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1.
Article in English | WPRIM | ID: wpr-739802

ABSTRACT

BACKGROUND: Chronic hyperglycemia has deleterious effects on pancreatic β-cell function and turnover. Recent studies support the view that cyclin-dependent kinase 5 (CDK5) plays a role in β-cell failure under hyperglycemic conditions. However, little is known about how CDK5 impair β-cell function. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. In this study, we examined the effect of myricetin on high glucose (HG)-induced β-cell apoptosis and explored the relationship between myricetin and CDK5. METHODS: To address this question, we subjected INS-1 cells and isolated rat islets to HG conditions (30 mM) in the presence or absence of myricetin. Docking studies were conducted to validate the interaction between myricetin and CDK5. Gene expression and protein levels of endoplasmic reticulum (ER) stress markers were measured by real-time reverse transcription polymerase chain reaction and Western blot analysis. RESULTS: Activation of CDK5 in response to HG coupled with the induction of ER stress via the down regulation of sarcoendoplasmic reticulum calcium ATPase 2b (SERCA2b) gene expression and reduced the nuclear accumulation of pancreatic duodenal homeobox 1 (PDX1) leads to β-cell apoptosis. Docking study predicts that myricetin inhibit CDK5 activation by direct binding in the ATP-binding pocket. Myricetin counteracted the decrease in the levels of PDX1 and SERCA2b by HG. Moreover, myricetin attenuated HG-induced apoptosis in INS-1 cells and rat islets and reduce the mitochondrial dysfunction by decreasing reactive oxygen species production and mitochondrial membrane potential (Δψm) loss. CONCLUSION: Myricetin protects the β-cells against HG-induced apoptosis by inhibiting ER stress, possibly through inactivation of CDK5 and consequent upregulation of PDX1 and SERCA2b.


Subject(s)
Animals , Apoptosis , Blotting, Western , Calcium-Transporting ATPases , Cyclin-Dependent Kinase 5 , Diabetes Mellitus, Type 2 , Down-Regulation , Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Gene Expression , Genes, Homeobox , Glucose , Hyperglycemia , Insulin-Secreting Cells , Membrane Potential, Mitochondrial , Polymerase Chain Reaction , Rats , Reactive Oxygen Species , Reticulum , Reverse Transcription , Up-Regulation
2.
Article in Chinese | WPRIM | ID: wpr-771676

ABSTRACT

The aim of this research is to investigate the effects of paeoniflorin and menthol on the physiological function of Calu-3 cell membrane during the transport of puerarin. Calu-3 cell was used as the cell model to simulate nasal mucosa tissues, and the cell membrane fluidity, Na⁺-K⁺-ATPase activity and Ca²⁺-ATPase activity were detected by fluorescence recovery after photobleaching(FRAP) and ultramicro enzyme activity testing, in order to explore the mechanism of compatible drugs on promoting puerarin transport. The results showed that when puerarin associated with low, middle and high concentration of menthol or both paeoniflorin and menthol, the fluorescence recovery rate was increased significantly, while Na⁺-K⁺-ATPase activity had no significant change and Ca²⁺-ATPase activity was enhanced significantly as compared with puerarin alone. Therefore, it was concluded that menthol had the abilit of promoting the transport and the mechanism might be related to increasing membrane fluidity and activating Ca²⁺-ATPase.


Subject(s)
Calcium-Transporting ATPases , Metabolism , Cell Line, Tumor , Cell Membrane , Glucosides , Chemistry , Humans , Isoflavones , Metabolism , Membrane Fluidity , Menthol , Chemistry , Monoterpenes , Chemistry , Sodium-Potassium-Exchanging ATPase , Metabolism
3.
Article in Chinese | WPRIM | ID: wpr-771667

ABSTRACT

This study aimed to observe the general state and changes in pathophysiological indexes of multiple cerebral infarction rat model with Qi-deficienty and Blood-stasis syndrome. Rats were randomly divided into 4 groups(with 30 in each group): the normal group, the sham group, the model group and the Yiqi Huoxue recipe group. Rats in the model group and Yiqi Huoxue group were provided with interruptable sleep deprivation for 7 days before the multiple cerebral infarction operation, and followed by another 4 weeks of sleep deprivation; rats in the Yiqi Huoxue group were intragastrically administrated with drug at a dose of 26 g·kg⁻¹, once a day for 4 weeks. The general state was observed, and the pathophysiological indexes were measured at 48 h, 2 weeks and 4 weeks after administration. The results showed that rats in the normal group and the sham group represented a good general state and behaviors, with a normal morphological structure of brain tissues; rats in the model group featured yellow fur, depression, accidie, loose stools and movement disorder, with obvious brain histomorphological damage, which became aggravated with the increase of modeling time; rats in the Yiqi Huoxue group showed release in the general state and above indexes. Compared with the sham group at three time points, rats in the model group showed decrease in body weight, exhaustive swimming time and RGB value of tongue surface image, and increase in whole blood viscosity of the shear rate under 5, 60 and 150 S⁻¹, reduction in cerebral cortex Na⁺-K⁺-ATPase, Ca²⁺-ATPase activity and contents of 5-HT, rise in TXB2 levels and decline in 6-keto-PGF1a in serum(<0.05, <0.01). Compared with the model group, rats in the Yiqi Huoxue group showed alleviations in the above indexes at 2 w and 4 w(<0.05, <0.01). The results showed that the characterization and pathophysiological indexes in the multiple cerebral infarction rat model with Qi-deficiency and blood-stasis syndrome were deteriorated; Yiqi Huoxue recipe could significantly alliviate the abnormal conditions, which suggested of the model was stable and reliable and the pathophysiologic evolutionary mechanism might be related to energy metabolism dysfunction, vasoactive substance abnormality and changes in neurotransmitters.


Subject(s)
Animals , Calcium-Transporting ATPases , Metabolism , Cerebral Infarction , Drugs, Chinese Herbal , Pharmacology , Energy Metabolism , Medicine, Chinese Traditional , Qi , Rats , Sodium-Potassium-Exchanging ATPase , Metabolism
4.
Article in Chinese | WPRIM | ID: wpr-815166

ABSTRACT

OBJECTIVE@#To investigate the effect of benzo(α)pyrene on the ATPase activity and content of Ca²⁺ in the hippocampus of neonatal SD rats.@*METHODS@#Sixty male and 60 female 4-days-old neonatal SD rats were randomly divided into 5 groups (n=24): a blank control group, a vehicle control group (peanut oil), 3 benzo(α)pyrene groups (0.02, 0.2 and 2 mg/kg, respectively). SD rats were given benzo(α)pyrene (dissolved in peanut oil) by gavage daily from postnatal day 4 (PND4) to PND20. The nerve reflex, the condition of neuro-muscle development and motion function were examined in the period of treatment. The colorimetric technique was used to detect the activity of Ca²⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in hippocampus after the treatment. The concentration of Ca²⁺ of synapse in the hippocampus of rats was detected by fluorescent labeling.@*RESULTS@#The results from the behavior tests showed that duration of surface reflex latency in rats with medium dose of benzo(α)pyrene was longer compared with that in the control group in PND12. The duration of surface reflex latency in rats with high dose of benzo(α) pyrene is longer in PND 14 and PND 16 compared with that in the control group (P<0.05). Compared with the rats in the control group, the activities of Ca²⁺-Mg²⁺-ATPase and Ca²⁺-ATPase in hippocampus in rats with high dose benzo(α) pyrene were significantly decreased, and the degree in the decrease of Ca²⁺-ATPase activity was dose-dependent (P<0.05). The contents of Ca²⁺ in the hippocampus in rats with medium or high dose of benzo(α) pyrene were significantly increased compared with that in the control group (P<0.05), which showed a dose-dependent manner (P<0.05).@*CONCLUSION@#Benzo(α)pyrene exposure led to the decrease in ATPase activity as well as Ca²⁺ overload of the synapse in the hippocampal tissue, which in turn results in the nerve damage of newborn SD rats.


Subject(s)
Animals , Benzo(a)pyrene , Toxicity , Ca(2+) Mg(2+)-ATPase , Metabolism , Calcium , Metabolism , Calcium-Transporting ATPases , Metabolism , Female , Hippocampus , Male , Rats , Rats, Sprague-Dawley
5.
Article in English | WPRIM | ID: wpr-727826

ABSTRACT

The etiology of periodontal disease is multifactorial. Exogenous stimuli such as bacterial pathogens can interact with toll-like receptors to activate intracellular calcium signaling in gingival epithelium and other tissues. The triggering of calcium signaling induces the secretion of pro-inflammatory cytokines such as interleukin-8 as part of the inflammatory response; however, the exact mechanism of calcium signaling induced by bacterial toxins when gingival epithelial cells are exposed to pathogens is unclear. Here, we investigate calcium signaling induced by bacteria and expression of inflammatory cytokines in human gingival epithelial cells. We found that peptidoglycan, a constituent of gram-positive bacteria and an agonist of toll-like receptor 2, increases intracellular calcium in a concentration-dependent manner. Peptidoglycan-induced calcium signaling was abolished by treatment with blockers of phospholipase C (U73122), inositol 1,4,5-trisphosphate receptors, indicating the release of calcium from intracellular calcium stores. Peptidoglycan-mediated interleukin-8 expression was blocked by U73122 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester). Moreover, interleukin-8 expression was induced by thapsigargin, a selective inhibitor of the sarco/endoplasmic reticulum calcium ATPase, when thapsigargin was treated alone or co-treated with peptidoglycan. These results suggest that the gram-positive bacterial toxin peptidoglycan induces calcium signaling via the phospholipase C/inositol 1,4,5-trisphosphate pathway, and that increased interleukin-8 expression is mediated by intracellular calcium levels in human gingival epithelial cells.


Subject(s)
Bacteria , Bacterial Toxins , Calcium , Calcium Signaling , Calcium-Transporting ATPases , Cytokines , Epithelial Cells , Epithelium , Gram-Positive Bacteria , Humans , Inflammation , Inositol 1,4,5-Trisphosphate Receptors , Interleukin-8 , Peptidoglycan , Periodontal Diseases , Phospholipases , Reticulum , Thapsigargin , Toll-Like Receptor 2 , Toll-Like Receptors , Type C Phospholipases
6.
Article in English | WPRIM | ID: wpr-92339

ABSTRACT

BACKGROUND: Phenylephrine (PE) produces tonic contraction through involvement of various calcium channels such as store-operated calcium channels (SOCCs) and voltage-operated calcium channels (VOCCs). However, the relative contribution of each calcium channel to PE-induced contraction has not been investigated in isolated rat aorta of early acute myocardial infarction (AMI). METHODS: Endothelium-denuded rat aortic rings from rats 3 days after AMI or sham-operated (SHAM) rats were prepared in an organ chamber with Krebs-Ringer bicarbonate solution for isometric tension recording. We assessed the PE dose-response relationships in 2.5 mM calcium medium for both groups. The same procedure was repeated using rings pretreated with the SOCC inhibitor 2-aminoethoxydiphenyl borate, sarco/endoplasmic-reticulum calcium ATPase inhibitor thapsigargin (TG), diacyl glycerol lipase inhibitor RHC80267, and sodium-calcium exchanger inhibitor 3,4-dichlorobenzamil hydrochloride for 30 minutes before addition of calcium. When ongoing tonic contraction was sustained, dose-response curves to the VOCC inhibitor nifedipine were obtained to assess the relative contribution of each calcium channel under various conditions. RESULTS: The effect of SOCC induction with TG pretreatment on PE-induced contraction was significantly lower in the AMI group compared to the SHAM group. In addition, there were significant decreases in the sensitivity and efficacy of the VOCC inhibitor nifedipine on PE-induced contraction in the AMI group. CONCLUSIONS: Results suggest that the change of vascular reactivity of PE in rat aorta 3 days after AMI is characterized by a decreased contribution of L-type VOCCs. The enhanced VOCC-independent calcium entry mechanisms after AMI can be mediated by enhanced capacitative calcium entry through the activation of SOCCs.


Subject(s)
Animals , Aorta , Calcium Channels , Calcium , Calcium-Transporting ATPases , Glycerol , Lipase , Myocardial Infarction , Nifedipine , Phenylephrine , Rats , Sodium-Calcium Exchanger , Thapsigargin
7.
Article in English | WPRIM | ID: wpr-52950

ABSTRACT

BACKGROUND AND OBJECTIVES: An increase in intracellular calcium concentration due to loss of Ca2+ homeostasis triggers arrhythmia or cardiac cell death in the heart. Paracrine factors released from stem cells have beneficial cardioprotective effects. However, the mechanism of modulation of Ca2+ homeostasis by paracrine factors in ischemic myocardium remains unclear. MATERIALS AND METHODS: We isolated rat bone marrow-derived mesenchymal stem cells (MSCs), and prepared paracrine media (PM) from MSCs under hypoxic or normoxic conditions (hypoxic PM and normoxic PM). We induced rat myocardial infarction by left anterior descending ligation for 1 hour, and treated PM into the border region of infarcted myocardium (n=6/group) to identify the alteration in calcium-regulated proteins. We isolated and stained the heart tissue with specific calcium-related antibodies after 11 days. RESULTS: The hypoxic PM treatment increased Ca2+-related proteins such as L-type Ca2+ channel, sarcoplasmic reticulum Ca2+ ATPase, Na+/K+ ATPase, and calmodulin, whereas the normoxic PM treatment increased those proteins only slightly. The sodium-calcium exchanger was significantly reduced by hypoxic PM treatment, compared to moderate suppression by the normoxic PM treatment. CONCLUSION: Our results suggest that hypoxic PM was significantly associated with the positive regulation of Ca2+ homeostasis in infarcted myocardium.


Subject(s)
Adenosine Triphosphatases , Animals , Antibodies , Arrhythmias, Cardiac , Calcium , Calcium-Transporting ATPases , Calmodulin , Cell Death , Heart , Homeostasis , Ligation , Mesenchymal Stem Cells , Myocardial Infarction , Myocardium , Paracrine Communication , Rats , Sarcoplasmic Reticulum , Sodium-Calcium Exchanger , Stem Cells
9.
Braz. j. med. biol. res ; 46(3): 227-234, 15/mar. 2013. graf
Article in English | LILACS | ID: lil-670900

ABSTRACT

Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.


Subject(s)
Animals , Male , Rats , Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Calmodulin/metabolism , Protein-Serine-Threonine Kinases/metabolism , Vas Deferens/metabolism , Muscle Contraction , Phosphorylation , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism
10.
Article in English | WPRIM | ID: wpr-320343

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular (rPT) cells.</p><p><b>METHODS</b>Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 μg/mL quercetin and/or cadmium (2.5, 5.0 μmol/L), in a serum-free medium at 37 °C at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress.</p><p><b>RESULTS</b>Exposure of rPT cells to cadmium acetate (2.5, 5.0 µmol/L) induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na+, K+-ATPase, Ca2+-ATPase, glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 µg/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants (non-enzymatic and enzymic) levels.</p><p><b>CONCLUSION</b>The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.</p>


Subject(s)
Animals , Antioxidants , Pharmacology , Therapeutic Uses , Apoptosis , Cadmium , Toxicity , Cadmium Poisoning , Calcium , Metabolism , Calcium-Transporting ATPases , Metabolism , Cells, Cultured , Kidney Tubules, Proximal , Metabolism , Malondialdehyde , Metabolism , Membrane Potential, Mitochondrial , Quercetin , Pharmacology , Therapeutic Uses , Rats , Reactive Oxygen Species , Metabolism , Sodium-Potassium-Exchanging ATPase , Metabolism
11.
Article in Korean | WPRIM | ID: wpr-102158

ABSTRACT

Recent studies indicate that reactive oxygen species (ROS) can act as modulators of neuronal activity, and are critically involved in persistent pain primarily through spinal mechanisms. In this study, we investigated the effects of NaOCl, a ROS donor, on neuronal excitability and the intracellular calcium concentration ([Ca2+]i) in spinal substantia gelatinosa (SG) neurons. In current clamp conditions, the application of NaOCl caused a membrane depolarization, which was inhibited by pretreatment with phenyl-N-tert-buthylnitrone (PBN), a ROS scavenger. The NaOCl-induced depolarization was not blocked however by pretreatment with dithiothreitol, a sulfhydryl-reducing agent. Confocal scanning laser microscopy was used to confirm whether NaOCl increases the intracellular ROS level. ROS-induced fluorescence intensity was found to be increased during perfusion of NaOCl after the loading of 2',7'-dichlorofluorescin diacetate (H2DCF-DA). NaOCl-induced depolarization was not blocked by pretreatment with external Ca2+ free solution or by the addition of nifedifine. However, when slices were pretreated with the Ca2+ ATPase inhibitor thapsigargin, NaOCl failed to induce membrane depolarization. In a calcium imaging technique using the Ca2+-sensitive fluorescence dye fura-2, the [Ca2+]i was found to be increased by NaOCl. These results indicate that NaOCl activates the excitability of SG neurons via the modulation of the intracellular calcium concentration, and suggest that ROS induces nociception through a central sensitization.


Subject(s)
Animals , Calcium , Calcium-Transporting ATPases , Central Nervous System Sensitization , Dithiothreitol , Fluoresceins , Fluorescence , Fura-2 , Humans , Membranes , Microscopy, Confocal , Neurons , Nociception , Perfusion , Rats , Reactive Oxygen Species , Substantia Gelatinosa , Thapsigargin , Tissue Donors
12.
Article in English | WPRIM | ID: wpr-358192

ABSTRACT

Recent studies in secretory pathway calcium ATPases (SPCA) revealed novel functions of SPCA2 in interacting with store-operated Ca(2+) channel Orai1 and inducing Ca(2+) influx at the cell surface. Importantly, SPCA2-mediated Ca(2+) signaling is uncoupled from its conventional role of Ca(2+)-ATPase and independent of store-operated Ca(2+) signaling pathway. SPCA2-induced store-independent Ca(2+) entry (SICE) plays essential roles in many important physiological processes, while unbalanced SICE leads to enhanced cell proliferation and tumorigenesis. Finally, we have summarized the clinical implication of SICE in oral cancer prognosis and treatment. Inhibition of SICE may be a new target for the development of cancer therapeutics.


Subject(s)
Calcium Channels , Physiology , Calcium Signaling , Physiology , Calcium-Transporting ATPases , Physiology , Cell Proliferation , Cell Transformation, Neoplastic , Metabolism , Humans , Neoplasms , ORAI1 Protein , Prognosis
13.
Article in English | WPRIM | ID: wpr-820024

ABSTRACT

OBJECTIVE@#To study the in vivo efficacy of these two ACTs in the treatment of Plasmodium falciparum (P. falciparum malaria) in Kolkata and to determine the prevalence of mutant S769N codon of the PfATPase6 gene among field isolates of P. falciparum collected from the study area.@*METHODS@#A total of 207P. falciparum positive cases were enrolled randomly in two study arms and followed up for 42 days as per WHO (2009) protocol. A portion of PfATPase6 gene spanning codon S769N was amplified and sequenced by direct sequencing method.@*RESULTS@#It was observed that the efficacy of both the ACT regimens were highly effective in the study area and no mutant S769N was detected from any isolate.@*CONCLUSIONS@#The used, combination AS+SP is effective and the other combination AM+LF might be an alternative, if needed.


Subject(s)
Adolescent , Adult , Aged , Antimalarials , Artemisinins , Base Sequence , Calcium-Transporting ATPases , Genetics , Child , Drug Resistance , Drug Therapy, Combination , Female , Genes, Protozoan , Humans , India , Malaria, Falciparum , Drug Therapy , Parasitology , Male , Middle Aged , Molecular Sequence Data , Mutation , Plasmodium falciparum , Genetics , Treatment Outcome
14.
Article in English | WPRIM | ID: wpr-727561

ABSTRACT

The receptor activator of NF-kappaB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-kappaB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) delta, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.


Subject(s)
Animals , Blood Proteins , Boron Compounds , Calcium-Transporting ATPases , Cell Membrane , Dentin , Estrenes , Inositol , Inositol 1,4,5-Trisphosphate , Inositol 1,4,5-Trisphosphate Receptors , Macrophages , Mice , NF-kappa B , Osteoclasts , Phosphoproteins , Proteins , Pyrrolidinones , Receptor Activator of Nuclear Factor-kappa B , Reticulum , Signal Transduction , Tumor Necrosis Factor-alpha , Type C Phospholipases
15.
Immune Network ; : 253-260, 2012.
Article in English | WPRIM | ID: wpr-20067

ABSTRACT

alpha-Mangostin is a xanthon derivative contained in the fruit hull of mangosteen (Garcinia mangostana L.), and the administration of alpha-Mangostin inhibited the growth of transplanted colon cancer, Her/CT26 cells which expressed Her-2/neu as tumor antigen. Although alpha-Mangostin was reported to have inhibitory activity against sarco/endoplasmic reticulum Ca2+ ATPase like thapsigargin, it showed different activity for autophagy regulation. In the current study, we found that alpha-Mangostin induced autophagy activation in mouse intestinal epithelial cells, as GFP-LC3 transgenic mice were orally administered with 20 mg/kg of alpha-Mangostin daily for three days. However, the activation of autophagy by alpha-Mangostin did not significantly increase OVA-specific T cell proliferation. As we assessed ER stress by using XBP-1 reporter system and phosphorylation of eIF2alpha, thapsigargin-induced ER stress was significantly reduced by alpha-Mangostin. However, coadministration of thapsigargin with alpha-Mangostin completely blocked the antitumor activity of alpha-Mangostin, suggesting ER stress with autophagy blockade accelerated tumor growth in mouse colon cancer model. Thus the antitumor activity of alpha-Mangostin can be ascribable to the autophagy activation rather than ER stress induction.


Subject(s)
Animals , Autophagy , Calcium-Transporting ATPases , Cell Proliferation , Colonic Neoplasms , Epithelial Cells , Fruit , Garcinia mangostana , Mice , Mice, Transgenic , Phosphorylation , Reticulum , Thapsigargin , Transplants , Xanthones
16.
Caracas; s.n; oct. 2011. 185 p. ^c30 cmilus. (LFT-4872011615789).
Thesis in Spanish | LILACS, LIVECS | ID: biblio-1152068

ABSTRACT

Los esfingolípidos, como la ceramida (Cer), la ceramida-1-fosfato (C-1-P), la esfingosina (Sph) y la esfingosina-1-fosfato (S-1P) estan relacionados con la señalización intracelular en procesos como crecimiento celular, movilización intracelular de Ca+2 y apoptósis. En este trabajo se evaluó el efecto de estos esfingolípidos en la homeostasis de Ca+2 intracelular y en la apoptósis en células de cáncer de mama MCF-7. Se utilizaron fluoróforos específicos para el Ca+2 y microscopía confocal. Se demostró que en estas células, la Sph (20 uM), la Cer (10uM), la S-P (2uM) y la C--P (uM) aumentaron la concentración intracelular ce Ca+2, induciendo su liberación desde el retículo endoplasmático (RE). Además, se observo que la esfingosina abrioun canal de Ca2+ en la membrana plasmática. También se demostró que la Cer inhibe parcialmente la actividad de la Ca2+-ATPasa del RE (SERCA), de forma dosis dependiente, mientras que la ceramina, su análogo no hidrolisable la inhibe totalmente. La Sph también inhibe completamente la actividad de la SERCA, a la misma concentración que induce la liberación del Ca+2 del RE. Asimismo, se evaluó el efecto de estos esfingolípidos sobre la inducción de la apotósis en células MCF-7 evidenciando que el tratamiento con la Cer, la ceramida, la Sph inducen toxicidad. También se observo que mientras la ceramida activo la caspasa 7 y la caspasa 8, el esfingolipido natural, la Cer no tuvo ningún efecto. Por su parte, la Sph activa la caspasa 8 sin modificar la activdad de la caspasa 7. Tanto la Cer, como la ceramida y la Sph, disminuyeron la expresión de la proteína Bcl-2 amti-apoptótica, y también indujeron la fragmentación de ADN, visualizada mediante la técnica de TUNEL, demostrando que estos esfingolípidos inducen apoptósis en MCF-7. La agelasina B, toxina purificada a partir de la esponja marina Agelas clathrodes tiene un efecto citotóxico un orden de magnitud mayor en MCF-7, en comparación con fibroplastos humanos. La agelasina B induce la liberación del Ca+2 almacenado en el RE en celulas MCF-7, ademas de inhibir la actividad de la SERCA en un 100%. También se demostró que esta toxina induce apoptosis, ya que disminuye el potencial de membrana mitocondrial, activa la caspasa 8, disminuye la expresion de la proteina Bcl-2 e induce fragmentación del ADN de las células MCF-7. Este mecanismo es similar al efecto de la tapsigargina.


Subject(s)
Humans , Animals , Sphingolipids/pharmacology , Breast Neoplasms/metabolism , Signal Transduction/drug effects , Calcium/metabolism , Apoptosis/drug effects , Agelas/chemistry , Purines/therapeutic use , Purines/pharmacology , Sphingolipids/toxicity , Sphingolipids/therapeutic use , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Ceramides/toxicity , Calcium-Transporting ATPases/adverse effects , In Situ Nick-End Labeling/methods , Receptors, Calcium-Sensing/therapeutic use , MCF-7 Cells , Naphthalenes/therapeutic use , Naphthalenes/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacology
17.
Chinese Medical Journal ; (24): 1061-1068, 2011.
Article in English | WPRIM | ID: wpr-239893

ABSTRACT

<p><b>BACKGROUND</b>The high mobility group A1 (HMGA1) proteins are architectural transcription factors found to be overexpressed in lung adenocarcinoma. Lentivirus-mediated RNA interference (RNAi) technology is a powerful tool for silencing endogenous or exogenous genes in human cancer cells. Our preliminary study shows that gemcitabine inhibits growth of the human lung cancer cell line SPCA-1 and induces apoptosis, and this effect might link with down-regulation of HMGA1 expression. This study aimed to investigate the chemosensitivity change of the lung adenocarcinoma cells SPCA-1 after HMGA1 inhibition by lentivirus-mediated RNAi.</p><p><b>METHODS</b>We studied a highly malignant lung adenocarcinoma cell line (SPCA-1 cells). Lentiviral short-hairpin RNA (shHMGA1) expression vectors targeting HMGA1 were used for generation of lentiviral particles. After being transfected into the lung adenocarcinoma cell line SPCA-1, the expression of HMGA1 was determined by retrotranscriptase polymerase chain reaction (RT-PCR) and Western blotting. The effect of gemcitabine on proliferation of positive and negative cells was observed by methyl thiazolyl tetrazolium (MTT) assay and clonogenic survival assay. Apoptosis was observed by flow cytometery. Chemosensitivity to gemcitabine was determined by IC50 analysis. Caspase activity was quantitated by a caspase colorimetric protease assay kit.</p><p><b>RESULTS</b>HMGA1-siRNA silenced its target mRNA specifically and effectively in SPCA-1 cells. The apoptotic rates of the scramble control group were (7.43 ± 0.21)%, (11.00 ± 0.20)%, and (14.93 ± 0.31)%, and the apoptotic rates in the silenced group were (9.53 ± 0.42)%, (16.67 ± 0.45)%, and (25.40 ± 0.79)% under exposure to 0.05, 0.5 and 5.0 µg/ml of gemcitabine (P < 0.05). The IC(50) of the silenced group was (0.309 ± 0.003) µg/ml which was significantly lower than in the scramble control group, (0.653 ± 0.003) µg/ml (P < 0.05). It reduced cancer cell proliferation and increased apoptotic cell death after being treated with gemcitabine compared with the scramble control group. HMGA1 silencing resulted in reduction in the phosphorylation of Akt, and promoted the activation of caspases 3, 8 and 9 upon exposure to gemcitabine.</p><p><b>CONCLUSIONS</b>Lentivirus-mediated RNA interference of HMGA1 enhanced chemosensitivity to gemcitabine in lung adenocarcinoma cells. The mechanism may be associated with the PI-3K/Akt signal pathway. HMGA1 may represent a novel therapeutic target in lung cancer.</p>


Subject(s)
Antimetabolites, Antineoplastic , Pharmacology , Blotting, Western , Calcium-Transporting ATPases , Genetics , Metabolism , Caspase 3 , Genetics , Metabolism , Caspase 8 , Genetics , Metabolism , Caspase 9 , Genetics , Metabolism , Cell Line, Tumor , Cell Proliferation , Deoxycytidine , Pharmacology , Flow Cytometry , Genetic Vectors , Genetics , HMGA Proteins , Genetics , Metabolism , Humans , Lentivirus , Genetics , RNA Interference , Physiology , Reverse Transcriptase Polymerase Chain Reaction
18.
Article in Chinese | WPRIM | ID: wpr-251209

ABSTRACT

<p><b>OBJECTIVE</b>To study characteristics of energy metabolism in the skeletal muscle of rats with postoperative fatigue syndrome (POFS) and the interventional effect of ginsenoside Rb1.</p><p><b>METHOD</b>We chose resection of 70% of the "middle" small intestine as the rat model for POFS. Ninety-six adult male SPF SD rats were randomly divided into the control group, the model group, and the ginsenoside Rb1-treated group by body weight. And then, each group was further randomly divided into four subgroups, according to different postoperative investigated time points, such as postoperative day 1, postoperative day 3, postoperative day 7 and postoperative day 10. So the animals were divided into twelve subgroups (n = 8 in each subgroup). Rats of the control group and the model group were injected intraperitoneally with saline at the dose of 10 mL x kg(-1) one hour before the operation and once a day during the postoperative days. Rats of the ginsenoside Rb1-treated group were administered 10 mg x kg(-1) ginsenoside Rb1 by the same method. The skeletal muscles were sampled on postoperative day 1, 3, 7 and 10. The contents of ATP, ADP, AMP in skeletal muscles were determined by HPLC, and the activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase were investigated by colorimetry.</p><p><b>RESULT</b>Compared with the control group, the content of ATP in skeletal muscle of rats of the model group decreased significantly on postoperative day 3 (P < 0.05), while the content of ADP significantly increased on postoperative day 7 and 10 (P < 0.05). The activity of Na(+)-K(+)-AT-Pase decreased on postoperative day 3 and 7 (P < 0.05), and the activity of Ca(2+)-ATPase decreased on postoperative day 7. After supplement of ginsenoside Rb1, on the investigated time points, all the negative changes of the indicators discovered above were significantly adjusted (P < 0.05) in rats of the ginsenoside Rb1-treated group, while no significant differences were investigated.</p><p><b>CONCLUSION</b>During a certain period of postoperative time, the activity of energy metabolism is depressed in the skeletal muscle of rats with POFS, but it can be improved by supplement of ginsenoside Rb1.</p>


Subject(s)
Animals , Calcium-Transporting ATPases , Physiology , Energy Metabolism , Fatigue , Drug Therapy , Metabolism , Ginsenosides , Pharmacology , Therapeutic Uses , Male , Muscle, Skeletal , Metabolism , Postoperative Complications , Drug Therapy , Metabolism , Rats , Rats, Sprague-Dawley , Sodium-Potassium-Exchanging ATPase , Physiology , Syndrome
19.
Article in English | WPRIM | ID: wpr-328517

ABSTRACT

<p><b>OBJECTIVE</b>To assess any direct effect of extract of Paris polyphylla Simth (EPPS), a Chinese plant, on a cardiomyocyte subject to ischemia-reperfusion injury and to further elucidate its protective effect against myocardium ischemia on the cellular level.</p><p><b>METHODS</b>Neonatal rat cardiomyocytes were isolated and subjected to an anoxia-reoxia injury simulating the ischemia-reperfusion injury in vivo in the presence or absence of EPPS or diltizem, a positive control. The lactate dehydrogenase (LDH) activities in culture supernatants and cell viabilities were analyzed using the enzymatic reaction kinetics monitoring-method and MTT method, respectively. Free intracellular calcium concentrations and activities of Na(+)-K(+) ATPase and Ca(2+) ATPase in cells were also measured with laser confocal microscopy and the inorganic phosphorus-transformation method, respectively.</p><p><b>RESULTS</b>In cardiomyocytes subject to anoxia-reoxia injury, EPPS at 50-400 mg/L showed a concentration-dependent inhibition on LDH leakage and maintenance of cell viability, and the effect was significant at 275 and 400 mg/L (both P<0.01). In addition, EPPS at 275 and 400 mg/L significantly inhibited the increase in intracellular free calcium (both P<0.01) as well as decreased the activities of Na(+)-K(+) ATPase and Ca(2+) ATPase (P<0.01, P<0.05).</p><p><b>CONCLUSIONS</b>EPPS prevents anoxia-reoxia injury in neonatal rat cardiomyocytes in vitro by preservation of Na(+)-K(+) ATPase and Ca(2+) ATPase activities and inhibition of calcium overload. The direct protective effect on cardiomyocytes may be one of the key mechanisms that underlie the potential therapeutic benefit of EPPS against myocardium ischemia.</p>


Subject(s)
Animals , Calcium , Metabolism , Calcium-Transporting ATPases , Metabolism , Cells, Cultured , Hypoxia , Metabolism , Liliaceae , Chemistry , Microscopy, Fluorescence , Myocardium , Plant Extracts , Pharmacology , Rats , Rats, Sprague-Dawley , Reperfusion Injury , Metabolism , Sodium-Potassium-Exchanging ATPase , Metabolism
20.
Chinese Medical Journal ; (24): 574-580, 2011.
Article in English | WPRIM | ID: wpr-241554

ABSTRACT

<p><b>BACKGROUND</b>Donor-pretreatment with ulinastatin may influence the liver graft during cold preservation. The aim of this research was to determine whether pretreatment of donor liver with Ulinastatin can attenuate cold preservation injury, and to explore the mechanism by which Ulinastatin affects the donor liver graft.</p><p><b>METHODS</b>One hundred and forty-four Wistar rats were divided into the Ulinastatin treatment group (T group) pretreated with Ulinastatin 50 000 U/kg and control group (C group) treated with 0.9% normal saline via peritoneal injection prior to the anesthetization. After the abdominal cavity was opened and perfused with cold Ringer's lactate solution, the liver was harvested. The harvested liver was preserved in cold Ringer's lactate solution for 0, 2, 6, 24 hours, at which time the liver tissue was sampled for determination of dry weight and wet weight, Na(+)-K(+)-ATPase and Ca(2+)-ATPase activity, lactic acid dehydrogenase (LDH) activity, lactic acid and malondialdehyde levels. Light microscopy and electron microscopy were used to observe liver morphology. The liver cold-preservation solution was taken for measurement of aspartate aminotransferase (AST) and alanine transaminase (ALT) levels. Correlation between ATPase activity and lactic acid level was analyzed by SPSS 13.0 for Windows.</p><p><b>RESULTS</b>The morphology in the T group had improved cell boundaries vs. the C group at each time point. Dry weight to wet weight in the T group was lower than in the C group at 6 hours (P < 0.05), but the difference was not significant at 24 hours. ALT levels in the T group were lower than that in the C group at 6 hours (P < 0.05) and 24 hours (P < 0.01). AST levels in the T group were lower than those in the C group at 2 hours (P < 0.05), 6 hours (P < 0.01) and 24 hours (P < 0.01). Na(+)-K(+)-ATPase activity in the T group was higher than in the C group and the mean difference between two groups was significant at 0 hour (P < 0.05) and 2 hours (P < 0.05). Ca(2+)-ATPase activity in the T group was higher than in the C group with the mean difference between two groups significant at 2 hours (P < 0.05). The T group had increased lactic acid levels at 0 hour (P < 0.01) and 2 hours (P < 0.05) compared with the C group, but there was no influence on the LDH activity at the same time. There were no obvious differences in the levels of malondialdehyde between the two groups at any time point. A linear correlation between Na(+)-K(+)-ATPase activity and lactic acid levels (r = 0.295, P < 0.05) was found.</p><p><b>CONCLUSIONS</b>Donor-pretreatment with ulinastatin may protect the cells in a liver graft from ischemia injury during cold preservation; the mechanism may be due to its promotion for cell glycolysis and its preservation of ATPase activity.</p>


Subject(s)
Animals , Calcium-Transporting ATPases , Metabolism , Cryopreservation , Methods , Glycoproteins , Pharmacology , Graft Survival , L-Lactate Dehydrogenase , Metabolism , Liver , Metabolism , Liver Transplantation , Methods , Malondialdehyde , Metabolism , Organ Preservation , Methods , Rats , Rats, Wistar , Sodium-Potassium-Exchanging ATPase , Metabolism , Tissue Donors
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