyl Transferase ◽

Transmission Electron ◽

Diphenyltetrazolium Bromide

Abstract Glioma is one of the most common types of primary brain tumors. Ivermectin (IVM), a broad-spectrum antiparasitic drug, has been identified as a novel anticancer agent due to its inhibitory effects on the proliferation of glioma cells in vitro and in vivo. However, the ability of IVM to induce autophagy and its role in glioma cell death remains unclear. The main objective of the present study was to explore autophagy induced by IVM in glioma U251 and C6 cells, and the deep underlying molecular mechanisms. In addition, we examined the effects of autophagy on apoptosis in glioma cells. In the present study, transmission electron microscopy (TEM), immunofluorescence, Western blot and immunohistochemistry were used to evaluate autophagy activated by IVM. Cell viability was measured by 3-(4,5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colony formation assay. The apoptosis rate was detected by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Meanwhile, autophagy inhibition was achieved by using chloroquine (CQ). U251-derived xenografts were established for examination of IVM-induced autophagy on glioma in vivo. Taken together, the results of the present study showed that autophagy induced by IVM has a protective effect on cell apoptosis in vitro and in vivo. Mechanistically, IVM induced autophagy through AKT/mTOR signaling and induced energy impairment. Our findings show that IVM is a promising anticancer agent and may be a potential effective treatment for glioma cancers.

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Effects of endothelin-1 on renal function and renin secretion in vivo and in vitro

European Journal of Pharmacology ◽

10.1016/0014-2999(90)92135-6 ◽

1990 ◽

Vol 183 (5) ◽

pp. 1814-1815

Author(s):

S. Morimoto ◽

K. Hisaki ◽

K. Nakase ◽

R. Ikegawa ◽

K. Hayashi ◽

...

Keyword(s):

Renal Function ◽

Renin Secretion ◽

Endothelin 1

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Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines againstStreptococcus pneumoniaeandSalmonella entericaSerovar Typhi

Infection and Immunity ◽

10.1128/iai.00211-18 ◽

2018 ◽

Vol 86 (9) ◽

Author(s):

Vivek Belde ◽

Matthew P. Cravens ◽

Dania Gulandijany ◽

Justin A. Walker ◽

Isabel Palomo-Caturla ◽

...

Keyword(s):

Antibody Response ◽

B Cell ◽

Salmonella Enterica ◽

Passive Immunization ◽

Terminal Deoxynucleotidyl Transferase ◽

Human Infants ◽

Content Type ◽

Serovar Typhimurium

ABSTRACTB cell antigen receptor (BCR) diversity increases by several orders of magnitude due to the action of terminal deoxynucleotidyl transferase (TdT) during V(D)J recombination. Unlike adults, infants have limited BCR diversity, in part due to reduced expression of TdT. Since human infants and young mice respond poorly to polysaccharide vaccines, such as the pneumococcal polysaccharide vaccine Pneumovax23 and Vi polysaccharide (ViPS) ofSalmonella entericaserovar Typhi, we tested the contribution of TdT-mediated BCR diversity in response to these vaccines. We found that TdT+/−and TdT−/−mice generated comparable antibody responses to Pneumovax23 and survivedStreptococcus pneumoniaechallenge. Moreover, passive immunization of B cell-deficient mice with serum from Pneumovax23-immunized TdT+/−or TdT−/−mice conferred protection. TdT+/−and TdT−/−mice generated comparable levels of anti-ViPS antibodies and antibody-dependent, complement-mediated bactericidal activity againstS. Typhiin vitro. To test the protective immunity conferred by ViPS immunizationin vivo, TdT+/−and TdT−/−mice were challenged with a chimericSalmonella entericaserovar Typhimurium strain expressing ViPS, since mice are nonpermissive hosts forS. Typhi infection. Compared to their unimmunized counterparts, immunized TdT+/−and TdT−/−mice challenged with ViPS-expressingS. Typhimurium exhibited a significant reduction in the bacterial burden and liver pathology. These data suggest that the impaired antibody response to the Pneumovax23 and ViPS vaccines in the young is not due to limited TdT-mediated BCR diversification.

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Estrogen modulates the recruitment of myelopoietic cell progenitors in rat through a stromal cell-independent mechanism involving apoptosis

Blood ◽

10.1182/blood.v87.7.2683.bloodjournal8772683 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2683-2692 ◽

Cited By ~ 33

Author(s):

NK Shevde ◽

JW Pike

Keyword(s):

Estrogen Receptor ◽

Ovarian Function ◽

Morphological Changes ◽

Estrogen Treatment ◽

Ovariectomized Rats ◽

Terminal Deoxynucleotidyl Transferase ◽

Protective Effects ◽

Hematopoietic Stem

Loss of ovarian function leads to a significant increase in the number of bone-resorbing osteoclasts. Estrogen replacement is known to manifest bone protective effects in the treatment of postmenopausal osteoporosis. In the present study, we used ovariectomized rats to examine the effects of estrogen loss at the osteoclast progenitor colony forming unit-granulocyte macrophage (CFU-GM) level. A significant increase in CFU-GM number was observed as early as 7 days following ovariectomy, and correlated directly with an increase in the number of osteoclast-like cells generated in marrow cultures. The increase in CFU-GM following ovariectomy was abrogated in animals that received estrogen treatment in vivo. A similar suppressive effect was observed on CFU-GM number when ovariectomized rat marrow was treated with estrogen in vitro. This effect was blocked in the presence of the estrogen antihormone ICI 164,384. Thus, the data suggest the possibility that estrogen exerts a direct effect on osteoclast progenitors, and does so through the estrogen receptor-mediated mechanism. Ovariectomy also led to an increase in the early hematopoietic stem/progenitor cell population (Thy 1.1+ cells) as determined by FLOW cytometry methods. Morphological changes as well as terminal deoxynucleotidyl transferase assays revealed that estrogen treatment negated growth factor-induced proliferation of these early progenitors by promoting apoptosis. The cellular effects of estrogen in vitro together with the immunocytochemical detection of the estrogen receptor in these cells, strongly support the contention that in addition to osteoclast progenitors such as CFU-GM, earlier hematopoietic progenitors are also unique cellular targets for estrogen action.

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Renal effects induced by prolonged mPGES1 inhibition

AJP Renal Physiology ◽

10.1152/ajprenal.00492.2013 ◽

2014 ◽

Vol 306 (1) ◽

pp. F68-F74 ◽

Cited By ~ 10

Author(s):

Francisco Salazar ◽

Michael L. Vazquez ◽

Jaime L. Masferrer ◽

Gabriel Mbalaviele ◽

Maria T. Llinas ◽

...

Keyword(s):

Renal Function ◽

Plasma Renin ◽

In Vivo Studies ◽

Renal Effects ◽

Excretory Function ◽

Membrane Bound ◽

Inflammatory Drugs ◽

Chronically Instrumented Dogs

The importance of membrane-bound PGE synthase 1 (mPGES1) in the regulation of renal function has been examined in mPGES1-deficient mice or by evaluating changes in its expression. However, it is unknown whether prolonged mPGES1 inhibition induces significant changes of renal function when Na+ intake is normal or low. This study examined the renal effects elicited by a selective mPGES1 inhibitor (PF-458) during 7 days in conscious chronically instrumented dogs with normal Na+ intake (NSI) or low Na+ intake (LSI). Results obtained in both in vitro and in vivo studies have strongly suggested that PF-458 is a selective mPGES1 inhibitor. The administration of 2.4 mg·kg−1·day−1 PF-458 to dogs with LSI did not induce significant changes in renal blood flow (RBF) and glomerular filtration rate (GFR). A larger dose of PF-458 (9.6 mg·kg−1·day−1) reduced RBF ( P < 0.05) but not GFR in dogs with LSI and did not induce changes of renal hemodynamic in dogs with NSI. Both doses of PF-458 elicited a decrease ( P < 0.05) in PGE2 and an increase ( P < 0.05) in 6-keto-PGF1α. The administration of PF-458 did not induce significant changes in renal excretory function, plasma renin activity, and plasma aldosterone and thromboxane B2 concentrations in dogs with LSI or NSI. The results obtained suggest that mPGES1 is involved in the regulation of RBF when Na+ intake is low and that the renal effects elicited by mPGES1 inhibition are modulated by a compensatory increment in PGI2. These results may have some therapeutical implications since it has been shown that prolonged mPGES1 inhibition has lower renal effects than those elicited by nonsteroidal anti-inflammatory drugs or selective cyclooxygenase-2 inhibitors.

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Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT1 receptors

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00701.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. H461-H467 ◽

Cited By ~ 45

Author(s):

Hai Ling Li ◽

Jun Suzuki ◽

Evelyn Bayna ◽

Fu-Min Zhang ◽

Erminia Dalle Molle ◽

...

Keyword(s):

Angiotensin Ii ◽

Ventricular Myocytes ◽

Annexin V ◽

Left Ventricular ◽

Terminal Deoxynucleotidyl Transferase ◽

Rat Ventricular Myocytes ◽

Adult Rat ◽

Cardiac Apoptosis

Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions. It was hypothesized that LPS directly induces cardiac apoptosis. In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h. Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT1) inhibitor (losartan), but not by inhibitors of AT2 receptors (PD-123319), tumor necrosis factor-α (TNFRII:Fc), or nitric oxide ( N G-monomethyl-l-arginine). Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects. LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1–3 days, which dissipated after 1–2 wk. Losartan (23 mg · kg−1 · day−1 in drinking water for 3 days) blocked LPS-induced in vivo apoptosis. In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT1 receptors in myocytes.

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Protective Effect of Eckol against Acute Hepatic Injury Induced by Carbon Tetrachloride in Mice

Marine Drugs ◽

10.3390/md16090300 ◽

2018 ◽

Vol 16 (9) ◽

pp. 300 ◽

Cited By ~ 8

Author(s):

Shulan Li ◽

Juan Liu ◽

Mengya Zhang ◽

Yuan Chen ◽

Tianxing Zhu ◽

...

Keyword(s):

Carbon Tetrachloride ◽

Liver Injury ◽

Protective Effect ◽

Acute Liver Injury ◽

Treated Group ◽

Terminal Deoxynucleotidyl Transferase ◽

Enhanced Expression ◽

Pre Treatment

Several in vitro studies have shown the potential hepatoprotective properties of eckol, a natural phlorotannin derived from the brown alga. However, the in vivo hepatoprotective potential of eckol has not been determined. In this study, we performed an in vivo study to investigate the protective effect of eckol and its possible mechanisms on the carbon tetrachloride (CCl4)-induced acute liver injury model in mice. Results revealed that eckol pre-treatment at the dose of 0.5 and 1.0 mg/kg/day for 7 days significantly suppressed the CCl4-induced increases of alanine transaminase (ALT) and aspartate aminotransferase (AST) levels in serum and meliorated morphological liver injury. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) analysis showed that the number of positive apoptotic hepatocytes in the eckol-treated group was lower than that in the CCl4 model group. Western blotting analysis also demonstrated the enhanced expression of bcl-2 and suppressed expression of cleaved caspase-3 by eckol. The CCl4-induced oxidative stress in liver was significantly ameliorated by eckol, which was characterized by reduced malondialdehyde (MDA) formations, and enhanced superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities and glutathione (GSH) content. Moreover, the CCl4-induced elevations of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were markedly suppressed in the eckol-treated group. However, eckol enhanced the level of IL-10, a potent anti-inflammatory cytokine, and recruited CD11c+ dendritic cells into the liver tissues of CCl4-treated mice. These results indicated that eckol has the protective effect on CCl4-induced acute liver injury via multiple mechanisms including anti-apoptosis, anti-oxidation, anti-inflammation and immune regulation.

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