In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.
Humans ; Trypsin/chemistry* ; Alkaloids/pharmacology*

Animals ; Aquaporins/metabolism* ; Disease Models, Animal ; Glycoproteins/pharmacology* ; Humans ; Lung/metabolism* ; Protective Agents/pharmacology* ; Respiratory Distress Syndrome/physiopathology* ; Sus scrofa ; Trypsin Inhibitors/pharmacology* ; Up-Regulation

PURPOSE: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). METHODS: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. RESULTS: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. CONCLUSION Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways.
Animals ; Cadherins ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation Mediators ; metabolism ; Injections, Intralesional ; Injections, Intravenous ; Intestinal Diseases ; drug therapy ; etiology ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; Leukocyte Elastase ; metabolism ; Male ; Mucin-2 ; metabolism ; Rats, Wistar ; Sepsis ; complications ; Trypsin ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

OBJECTIVETo identify the ileum tissue proteins differentially expressed in Pi-qi deficiency syndrome rats using proteomic approach.

METHODSThirty-seven rats were randomly divided into 3 groups, i. e., the normal control group (Group 1), the Pi-qi deficiency syndrome model group (Group 2), and the reserpine group (Group 3). The Pi-qi deficiency syndrome model was established using excessive exerting combined with irregular diet, and peritoneally injecting Reserpine Injection (1 mg/mL) respectively. The ileum tissues were separated after identified fuzzy method. The differentially expressed proteins were separated with two dimensional electrophoresis (2-DE), analyzed by Mass Spectrometry, and identified by MASCOT Software.

RESULTSThree proteins were differentially expressed in Group 2 and nine proteins were differentially expressed in Group 3 (P < 0.05).

CONCLUSIONPi-qi deficiency syndrome was closely related with decreased expression of albumin as well as increased expressions of trypsin and glucose-regulated protein 78.

Albumins ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Heat-Shock Proteins ; metabolism ; Ileum ; drug effects ; metabolism ; Male ; Medicine, Chinese Traditional ; Proteome ; analysis ; Proteomics ; Qi ; Rats ; Rats, Sprague-Dawley ; Syndrome ; Trypsin ; metabolism

OBJECTIVETo study the effects of ulinastatin on coagulation in children who underwent open-heart surgery with cardiopulmonary bypass (CPB).

METHODSFifty children who underwent open-heart surgery for ventricular septal defect were randomly divided into two groups: ulinastatin treatment and control. Before CPB, ulinastatin (1.0×10(4) U/kg) was added to CPB priming fluid only in the ulinastatin treatment group. Activated partial thromboplasin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen and international normalized ratio (INR) were measured both before and at 1 hr, 6 hrs and 24 hrs after CPB.

RESULTSThe PT in the ulinastatin group was more prolonged than in the control group at 1 hr after CPB (18.7 ± 0.7 s vs 15.5 ± 0.5 s) and 6 hrs after CPB (17.5 ± 0.6 s vs 15.0 ± 0.6 s). The APTT in the ulinatatin group was also significantly more prolonged than in the control group at 6 hrs after CPB (38.7 ± 3.1 s vs 35.3 ± 3.1 s) and 24 hrs after CPB (34.2 ± 3.0 s vs 31.1 ± 2.6 s).

CONCLUSIONSUlinastatin may prolong PT and APTT after CPB, and thus affects coagulation in children.

Blood Coagulation ; drug effects ; Cardiac Surgical Procedures ; Cardiopulmonary Bypass ; Female ; Glycoproteins ; pharmacology ; Humans ; Infant ; Male ; Partial Thromboplastin Time ; Prothrombin Time ; Trypsin Inhibitors ; pharmacology

OBJECTIVETo study the changes of tissue composition and immunogenicity of porcine and human aortic valves after decellularization.

METHODSThree cryopreserved human aortic valves and 4 porcine valves were decellularized with trypsin, and the leaflet tissue was homogenized for SDS-PAGE protein electrophoresis and U-937 migration assay.

RESULTSTrypsin effectively removed the cells from the valve. SDS-PAGE demonstrated an obvious difference in the tissue composition between porcine and human valves. Although decellularization significantly diminished the differences between the valves, decellularized procine aortic valve stilled contained more protein components (between 26 000 and 43 000) than human valve. U-937 migration assay showed an obvious decrease of cell migration in the valves by decellularization (from 832.7×10(3) to 152.4∓31.1×10(3) for porcine valves, P<0.01, and from 644.9×10(3) to 91.2×10(3) for the human valves, P<0.01). Decellularized porcine valves induced a significantly greater cell migration than decellularized human valves (P<0.05).

CONCLUSIONDecellularization with trypsin can effectively decrease the immunogenicity of human or porcine heart valve, but can not completely eliminate the antigen, and decellularized porcine valve still retain strong immunogenicity.

Animals ; Antigens ; isolation & purification ; Aortic Valve ; cytology ; immunology ; Bioprosthesis ; Heart Valve Prosthesis ; Humans ; Swine ; Tissue Engineering ; methods ; Tissue Scaffolds ; Trypsin ; pharmacology

OBJECTIVETo investigate the effect of intraluminal administration of ulinastatin (a protease inhibitor) in the intestine on intestinal inflammation in rats with hemorrhagic shock.

METHODSTwenty-eight Wistar rats were randomized into control group (A), intestinal saline perfusion group (B), ulinastatin intestinal perfusion group (C), and intravenous ulinastatin injection group (D) (n=7). The mean arterial blood pressure (MAP) and survival time of the rats were recorded. The changes in human polymorphonuclear cell (PMN) CD11b expression were detected by flow cytometry. The leukocyte count was recorded at different time points after the treatment, and the pathology of the intestinal mucosa was observed comparatively.

RESULTSGroups C and D showed significantly slower reduction of the MAP than groups A and B after hemorrhagic shock (P<0.05). The survival time of the rats was the longest in group C (P<0.05). CD11b expression increased gradually during hemorrhagic shock in all the groups, but the expression level was the lowest in group C (P<0.05). Hemorrhagic shock caused a reduction in leukocyte counts, which remained the highest in group C (P<0.05). Group C also showed the least intestinal pathology among the 4 groups.

CONCLUSIONIntestinal perfusion of ulinastatin can lower the reduction rate of MAP, attenuate plasma activation and intestinal inflammation, and prolong the survival of rats with hemorrhagic shock. These results indicate an important role of protease in intestinal inflammation during hemorrhagic shock.

Animals ; Arterial Pressure ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation ; enzymology ; metabolism ; Intestines ; enzymology ; metabolism ; Plasma ; metabolism ; Rats ; Rats, Wistar ; Shock, Hemorrhagic ; blood ; enzymology ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.
Amino Acid Sequence ; Animals ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Spider Venoms ; biosynthesis ; genetics ; pharmacology ; Spiders ; Trypsin Inhibitors ; pharmacology

Lyophylized antler powder was hydrolyzed by pepsin and trypsin separately and also simultaneously to give hydrolysates with special physical activities. Complete hydrolysis peptides with MW lower than 1 x 10(3) were collected for assay of angiotensin I-converting enzyme (ACE) inhibitory activity, antioxidant activity and proliferative activity toward UMR-106 osteoblast cells. The results of the experiments revealed that all hydrolysates exhibited potent hydroxyl radical scavenging activity with an IC50 value less than 1 mg/ml which was much lower than the value of 5.5 g x L(-1) for vitamin C. The peptic and peptic tryptic hydrolysates demonstrated strong angiotensin I-converting enzyme (ACE) inhibitory activity. The tryptic hydrolysate increased the proliferation of the UMR-106 cells by 73.43%. The results verified the traditional use of antler in bone-strengthening, anti-aging. The exploratory studies on the ACE inhibitory activity of antler hydrolysates indicated that the hydrolysates might be potentially useful in prevention and treatment of hypertension. Further purification of peptides contributing to the antioxidant activity, angiotensin I-converting enzyme-inhibitory activity and proliferative activity toward osteoblasts from antler hydrolysates is warranted.
Angiotensin-Converting Enzyme Inhibitors ; metabolism ; Animals ; Antioxidants ; metabolism ; Antlers ; chemistry ; metabolism ; Biphenyl Compounds ; metabolism ; Cell Proliferation ; drug effects ; Deer ; Endopeptidases ; metabolism ; Free Radical Scavengers ; Hydrolysis ; Hypertension ; chemically induced ; Pepsin A ; metabolism ; Peptides ; pharmacology ; Peptidyl-Dipeptidase A ; metabolism ; Picrates ; metabolism ; Trypsin ; metabolism

OBJECTIVE: To study the effect of urinary trypsin inhibitor (UTI) on STR genotyping with urinary samples. METHODS: Midstream urine samples of 5 male and 5 female volunteers were collected respectively, sub-packaged, added with different concentration of UTI and stored at -80 degrees C. Genomic DNA was extracted from those urinary samples, of which STR profiles were genotyped with IdentifilerTM kit at 8 different time points. Results of genotyping in urinary samples were compared with those of the homogenous blood control samples and the successful rate of genotyping in different group of urinary samples treated with UTI was determined. RESULTS: Fifteen STR loci included in Identifiler system were all detected in control blood samples and urinary samples stored for 1 day. STR locus loss was observed and all 15 STR loci disappeared in female urinary samples untreated with UTI while those storage periods prolonged to 3 and 9 days, respectively. However, all 15 STR loci could be detected in female urinary samples treated with UTI and stored for as long as 9 days. No STR loci could be detected in male urinary samples preserved without UTI for 7 days while 9 STR loci detected preserved with UTI for 9 days. There was no significant difference among the average detection ratios of STR loci in female urinary samples treated with UTI at concentrations of 0.2, 0.4 or 0.6 microg/mL and stored for 30 days, mean of which was as high as 0.8400 +/- 0.0423, statistically higher than that in male urinary samples (0.1600 +/- 0.0423). CONCLUSION Detection rate of STR loci in urinary samples preserved with UTI was increased significantly, which results in prolonging the storage periods of urinary samples for personal identification.
DNA/urine* ; DNA Fingerprinting/methods* ; Female ; Forensic Genetics/methods* ; Genetic Loci/genetics* ; Genotype ; Glycoproteins/pharmacology* ; Humans ; Male ; Microsatellite Repeats ; Polymerase Chain Reaction ; Specimen Handling/methods* ; Time Factors ; Trypsin Inhibitors/pharmacology*