OBJECTIVETo observe the effect of tetrandrine on activity of collagenase derived from human hypertrophic scar for the sake of clarifying the mechanism as tetrandrine acting on scar.

METHODSThe experimental concentration was controlled below that of cell proliferation inhibited, SDS-PAGE electrophoresis was adopted to separate collagenase from extracellular matrix, and then activated by trypsin analyzed the activity of collagenase with density scanning apparatus. At the same time quantity of extracellular collagen was measured using improved chloraseptine T oxidizing assay, moreover analyzed correlation between activity of collagenase and quantity of extracellular collagen.

RESULTSIn the concentration below the lever of inhibiting fibroblast proliferation, the total activity of collagenase could be significantly increased by tetrandrine with dosage-dependence associated with quantity of extracellular collagen reduced, which was much greater than that of triamcinolone.

CONCLUSIONIncreasing activity of collagenase on degradation of collagen even in a lower concentration was one of the mechanisms of tetrandrine treating hypertrophic scar.

Benzylisoquinolines ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagenases ; metabolism ; Fibroblasts ; cytology ; Humans

OBJECTIVETo investigate the effect of baicalein and quercetin on the enzymatic resistance of dentin matrix collagen.

METHODSBaicalein, quercetin and proanthocyanidin were dissolved in 20% dimethyl sulfoxide (DMSO) ethanol and prepared into pretreatment agents with a concentration of 50 g/L. Demineralized dentin specimens were prepared and immersed in pretreatment agents at 37 °C for 24 h, then they were digested in solution containing type?collagenase. The pretreatment agents of blank control group and negative control group were 20% DMSO ethanol, blank control group were digested in solution without collagenase. The ultimate tensile strength (UTS) and the hydroxyproline content of enzymolysis liquid in each group were measured respectively after collagenase digestion for 24 h, the dentin collagen morphology were observed under a field emission scanning electron microscopic (FE-SEM) after collagenase digestion for 12 h.

RESULTSAfter collagenase digestion for 24 h, the baicalein group had the highest UTS [(16.00±1.31) MPa], followed by proanthocyanidin group [(12.64±0.91) MPa], blank control group [(7.84±1.18) MPa], quercetin group [(3.20±1.07) MPa], and negative control group (0 MPa). Significant differences were detected among the UTS in each two group (P < 0.01). The hydroxyproline content in blank control group was the lowest [(0.40 ± 0.16) mg/L], followed by baicalein group[(2.95 ± 0.18) mg/L], proanthocyanidin group [(4.78±0.38) mg/L], quercetin group[(28.22±1.53) mg/L], and negative control group [(34.39±0.39) mg/L]. There were significant differences among the hydroxyproline contents in each group (P < 0.01). After collagenase digestion for 12 h, intact collagen network could be seen in blank control group under a FE-SEM. Collagen network in negative control group suffered nearly complete destruction and collapsed. In quercetin group, most of collagen collapsed. In proanthocyanidin group, a small portion of collagen destruction and collapse could be seen. In baicalein group, collagen network remained intact.

CONCLUSIONSThe use of baicalein and quercetin could improve enzymatic resistance of dentin matrix collagen at a concentration of 50 g/L. The effect of baicalein was better than that of proanthocyanidin while the effect of quercetin was weaker than that of proanthocyanidin.

Antioxidants ; pharmacology ; Collagen ; metabolism ; Collagenases ; metabolism ; Dental Bonding ; Dentin ; drug effects ; metabolism ; Dentin-Bonding Agents ; Flavanones ; pharmacology ; Proanthocyanidins ; Quercetin ; pharmacology ; Tensile Strength

OBJECTIVETo observe the effect of isolating the chondrocytes from articular cartilage with the method of three-step enzymatic digestion, and the biological characteristics of the isolated chondrocytes during cultivation in vitro in order to evaluate their biological activity.

METHODSThe method of three-step enzymatic digestion was designed that the articular cartilage was digested one by one with the 1 g/L trypsin and 1 g/L EDTA, 1 g/L hyaluronidase and 2 g/L collagenase I in the culture medium to isolate chondrocytes. The harvesting and viability rate of the primary chondrocytes were detected. During the passage cultivation in vitro, the changes of the chondrocytes shape and growth were observed, the changes of the collagen type I and II and aggrecan in the extracellular matrix were investigated and detected.

RESULTS(1) The extracellular matrix of articular cartilage was completely dissolved by the three-step enzymatic digestion, and the chondrocytes were completely isolated from the solid matrix. The number of the harvested chondrocytes from every gram of wet cartilage was 50.3 x 10(6) on average, and their viability rate was 98.8% on average. (2) The primary and first passage chondrocytes had triangle or multi-angle shape, and became elliptic shape at the growing confluence with the positive immunohistochemical stain of collagen type II and the strong heterochromia to toluidine blue. The content of sulfate glycosaminoglycans (GAG) in the extracellular matrix of the primary passage cells was (92 +/- 10) microg/cm(2). The chondrocytes after the third passaging gradually became spindle shape with the negative stain of collagen type II and the weak heterochromia to toluidine blue. The content of sulfate GAG of the fourth passage cells was (48 +/- 12) microg/cm(2).

CONCLUSION(1) The method of three-step enzymatic digestion can make the extracellular matrix of articular cartilage completely degraded, and has advantages in the high efficiency of harvesting primary chondrocytes, high cellular viability rate and simple manipulation. (2) The primary and first passage chondrocytes have fine biological activity, and the chondrocytes after the third passaging have lost their special biological activity.

Animals ; Cartilage, Articular ; cytology ; drug effects ; Cell Culture Techniques ; Cell Separation ; methods ; Cells, Cultured ; Chondrocytes ; cytology ; Collagenases ; pharmacology ; Female ; Hyaluronoglucosaminidase ; pharmacology ; Male ; Rabbits ; Trypsin ; pharmacology

BACKGROUNDAn ideal aneurysm model of cerebral aneurysm is of great importance for studying the pathogenesis of the lesion and testing new techniques for diagnosis and treatment. Several models have been created in rabbits and are now widely used in experimental studies; however, every model has certain intrinsic limitations. Here we report the development of a novel saccular aneurysm model in rabbits using an arterial pouch that is subject to in vitro pre-digestion with combined elastase and collagenase.

METHODSA segment of right common carotid artery (CCA) was dissected out and treated with elastase (60 U/ml, 20 min) followed by type I collagenase (1 mg/ml, 15 min) in vitro. The graft was anastomosed to an arterial arch built with the left CCA and the remaining right CCA, while the other end of the graft was ligated. The dimension and tissue structure of the pouch were analysed immediately, 2 or 8 weeks after operation.

FINDINGSTen terminal aneurysms were produced. The gross morphology of the aneurysm resembles the human cerebral terminal aneurysms. We have observed the following pathological changes: (1) growth of the aneurysm (mean diameter increased from (2.0+/-0.1) to (3.2+/-0.3) mm at 2 weeks, P<0.001, n=7-10); (2) thinning of the aneurysmal wall (the mean wall thickness decreased to 44% at 2 weeks), which was accompanied by significant losses of elastic fibres, collagen and the cellular component; and (3) spontaneous rupture (3 out of 9, one aneurysm ruptured 24 h after operation with the other two at 2 and 4 weeks respectively).

CONCLUSIONThis rabbit arterial pouch model mimics human cerebral aneurysms in relation to morphology and histology. In particular, this model exhibited an increased tendency of spontaneous rupture.

Animals ; Carotid Artery, Common ; drug effects ; pathology ; physiopathology ; Collagenases ; Disease Models, Animal ; In Vitro Techniques ; Intracranial Aneurysm ; chemically induced ; pathology ; physiopathology ; Pancreatic Elastase ; Rabbits

The equilibrium between deposition and degradation of extracellular matrix(ECM) is essential to normal tissue development and repair of wound or inflammatory responses. It has recently become apparent that several cytokines and growth factors are capable of modulating fibroblast proliferation and biosynthetic activity. To understand the role of these factors in connective tissue regulation, we examined the effect of interferon-gamma (IFN-gamma) on stromelysin-1 gene expression in cultured human dermal fibroblasts. The steady-state levels of stromelysin-1 mRNA were increased in IFN-gamma treated cultured dermal fibroblasts. In the CAT assay, the stromelysin-1 promoter activity was increased 2.8-fold compared with untreated control. Therefore IFN-gamma stimulates the stromelysin-1 promoter activity, resulting in transcriptional enhancement of gene expression. Transforming growth factor-beta (TGF-beta) showed the antagonistic action to the effects of IFN-gamma in cultured dermal fibroblasts. Furthermore, gel mobility shift assays demonstrated enhanced AP-1 binding activities in nuclear extracts from cells incubated with IFN-gamma. These data suggest that IFN-gamma is an up-regulator and TGF-beta is a down regulator on the stromelysin-1 gene expression, respectively, and the AP-1 binding site may be necessary for gene response.
Cell Nucleus ; Cells, Cultured ; Chloramphenicol O-Acetyltransferase/metabolism ; Chloramphenicol O-Acetyltransferase/genetics ; Collagenases/genetics ; Collagenases/drug effects ; Fibroblasts/metabolism ; Fibroblasts/drug effects* ; Gene Expression Regulation/drug effects ; Human ; Interferon Type II/pharmacology* ; Promoter Regions (Genetics) ; Recombinant Proteins/metabolism ; Recombinant Proteins/genetics ; Skin/cytology* ; Stromelysin 1/metabolism* ; Stromelysin 1/genetics* ; Stromelysin 1/drug effects ; Transcription Factor AP-1/metabolism ; Transcription, Genetic ; Transforming Growth Factor beta/pharmacology ; Up-Regulation (Physiology)

To investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody Fab' fragment and lidamycin (LDM) prepared with different linkers. The immunoconjugates were prepared by linking Fab' to lysine-69 of LDM apoprotein by SPDP, LCSPDP, SMBS or SSMPB as the intermediate drug linkers. Immunoreactivities of the conjugates were determined by ELISA. The cytotoxicities of the conjugates were examined by clonogenic assay. In vivo antitumor effects of the conjugates were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the conjugates retained part of the immunoreactivity of 3G11 against the antigen. The cytotoxicities of the Fab'-SMBS-LDM and Fab'-SSMPB-LDM to HT-1080 cells were significantly potent, compared with Fab'-SPDP-LDM, Fab'-LCSPDP-LDM and free LDM. In animal models at the same condition, free LDM, Fab'-SPDP-LDM and Fab'-LCSPDP-LDM inhibited the growth of HT-1080 tumor by 70.9%, 74.8% and 72.3%, while Fab'-SMBS-LDM and Fab'-SSMPB-LDM reached 78.0% and 87.7%, respectively. The median survival time of the mice treated with free LDM, Fab'-SPDP-LDM and Fab'-LCSPDP-LDM were prolonged by 71.9%, 82.2% and 107.5%, respectively, compared with that of untreated group. Whereas, the median survival time of Fab'-SMBS-LDM and Fab'-SSMPB-LDM were prolonged by 145.2% and 165.8%, respectively, indicating that Fab'-SSMPB-LDM was more effective than Fab'-SMBS-LDM in tumor suppression and life span prolongation. Fab'-SSMPB-LDM has more marked selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.
Aminoglycosides ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antibodies, Monoclonal ; immunology ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; drug effects ; Collagenases ; immunology ; Enediynes ; pharmacology ; Fibrosarcoma ; pathology ; Humans ; Immunoconjugates ; pharmacology ; Immunoglobulin Fab Fragments ; immunology ; Matrix Metalloproteinase Inhibitors ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Tumor Burden ; drug effects

OBJECTIVETo investigate the effect of epigallocatechin-3-gallate (EGCG) on biomodification of demineralized dentine substrate, in its permeability, hydrophobicity, and inhibition ability to collagen enzymatic degradation.

METHODSThe dentine substrates were treated with simulated pulpal pressure created by mixtures of 0.02%, 0.1% EGCG/bovine serum albumin (BSA) in acidic environment (pH4.4) for 48 h. A fluid-transport model was used to measure the fluid permeability through demineralized dentine substrate. Positive replicas of dentine substrate were fabricated before and after being subjected to acidic environment for scanning electron microscope (SEM) examination. The blank group contained no EGCG and the positive group were treated with Gluma desensitizer. Static contact angle measurements on demineralized dentin and 0.1% EGCG primed dentin were performed by contact angle analyzer. The priming time were 60 s, 120 s, 0.5 h, 1 h. Dentine specimens bonded with Adper single bond 2 were subjected to 100 mg/L collagenase and observed under SEM. Resin-bonded specimens (with 0.02%, 0.1%, 0.5% EGCG priming, or without EGCG priming) were created for micro-tensile bond strength evaluation (MTBS). Resin-bonded specimens after thermol cycling were created for MTBS evaluation.

RESULTSThe fluid permeability in the blank control group increased ([151.3±22.3]%), the fluid permeability in 0.1% EGCG/BSA group decreased ([23.7±6.3]%). Compared to the blank control group, the contact angle of 120 s, 0.5 h, 1 h groups increased by 31.0%, 53.5%, 57.8% in deep dentin and 37.4%, 59.3%, 62.4% in shallow dentin. The SEM examination showed that 0.1% and 0.5% EGCG priming for 120 s significantly increased dentin collagen's resistance to collagenase. The immediate MTBS of 0.1% and 0.5% EGCG groups were (29.4±4.8) and (19.8± 4.9) MPa. After thermol cycling, the MTBS of 0.1% and 0.5% EGCG groups were (19.9±5.1) and (15.3± 6.3) MPa.

CONCLUSIONSUnder acidic environment (pH4.4), the 0.1% EGCG can reduce dentine permeability under acidic environment. The 0.1% EGCG can increase hydrophobicity of dentin substrate, and strengthen dentin substrate's resistance to collagenase hydrolysis, thus increased the resin-dentin bonding durability.

Acid Etching, Dental ; Catechin ; analogs & derivatives ; pharmacology ; Collagen ; chemistry ; drug effects ; Collagenases ; pharmacology ; Composite Resins ; Dental Bonding ; Dental Cements ; Dental Pulp ; Dentin ; chemistry ; drug effects ; Dentin Permeability ; drug effects ; Dentin-Bonding Agents ; Glutaral ; pharmacology ; Hydrogen-Ion Concentration ; Hydrolysis ; Methacrylates ; pharmacology ; Microscopy, Electron, Scanning ; Pressure ; Resin Cements ; Serum Albumin, Bovine ; pharmacology ; Tensile Strength ; Time Factors

This study is to investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody 3G11 and lidamycin (LDM) prepared by different methods. The immunoconjugates were prepared by linking 2-iminothiolane modified 3G11 to lysine-69 of LDM apoprotein by SPDP and SMBS as the intermediate drug linker. Immunoreactivity of the conjugates was determined by ELISA. The cytotoxicity of the conjugates was examined by clonogenic assay. Antitumor effects of the conjugates in vivo were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the immunoconjugates retained the immunoreactivity of 3G11 against type IV collagenase. The cytotoxicity of the 3G11-SMBS-LDM to HT-1080 cells was significantly more potent than that of free LDM and 3G11-SPDP-LDM. In animal model at the same condition, free LDM inhibited the growth of HT-1080 tumor by 71.2%, while 3G11-SPDP-LDM and 3Gl1-SMBS-LDM reached 77.1% and 86.1%, respectively. The median survival time of the mice treated with free LDM was prolonged by 71.9% compared with that of untreated group. Whereas, the median survival time of 3G11-SPDP-LDM and 3G11-SMBS-LDM was prolonged by 125.3% and 163.7%, respectively, indicating that 3G11-SMBS-LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy. LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.
Aminoglycosides ; therapeutic use ; Animals ; Antibiotics, Antineoplastic ; therapeutic use ; Antibodies, Monoclonal ; immunology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Collagenases ; immunology ; Enediynes ; therapeutic use ; Fibrosarcoma ; pathology ; therapy ; Humans ; Immunoconjugates ; therapeutic use ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Tumor Burden ; drug effects

The neuroprotective effect of propofol against intracerebral hemorrhage (ICH) in rats was investigated. ICH was induced in rats by infusion of collagenase (Type VII) 0.5 U (1 U x microL(-1)) into the left caudate nucleus. Three doses of propofol were given intraperitoneally (i.p.) 10 min before collagenase infusion. Effects of propofol on neurological behavioral scores, brain water content (BWC), activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in brain tissue, expression level of caspase-3 were studied. In propofol groups (30 and 100 mg x kg(-1)), the neurological behavioral score, BWC and the content of MDA were significantly lower than those in ICH group (P < 0.05, P < 0.01), whereas the activity of SOD was higher than that in ICH group (P < 0.05). Meanwhile, propofol (15, 30, and 100 mg x kg(-1)) inhibited caspase-3 expression in dose-dependent manner (r = 0.877). Brain damages caused by ICH in rats can be alleviated by propofol, which mechanism might be attributed to its antioxidant activity.
Animals ; Behavior, Animal ; drug effects ; Brain ; metabolism ; Brain Edema ; drug therapy ; etiology ; Caspase 3 ; metabolism ; Cerebral Hemorrhage ; chemically induced ; complications ; metabolism ; physiopathology ; Collagenases ; Male ; Malondialdehyde ; metabolism ; Neuroprotective Agents ; pharmacology ; therapeutic use ; Propofol ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

This study is to investigate if batroxobin has the protective effect against nerve injury caused by cerebral hemorrhage in rats and its possible mechanism. Animals were divided into sham group, model group, batroxobin 4, 8, and 16 BU x kg(-1) groups and nimodipine positive control group. On the brain stereotaxic apparatus, the rat intracerebral hemorrhage model was established by injecting collagenase with microinjector into the brain caudate nucleus which was located according to the brain stereotaxic atlas. Neuroethology of the rats was estimated. The brain tissue pathomorphology was observed with electron microscope. The water content of the brain tissue was quantitated with wet/dry weight measurement. SOD and MDA were determined according to the kit procedure, and free Ca2+ concentration in neurocyte was measured by fluorospectrophotometer. As shown in results, batroxobin could improve neuroethology scale of the rats, relieve histiocyte edema and bleeding degree. The water content of the brain tissue, MDA and free Ca2+ concentration were reduced and SOD activity was raised in batroxobin treatment groups. Therefore, it is possible that batroxobin has some protective effect against nerve injury caused by cerebral hemorrhage in rats, and its mechanism maybe relate to lessening brain edema, reducing MDA content, raising SOD activity, and inhibiting calcium overload.
Animals ; Batroxobin ; pharmacology ; therapeutic use ; Behavior, Animal ; drug effects ; Brain ; pathology ; Brain Edema ; drug therapy ; etiology ; Calcium ; metabolism ; Cerebral Hemorrhage ; chemically induced ; complications ; metabolism ; physiopathology ; Collagenases ; Female ; Male ; Malondialdehyde ; metabolism ; Neurons ; metabolism ; Neuroprotective Agents ; pharmacology ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism