Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.
Acrylamides/pharmacology* ; Animals ; Apoptosis/drug effects* ; Cryopreservation ; Disaccharides/pharmacology* ; Electrolytes/pharmacology* ; Glutamates/pharmacology* ; Glutathione/pharmacology* ; Heart/physiology* ; Heart Transplantation/methods* ; Hepatocyte Growth Factor/antagonists & inhibitors* ; Histidine/pharmacology* ; Histone Deacetylase Inhibitors/pharmacology* ; Intracellular Signaling Peptides and Proteins/antagonists & inhibitors* ; Male ; Mannitol/pharmacology* ; Oxidative Stress/drug effects* ; Phenylenediamines/pharmacology* ; Proto-Oncogene Proteins/antagonists & inhibitors* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; YAP-Signaling Proteins

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.
Animals ; *Gene Expression Regulation/drug effects ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Male ; Mannitol/pharmacology ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Reactive Oxygen Species/*metabolism ; Rotenone/pharmacology ; Sodium Chloride/pharmacology ; Toll-Like Receptors ; Transcription Factors/genetics/*metabolism

OBJECTIVETo study the protective effect of cold autologous blood cardioplegic solution on the heart of infants with cyanotic congenital heart disease (CCHD).

METHODSNinety-six infants with CCHD who underwent cardiopulmonary bypass (CPB) were randomly and equally divided into three groups: histidine-tryptophan-ketoglutarate (HTK) solution, cold non-autologous blood cardioplegic solution, and cold autologous blood cardioplegic solution. The right auricular tissues were taken before aortic cross-clamping and at 30 minutes after aortic declamping, and ATP level and energy charge (EC) in the myocardium were measured. Venous blood was collected before and immediately after CPB, and the serum levels of creatine kinase (CK)-MB and cardiac troponin I (cTnI) were measured. The clinical parameters, such as the re-beat time and re-beat rate during CPB, cardiac index, dependence on positive inotropic agents, and left ventricular ejection fraction (LVEF) at 2 hours after CPB, the incidence rate of arrhythmia within 24 hours after CPB, and postoperative complications and mortality, were recorded.

RESULTSAt 30 minutes after aortic declamping, the three groups showed significantly decreased ATP and EC levels (P<0.05), and the cold autologous blood group had significantly higher ATP and EC levels than the other two groups (P<0.05). Immediately after CPB, the three groups showed significantly increased serum levels of CK-MB and cTnI (P<0.05), and the cold autologous blood group had significantly lower serum levels of CK-MB and cTnI than the other two groups (P<0.05). The cold autologous blood group had significantly better outcomes than the other two groups in terms of the re-beat time during CPB and the dependence on positive inotropic agents and LVEF at 2 hours after CPB (P<0.05).

CONCLUSIONSCold autologous blood cardioplegic solution is superior to HTK and cold non-autologous blood cardioplegic solutions in preserving myocardial energy and reducing myocardial injury in infants with CCHD who undergo CPB, thus providing a better protective effect on the heart.

Cardioplegic Solutions ; pharmacology ; Cardiopulmonary Bypass ; Energy Metabolism ; Female ; Glucose ; pharmacology ; Heart Defects, Congenital ; metabolism ; surgery ; Humans ; Infant ; Infant, Newborn ; Male ; Mannitol ; pharmacology ; Myocardium ; metabolism ; Potassium Chloride ; pharmacology ; Procaine ; pharmacology ; Ventricular Function, Left

The aim of the study is to investigate the effects of concentration, intestinal segments, pH, inhibitors of proteins (P-gp), Na(+)-dependent glucose transporter (SGLT1) on the intestinal absorption of berberine, and to compare intestinal absorption of berberine in combinations. With phenol red as the indicator, in situ single pass intestinal perfusion (SPIP) model was used and intestinal absorption of pure berberine at concentrations of 36.70, 46.17 and 92.33 microg x mL(-1), simulated system of HLJDT (mixture of berberine, baicalin and geniposide), HLJDT with the concentration of berberine 92.33 microg x mL(-1) in perfusion solution of different intestinal segments (duodenum, jejunum, ileum, and colon) were determined by HPLC in combination with diode array detection (DAD). The results indicated that Ka values ofberberine at different concentrations had little significant difference among that obtained after perfusing via duodenum, jejunum, ileum and colon indicating that the absorption of berberine was mainly the passive diffusion. It was also suggested that SGLT1 and P-gp might exert some effects on the absorption of berberine. Ka and Peff values of berberine in a mixture of pure compounds and HLJDT for different intestine segments of rat showed an increasing tendency and was significantly different (P < 0.05) indicating that berberine in a mixture of pure compounds and HLJDT was assimilated better in small intestine. These results indicate that the intestinal absorption of berberine may be affected by compatibility of compounds. Additionally, berberine has wide absorption window and better absorption in colon.
ATP-Binding Cassette, Sub-Family B, Member 1 ; physiology ; Animals ; Berberine ; administration & dosage ; pharmacokinetics ; Colon ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Duodenum ; metabolism ; Ileum ; metabolism ; Intestinal Absorption ; Intestine, Small ; metabolism ; Jejunum ; metabolism ; Male ; Mannitol ; pharmacology ; Perfusion ; Rats ; Rats, Sprague-Dawley ; Sodium-Glucose Transporter 1 ; physiology ; Verapamil ; pharmacology

A new method that uses the reduced scattering coefficients (micro'(s)) measured in rat's brain tissue in vivo after administration of hyperosmotic solution by bifurcated fiber optic probe is proposed in this paper. 60 SD rats were divided into three groups by randomization method, and then were treated by 0.9% NaCl, 20% Mannitol and 7.5% NaCl through vena caudalis, respectively. The changes of micro'(s) in every rat's local cortex were observed continuously by a bifurcated fiber optic probe in vivo in a mini-invasive way. No changes of micro'(s) were observed in the control group which was given by 0.9% NaCl, while the micro'(s), relative changes of the 20% mannitol group and 7.5% NaCl group increased by 7.3% +/- 1.7% and 12.8% +/- 2.9%, respectively. The results showed that there were significant differences among the three groups (P < 0.05). The micro'(s) of rat's local cortex observed by bifurcated fiber optic probe can be used for shedding light on the anhydration induced by hyperosmotic solution.
Animals ; Cerebral Cortex ; drug effects ; Female ; Male ; Mannitol ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saline Solution, Hypertonic ; pharmacology ; Scattering, Radiation

This study is to investigate the effects of concentration, intestinal section, pH, paracellular route, substrate/inhibitor of enzyme (CYP3A) and proteins (P-gp, MRP2, SGL1) on the absorption of forsythoside A. The absorption of three concentrations (2.6, 5.2, and 10.4 microg x mL(-1)) of forsythoside A in different intestinal segments was studied with phenol red as the marker by rat circulation in situ. The results showed that the residue of forsythoside A with different concentrations had little significant difference from that obtained after perfusing via duodenum, jejunum, ileum and colon, which indicated that the absorption of forsythoside A was passive diffusion and had no difference in different segments of rat intestine. The residue of forsythoside A increased to 466.160 and 463.429 microg respectively when cyclosporine (4 microg x mL(-1)) or midazolam (50 micromol x L(-1)) was added to the circulation fluid, which showed significant difference compared to the control group (P < 0.05). Moreover, the residue of forsythoside A showed a tendency of increase with the increase of cyclosporine or midazolam. When digoxin (50 micromol x L(-1)) or EDTA (10 microg x mL(-1)) was added to the circulation fluid, the residue of forsythoside A decreased to 325.110 and 369.888 microg respectively, which showed significant difference as compared to the control group (P < 0.05). Besides, the residue of forsythoside A showed a tendency of reduction with the increase of digoxin or EDTA. However, there is no significant change in the absorption of forsythoside A when the different concentrations of mannitol were added to the circulation fluid. The results above indicated that the absorption of forsythoside A was mainly passive diffusion and involved paracellular route at the same time. In addition, the substrates of P-gp or CYP3A had dose-dependent effect on the absorption of forsythoside A.
Animals ; Colon ; metabolism ; Cyclosporine ; pharmacology ; Digoxin ; pharmacology ; Dose-Response Relationship, Drug ; Duodenum ; metabolism ; Edetic Acid ; pharmacology ; Glycosides ; administration & dosage ; pharmacokinetics ; Hydrogen-Ion Concentration ; Ileum ; metabolism ; Intestinal Absorption ; Jejunum ; metabolism ; Male ; Mannitol ; pharmacology ; Midazolam ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the change of intestinal mucosa barrier in chronic severe hepatitis B patients and clinical intervention.

METHOD(1) 30 normal healthy controls and 60 chronic severe hepatitis B patients were enrolled in this study. The change of intestinal permeability was determined by urine lactulose/ mannitol ratio (L/M), and the serum diamine oxidase (DAO) was measured. (2) 60 chronic severe hepatitis B patients were randomly divided into two groups: the control group and the treated group, each group has 30 cases. Patients in the control group received standard treatment for 2 weeks, however, in addition to standard treatment, patients in the treated group also received glutamine 10g tid. Endotoxin (ET), DAO and L/M were compared between the two group.

RESULTS(1) Compared to healthy controls, the level of L/M and DAO was significantly increased in chronic severe hepatitis B patients (t = 2.762, P less than 0.01 or t = 6.326, P less than 0.01). (2) Compared to the control group, ET, DAO and L/M were significantly lower 2 weeks after treatment (F = 11.662, P less than 0.01; F = 12.699, P less than 0.01; F = 19.981, P less than 0.01).

CONCLUSION(1) There is an early intestinal mucosa barrier damage in chronic severe hepatitis B patients. (2) Compared to standard treatment, adding glutamine can reverse intestinal mucosa barrier damage.

Administration, Oral ; Adolescent ; Adult ; Amine Oxidase (Copper-Containing) ; blood ; Antiviral Agents ; pharmacology ; therapeutic use ; Child ; Endotoxins ; blood ; Female ; Glutamine ; pharmacology ; therapeutic use ; Hepatitis B, Chronic ; drug therapy ; metabolism ; physiopathology ; Humans ; Intestinal Mucosa ; drug effects ; metabolism ; physiopathology ; Intestine, Small ; Lactulose ; urine ; Male ; Mannitol ; urine ; Middle Aged ; Permeability ; Treatment Outcome ; Young Adult

OBJECTIVETo study the conditions on separation and regeneration of protoplast from Phellinus igniarius.

METHODThe effects of enzymolysis conditions of P. igniarius mycelia on yield of protoplast and culturing conditons on regeneration ratio of protoplast were investigated.

RESULTWhen the 8 days-old mycelia was hydrolysed by 1.5% of lywallzyme adding to driselase of 0. 5% and at 30 degrees C for 3 h and enzymolysis was stablized by sucrose as a stablisher of osmotic pressure, higher yield of P. igniarius protoplast was obtained. If 10 days-old mycelia was used as raw material of enzymolysis and manntol was selected as stablisher of osmotic pressure of enzymolysis, higher regeneration ratio of P. igniarius protoplast also would be obtained in following regeneration step at same time keeping higher yield. For the regeneration processing, it was beneficial for the regeneration of P. igniarius protoplast that PDA plusing mulberry ramulus was used as the culture medium of regeneration and manntol was selected as the osmotic pressure establisher of regeneration culture medium.

CONCLUSIONThe method and conditions to keep both higher yield and regeneration ratio of P. igniarius protoplast were obtained.

Culture Media ; pharmacology ; Fungal Proteins ; pharmacology ; Glucan Endo-1,3-beta-D-Glucosidase ; pharmacology ; Glycoside Hydrolases ; pharmacology ; Mannitol ; pharmacology ; Multienzyme Complexes ; pharmacology ; Osmotic Pressure ; Peptide Hydrolases ; pharmacology ; Polyporaceae ; drug effects ; physiology ; Protoplasts ; drug effects ; physiology ; Regeneration ; drug effects ; Sucrose ; pharmacology ; Temperature

OBJECTIVEStudy blood vessel injury and gene expression indicating vascular endothelial cell apoptosis induced by mannitol with and without administration of anti-oxidative vitamins.

METHODSHealthy rabbits were randomly divided into four groups. Mannitol was injected into the vein of the rabbit ear in each animal. Pre-treatment prior to mannitol injection was performed with normal saline (group B), vitamin C (group C) and vitamin E (group D). Blood vessel injury was assessed under electron and light microscopy. In a second experiment, cell culture specimen of human umbilical vein endothelial cells were treated with mannitol. Pre-treatment was done with normal saline (sample B), vitamin C (sample C) and vitamin E (sample D). Total RNA was extracted with the original single step procedure, followed by hybridisation and analysis of gene expression.

RESULTSIn the animal experiment, serious blood vessel injury was seen in group A and group B. Group D showed light injury only, and normal tissue without pathological changes was seen in group C. Of all 330 apoptosis-related genes analysed in human cell culture specimen, no significant difference was seen after pre-treatment with normal saline, compared with the gene chip without pre-treatment. On the gene chip pre-treated with vitamin C, 45 apoptosis genes were down-regulated and 34 anti-apoptosis genes were up-regulated. Pre-treatment with vitamin E resulted in the down-regulation of 3 apoptosis genes.

CONCLUSIONVitamin C can protect vascular endothelial cells from mannitol-induced injury.

Animals ; Antioxidants ; pharmacology ; Apoptosis ; Endothelial Cells ; cytology ; pathology ; Gene Expression Regulation ; Humans ; Mannitol ; chemistry ; pharmacology ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; chemistry ; Oxidation-Reduction ; Rabbits ; Vitamins ; metabolism

AIMTo study the effect of negatively charged and positively charged self-microemulsifying systems (SMES) on the cellular tight junction complex was to be investigated at molecular cell level.

METHODSHuman intestinal epithelial Caco-2 cell model was established. Effect of formulations on the transepithelial electrical resistance (TEER) and permeability of the paracellular transport marker mannitol were measured to evaluate the cell integrity. Changes in subcellular localization of the tight junction protein zona occludens 1 (ZO-1) and cytoskeleton protein actin by immunofluorescence were also assessed after treatment of two SMESs in different dilutions.

RESULTSThe TEER of cell monolayers was not markedly affected by negatively charged SMES in different dilutions. The positively charged SMES could significantly decrease the TEER (P < 0.05) in three dilutions. The full recovery of TEER was found after the treatment of lower dilution for 2 h, then cultured for 48 h, while the recovery of TEER was 81.3% of control in 1 : 50 dilution. Two SMESs could enhance the apparent permeability coefficient of mannitol (2.9 - 64.6 folds), which depended on the dilution times. The immunofluorescent results indicated that the distribution of ZO-1 and actin were discrete in cell membrane after the treatment of formulation. Since the positively charged microemulsion could bind to the epithelial cell membrane by electrostatic interaction, the actin of the cells undergone some kind of stress stimulated by the higher concentration of microemulsion was more markedly affected than the negatively charged SMES. Effect of formulations on ZO-1 and actin relied on the dilution.

CONCLUSIONSMES is able to enhance the paracellular transport marker mannitol. The mechanism of opening of tight junctions by SMES might be the change of distribution of ZO-1 and actin.

Actins ; metabolism ; Caco-2 Cells ; Cell Membrane ; drug effects ; metabolism ; Cell Survival ; drug effects ; Drug Delivery Systems ; Electric Impedance ; Emulsions ; administration & dosage ; pharmacology ; toxicity ; Ethylene Glycols ; pharmacology ; toxicity ; Glycerides ; Glycerol ; analogs & derivatives ; pharmacology ; toxicity ; Humans ; Mannitol ; pharmacokinetics ; Membrane Proteins ; metabolism ; Organic Chemicals ; pharmacology ; toxicity ; Phosphoproteins ; metabolism ; Protein Transport ; Tight Junctions ; drug effects ; metabolism ; Zonula Occludens-1 Protein