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Objective To explore the effect of bacterial endotoxin lipopolysaccharide (LPS) on wound healing by observation of change in expression of matrix metalloproteinases (MMPs) and specific tissue inhibitors of metalloproteinases (TIMPs) in fibroblasts after the challenge of lipopolysaccharide. Methods Human dermal fibroblasts were cultured in RPMI medium containing 10% fetal calf serum (FCS). At cellular confluence the medium was changed, and the cells were cultured under serum free conditions with 0.1% bovine serum albumin (BSA) in the presence of LPS (0, 10?g/ml, 100?g/ml and 1000?g/ml). All cultures were maintained at 37℃ in a humidified atmosphere of 5% CO_2 in air. After 48-hour treatment, the medium was collected for measurement of MMPs by fluorescence labeled gelatin zymography and for measurement of TIMPs by fluorescence labeled reverse gelatin zymography. The expressions of MMPs and TIMPs were quantitatively analyzed with the band's area and density from the gel image using the Phoretix software. Results LPS up-regulated the expression of both MMPs and TIMPs in fibroblasts, and expression of TIMPs was higher than that of MMPs. Compared with base level, MMPs respectively increased 1.3, 1.7 and 1.6 times, TIMP-1 respectively increased 3, 4.5 and 3.6 times, TIMP-2 respectively increased 3, 6 and 5.6 times. Conclusion It seems that LPS showed no damaging effect on fibroblasts in regulation of matrix accumulation in wound healing.
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Objective To investigate and evaluate the changes of PYK2 expression in hippocampal neurons and microglial cells in Kainate acid-induced status epilepticus. Methods Kainate acid-induced status epilepticus was established in rats, and by using immunostaining, changes of PYK2 expression in hippocampal neurons and microglial cells was investigated. Results Expression of PYK2 in hippocampal pyramidal neurons was markedly decreased after kainate acid-induced status epilepticus. However, 24h after the epileptic onset, a pronounced up-regulation of PYK2 and phosphor-PYK2 immunoreactivities were evident in amoeboid microglial cells. The upregulation of PYK2 and phosphor-PYK2 was in accordance with the morphological changes of the activated microglial cells. Conclusion PYK2 was activated in microglial cells after seizure. Furthermore, the activation of PYK2 in microglial cells after seizure might be related to the morphological and behavioral changes of microglial cells after activation.
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Objective To explore the potential mechanisms of therapeutic effect of propolis in the treatment of diabetic wound. Methods Human fibroblasts(Fb) and monocytic THP-1 cells were routinely cultured at 37℃ in an environment of 95% air and 5% CO2 in RPMI medium containing 11mmol/l glucose, 15mmol/l HEPES and 5% fetal calf serum. To a portion of culturing THP-1 monocytes phorbol myristate actetate (PMA; 2?10 -7mol/L) was added. After 24 hours when approximately 75% of the monocytes became macrophage which attached to the plate. Propolis was added to a culture system of human fibroblasts(Fb), THP-1 monocyte and THP-1 monocyte-derived macrophage(Mac) with different concentration of propolis(10, 50, 100, 200?g/ml) and different glucose(5mmol/L; 25mmol/L), For each experiment the cells were cultured for 48 hours in the presence of Propolis. Then the media were collected and the protein expression of matrix metalloproternase-9 (MMP-9) were determined by gelatin zymography using a MDPF- labeled gelatin as substrate. The media were loaded onto an 8% SDS PAGE gel containing MDPF labeled gelatin (1%) after electrophoresis, the gels were placed on a long wave ultra violet light box and photographed. The area degraded by the MMPs was observed as dark bands on a light background. The MMPs were quantified by measurement of the band area using the Phoretix_1D_Advanced Digital Science Electrophoresis Documentation and Analysis System. Results the high glucose has a tendency to increase the expression of MMP-9, The propolis can decreased the expression of MMP-9, and has a dependent on doses tendency. F test P
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Objective To investigate and evaluate the changes of phospho PYK2 and phospho p38MAPK expression in neurons after focal cerebral ischemia. Methods Focal ischemia reperfusion model was established in rats, and by using immunostaining, the changes of phospho PYK2 and phospho p38MAPK expression in neurons was observed. Results Faint phospho PYK2 and phospho p38MAPK immunoreactivity were revealed in normal cortical neurons. Fifteen minutes after the ischemia onset, a pronounced upregulation of phospho PYK2 and phospho p38MAPK immunoreactivities were evident in these ischemia attacked neurons. The immunoreactivities of phospho PYK2 and phospho p38MAPK reached its peak at 30min after ischemia, and decreased 60min after ischemia. Conclusion Cerebral ischemia was able to induce neuronal PYK2 phosphorylation. The activation of PYK2 might link ischemia attack to the p38MAPK signaling pathway to initiate the neuronal response to the stress stimuli