OBJECTIVE: To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. METHODS: Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 μg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. RESULTS: Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury ( < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury ( < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury ( < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma ( < 0.05) and increased further at 2 days ( < 0.01); the water content of the brain did not change significantly at 2 h ( > 0.05) but increased significantly 2 d after the injury ( < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma ( < 0.01). CONCLUSIONS Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.
Animals ; Brain Edema ; drug therapy ; etiology ; Brain Injuries, Traumatic ; complications ; drug therapy ; Gene Expression Regulation, Enzymologic ; drug effects ; Indazoles ; pharmacology ; therapeutic use ; MAP Kinase Signaling System ; drug effects ; Matrix Metalloproteinase 9 ; genetics ; Piperazines ; pharmacology ; therapeutic use ; Protein Kinase Inhibitors ; pharmacology ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The objective of this study was to assess the angiogenic potential expressed as a quotient of vascular endothelial growth factor A (VEGF-A), as an indicator of proangiogenic activity, and the circulating receptors (soluble VEGF receptor protein R1 (sVEGFR-1) and sVEGFR-2), as indicators of the effect of angiogenic inhibition, depending on the concentrations of matrix metalloproteinase 2 (MMP-2) and MMP-9 and their tissue inhibitor 1 (TIMP-1) and TIMP-2 in the plasma of patients with lower extremity artery disease (LEAD). These blood parameters in patients with intermittent claudication (IC) and critical limb ischemia (CLI) were compared for select clinical and biochemical features. Stimulation of angiogenesis in the plasma of individuals with LEAD was evident as indicated by the significant increase in VEGF-A concentration along with reduced inhibition depending on circulating receptors sVEGFR-1 and sVEGFR-2. Critical ischemia was associated with higher VEGF-A, MMP-9, TIMP-1, and TIMP-2 concentrations than in the case of IC.
Aged ; Angiogenesis Inhibitors/pharmacology* ; Female ; Gene Expression Regulation ; Humans ; Intermittent Claudication/drug therapy* ; Ischemia/drug therapy* ; Lower Extremity/blood supply* ; Male ; Matrix Metalloproteinase 9/blood* ; Middle Aged ; Neovascularization, Pathologic ; Tissue Inhibitor of Metalloproteinase-1/blood* ; Tissue Inhibitor of Metalloproteinase-2/blood* ; Vascular Endothelial Growth Factor A/blood* ; Vascular Endothelial Growth Factor Receptor-1/blood* ; Vascular Endothelial Growth Factor Receptor-2/blood*

To investigate effects of verapamil on primary cultured human urethral scar fibroblasts (USFs) and to provide basis for protecting the formation of urethra scar.
 Methods: The cell proliferation was evaluated with the cell counting kit (CCK)-8 method after USFs were incubated various verapamil concentrations (50, 100, 150, 200, or 250 μmol/L) or solvent for 12, 24, or 48 h. The protein level of matrix metalloproteinase (MMP) was evaluated with ELISA after cells were incubated with verapamil (100 μmol/L) or solvent (control cells) for 24 h.
 Results: The proliferation of USFs was obviously suppressed after verapamil treatment, which was in a dose-dependent and time-dependent manner. Meanwhile, the protein levels of MMP-2 and MMP-9 in the verapamil treatment group increased obviously compared with those of the control groups (P<0.05).
 Conclusion: Calcium channel blockers may prevent the excessive formation of urethra scar by inhibiting the proliferation of urethral scar fibroblasts and enhancing the activity of MMP.
Calcium Channel Blockers ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; prevention & control ; Fibroblasts ; drug effects ; Humans ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Matrix Metalloproteinase Inhibitors ; pharmacology ; Up-Regulation ; drug effects ; Urethra ; cytology ; pathology ; Verapamil ; pharmacology

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo study the effect and mechanism of bevacizumab on proliferation and invasion of human lung cancer A549 cells.

METHODSA549 cells were treated with bevacizumab. Proliferation and invasion of the bevacizumab-treated A549 cells were detected using cell counting kit CCK-8 and Transwell assay, respectively. The expression of the mRNA and protein of MMP-2, MMP-9 and c-Met were detected by real-time PCR and Western blotting, respectively.

RESULTSProliferation activity was inhibited at the concentration of 10 µg/ml and promoted at the concentration of 100 µg/ml bevacizumab. Bevacizumab in the concentration of 50 µg/ml had a stronger inhibitory effect on the invasion of A549 cells (16 406.19 ± 5 674.23 penetrated cells) than that of control group (36 108.68 6 263.83, P<0.05). The real-time PCR showed that bevacizumab had a stronger inhibitory effect on the expression of MMP-2 and MMP-9 mRNA at the concentration of 50 µg/ml and on the expression c-Met mRNA at the concentration of 10 µg/ml bevacizumabin the A549 cells. However bevacizumab at the concentration of 100 µg/ml showed a promoting effect on the expression of MMP-2, MMP-9 and c-Met mRNA (1.82 ± 0.31, 1.60 ± 0.25, 2.63 ± 0.48), significantly higher than that of the control group (1.00 ± 0.19, 1.00 ± 0.23, 1.00 ± 0.22, P<0.05). The expression of MMP-2, MMP-9 and c-Met mRNA and protein was inhibited by 10 µg/ml bevacizumab in a time-dependent manner. The Western blot assay showed that bevacizumab had a bi-directional effect on the expression of MMP-2 and c-Met proteins in the A549 cells: a promoting effect at 100 µg/ml and inhibitory effect on the expression of MMP-2 at 50 µg/ml bevacizumab, and inhibitory effect on the expression of c-Met protein at 10 µg/ml bevacizumab.

CONCLUSIONSOur findings indicate that in a certain range of concentrations, bevacizumab has prominent inhibitory effect on the proliferation and invasion of A549 cells. However,over the concentration of 100 µg/ml, bevacizumab shows a weakening anti-invasion effect, even has a promoting effect on cell proliferation. This phenomenon may be related to the inhibiting effect on the expression of MMP-2 and c-Met proteins in a non-concentration-dependent manner by bevacizumab.

Angiogenesis Inhibitors ; pharmacology ; Bevacizumab ; pharmacology ; Blotting, Western ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-met ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo explore the effect of the Hsp90 inhibitor anacardic acid on cell proliferation, invasion and migration of breast cancer MDA-MB-231 cells.

METHODSThe inhibitory effect of anacardic acid on Hsp90 was assessed with in vitro ATPase inhibition assay and ATP-sepharose binding assay. MTT assay was used to detect the growth inhibition induced by anacardic acid in MDA-MB-231 cells. Transwell assays were used to evaluate MDA-MB-231 cell invasion and migration. Western blotting was performed to assess the effect of anacardic acid in triggering the degradation of MMP-9, TIMP-1, Hsp90, and Hsp70.

RESULTSAnacardic acid exhibited a modest activity of ATPase inhibition with an IC50 value of 82.5 µmol/L. Anacardic acid significantly suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner (IC50 value of 29.3 µmol/L). Treatment with 12.5, 25, and 50 µmol/L anacardic acid for 36 h caused inhibition of cell invasion by 23.6%, 56.6%, and 67.0% in MDA-MB-231 cells, respectively (P<0.05), and anacardic acid treatment for 24 h inhibited the cell migration by 30.0%, 45.5%, and 77.5%, respectively (P<0.05). Anacardic acid dose-dependently induced MMP-9 degradation, but did not obviously affect Hsp90 or Hsp70 expressions.

CONCLUSIONAnacardic acid can significantly inhibit the proliferation, invasion, and migration of MDA-MB-231 cells, the mechanism of which may involve the inhibition of Hsp90 ATPse activity and down-regulation of MMP-9 expression.

Anacardic Acids ; pharmacology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; drug effects ; Cell Movement ; Cell Proliferation ; Down-Regulation ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; metabolism ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

This study investigated effects of Ginsenoside Ro (Ro) on interleukin-1β (IL-1β)-induced apoptosis and inflammation in rat chondrocytes. The rat chondrocytes were co-treated with IL-1β (10 ng·kg(-1)) and Ro (50, 100 and 200 μmol·L(-1)) for 48 h. Chondrocytes viability was detected by the MTT assay and Annexin V-FITC/PI dual staining assay. Caspase 3 activity was measured by using caspase 3 colorimetric assay kit. Apoptosis related proteins Bax, Bad, Bcl-xL, PCNA, p53 and phospho-p53, along with inflammation related protein MMP 3, MMP 9 and COX-2, and the expression of phospho-NF-κB p65 were assayed by western blotting analyses. Ro could improve IL-1β-induced chondrocytes viability. Ro could suppress IL-1β-induced apoptosis by inhibiting levels of Bax and Bad, decreasing p53 phosphorylation and promoting the expression of Bcl-xL and PCNA. Ro inhibited caspase 3 activity. IL-1β-induced inflammation and matrix degration were also alleviated by Ro with down-regulating the expression of MMP 3, MMP 9 and COX-2. Moreover, Ro inhibited NF-κB p65 phosphorylation induced by IL-1β. In conclusion, these results suggested Ro exerted anti-apoptosis and anti-inflammation in IL-1β-induced rat chondrocytes, which might be related to NF-κB signal pathway. Therefore, we propose that Ro might be a potential novel drug for the treatment of osteoarthritis.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; drug effects ; metabolism ; Caspase 3 ; metabolism ; Cell Survival ; drug effects ; Chondrocytes ; cytology ; drug effects ; Cyclooxygenase 2 ; drug effects ; metabolism ; Down-Regulation ; drug effects ; Drug Evaluation, Preclinical ; Ginsenosides ; pharmacology ; Inflammation ; chemically induced ; drug therapy ; Interleukin-1beta ; antagonists & inhibitors ; pharmacology ; Matrix Metalloproteinase 3 ; drug effects ; metabolism ; Matrix Metalloproteinase 9 ; drug effects ; NF-kappa B ; antagonists & inhibitors ; drug effects ; metabolism ; Phosphorylation ; drug effects ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects

OBJECTIVE: To study the effect of quercetin, an inhibitor of matrix metalloproteinases 9 (MMP-9), on the growth and metastasis of lung cancer cells and the underlying mechanisms.
 METHODS: We evaluated the inhibitory effect and the inhibitory kinetics of quercetin on MMP-9 by ELISA and enzyme inhibition kinetics, and the inhibitory effect of quercetin on the growth of lung cancer cell (A549) by MTT. The effect of quercetin on levels of MMP-9 (mRNA and protein) and TGF-β1 (protein) in A549 were measured by RT-PCR and Western blot, respectively. The synergistic inhibition effect of quercetin plus TIMP-1 on the growth of lung cancer cell A549 was discussed.
 RESULTS: Quercetin induced the apoptosis of A549. It was a reversible competitive inhibitor of MMP-9 (half inhibition rate IC50 of 5.25 μmol/L, inhibition constant Ki was 2.18 μmol/L). With the increase in quercetin concentration, the levels of MMP-9 (mRNA and protein) and TGF-β1 (protein) were decreased, and the number of tumor cells on wear filter membrane was reduced. The combination of quercetin (at low concentrations) with TIMP-1 showed synergistic inhibitory effect on the growth of A549 cells. 
 CONCLUSION Quercetin is a competitive inhibitor of MMP-9 and could downregulate the expression of MMP-9 and TGF-β1, which plays an important role in A549 apoptosis.
Apoptosis ; Cell Line, Tumor ; drug effects ; Down-Regulation ; Humans ; Lung Neoplasms ; pathology ; Matrix Metalloproteinase 9 ; metabolism ; Matrix Metalloproteinase Inhibitors ; pharmacology ; Neoplasm Metastasis ; Quercetin ; pharmacology ; RNA, Messenger ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

Our previous studies showed that biomodification of demineralized dentin collagen with proanthocyanidin (PA) for a clinically practical duration improves the mechanical properties of the dentin matrix and the immediate resin-dentin bond strength. The present study sought to evaluate the ability of PA biomodification to reduce collagenase-induced biodegradation of demineralized dentin matrix and dentin/adhesive interfaces in a clinically relevant manner. The effects of collagenolytic and gelatinolytic activity on PA-biomodified demineralized dentin matrix were analysed by hydroxyproline assay and gelatin zymography. Then, resin-/dentin-bonded specimens were prepared and challenged with bacterial collagenases. Dentin treated with 2% chlorhexidine and untreated dentin were used as a positive and negative control, respectively. Collagen biodegradation, the microtensile bond strengths of bonded specimens and the micromorphologies of the fractured interfaces were assessed. The results revealed that both collagenolytic and gelatinolytic activity on demineralized dentin were notably inhibited in the PA-biomodified groups, irrespective of PA concentration and biomodification duration. When challenged with exogenous collagenases, PA-biomodified bonded specimens exhibited significantly less biodegradation and maintained higher bond strengths than the untreated control. These results suggest that PA biomodification was effective at inhibiting proteolytic activity on demineralized dentin matrix and at stabilizing the adhesive/dentin interface against enzymatic degradation, is a new concept that has the potential to improve bonding durability.
Chlorhexidine ; chemistry ; pharmacology ; Collagenases ; pharmacology ; Dental Bonding ; Dental Cements ; chemistry ; Dental Stress Analysis ; instrumentation ; Dentin ; drug effects ; ultrastructure ; Dentin-Bonding Agents ; chemistry ; Gelatinases ; pharmacology ; Humans ; Hydroxyproline ; analysis ; Matrix Metalloproteinase 8 ; pharmacology ; Matrix Metalloproteinase Inhibitors ; chemistry ; pharmacology ; Proanthocyanidins ; chemistry ; pharmacology ; Stress, Mechanical ; Surface Properties ; Tensile Strength ; Tooth Demineralization ; pathology ; physiopathology

In this study, the human umbilical vein endothelial cell model was used to study the regulating effect of lipophilic components in Salvia miltiorrhiza on angiogenesis, and explore its possible mechanism. The cell model was established to determine the effect of lipophilic components in S. miltiorrhiza on the proliferative activity and migration capacity of endothelial cells. Then the realtime fluorescence quantification PCR technology was applied to detect the changes in the gene expressions of angiogenesis-related cytokines VEGF-A, VEGF-C and MMP-9. The results showed that 5 mg x L(-1) lipophilic components in S. miltiorrhiza could inhibit the proliferation and migration of endothelial cells, and reduce the expression of VEGF-A and MMP-9 genes. It indicated that lipophilic components in S. miltiorrhiza may inhibit the proliferation and migration of endothelial cells by inhibiting the expression of VEGF-A and MMP-9 genes, so as to show the inhibitory effect on angiogenesis.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; Vascular Endothelial Growth Factor A ; genetics ; metabolism