Hypotonic Research Articles

Simultaneous determination of eight B-vitamins in rat intestinal perfusate to identify effects of osmotic pressures on absorptions.

A rapid and accurate HPLC-DAD method was developed and validated to simultaneously determine eight B-vitamins (VBs, namely thiamine, riboflavin, niacinamide, calcium pantothenic, pyridoxine, biotin, folic acid and cyanocobalamin) and phenolsulfonphthalein in rat intestinal perfusate. Chromatographic separation was achieved using an Inertsil ODS-3 column (250 × 4.6 mm i.d., 5 μm) at a temperature of 40°C. Gradient elution mode was applied at the flow rate of 1.0 mL/min with the mobile phase of acetonitrile-30 mm K2 HPO4 (pH 5.80). The method was successfully applied to identify the effects of osmotic pressures on the absorption of the VBs. The absorption profiles of single and mixed VBs were also compared. Histological section technology was applied to observe the microstructure of small bowel mucosa after perfusion. The results indicated that each compound possessed a better absorption profile under isotonic conditions than under hypotonic or hypertonic conditions for single or mixed solutions. Compared with single VBs, better absorptions in mixed VBs were observed. Pathological tissue slice test suggested that hypotonic and hypertonic solutions changed or damaged the microstructure of mucosa to varying degrees. Taken together, the investigations indicated that multi-VBs administered orally under isotonic condition could generate fast and complete absorption profiles for VBs.

Expression and localization of pannexin-1 and CALHM1 in porcine bladder and their involvement in modulating ATP release.

ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize calcium homeostasis modulator 1 (CALHM1) and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. To determine gene expression and cellular distribution, PCR and immunohistochemistry were performed, respectively, in the porcine bladder. CALHM1 and pannexin-1-mediated ATP release in response to hypotonic solution (0.45% NaCl)-induced stretch, and extracellular Ca2+ depletion ([Ca2+]0) was measured in isolated urothelial, suburothelial, and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial, and detrusor muscle layers, with the highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor, 10Panx1, and by the CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a nonselective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca2+]0 caused a marked, but transient, elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca2+]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells were insignificant. 10Panx1 showed no significant inhibition of [Ca2+]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1, which are densely expressed in the porcine bladder, function as ATP release channels in response to bladder distension. Modulation of extracellular Ca2+ may also regulate ATP release in the porcine bladder through voltage-gated CALHM1 ion channels.

Low osmolality and shear stress during liposuction impair cell viability in autologous fat grafting

Liposuction and subsequent autologous fat grafting have become essential techniques for fat augmentation in plastic surgery. However, standard harvesting techniques that ensure the survival of adipocytes and stromal vascular fraction (SVF) cells and thus preserve the transplanted fat volume are lacking. In particular, the effect of different parameters of the tumescent solution has not been studied in this context. We hypothesized that the osmolality of the tumescent solution could have a significant effect on the survival of adipocytes and SVF cells. We developed two distinct invitro models based on freshly harvested excision fat from patients undergoing surgical treatment. First, we investigated the effect of osmolality by incubating excision fat in different tumescent solutions and analyzed the total cell survival and the differentiation potential of SVF cells. Vital whole-mount staining, isolation yield of SVF cells, clonogenicity, and osteogenic and adipogenic differentiation capacities were analyzed. Second, we addressed the additional effect of mechanical stress by simulating a liposuction on pieces of excision fat after incubation with the tumescent solutions. Osmolality of the tumescent solution by itself did not have a significant effect on adipocyte and SVF viability or SVF differentiation. However, when osmolality was combined with liposuction, a significant trend toward lower viability and more lipid droplets with lower osmolality was observed. Especially, SVF viability was significantly lower after liposuction with a hypotonic (150mOsm/kg) solution. This study demonstrates the considerable effect of osmolality during liposuction and may lead to the development of "cell-protective" tumescent solutions.

Regulation of osmolality for cancer treatment.

Disseminated metastasis is associated with a poor prognosis, and its management in the peritoneal or pleural cavity is crucial in the treatment of cancer. Recent studies show that ion and water transporters play important roles in fundamental cellular functions, including the regulation of cell volume that would be involved in the cancer process. Here, we review the evidence for hypotonic treatments of cancer and evaluate the potential of the cellular physiological approach in clinical management. The regulation of extracellular osmolality is a promising method, with several studies demonstrating the cytocidal effects of hypotonic solution on cancer cells. Peritoneal lavage with distilled water (DW) during surgery is reported to improve the survival rate of patients with spontaneously ruptured hepatocellular carcinoma. The in vitro studies included in this review also indicate the cytocidal effects of hypotonic shock on esophageal, gastric, colonic, pancreatic, and liver cancer cells with several unique methods and apparatuses, such as a differential interference contrast microscope connected to a digital video camera, a high-resolution flow cytometer and re-incubation analysis. The in vivo studies demonstrate the safeness of a peritoneal injection of DW into mice and indicate that the development of dissemination nodules can be prevented by the pre-incubation of cancer cells with DW or the peritoneal injection of DW. We also demonstrate that the blockade of Cl- channels/transporters enhances the cytocidal effects of hypotonic shock by inhibiting regulatory volume decrease in various cancer cells. A deeper understanding of molecular mechanisms may lead to the discovery of these cellular physiological approaches as a novel therapeutic strategy for disseminated metastasis.

Flow cytometric osmotic fragility test: Increased assay sensitivity for clinical application in pediatric hematology.

Osmotic fragility test (OFT) is widely considered as a sensitive indicator of red blood cells' sensitivity to the hypotonic solution. It is often used as a screening test for the diagnosis of hereditary spherocytosis (HS). Nowadays, the osmotic fragility test based on flow cytometric analysis (FCM OF) is widely used in laboratory practice. The purpose of this study was to optimize the assay sensitivity and to validate its clinical application in the diagnostic screening of childhood anemias. The study was conducted on 175 children suffering from various types of anemia (including 30 children with proven hereditary spherocytosis, HS) and 16 healthy subjects. All children were aged between 3months and 17years, including 94 boys and 97 girls. FCM OF was performed on every subject according to two different analysis time patterns (hemolysis was analyzed for 214 or 300s) using Cytomics FC500 flow cytometer. Significant higher sensitivity was demonstrated by the tests carried out according to the longer analysis time pattern (90.0 vs. 83.33%). The level of specificity of both the analysis patterns was similar. When an extended analysis time was used, the percentage of red cell survival levels in HS patients were significantly lowered compared to the same cases analyzed with shorter incubation times and all other non-HS anemic cases (9.31±4.69 vs. 35.59±15.30%, P<0.05). During the shorter analysis time, the values obtained were 13.76±7.92% for HS and 48.18±19.04% for non-HS, P<0.0001. The 300-s test is very useful in distinguishing thalassemia patients from patients with other types of anemias (94.74% sensitivity and 90.12% specificity) and provided the values of remaining red blood cells as 70.46±12.29% for thalassemia and 27.16±13.01% for nonthalassemia subjects, P<0.0001. Flow cytometric osmotic fragility test with a longer (300-s) analysis time demonstrated an increased sensitivity in detecting HS in anemic children. © 2017 International Clinical Cytometry Society.

English

The crude methanol extract of leaves of Jasminum matthewii as well as its hexane, carbon tetrachloride, chloroform and aqueous soluble partitionates were subjected to screening for antioxidant, cytotoxic, thrombolytic, membrane stabilizing, antimicrobial, analgesic, anti-diarrheal and central nervous system depressant activity assays. The antioxidant potential was evaluated in terms of total phenolic content and free radical scavenging activity using butylated hydroxytolune (BHT) and ascorbic acid as standards. Among the test samples of J. matthewii, the highest free radical scavenging activity was demonstrated by the aqueous soluble fraction (IC50= 41.55&plusmn;0.51 &micro;g/ml), whereas in case of brine shrimp lethality bioassay, the hexane soluble fraction revealed the highest cytotoxic activity with LC50 value 0.19&plusmn;0.32 &mu;g/ml. In thrombolytic activity assay, the aqueous soluble fraction of J. matthewii extractives showed 62.29&plusmn;0.29% of clot lysis, whereas standard streptokinase demonstrated 66.77% clot lysis. Among the test samples, the crude methanol extract inhibited 70.15&plusmn;0.39% haemolysis of red blood cells induced by hypotonic solution. In case of heat-induced condition, the aqueous soluble fraction demonstrated 25.25&plusmn;0.31% inhibition of haemolysis of red blood cells. None of the test samples revealed any zone of inhibition in disc diffusion assay. In peripheral analgesic activity assay, the crude methanol extract of J. matthewii demonstrated 51.02% inhibition of writhing at a dose of 400 mg/kg body weight dose compared to 74.49% inhibition by standard diclofenac sodium. In anti-diarrheal activity assay, the methanolic crude extract reduced diarrheal feces by 89.00&plusmn;0.15% at 400 mg/kg dose. J. matthewii extractives potentiated phenobarbitone sodium-induced sleeping time in a dose dependent manner. Key words: Jasminum matthewii, free radical scavenging activity, brine shrimp lethality, thrombolysis, membrane stabilization, hypotonic solution, zone of inhibition, writhing.

Elucidation of membrane stabilizing Potentials of Methanolic Extract of Vigna unguiculata (cowpea) Linn (seed)

Different concentration of methanolic extracts of seeds of Vigna unguiculata (cowpea) Linn collected from the local area of Noakhali, Bangladesh were studied for membrane Stabilizing Assay. V.unguiculata Linn seeds were initially collected, processed and extracted with methanol. Then, five different concentration (1mg/ml, 3mg/ml, 5mg/ml, 7mg/ml, 9mg/ml) of methanolic extract of Cowpea (V.unguiculata) were subjected for determination of membrane stabilizing activity. In the assay for membrane stabilizing activity, five different concentration of crude methanol extract capable to inhibit hemolysis of erythrocyte membrane dose dependently in hypotonic solution and heat- induced conditions, which indicates the anti-inflammatory property of the samples. Where, Acetyl salicylic acid was used as standard drug. From the above discussion it is clear that Vigna unguiculata Linn seeds methanolic extract showed significant anti-inflammatory potentials. So, it will be very much possible source for isolating lead compound for curing inflammatory disorder.

Biomechanical Strengthening of the Human Cornea Induced by Nanoplatform-Based Transepithelial Riboflavin/UV-A Corneal Cross-Linking

The purpose of this study was to investigate the biomechanical stiffening effect induced by nanoplatform-based transepithelial riboflavin/UV-A cross-linking protocol using atomic force microscopy (AFM). Twelve eye bank donor human sclerocorneal tissues were investigated using a commercial atomic force microscope operated in force spectroscopy mode. Four specimens underwent transepithelial corneal cross-linking using a hypotonic solution of 0.1% riboflavin with biodegradable polymeric nanoparticles of 2-hydroxypropyl-β-cyclodextrin plus enhancers (trometamol and ethylenediaminetetraacetic acid) and UV-A irradiation with a 10 mW/cm2 device for 9 minutes. After treatment, the corneal epithelium was removed using the Amoils brush, and the Young's modulus of the most anterior stroma was quantified as a function of scan rate by AFM. The results were compared with those collected from four specimens that underwent conventional riboflavin/UV-A corneal cross-linking and four untreated specimens. The average Young's modulus of the most anterior stroma after the nanoplatform-based transepithelial and conventional riboflavin/UV-A corneal cross-linking treatments was 2.5 times (P < 0.001) and 1.7 times (P < 0.001) greater than untreated controls respectively. The anterior stromal stiffness was significantly different between the two corneal cross-linking procedures (P < 0.001). The indentation depth decreased after corneal cross-linking treatments, ranging from an average of 2.4 ± 0.3 μm in untreated samples to an average of 1.2 ± 0.1 μm and 1.8 ± 0.1 μm after nanoplatform-based transepithelial and conventional cross-linking, respectively. The present nanotechnology-based transepithelial riboflavin/UV-A corneal cross-linking was effective to improve the biomechanical strength of the most anterior stroma of the human cornea.

Hepatoprotective effect of the solvent extracts of Viola canescens Wall. ex. Roxb. against CCl4 induced toxicity through antioxidant and membrane stabilizing activity

BackgroundViola canescens Wall. ex. Roxb. exhibits analgesic, antimalarial and antispasmodic activities. It is used folklorically for the treatment of liver diseases, hypertension, malaria and cancer. The current study investigates phytochemical constituents, antioxidant and hepatoprotective activity of solvent extracts of whole plant of Viola canescens.MethodsPhytochemicals, acute toxicity study and antioxidant activity of Viola canescens methanolic extract (VCME), ethyl acetate fraction (EAF), and partially purified EAF (90% EAF and combination of 80% EAF + 20% methanol fraction (EAF + Me) was carried out. Hepatoprotective activity of VCME, EAF (200 and 400 mg/kg body weight) and partially purified EAF (50 mg/kg body weight) was investigated in carbon tetrachloride (CCl4) intoxicated BALB/c mice for 7 days. Membrane stabilization effect was determined by hypotonic solution induced hemolysis while DNA ladder assay was carried out by polyacrylamide gel electrophoresis.ResultsPhytochemical screening of VCME showed the presence of alkaloids, phenols, flavonoids, saponins, carbohydrates, tannins and triterpenes. VCME, EAF (at 200 and 400 mg/kg body weight) and partially purified EAF (90% EAF and EAF + Me) at 50 mg/kg body weight significantly reduced the level of ALT, ALP, total bilirubin and restored the level of serum protein in comparison to CCl4 treated group. A significant reduction in malondialdehyde (MDA) and elevation in catalase (CAT) and superoxide dismutase (SOD) level was observed in extract treated animals as compared to CCl4 (p < 0.05). The IC50 values in membrane stabilization potential for VCME, EAF and sodium salicylate were 3.7 ± 0.11, 3.4 ± 0.15 and 3.2 ± 0.09 mg/ml, respectively. Similarly, CCl4 induced degradation of DNA was counteracted by VCME and EAF. The liver biopsy of mice treated with the solvent extracts showed remarkable restoration of normal histological archeitecture.ConclusionsViola canescens showed significant hepatoprotective potential due to its antioxidant and membrane stabilization effect.

High-throughput Screening of Erratic Cell Volume Regulation Using a Hydrogel-based Single-cell Microwell Array.

Here, we report that a single-cell microwell array based on photocrosslinked hydrogel can be used to screen cells exhibiting a defective regulatory volume decrease (RVD) in high-throughput. The RVD is a regulatory function of cells that maintains cell volume homeostasis in a hypotonic medium. Single Madin-Darby canine kidney (MDCK) cells grown in the microwells were loaded with a volume-sensitive fluorescence dye. Changes in the volume of discrete single cells were traced for 20 min in a hypotonic solution using a wide-field fluorescence microscopy. The volume changes of more than 100 single cells were analyzed simultaneously using time-lapse fluorescence micrographs. Cells showing erratic RVD could be easily screened from the image analysis. Nearly 40% of the MDCK single cells exhibited weak, or no, RVD. Since other previously reported methods could not detect as many changes in the volume of discrete singles cells as the method used in this report, we anticipate that our reported method will provide an efficient way of elucidating the RVD mechanisms of cells that have not yet been completely understood.

Biological Investigation of Jatropha gossypiifolia: A Stiff Medicinal Plant in Bangladesh

This study describes a biological investigations of methanolic extract of Jatropha gossypiifolia, a plant belonging to the family Euphorbiaceae. The aim of this investigation was to expose the viable phytochemical constituents properly as their contribution towards the antioxidant, anthelmintic and membrane stabilizing activities. The anthelmintic test was conducted on earthworm Pheritima phosthuma using five unique concentrations of the extract and albendazole as standard drug. The extracts were used for the investigation of antioxidant activity followed by DPPH assay and using BHT as a standard. The crude methanolic stem and leaves extract of J. gossypiifolia was conducted to attempt for membrane stabilizing activities using standard protocols. The leaves and the stems portion considerably have the antioxidant property of 3.186 µg/ml and 10.56 µg/ml respectively. According to the absorbance values of the various extract solutions, results of the colorimetric analysis of total phenolic are that the leaves cover the highest Phenolic content (65.66mg/g in GAE) rather than the stem portion (33.332mg/g in GAE). The leaves and stem methanolic extract was found both at hypotonic solution and heat induced conditions, inhibit 39.19±0.26%, 38.17±0.14 and 27.39±0.15%, 26.97±0.47% hemolysis of erythrocyte membrane respectively, when comparing standard acetyl salicylic acid. The preliminary phytochemical evaluation of methanolic extract of J. gossypiifolia confirmed the presence of glycosides, tannins, phytosterols, triterpenoids, diterpenes, saponins and phenols both at its leaves and stem portions. The results of the study showed that the plant extract has potential membrane stabilizing, anthelmintic, antioxidant activities with significant phytochemicals.

Spermatocyte Spreading during Meiotic Cell Preparation is a Two Step Process

Cytological techniques have been instrumental for the investigation of meiosis and gametogenesis. Especially high resolution chromatin spreads of male and female germline cells provide for detailed insights into the molecular mechanisms acting during germ cell differentiation. Most spreading techniques for germ cells are done with a hypotonic detergent solution followed by an extended drying period. Using video microscopy and we monitored the course of mouse spermatocyte spreading after exposure to graded concentrations of the ionic detergent mixture Lipsol that is known to efficiently spread meiocytes. Our analysis disclosed that spreading of meiotic cells is optimal at a final detergent concentration of about 0.7% and occurs in two phases. First, cells undergo slow swelling in the dilute detergent solution (lasting up to >1 h) followed by rapid dispersion of the nuclear chromatin over the glass surface just prior to the final evaporation of the water in the solution. These results provide a better understanding of an important technique in meiosis research and identify factors determining the spreading process.

Enteral electrolyte solutions with different osmolarities administered in a continuous flow in newborn calves

ABSTRACT: This study compared the effects of enteral electrolyte solutions with different osmolarities in Holstein cattle. Eighteen newborn calves were evenly divided into three groups and administered the following treatments: hypotonic electrolyte solution (ESHYPO) containing 4g NaCl, 0.5g KCl, 1g sodium acetate, and 7.5g dextrose diluted in 1,000mL water; isotonic electrolyte solution (ESISO) containing 5g NaCl, 1g KCl, 2g sodium acetate, and 10g dextrose diluted in 1,000mL water; and hypertonic electrolyte solution (ESHYPER) containing 6g NaCl, 1g KCl, 3g sodium acetate, and 15g dextrose diluted in 1,000mL water. Solutions were administered at a rate of 15mL kg-1hr-1 for 12 hours in a continuous flow. All three solutions increased the concentration of plasma sodium, but ESHYPO did not alter the serum osmolarity. Both ESISO and ESHYPO resulted in an increase in volemia.

Rapid Profiling Cell Cycle by Flow Cytometry Using Concurrent Staining of DNA and Mitotic Markers.

The flow cytometric quantitation of DNA content by DNA-binding fluorochrome, propidium iodide (PI) is the most widely used method for cell cycle analysis. However, the commonly used methods are time-consuming and labor-intensive and are incompatible with staining of mitotic markers by fluorescent-labeled antibodies. Here, we report an optimized simple protocol for rapid and simultaneous analysis of characteristic mitotic phosphorylated proteins and DNA content, permitting the quantification of cells in mitosis, G1, S and G2 stage in a single assay. The protocol detailed here employs detergent-based hypotonic solution to rapidly permeabilize cells and allows simultaneous staining of DNA with PI and mitotic marker, phospho-Histone H3, with specific antibody within 20 min. The protocol requires only inexpensive and commercial available reagents and also enables a rapid and complete analysis of cell cycle profile.

Does Accidental Overcorrection of Symptomatic Hyponatremia in Chronic Heart Failure Require Specific Therapeutic Adjustments for Preventing Central Pontine Myelinolysis?

This review aims at summarizing essential aspects of epidemiology and pathophysiology of hyponatremia in chronic heart failure (CHF), to set the ground for a practical as well as evidence-based approach to treatment. As a guide through the discussion of the available evidence, a clinical case of hyponatremia associated with CHF is presented. For this case, the severe neurological signs at presentation justified an emergency treatment with hypertonic saline plus furosemide, as indicated. Subsequently, as the neurological emergency began to subside, the reversion of the trend toward hyponatremia overcorrection was realized by continuous infusion of hypotonic solutions, and administration of desmopressin, so as to prevent the very feared risk of an osmotic demyelination syndrome. This very disabling complication of the hyponatremia correction is then briefly outlined. Moreover, the possible advantages related to systematic correction of the hyponatremia that occurs in the course of CHF are mentioned. Additionally, the case of tolvaptan, a vasopressin receptor antagonist, is concisely presented in order to underline the different views that have led to different norms in Europe with respect to the USA or Japan as regards the use of this drug as a therapeutic resource against the hyponatremia.

Comparison of the Incidence of Postoperative Hyponatremia after Infusion of Hypotonic Versus Isotonic Intravenous Solutions in Children

Introduction: Hyponatremia is the most common electrolyte disorder in patients following surgical interventions (19-50%). Hospital acquired hyponatremia is often due to using hypotonic solution s and can be lethal.Materials and Methods:Between January and December 2014, 190 children (1 month to 12 years) who were admitted in the urology department of Children’s Hospital Medical Center for elective surgical procedures were enrolled in the study. The patients were randomly divided into two groups: group I received 50 mEq/L sodium and 20 mEq/L potassium in D/W 5% and group II received 154 mEq/l sodium and 20 mEq/L potassium in D/W 5% at the maintenance dose for a period of 6 hours following the operation. The patients did not have any oral fluid intake 6 hours postoperatively. The incidence of hyponatremia before and after maintenance IV fluid therapy was analyzed. Other characteristics of the patients such as age, gender, duration of hospitalization, other concomitant electrolyte disturbances, and symptoms of hypervolemia were also evaluated. The incidence of fluid-IV therapy-induced hyponatremia was investigated and analyzed in different categories of patients. Results: One hundred and ninety patients were enrolled. The mean age was 3.75 years (ranging from 1 month to 12years). One hundred and thirty-three patients (70%) were boys. The incidence of hyponatremia before and after maintenance IV fluid therapy was 9.5% and 36%, respectively. After the therapy, the incidence of hyponatremia was 54% and 17% in hypotonic and isotonic groups, respectively. Final multivariate logistic analysis showed that hyponatremia was common in patients that received hypotonic solution after surgery.Conclusions: Hyponatremia was markedly induced in patients receiving hypotonic solution after surgery. It seems isotonic fluid therapy after surgery protects the patients from hyponatremia.Keywords: Hyponatremia; Isotonic solutions; Hypotonic solutions; Child.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Medicinal properties of the sesbania grandiflora leaves

Background: The leaves of Sesbania grandiflora have been used in local traditional medicine since ancient times. In the present study we investigated, in vivo and in vitro, the potential health benefits of various fractions of the ethanolic extract of these leaves. Materials and methods: Crude ethanolic extract (CEE) of S. grandiflora leaves was partitioned into ethyl acetate soluble fraction (EASF), petroleum ether soluble fraction (PSF), carbon tetrachloride soluble fraction (CTSF), chloroform soluble fraction (CSF), and water soluble fraction (WSF). The extracts were evaluated for their thrombolytic, membrane stabilizing (anti-inflammatory), antimicrobial, and antidiarrheal activities. The results were compared to the effects of standard drugs: streptokinase for the thrombolytic, acetylsalicylic acid (ASA) for the membrane stabilizing, kanamycin for antimicrobial, and loperamide for the antidiarrheal activities. Results: For thrombolysis, EASF showed the highest % of clot lysis (59.6%) among all fractions, while streptokinase and water resulted in 69.2% and 3.1% clot lysis respectively. With respect to the membrane stabilizing activity, the EASF significantly inhibited the hemolysis of human erythrocytes induced by hypotonic solution (64.3±0.6%) or by heat (57.2±0.7%). The other fractions exhibited no membrane stabilizing effect. By contrast ASA resulted in 73.9±0.3% inhibition of osmotically induced hemolysis and a slightly lower level of inhibition in the case of heat-induced hemolysis (70.1±0.3%). The antidiarrheal activity was evaluated in the mouse model. The unfractionated, CEE reduced the number of defecation episodes by 25.0% at a dose of 200 mg/kg and by 41.1% at dose 400 mg/kg body weight. All extract fractions exhibited significant antibacterial activity, which was higher against Gram negative bacteria than Gram positive bacteria. Since the pharmacological activities of S. grandiflora are due to the presence of bioactive compounds we detected and quantified the presence of significant levels of flavonoid and tannin substances. Conclusion: Leaves of Sesbania grandiflora have the potential to be used as a remedy for thrombosis, diarrhea, and inflammatory diseases and against few important bacterial pathogens.

High Dose Oral Furosemide with Salt Ingestion in the Treatment of Refractory Ascites of Liver Cirrhosis

We aimed to evaluate and compare the efficacy and safety of high-dose furosemide+salt orally by comparing HSS+ furosemide (i.v.) and repeated paracentesis in patients with RA. This was a prospective study of 78 cirrhotic patients with RA, randomized into three groups: Group A (n= 25) i.v. furosemide (200-300 mg bid) and 3% hypotonic saline solution (HSS) (once or twice a day); Group B (n= 26) oral furosemide tablets (360-520 mg bid) and salt (2.5 g bid); and, Group C (n= 27) repeated large-volume-paracentesis (RLVP) with albumin infusion. Patients without hyperkalemia were administrated 100 mg of spironolactone/day. During the follow-up; INR, creatinine, and total bilirubin levels were measured to determine the change in MELD (model of end stage liver disease) score. Hepatic encephalopathy (HE), severe episodes of spontaneous bacterial peritonitis (SBP) and pleural effusions (PE) occurred more frequently in Group C. Improvement in Child-Pugh and MELD score was better in Group A and B than Group C. In Group B, improvements were seen in the Child-Pugh and MELD score, reduction in body weight, duration and number of hospitalization. In Groups A and B, remarkable increases in diuresis were observed (706±116 to 2425±633 mL and 691±111 to 2405±772 mL) and serum sodium levels also improved. HE and SBP were occurred more often in group C (p<0.002). Hospitalization decreased significantly in Group B (p<0.001). There was no significant difference in survival among groups. High dose oral furosemide with salt ingestion may be an alternative, effective, safe and well-tolerated method of therapy for RA.

AB276. SPR-03 In vitro evaluation of hydrostatic pressure on ATP release and Caspase-1 activation in rat urothelial cells

ObjectiveBladder outlet obstruction (BOO) is projected to affect approximately 1.1 billion men and women by 2018. While BOO results from a multitude of etiologies, they are all characterized by chronically elevated storage and voiding pressures, along with inflammation in the bladder tissue, which can lead to bladder fibrosis and overactive bladder symptoms. Previous research in our lab demonstrated that ATP release by rat primary urothelial cells increased in response to elevated pressure (10–15 cmH2O) in vitro. Thus, we hypothesize that the release of ATP by urothelial cells upon exposure to pathological pressures in BOO initiates a cascade of events that includes the formation of the NLRP3 inflammasome and caspase-1 activation. In the present study, we examined the effects of elevated pressure on ATP-purinergic signaling and caspase-1 activation in rat urothelial cells in vitro.MethodsUsing a custom built pressure system, rat urothelial cell line MYP3 cells (1.2×106 cells/well in F-12 K medium) were exposed to pressure conditions that represent both pathological storage and voiding pressures: 15 cmH2O 60 min, 15, 40 and 75 cmH2O 1 min. Cells that were prepared in a similar manner but maintained under atmospheric pressure served as a control. In addition, MYP3 cells that were exposed to a hypotonic condition (240 mOsm) and cells treated with 1.25 mM ATP served as two positive controls. After exposure to pressure, the supernatant media was collected and the extracellular ATP concentration was measured using a luciferin-luciferase assay kit (Life Technologies). Cells were then lysed and intracellular caspase-1 activity was measured using an established method. The pressure experiments (40 cmH2O 1 min) were repeated in the presence of a P2X7 antagonist [100 µM A-438079 (R&D Systems)] to determine the mechanism for pressure-induced caspase-1 activation.ResultsExposure of MYP3 cells to hydrostatic pressure for 1 min at 15, 40 and 75 cmH2O resulted in a 3-, 3.5- and 4.5-fold increase in extracellular ATP levels, respectively, compared to the 0 cmH2O control. When these cells were exposed to a hypotonic solution, ATP release increased by 6-fold compared to the isotonic control. Exposure of MYP3 cells to pressure also resulted in up to 1.3-fold increases in caspase-1 levels, which were similar to the positive controls, indicating caspase-1 levels are maximized after exposure to pathologic pressure conditions.ConclusionsThe significant increase in ATP release indicates that hydrostatic pressure is a good mechanical stimulus to model BOO in vitro. The acute caspase-1 responses after 1-minute exposure to pressure suggest that high-pressure voiding may be an important trigger of the NLRP3 inflammasome in BOO. The results that demonstrate exposure to pressure and treatment with ATP both result in caspase-1 activation implicate autocrine purinergic signaling as a mediator of NLRP inflammasome formation.Funding Source(s)NIH (R01DK103534, P20GM103444), NSF (1264579)