Anti-Inflammatory Effects of Rhamnetin on Bradykinin-Induced Matrix Metalloproteinase-9 Expression and Cell Migration in Rat Brain Astrocytes

Bradykinin (BK) has been shown to induce matrix metalloproteinase (MMP)-9 expression and participate in neuroinflammation. The BK/MMP-9 axis can be a target for managing neuroinflammation. Our previous reports have indicated that reactive oxygen species (ROS)-mediated nuclear factor-kappaB (NF-κB) activity is involved in BK-induced MMP-9 expression in rat brain astrocytes (RBA-1). Rhamnetin (RNT), a flavonoid compound, possesses antioxidant and anti-inflammatory effects. Thus, we proposed RNT could attenuate BK-induced response in RBA-1. This study aims to approach mechanisms underlying RNT regulating BK-stimulated MMP-9 expression, especially ROS and NF-κB. We used pharmacological inhibitors and siRNAs to dissect molecular mechanisms. Western blotting and gelatin zymography were used to evaluate protein and MMP-9 expression. Real-time PCR was used for gene expression. Wound healing assay was applied for cell migration. 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCF-DA) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) were used for ROS generation and NOX activity, respectively. Promoter luciferase assay and chromatin immunoprecipitation (ChIP) assay were applied to detect gene transcription. Our results showed that RNT inhibits BK-induced MMP-9 protein and mRNA expression, promoter activity, and cell migration in RBA-1 cells. Besides, the levels of phospho-PKCδ, NOX activity, ROS, phospho-ERK1/2, phospho-p65, and NF-κB p65 binding to MMP-9 promoter were attenuated by RNT. In summary, RNT attenuates BK-enhanced MMP-9 upregulation through inhibiting PKCδ/NOX/ROS/ERK1/2-dependent NF-κB activity in RBA-1.

Substrate stiffness modulates integrin α5 expression and ECM-associated gene expression in fibroblasts

Biomaterials, like polydimethylsiloxane (PDMS), are soft, biocompatible, and tuneable, which makes them useful to delineate specific substrate factors that regulate the complex landscape of cell-substrate interactions. We used a commercial formulation of PDMS to fabricate substrates with moduli 40 kPa, 300 kPa, and 1.5 MPa, and cultured HMF3S fibroblasts on them. Gene expression analysis was performed by RT-PCR and Western blotting. Cellular and nuclear morphologies were analyzed using confocal imaging, and MMP-2 and MMP-9 activities were determined with gelatin zymography. Results, comparing mechanotransduction on PDMS substrates with control petridishes, show that substrate stiffness modulates cell morphologies and proliferations. Cell nuclei were rounded on compliant substrates and correlated with increased tubulin expression. Proliferations were higher on stiffer substrates with cell cycle arrest on softer substrates. Integrin alpha5 expression decreased on lower stiffness substrates, and correlated with inefficient TGF-beta; activation. Changes to the activated state of the fibroblast on higher stiffness substrates were linked to altered TGF-beta; secretion. Collagen I, collagen III, and MMP-2 expression levels were lower on compliant PDMS substrates as compared to stiffer ones; there was little MMP-9 activity on substrates. These results demonstrate critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts which is highly relevant in diseases like tissue fibrosis.

Lidocaine Alleviates Sepsis-Induced Acute Lung Injury in Mice by Suppressing Tissue Factor and Matrix Metalloproteinase-2/9

Acute lung injury (ALI) is one of the fatal symptoms of sepsis. However, there were no effective clinical treatments. TF accumulation-induced fibrin deposit formations and coagulation abnormalities in pulmonary vessels contribute to the lethality of ALI. Suppressor of cytokine signaling 3 (SOCS3) acts as an endogenous negative regulator of the TLR4/TF pathway. We hypothesized that inducing SOCS3 expression using lidocaine to suppress the TLR4/TF pathway may alleviate ALI. Hematoxylin and eosin (H&E), B-mode ultrasound, and flow cytometry were used to measure the pathological damage of mice. Gelatin zymography was used to measure matrix metalloproteinase-2/9 (MMP-2/9) activities. Western blot was used to assay the expression of protein levels. Here, we show that lidocaine could increase the survival rate of ALI mice and ameliorate the lung injury of ALI mice including reducing the edema, neutrophil infiltration, and pulmonary thrombosis formation and increasing blood flow velocity. Moreover, in vitro and in vivo, lidocaine could increase the expression of p-AMPK and SOCS3 and subsequently decrease the expression of p-ASK1, p-p38, TF, and the activity of MMP-2/9. Taken together, our study demonstrated that lidocaine could inhibit the TLR4/ASK1/TF pathway to alleviate ALI via activating AMPK-SOCS3 axis.

Compliance of the fish outflow tract is altered by thermal acclimation through connective tissue remodelling

To protect the gill capillaries from high systolic pulse pressure, the fish heart contains a compliant non-contractile chamber called the bulbus arteriosus which is part of the outflow tract (OFT) which extends from the ventricle to the ventral aorta. Thermal acclimation alters the form and function of the fish atria and ventricle to ensure appropriate cardiac output at different temperatures, but its impact on the OFT is unknown. Here we used ex vivo pressure–volume curves to demonstrate remodelling of passive stiffness in the rainbow trout ( Oncorhynchus mykiss ) bulbus arteriosus following more than eight weeks of thermal acclimation to 5, 10 and 18°C. We then combined novel, non-biased Fourier transform infrared spectroscopy with classic histological staining to show that changes in compliance were achieved by changes in tissue collagen-to-elastin ratio. In situ gelatin zymography and SDS-PAGE zymography revealed that collagen remodelling was underpinned, at least in part, by changes in activity and abundance of collagen degrading matrix metalloproteinases. Collectively, we provide the first indication of bulbus arteriosus thermal remodelling in a fish and suggest this remodelling ensures optimal blood flow and blood pressure in the OFT during temperature change.

Posidonia oceanica (L.) Delile Dampens Cell Migration of Human Neuroblastoma Cells

Neuroblastoma (NB) is a common cancer in childhood, and lethal in its high-risk form, primarily because of its high metastatic potential. Targeting cancer cell migration, and thus preventing metastasis formation, is the rationale for more effective cancer therapy against NB. Previous studies have described the leaf extract from Posidonia oceanica marine plant (POE) as an antioxidant, anti-inflammatory agent and inhibitor of cancer cell migration. This study aims to examine the POE anti-migratory role in human SH-SY5Y neuroblastoma cells and the underlying mechanisms of action. Wound healing and gelatin zymography assays showed that POE at early times inhibits cell migration and reduces pro-MMP-2 release into culture medium. By monitoring expression level of key autophagy markers by Western blot assay, a correlation between POE-induced cell migration inhibition and autophagy activation was demonstrated. Cell morphology and immunofluorescence analyses showed that POE induces neurite formation and neuronal differentiation at later times. These results suggest POE might act against cell migration by triggering early nontoxic autophagy. The POE-induced cellular morphological change toward cell differentiation might contribute to prolonging the phytocomplex anti-migratory effect to later times. Overall, these results encourage future in vivo studies to test POE applicability in neuroblastoma treatment.

Staurosporine-induced cleavage of apoptosis-inducing factor in human fibrosarcoma cells is independent of matrix metalloproteinase-2

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS)-induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduced AIF proteolysis. However, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25-24 hr). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after 6 hr STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (~60 and 45 kDa) in the cytosol which were significantly increased at 6 hr. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.

Curcumin Analogs, PGV-1 and CCA-1.1 Exhibit Anti-migratory Effects and Suppress MMP9 Expression on WiDr Cells

BACKGROUND: Colon cancer is still a crucial concern in the development of chemotherapeutic drugs due to the drug resistance phenomenon and various side effects to patients. One of the newest compound that show anticancer activities against several cancer cells, Chemoprevention Curcumin Analog 1 (CCA-1.1), has increasingly been explored to overcome the limitation of conventional drugs.METHODS: We evaluated the anti-migratory effect of CCA-1.1 and Pentagamavunone-1 (PGV-1) by using WiDr colon cancer cells. The expression profiles of Tumor Protein 53 (TP53) and Matrix Metalloproteinase-9 (MMP9) in colon cancer were obtained from the UALCAN database. Survival outcomes of TP53 and MMP9 in colon cancer patients were analyzed using the Kaplan-Meier method. We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), scratch wound healing, and gelatin zymography assays to observe the cytotoxic effect, anti-migratory activity, and MMP9 expression, respectively, in CCA-1.1 or PGV-1-treated cells.RESULTS: Level of MMP9 was found significantly overexpressed in the primary tumor and metastasis nodal, while TP53 mutation sample types were observed and influenced the survival outcome in colon cancer patients. CCA-1.1 and PGV-1 exhibited strong cytotoxic activity after 24 and 48 h treatment against WiDr cells. The migration assay demonstrated that PGV-1 and CCA-1.1 at 1 mM inhibited cell migration up to 40% after 48 h in single and combination with doxorubicin. The MMP9 expression was significantly inhibited by 0.5 mM CCA-1.1.CONCLUSION: This study emphasizes that the anti-migratory effect of CCA-1.1 is better than PGV-1 via MMP9 suppression on WiDr. Thus, CCA-1.1 is prominent to be developed as an anti-metastatic agent.KEYWORDS: chemopreventive curcumin analog 1.1 (CCA-1.1), PGV-1, WiDr cells, anti-migration, MMP9

Matrix Metalloproteinase-9 is Involved in the Fibrotic Process in Denervated Muscles after Sciatic Nerve Trauma and Recovery

Fibrosis of the injured muscles is a problem of recovery from trauma and denervation. The aim of the work was to investigate the interconnection of matrix metalloproteinase-9 (ММР-9) activity in denervated muscles with fibrosis and to estimate its role in nerve restoration by the epineurial suture, fibrin-based glue, and polyethylene glycol hydrogel. The activity of matrix metalloproteinases was estimated by gelatin zymography. Collagen density in muscles was determined histochemically. An increased level of the active MMP-9 is associated with the fibrous changes in the denervated skeletal muscles and after an epineurial suture. The use of fibrin glue and polyethylene glycol hydrogel resulted in a lower level of collagen and ММР-9 activity, which may be a therapeutic target in the treatment of neuromuscular lesions, and has value in fibrosis analysis following microsurgical intervention for peripheral nerve reconstruction.

Overexpressed HGF promotes metastasis of squamous cell carcinoma of the head and neck through the PI3K/Akt and JNK signaling pathways

Background: The role of HGF in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Methods: Reverse transcription PCR, western blotting, gelatin zymography, immunohistochemistry, actin polymerization, chemotaxis and migration assays were used in the authors' study. Results: HGF expression level was upregulated in SCCHN cells, which was associated with clinical stage; tumor, node, metastasis classification; and lymphatic invasion. SCCHN cells with high Met expression were sensitive to cell invasion, which was blocked by inhibiting PI3K/Akt and JNK. HGF induced MMP9 expression and enhanced its activity. Akt induced the activation of JNK through the PI3K/Akt and JNK signaling pathways. Conclusion: HGF upregulates MMP9 through the activation of the PI3K/Akt and JNK signaling pathways in SCCHN cells.

Proteases Production and Chitin Preparation from the Liquid Fermentation of Chitinous Fishery By-Products by Paenibacillus elgii

Chitinous fishery by-products have great application in the production of various bioactive compounds. In this study, Paenibacillus elgii TKU051, a protease-producing bacterial strain, was isolated using a medium containing 1% squid pens powder (SPP) as the sole carbon/nitrogen (C/N) source. P. elgii TKU051 was found to produce at least four proteases with molecular weights of 100 kDa, 57 kDa, 43 kDa, and 34 kDa (determined by the gelatin zymography method). A P. elgii TkU051 crude enzyme cocktail was optimally active at pH 6–7 and 60 °C. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and α-glucosidase inhibitory activity of the hydrolysates obtained from the hydrolysis of shrimp shell powder, shrimp head powder, shrimp meat powder, fish head powder and soya bean powder catalyzed by the P. elgii TkU051 crude enzyme cocktail were also evaluated. P. elgii TKU051 exhibited a high deproteinization capacity (over 94%) on different kinds of shrimp waste (shrimp heads and shells; fresh and cooked shrimp waste; shrimp waste dried by oven and lyophilizer), and the Fourier-transform infrared spectroscopy profile of the chitin obtained from the deproteinization process displayed the characteristic of chitin. Finally, the obtained chitin exhibited an effect comparable to commercial chitin in terms of adsorption against Congo Red (90.48% and 90.91%, respectively). Thus, P. elgii TKU051 showed potential in the reclamation of chitinous fishery by-products for proteases production and chitin extraction.