Thrombin Induces Protease Activated Receptor (PAR)-3/-4 Interaction in Human Podocytes

Abstract INTRODUCTION Nephrotic Syndrome, one of the most common forms of glomerular disease, is characterized by massive proteinuria with structural and functional injury of specialized glomerular cells called podocytes.We and others have shown that thrombin generation is enhanced in nephrotic syndrome. Recent in vitro studies have demonstrated that exposure to high concentrations of thrombin can injure podocytes, suggesting that thrombin may exacerbate glomerular injury. However, the molecular mechanisms by which thrombin induces podocyte injury are not yet known. Thrombin activates platelets, leukocytes, and other cells via the protease activated receptor (PAR) system. Thus, we hypothesized that thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner. METHODS Experiments were performed with differentiated, conditionally immortalized human podocytes. After 36 hours of thrombin (20nM) exposure podocyte apoptosis was determined by TUNEL assay. Specific PAR antibodies and activating peptides were utilized to determine which PARs mediate thrombin-induced podocyte apoptosis. Specific PAR antibodies (ab) included hPAR1ab (ATAP2), hPAR2 ab (SAM11), hPAR3 ab (8E8), hPAR4 ab (H-120). Activation peptides (AP) included PAR1 AP (TFFLRNPNDKNH2), PAR2 AP (SLIGRLNH2); PAR3 AP (TFRGAPOH); PAR4 AP (AYPGKFNH2) and a control peptide (FSLLRNNH2). Phalloidin assays were used to evaluate structural changes in the actin-cytoskeleton as a marker of podocyte stress. Proximity ligation assays (PLAs) allow detection of protein-protein interactions at the molecular level. PLAs were performed on podocytes grown in an 8-well chamber slide system. PLA was carried out using the DUOLinkTM kit (Sigma-Aldrich) according to the manufacturer's instructions and as described previously. Oligonucleotide-conjugated secondary antibodies against mouse and rabbit primary antibodies were used and protein-protein associations were detected by microscopy as bright red dots. One-Way ANOVA and t-tests were used to determine statistical significance (SigmaPlotTM). RESULTS Thrombin exposure induced a significant increase in apoptosis of human podocytes from 1.8% to 42.87% (p <0.05). Blockade of PAR-3 or PAR-4 resulted in a significant decrease in apoptosis [9.2% with hPAR-3 ab and 11.7% with hPAR-4ab] (p <0.05). Inhibition of thrombin enzymatic activity with hirudin, a direct thrombin inhibitor, also resulted in a decrease in apoptosis to 2.1% (p <0.05). In comparison to a control peptide, PAR-4 activation peptide significantly increased apoptosis from 1.7% to 40.1% (p <0.05), while PAR-3 activation peptide did not. Analogous results were seen with the phalloidin assay. Thrombin caused actin cytoskeletal changes, while PAR-3 and PAR-4 blockade ameliorated these changes. In addition, only activation with PAR-4 activating peptide resulted in loss of actin stress fibers. These findings suggest that, in human podocytes, thrombin signaling is mediated via PAR-4, in a PAR-3-dependent manner. Thus to directly assess this hypothesis, we performed proximity ligation assays with PAR-3/PAR-4 antibodies in the presence and absence of thrombin which revealed the presence of PAR-3/PAR-4 interactions in thrombin exposed podocytes, but not in control podocytes. The quantification of the in situ PLA signals per cell was performed with a BZ900 microscope and software (Keyence, Osaka, Japan) equipped with automated 'dynamic cell count" feature (p =0.03). CONCLUSIONS Thrombin-induced injury is mediated in a PAR-dependent fashion in human podocytes. Specifically, in this in vitro model, thrombin-induced apoptosis appears to be mediated in a PAR-3/-4 dependent manner. Furthermore, these data suggest that thrombin induced podocyte injury may be mediated in a manner dependent on interactions between PAR-3 and -4. This is a novel finding, as PAR-3/-4 interactions are known to mediate mouse platelet signaling, but have not been previously described in human cells. Collectively, our findings suggest that interrupting thrombin-mediated podocyte injury may offer a novel therapeutic approach to reduce podocyte injury during nephrotic syndrome. Disclosures No relevant conflicts of interest to declare.

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Thrombin Induces Apoptosis in Human and Rat Podocytes in a Protease Activated Receptor (PAR)-Dependent Manner

Blood ◽

10.1182/blood.v124.21.2808.2808 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2808-2808

Author(s):

Ruchika Sharma ◽

Amanda P Waller ◽

Adam Guess ◽

Shipra Agrawal ◽

Berend Isermann ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Dependent Manner ◽

Glomerular Injury ◽

Podocyte Injury ◽

Control Peptide ◽

Increased Risk ◽

Human Thrombin ◽

Activation Peptide ◽

Induced Apoptosis

Abstract INTRODUCTION Nephrotic Syndrome, one of the most common forms of glomerular disease, is characterized by massive proteinuria with structural and functional injury of specialized glomerular cells called podocytes. There is evidence to indicate that thrombin generation is enhanced in nephrotic syndrome. The massive protein loss in nephrotic syndrome includes loss of key coagulation regulators, leading to a complex coagulopathy, enhanced thrombin activating capacity and, consequently, increased risk for devastating thromboembolic complications. Recent in vitro studies have demonstrated that exposure to high concentrations of thrombin can injure podocytes, suggesting that thrombin may exacerbate glomerular injury. However, the molecular mechanisms by which thrombin induces podocyte injury are not yet known. Thrombin activates platelets, leukocytes, and other cells via the protease activated receptor (PAR) system. Thus, we hypothesized that thrombin exacerbates glomerular injury by enhancing podocyte apoptosis in a PAR-dependent manner. METHODS Experiments were performed with differentiated, conditionally immortalized human and rat podocytes. After 36 hours of thrombin (20nM) exposure podocyte apoptosis was determined by TUNEL assay. In human podocytes, specific PAR antibodies and activating peptides were utilized to determine which PARs mediate thrombin-induced podocyte apoptosis. Specific PAR antibodies (ab) included hPAR1ab (ATAP2), hPAR2 ab (SAM11), hPAR3 ab (8E8), hPAR4 ab (H-120). Activation peptides (AP) included PAR1 AP (TFFLRNPNDKNH2), PAR2 AP (SLIGRLNH2); PAR3 AP (TFRGAPOH); PAR4 AP (AYPGKFNH2) and a control peptide (FSLLRNNH2). Phalloidin assays were used to evaluate structural changes in the actin-cytoskeleton as a marker of podocyte stress. We have established that human thrombin is hemostatically active and regulated in rat plasma. Thus, rat podocytes were exposed to human thrombin, to determine toxicity, and specific PAR antibodies to determine if blockade of PARs could ameliorate thrombin mediated toxicity. One-Way ANOVA and t-tests were used to determine statistical significance (SigmaPlotTM). RESULTS Thrombin exposure induced a significant increase in apoptosis of human podocytes from 1.8% to 42.87% (p<0.05). Blockade of PAR-3 or PAR-4 resulted in a significant decrease in apoptosis [9.2% with hPAR-3 ab and 11.7% with hPAR-4ab] (p<0.05). Inhibition of thrombin enzymatic activity with hirudin, a direct thrombin inhibitor, also resulted in a decrease in apoptosis to 2.1% (p<0.05). In comparison to a control peptide, PAR-4 activation peptide significantly increased apoptosis from 1.7% to 40.1% (p<0.05), while PAR-3 activation peptide did not. Analogous results were seen with the phalloidin assay. Thrombin caused actin cytoskeletal changes, while PAR-3 and PAR-4 blockade ameliorated these changes. In addition, only activation with PAR-4 activating peptide resulted in loss of actin stress fibers. Figure 1 Figure 1. Figure 2 Figure 2. In rat podocytes human thrombin had a similar effect with increased apoptosis from 1% to 33.6% (p<0.05). We also demonstrated that this may be a PAR-mediated mechanism as blockade of PAR-1 and PAR-4 with specific antibodies ameliorated thrombin induced apoptosis [1.94% with PAR-1 ab and 3.5% with PAR-4ab] while blockade of PAR-2 and PAR-3 did not have a similar effect (p<0.05). Figure 3 Figure 3. CONCLUSIONS Thrombin-induced injury is mediated in a PAR-dependent fashion in both humans and rats. Specifically, in this in vitro model, thrombin induced apoptosis appears to be mediated in a PAR-3/-4 dependent manner in human podocytes but in a PAR-1/-4 manner in rat podocytes. Furthermore, these data suggest that thrombin induced podocyte injury may be mediated in a manner dependent on PAR heterodimerization. Our findings collectively suggest that interrupting thrombin-mediated podocyte injury may offer a novel future therapeutic approach to reduce podocyte injury in nephrotic syndrome. Disclosures No relevant conflicts of interest to declare.

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VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520617666170912115441 ◽

2018 ◽

Vol 18 (2) ◽

pp. 255-262 ◽

Cited By ~ 5

Author(s):

Aikebaier Maimaiti ◽

Amier Aili ◽

Hureshitanmu Kuerban ◽

Xuejun Li

Keyword(s):

Western Blot ◽

Cell Viability ◽

Gallic Acid ◽

Molecular Mechanisms ◽

A549 Cells ◽

Dependent Manner ◽

Lung Carcinoma Cells ◽

Induced Apoptosis ◽

The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

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Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

10.1101/2021.10.27.466054 ◽

2021 ◽

Author(s):

Jitske Jansen ◽

Bartholomeus T van den Berge ◽

Martijn van den Broek ◽

Rutger J Maas ◽

Brigith Willemsen ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Idiopathic Nephrotic Syndrome ◽

Super Resolution ◽

Induced Pluripotent Stem Cell ◽

Glomerular Injury ◽

Kidney Organoids

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. Here, we report human induced pluripotent stem cell derived kidney organoids containing a podocyte population that heads towards adult podocytes and were superior compared to 2D counterparts, based on scRNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies.

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Suppression of the TGF-β pathway by a macrolide antibiotic decreases fibrotic responses by ocular fibroblasts in vitro

Royal Society Open Science ◽

10.1098/rsos.200441 ◽

2020 ◽

Vol 7 (9) ◽

pp. 200441

Author(s):

Thomas Stahnke ◽

Beata Gajda-Deryło ◽

Anselm G. Jünemann ◽

Oliver Stachs ◽

Katharina A. Sterenczak ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Macrolide Antibiotic ◽

Drug Repositioning ◽

Dependent Manner ◽

Glaucoma Filtration Surgery ◽

Concentration Dependent Manner ◽

Device Implantation

To elucidate and to inhibit post-surgical fibrotic processes after trabeculectomy in glaucoma therapy, we measured gene expression in a fibrotic cell culture model, based on transforming growth factor TGF-β induction in primary human tenon fibroblasts (hTFs), and used Connectivity Map (CMap) data for drug repositioning. We found that specific molecular mechanisms behind fibrosis are the upregulation of actins, the downregulation of CD34, and the upregulation of inflammatory cytokines such as IL6, IL11 and BMP6 . The macrolide antibiotic Josamycin (JM) reverses these molecular mechanisms according to data from the CMap, and we thus tested JM as an inhibitor of fibrosis. JM was first tested for its toxic effects on hTFs, where it showed no influence on cell viability, but inhibited hTF proliferation in a concentration-dependent manner. We then demonstrated that JM suppresses the synthesis of extracellular matrix (ECM) components. In hTFs stimulated with TGF-β1, JM specifically inhibited α-smooth muslce actin expression, suggesting that it inhibits the transformation of fibroblasts into fibrotic myofibroblasts. In addition, a decrease of components of the ECM such as fibronectin, which is involved in in vivo scarring, was observed. We conclude that JM may be a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo .

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ROS-Induced GATA4 and GATA6 Downregulation Inhibits StAR Expression in LPS-Treated Porcine Granulosa-Lutein Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/5432792 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 3

Author(s):

Xiaolu Qu ◽

Leyan Yan ◽

Rihong Guo ◽

Hui Li ◽

Zhendan Shi

Keyword(s):

Molecular Mechanisms ◽

Cell Model ◽

Animal Husbandry ◽

Dependent Manner ◽

Progesterone Production ◽

Progesterone Synthesis ◽

Porcine Granulosa Cell ◽

Underlying Mechanisms ◽

Dose Dependent

LPS is a major endotoxin produced by gram-negative bacteria, and exposure to it commonly occurs in animal husbandry. Previous studies have shown that LPS infection disturbs steroidogenesis, including progesterone production, and subsequently decreases animal reproductive performance. However, little information about the underlying mechanisms is available thus far. In the present study, an in vitro-luteinized porcine granulosa cell model was used to study the underlying molecular mechanisms of LPS treatment. We found that LPS significantly inhibits progesterone production and downregulates the expressions of progesterone synthesis-associated genes (StAR, CYP11A1, and 3β-HSD). Furthermore, the levels of ROS were significantly increased in an LPS dose-dependent manner. Moreover, transcriptional factors GATA4 and GATA6, but not NR5A1, were significantly downregulated. Elimination of LPS-stimulated ROS by melatonin or vitamin C could restore the expressions of GATA4, GATA6, and StAR. In parallel, StAR expression was also inhibited by the knockdown of GATA4 and GATA6. Based on these data, we conclude that LPS impairs StAR expression via the ROS-induced downregulation of GATA4 and GATA6. Collectively, these findings provide new insights into the understanding of reproductive losses in animals suffering from bacterial infection and LPS exposure.

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Nicotinamide Enhances the Polyploidization of Primary Megakaryocytes.

Blood ◽

10.1182/blood.v106.11.1366.1366 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1366-1366

Author(s):

Lisa M. Giammona ◽

Eleftherios Papoutsakis ◽

William M. Miller

Keyword(s):

Dna Content ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Annexin V ◽

Dependent Manner ◽

Vitamin B3 ◽

Ploidy Levels ◽

High Ploidy

Abstract Megakaryocyte (Mk) maturation includes the development of polyploid cells via endomitosis. In vitro models of Mk differentiation can be used to gain a better understanding of the molecular mechanisms controlling this process. However, it is challenging to achieve ploidy levels in cultured human cells that are as high as those observed in vivo. Others have recently reported the use of chemical inhibitors to increase Mk ploidy (Lannutti et al., Blood 105:3875, 2005). Here, we show that nicotinamide (NIC), a form of vitamin B3, enhances the normal process of Mk polyploidization and leads to both a greater fraction of high ploidy cells and a greater degree of polyploidization. Human mobilized peripheral blood CD34+ cells were cultured in serum-free medium supplemented with thrombopoietin (TPO) to induce Mk differentiation. Beginning on day 5 of culture, cells were treated with nicotinamide (3 and 6.25 mM) and monitored for DNA content, growth, apoptosis, and surface marker expression. NIC treatment resulted in a greater fraction of Mks with high ploidy (DNA content greater than or equal to 8N). The ploidy of NIC treated cells continued to increase over the duration of the 13-day culture, whereas the ploidy of untreated cells peaked at day 9. On day 13 (8 days of NIC exposure), the percentages of high ploidy Mks for the untreated, 3 mM NIC, and 6.25 mM NIC conditions were 23%, 48%, and 63%, respectively. Furthermore, cells treated with NIC reached ploidy levels of 64N and 32N for 6.25 and 3 mM NIC, respectively, compared to 16N for untreated cells. NIC-treated cells also displayed dramatic differences in morphology - characterized by an increase in cell size, the presence of a more highly lobated nucleus, and an increased frequency of proplatelet-forming cells. Nicotinamide is known to inhibit poly(ADP-ribose) polymerase (PARP) and Sir2, which are both NAD+ dependent enzymes. Preliminary experiments show that PARP activity is low in cultured Mks and is not affected by addition of 6.25 mM NIC. Continued exposure (beginning at day 5) to the PARP inhibitors (and nicotinamide analogs) 3-aminobenzamide (3-AB) and benzamide at concentrations of 1, 3, and 6.25 mM was toxic to cells in a dose dependent manner. Interestingly, high doses of NIC (25 and 50 mM) were also toxic to cells. Remarkably, while Mk polyploidization and apoptosis are typically correlated, the increase in DNA content observed for NIC-treated cells occurred without significantly affecting the percentage of apoptotic Mks (assessed by Annexin V staining). These data suggest that it may be possible to partially decouple Mk apoptosis and polyploidization. Furthermore, while 6.25 mM NIC inhibited cell proliferation by ~35%, total expansion of cells cultured with 3 mM NIC was similar to that of untreated cells. This, combined with similar Mk commitment, as defined by a similar percentage of CD41+ cells, resulted in a greater overall number of high ploidy Mks in cultures treated with NIC. Since there is a direct correlation between Mk DNA content and platelet production (Mattia et al., Blood 99:888, 2002), these results suggest a possible therapeutic benefit of NIC for the management of thrombocytopenia. Similarly, NIC could also be used as an additive to ex vivo Mk cultures destined for transplantation. Figure Figure

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MC1R is dispensable for the proteinuria reducing and glomerular protective effect of melanocortin therapy

Scientific Reports ◽

10.1038/srep27589 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 10

Author(s):

Yingjin Qiao ◽

Anna-Lena Berg ◽

Pei Wang ◽

Yan Ge ◽

Songxia Quan ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Protective Effect ◽

Dominant Negative ◽

Melanocortin Receptor ◽

Hair Color ◽

Glomerular Injury ◽

Glomerular Diseases ◽

Red Hair ◽

Filtration Barrier

Abstract Melanocortin therapy by using adrenocorticotropic hormone (ACTH) or non-steroidogenic melanocortin peptides attenuates proteinuria and glomerular injury in experimental glomerular diseases and induces remission of nephrotic syndrome in patients with diverse glomerulopathies, even those resistant to steroids. The underlying mechanism remains elusive, but the role of melanocortin 1 receptor (MC1R) has been implicated and was examined here. Four patients with congenital red hair color and nephrotic syndrome caused by idiopathic membranous nephropathy or focal segmental glomerulosclerosis were confirmed by gene sequencing to bear dominant-negative MC1R mutations. Despite prior corticosteroid resistance, all patients responded to ACTH monotherapy and ultimately achieved clinical remission, inferring a steroidogenic-independent and MC1R-dispensable anti-proteinuric effect of melanocortin signaling. In confirmatory animal studies, the protective effect of [Nle4, D-Phe7]-α-melanocyte stimulating hormone (NDP-MSH), a potent non-steroidogenic pan-melanocortin receptor agonist, on the lipopolysaccharide elicited podocytopathy was completely preserved in MC1R-null mice, marked by reduced albuminuria and diminished histologic signs of podocyte injury. Moreover, in complementary in vitro studies, NDP-MSH attenuated the lipopolysaccharide elicited apoptosis, hypermotility and impairment of filtration barrier function equally in primary podocytes derived from MC1R-null and wild-type mice. Collectively, our findings suggest that melanocortin therapy confers a proteinuria reducing and podoprotective effect in proteinuric glomerulopathies via MC1R-independent mechanisms.

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Synergistic integration of Netrin and ephrin axon guidance signals by spinal motor neurons

eLife ◽

10.7554/elife.10841 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 36

Author(s):

Sebastian Poliak ◽

Daniel Morales ◽

Louis-Philippe Croteau ◽

Dayana Krawchuk ◽

Elena Palmesino ◽

...

Keyword(s):

Motor Neurons ◽

Molecular Mechanisms ◽

Neural Circuit ◽

Growth Cones ◽

In Vitro Assays ◽

Dependent Manner ◽

Motor Axons ◽

Spinal Motor Neurons ◽

Spinal Motor

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin–ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways.

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Spontaneous maturation in Bufo arenarum oocytes: participation of protein kinase C

Zygote ◽

10.1017/s0967199400003191 ◽

1996 ◽

Vol 4 (04) ◽

pp. 257-262 ◽

Cited By ~ 10

Author(s):

L. Zelarayán ◽

J. Oterino ◽

M.I. Bühler

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Molecular Mechanisms ◽

Respiratory Activity ◽

Follicle Cells ◽

Dependent Manner ◽

Germinal Vesicle Breakdown ◽

Kinase C ◽

Bufo Arenarum

SummaryAlthough progesterone is the maturation inducer in amphibians, it has been demonstrated that inBufo arenarumoocytes resumed meiosis with no need of exogenous hormonal stimulus if derived of their enveloping, follicle cells. This phenomenon, called spontaneous maturation, is quite rare in amphibians. InB. arenarum, spontaneous maturation took place only in oocytes obtained during the reproductive period (spring-summer). During this period the oocytes also demonstrated a respiratory activity characteristic of mature oocytes. Interestingly, full-grownB. arenarumoocytes always responded to progesterone regardless of the season in which they were obtained and of their respiratory activity. The disposition of oocytes competent or not competent to mature spontaneously provides a useful system for the study of molecular mechanisms involved in the maturation process. The data presented here indicate that the activation of protein kinase C (PKC) induces germinal vesicle breakdown (GVBD) in denuded oocytes unable to mature spontaneously (winter oocytes) and is involved in the in vitro spontaneous maturation ofB. arenarumfull-grown oocytes. The inhibition of PKC by 1-(5-isoquinolynyl-sulphonyl-2-methyl-piperazine (H-7) impeded spontaneous maturation in a dose-dependent manner, thus supporting the participation of the PKC pathway during this process. Interestingly phorbol 12-myristate-13-acetate (PMA)-induced GVBD is inhibited by the incubation of the oocytes in dibutyryl cAMP (dbcAMP), indicating that both pathways, PKC and protein kinase A (PKA), are related at a certain point. However, spontaneous GVBD is less sensitive than PMA-induced GVBD to dbcAMP. This fact would support the suggestion that in spontaneous GVBD mechanisms different from activation of PKC are at work.

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Polygonatum sibiricum Polysaccharides Protect against MPP-Induced Neurotoxicity via the Akt/mTOR and Nrf2 Pathways

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/8843899 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Si Huang ◽

Haiyan Yuan ◽

Wenqun Li ◽

Xinyi Liu ◽

Xiaojie Zhang ◽

...

Keyword(s):

Molecular Mechanisms ◽

Neuronal Loss ◽

Heme Oxygenase 1 ◽

Related Factor ◽

Dependent Manner ◽

Quinone Oxidoreductase ◽

Glutamate Cysteine Ligase ◽

Dopaminergic Neuronal Loss

Polygonatum sibiricum, a well-known life-prolonging tonic in Chinese medicine, has been widely used for nourishing nerves in the orient, but the underlying molecular mechanisms remain unclear. In this study, we found that P. sibiricum polysaccharides (PSP) ameliorated 1-methyl-4-phenyl-1,2.3,6-tetrahydropyridine- (MPTP-) induced locomotor activity deficiency and dopaminergic neuronal loss in an in vivo Parkinson’s disease (PD) mouse model. Additionally, PSP pretreatment inhibited N-methyl-4-phenylpyridine (MPP+) induced the production of reactive oxygen species, increasing the ratio of reduced glutathione/oxidized glutathione. In vitro experiments showed that PSP promoted the proliferation of N2a cells in a dose-dependent manner, while exhibiting effects against oxidative stress and neuronal apoptosis elicited by MPP+. These effects were found to be associated with the activation of Akt/mTOR-mediated p70S6K and 4E-BP1 signaling pathways, as well as nuclear factor erythroid 2-related factor 2- (Nrf2-) mediated NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutamate-cysteine ligase catalytic subunit (Gclc), and glutamate-cysteine ligase modulatory subunit (Gclm), resulting in antiapoptotic and antioxidative effects. Meanwhile, PSP exhibited no chronic toxicity in C57BJ/6 mice. Together, our results suggest that PSP can serve as a promising therapeutic candidate with neuroprotective properties in preventing PD.

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