Interleukin-9 Facilitates Osteoclastogenesis in Rheumatoid Arthritis.

In rheumatoid arthritis (RA), inflammatory cytokines play a pivotal role in triggering abnormal osteoclastogenesis leading to articular destruction. Recent studies have demonstrated enhanced levels of interleukin-9 (IL-9) in the serum and synovial fluid of patients with RA. In RA, strong correlation has been observed between tissue inflammation and IL-9 expression in synovial tissue. Therefore, we investigated whether IL-9 influences osteoclastogenesis in patients with RA. We conducted the study in active RA patients. For inducing osteoclast differentiation, mononuclear cells were stimulated with soluble receptor activator of NF-kB ligand (sRANKL) and macrophage-colony-stimulating factor (M-CSF) in the presence or absence of recombinant (r) IL-9. IL-9 stimulation significantly enhanced M-CSF/sRANKL-mediated osteoclast formation and function. Transcriptome analysis revealed differential gene expression induced with IL-9 stimulation in the process of osteoclast differentiation. IL-9 mainly modulates the expression of genes, which are involved in the metabolic pathway. Moreover, we observed that IL-9 modulates the expression of matrix metalloproteinases (MMPs), which are critical players in bone degradation. Our results indicate that IL-9 has the potential to influence the structural damage in the RA by promoting osteoclastogenesis and modulating the expression of MMPs. Thus, blocking IL-9 pathways might be an attractive immunotherapeutic target for preventing bone degradation in RA.

Doxycycline host-directed therapy in human pulmonary tuberculosis.

BACKGROUNDMatrix metalloproteinases (MMPs) are key regulators of tissue destruction in tuberculosis (TB) and may be targets for host-directed therapy. We conducted a phase II double-blind, randomized, controlled trial investigating doxycycline, a licensed broad-spectrum MMP inhibitor, in patients with pulmonary TB.METHODSThirty patients with pulmonary TB were enrolled within 7 days of initiating anti-TB treatment and randomly assigned to receive either 100 mg doxycycline or placebo twice a day for 14 days, in addition to standard care.RESULTSWhole blood RNA-sequencing demonstrated that doxycycline accelerated restoration of dysregulated gene expression in TB towards normality, rapidly down-regulating type I and II interferon and innate immune response genes, and up-regulating B-cell modules relative to placebo. The effects persisted for 6 weeks after doxycycline discontinuation, concurrent with suppressed plasma MMP-1. Doxycycline significantly reduced sputum MMP-1, -8, -9, -12 and -13, suppressed type I collagen and elastin destruction, reduced pulmonary cavity volume without altering sputum mycobacterial loads, and was safe.CONCLUSIONAdjunctive doxycycline with standard anti-TB treatment suppressed pathological MMPs in PTB patients. Larger studies on adjunctive doxycycline to limit TB immunopathology are merited.TRIAL REGISTRATIONClinicalTrials.gov NCT02774993.FUNDINGSingapore National Medical Research Council (NMRC/CNIG/1120/2014, NMRC/Seedfunding/0010/2014, NMRC/CISSP/2015/009a); the Singapore Infectious Diseases Initiative (SIDI/2013/013); National University Health System (PFFR-28 January 14, NUHSRO/2014/039/BSL3-SeedFunding/Jul/01); the Singapore Immunology Network Immunomonitoring platform (BMRC/IAF/311006, H16/99/b0/011, NRF2017_SISFP09); an ExxonMobil Research Fellowship, NUHS Clinician Scientist Program (NMRC/TA/0042/2015, CSAINV17nov014); the UK Medical Research Council (MR/P023754/1, MR/N006631/1); a NUS Postdoctoral Fellowship (NUHSRO/2017/073/PDF/03); The Royal Society Challenge Grant (CHG\R1\170084); the Sir Henry Dale Fellowship, Wellcome Trust (109377/Z/15/Z); and A*STAR.

The TLR-2/TonEBP signaling pathway regulates 29-kDa fibronectin fragment-dependent expression of matrix metalloproteinases.

Tonicity-responsive enhancer-binding protein (TonEBP; nuclear factor of activated T cells 5) is a transcription factor that responds to changes in osmolality. However, recent studies have shown that it also modulates immune responses under inflammatory conditions independently of hyperosmolality. Fibronectin fragments (FN-fs), which are abundant in the synovial fluid of patients with osteoarthritis (OA), induce expression of matrix metalloproteinases (MMPs) via the toll-like receptor-2 (TLR-2) signaling pathway. In this study we examined whether TonEBP is involved in 29-kDa FN-f-induced expression of MMPs. The expression of TonEBP was significantly higher in human osteoarthritis compared with normal cartilage samples. 29-kDa FN-f affected the expression of MMPs 1, 3, and 13 via TonEBP, and expression and nuclear accumulation of TonEBP were induced by activation of the phospholipase C/NF-κB/MAPK signaling pathway and, in particular, modulated by TLR-2. In addition, 29-kDa FN-f induced the expression of osmoregulatory genes, including Tau-T, SMIT, and AR, as well as voltage-dependent calcium channels via the TonEBP/TLR-2 signaling pathway. These results show that 29-kDa FN-f upregulates MMPs in chondrocytes via the TLR-2/TonEBP signaling pathway.

Granulocyte-macrophage colony-stimulating factor initiates amniotic membrane rupture and preterm birth in a mouse model.

OBJECTIVE: Preterm premature rupture of membranes is associated with 30% of all preterm births. The weakening of amniotic membranes is associated with an increase in matrix metallopeptidases (MMPs) along with a decrease in their inhibitors, tissue inhibitor metallopeptidases (TIMPs). Additionally, granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to weaken fetal membranes in-vitro. We hypothesize pregnant mice treated with GM-CSF lead to increased MMPs:TIMPs resulting in membrane rupture and preterm birth. STUDY DESIGN: Pregnant CD-1 mice on gestational day 17 received either an intrauterine injection of GM-CSF or vehicle control. A second series of mice were administered an intrauterine injection of Lipopolysaccharide along with either anti-mouse GM-CSF or control antibody. Mice were evaluated for rupture of membranes and/or preterm birth and the uterus, amniotic fluid, and serum were collected for analysis. RESULTS: 87.5% of GM-CSF mice exhibited evidence of membrane rupture or preterm birth, compared with 0% in control mice (p < .001). Treatment with GM-CSF decreased the expression of TNFα (p < .05) while increasing the ratio of MMP2:TIMP1 (p < .05), MMP2:TIMP2 (p < .05), MMP2:TIMP3 (p < .001), MMP9:TIMP1 (p < .01), MMP9:TIMP2 (p < .05), MMP9:TIMP3 (p < .001), and MMP10:TIMP1 (p < .05). Mice treated with LPS and the GM-CSF antibody resulted in a decrease in the ratio of MMP2:TIMP1 (p < .0001) compared with controls. CONCLUSION: These studies demonstrate GM-CSF will result in membrane rupture and preterm birth by increasing the ratio MMPs:TIMPs in our animal model. By increasing our understanding of the molecular pathways associated with GM-CSF, we may be able to develop future therapies to prevent preterm birth and reduce neonatal morbidity.

The Biological and Clinical Role of the Long Non-Coding RNA LOC642852 in Ovarian Carcinoma.

The objective of the present study was to analyze the biological and clinical role of the long non-coding RNA LOC642852 in ovarian carcinoma (OC). LOC642852 expression was analyzed in seven OC cell lines (OVCAR-3, OVCAR-8, OVCA 433, OVCA 429, OC 238, DOV13, ES-2) and 139 high-grade serous carcinoma (HGSC) specimens (85 effusions, 54 surgical specimens). Following LOC642852 knockout (KO) using the CRISPR/Cas9 system, OVCAR-8 HGSC cells were analyzed for spheroid formation, migration, invasion, proliferation, matrix metalloproteinase (MMP) activity, and expression of cell signaling proteins. OVCAR-8 cells with LOC642852 KO were significantly less motile and less invasive compared to controls, with no differences in spheroid formation, proliferation, or matrix metalloproteinase (MMP) activity. Total Akt and Erk levels were comparable in controls and KO cells, but their phosphorylation was significantly increased in the latter. In clinical specimens, LOC642852 was overexpressed in ovarian tumors and omental/peritoneal metastases compared to effusion specimens (p = 0.013). A non-significant trend for shorter overall (p = 0.109) and progression-free (p = 0.056) survival was observed in patients with HGSC effusions with high LOC642852 levels. Bioinformatics analysis showed potential roles for LOC642852 as part of the TLE3/miR-221-3p ceRNA network and in relation to the FGFR3 protein. In conclusion, LOC642852 inactivation via CRISPR/Cas9 affects cell signaling, motility, and invasion in HGSC cells. LOC642852 is differentially expressed in HGSC cells at different anatomical sites. Its potential role in regulating the TLE3/miR-221-3p ceRNA network and FGFR3 merits further research.

Effects of 1,25-Dihydroxy vitamin D3 on TNF-α induced inflammation in human chondrocytes and SW1353 cells: a possible role for toll-like receptors.

The purpose of this study is to evaluate anti-inflammatory and chondro-protective effects of 1,25(OH)2D3 in human chondrocytes and SW1353 cells via investigating expressions of MMPs, TIMPs, VDR, and intracellular signalling pathway mediators such as TLR-2 and -4. The HC and SW1353 cells were treated with 1,25(OH)2D3 at 10, 100, and 1000 nM concentrations in the absence/presence of TNF-α (20 ng/mL) for 48 h. The mRNA expressions of MMP-1, -2, -3, -9, and -13, TIMP-1 and -2, VDR, TLR-2 and -4 in HC and SW1353 cells were detected by qPCR after treatments. The cytotoxicity and cell proliferation analyses were assessed by LDH and WST-1 assay, respectively. Protein levels of MMPs, TIMPs, and VDR were analysed by immunocytochemistry and ELISA methods. TNF-α markedly increased cytotoxicity for 24, 48, 72 h (p < 0.05) and vitamin D treatment was shown to diminish the cytotoxic effect of TNF-α. Cell proliferations increased by Vitamin D in a dose-dependent manner. mRNA expressions of MMP-1, -2, -3, -9, and -13, TLR-2 and -4 genes decreased with 1,25(OH)2D3 treatment (p < 0.05). VDR, TIMP-1 and -2 levels elevated after TNF-α exposure compared with the control group in HC cells (p < 0.05). Protein expression levels were determined using Western blotting, ELISA and immunocytochemistry. 1,25(OH)2D3 via binding to VDR, reversed the effects of TNF-α by inhibiting TLR-2 and 4. Decreased levels of VDR, TIMP-1 and -2 after TNF-α treatment were elevated by 1,25(OH)2D3 proportional with increasing 1,25(OH)2D3 doses. 1,25(OH)2D3 and TNF-α co-treatment decreased MMP-1, -2, -3, -9, and -13 levels were after TNF-α exposure.

Effect of proinflammatory cytokines on endometrial collagen and metallopeptidase expression during the course of equine endometrosis.

Equine endometrosis (endometrial fibrosis) is a degenerative chronic process that occurs in the uterus of the mare and disturbs proper endometrial function. Fibrosis is attributed to excessive deposition of extracellular matrix (ECM) components. The turnover of ECM is mediated by matrix metallopeptidases (MMP). Previously, it was shown that cytokines modulate MMP expression in other tissues and may regulate fibrosis indirectly by attracting inflammatory cells to the site of inflammation and directly on various tissues. However, the regulation of MMP expression in equine endometrosis is still relatively unknown. Thus, our aim was to determine if interleukin (IL)-1ß and IL-6 regulate ECM, MMPs, or their inhibitors (TIMPs) and whether this regulation differs during endometrosis in the mare. Endometrial fibrosis was divided into four categories according to severity: I (no degenerative changes), IIA (mild degenerative changes), IIB (moderate degenerative changes) and III (severe degenerative changes) according to Kenney and Doig classification. Endometrial explants (n = 5 for category I, IIA, IIB and III according to Kenney and Doig) were incubated with IL-1ß (10 ng/ml) or IL-6 (10 ng/ml) for 24 h. Secretion and mRNA transcription of collagen type 1 (Col1a1) and type 3 (Col3a1), fibronectin (Fn1), Mmp-1, -2, -3, -9, -13, Timp-1, -2 were analyzed by real-time PCR and ELISA, respectively. IL-1ß treatment up-regulated secretion of COL1, MMP-2, TIMP1, and TIMP2 in category I endometrial fibrosis tissues (P < 0.05). IL-6 treatment up-regulated secretion of ECM, MMP-2, and MMP-3 and down-regulated secretion of MMP-9 in category I tissues (P < 0.05). In category IIA tissues, IL-1ß and IL-6 treatment up-regulated secretion of COL3 (P < 0.05; P < 0.05), and IL-6 treatment also down-regulated secretion of MMP-9 (P < 0.05). In category IIB tissues, IL-1ß treatment down-regulated secretion of COL3 (P < 0.05) and up-regulated secretion of MMP-3 (P < 0.01), while IL-6 treatment up-regulated secretion of MMP-3, MMP-9, and MMP-13 (P < 0.05). In category III tissues, IL-1ß treatment up-regulated secretion of COL1, MMP-1, MMP-9 and TIMP-2 (P < 0.05), and IL-6 up-regulated secretion of all investigated ECM components, MMPs and TIMPs. These results reveal that the effect of IL-1ß and IL-6 on equine endometrium differs depending on the severity of endometrial fibrosis. Our findings indicate an association between inflammation and development of endometrosis through the effect of IL-1ß and IL-6 on expression of ECM components, MMPs, and TIMPs in the mare.

Effects of proteasome inhibitors on cytokines, metalloproteinases and their inhibitors and collagen type-I expression in periprosthetic tissues and fibroblasts from loose arthroplasty endoprostheses.

Objective: Aseptic loosening is a major problem in total joint replacement. Implant wear debris provokes a foreign body host response and activates cells to produce a variety of mediators and ROS, leading to periprosthetic osteolysis. Elevated ROS levels can harm proteasome function. Proteasome inhibitors have been reported to alter the secretory profile of cells involved in inflammation and also to induce ROS production. In this work, we aimed to document the effects of proteasome inhibitors MG-132 and Epoxomicin, on the production of factors involved in aseptic loosening, in periprosthetic tissues and fibroblasts, and investigate the role of proteasome impairment in periprosthetic osteolysis. Materials and methods: IL-6 levels in tissue cultures were determined by sandwich ELISA. MMP-1, -3, -13, -14 and TIMP-1 levels in tissue or cell cultures were determined by indirect ELISA. Results for MMP-1 and TIMP-1 in tissue cultures were confirmed by Western blotting. MMP-2 and MMP-9 levels were determined by gelatin zymography. Gene expression of IL-6, MMP-1,-3,-14, TIMP-1 and collagen type-I was determined by RT-PCR. Results: Results show that proteasome inhibition induces the expression of ΜΜΡ-1, -2, -3, -9 and suppresses that of IL-6, MMP-14, -13, TIMP-1 and collagen type I, enhancing the collagenolytic and gelatinolytic activity already present in periprosthetic tissues, as documented in various studies. Conclusions: These findings suggest that proteasome impairment could be a contributing factor to aseptic loosening. Protection and enhancement of proteasome efficacy could thus be considered as an alternative strategy toward disease treatment.

The Collagenase of the Bacterium Clostridium histolyticum in the Treatment of Irradiation-Induced Capsular Contracture.

BACKGROUND: Irradiation therapy is an important pillar in the treatment of breast cancer. However, it can trigger capsular fibrosis, the most significant complication of implant-based breast reconstruction. As collagen is the main component of fibrotic capsules, the collagenase of the bacterium Clostridium histolyticum poses a potential treatment option for this pathological condition. METHODS: Thirty-six rats received miniature silicone implants on their backs. On day 1, the implant sites of two groups were irradiated with 10 Gy. On day 120, one irradiated group received collagenase injections into the implant pockets (n = 12). Non-irradiated (n = 12) and irradiated capsules (n = 12) were injected with plain solvent solution serving as controls. Data were analyzed by means of in vivo imaging, histology, immunohistochemistry and gene expression analysis. RESULTS: Compared with both controls, the injection of collagenase led to significantly thinner capsules. This was verified by in vivo imaging and histology. Although irradiation provoked alterations in capsule collagen structure and vessel wall thickness, the application of collagenase resulted in a significant reduction of collagen density. This was accompanied by an up-regulation of VEGF-A gene expression. Of note, hematoma formation inside the implant pocket occurred in two cases after collagenase injection. CONCLUSIONS: The collagenase of the bacterium Clostridium histolyticum is effective in degrading irradiation-induced capsular fibrosis around silicone implants. Hematoma formation occurred most likely because of irradiation-induced alterations in vessel wall architecture and capsule vascularization. Further studies need to be performed to address the clinical safety of this novel treatment option.

Local intra­articular injection of vascular endothelial growth factor accelerates articular cartilage degeneration in rat osteoarthritis model.

In the pathophysiology of osteoarthritis (OA), articular cartilage degeneration exhibits a significant role. Vascular endothelial growth factor (VEGF) is considered to be an effective angiogenic factor and a crucial regulator of articular cartilage degeneration in the development of OA. Therefore, the present study aimed to investigate the underlying influences of exogenous VEGF on articular cartilage degeneration in OA model rat. A total of 24 male Sprague­Dawley rats were randomly allocated into 3 groups. In the normal saline (NS) and VEGF groups, animals received bilateral anterior cruciate ligament (ACL) transection to establish the OA model; at 4 weeks post­surgery, the rats received local intra­articular injections of 100 µl NS or VEGF solution, respectively, every week for 4 weeks. The Control group received neither surgery nor injections. All animals were sacrificed at 12 weeks following surgery. Prominent cartilage degeneration was observed in rats in the NS­ and VEGF­injected groups. The extent and the grade of cartilage damage in the VEGF­injected group were notably more severe compared with those in the NS­treated group. Western blotting results demonstrated that the expression levels of aggrecan and type II collagen were significantly reduced in OA model rats that were treated with VEGF. In addition, the expression levels of matrix metalloproteinase (MMP)­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (a disintegrin and metalloproteinase; ADAMTS)­4, ­5 and ­12, type III collagen and transforming growth factor­ß1 were significantly increased following VEGF administration. Results from the present study indicated that VEGF may exhibit a promoting role in the development of OA by destroying articular cartilage matrix.

Link protein N-terminal peptide and fullerol promote matrix production and decrease degradation enzymes in rabbit annulus cells.

PURPOSE: Intervertebral disc degeneration is a major cause of back pain. Novel therapies for prevention or reversal of disc degeneration are needed. It is desirable for potential therapies to target both inflammation and matrix degeneration. MATERIALS AND METHODS: The combined regenerative potential of link protein N-terminal peptide (LN) and fullerol on annulus fibrosus (AF) cells was evaluated in a 3D culture model. RESULTS: Interleukin-1α (IL-1α)-induced AF cell degeneration was counteracted by fullerol, LN, and fullerol + LN, with the latter having the greatest effect on matrix production as evaluated by real-time polymerase chain reaction and glycosaminoglycan assay. IL-1α-induced increases in pro-inflammatory mediators (interleukin-6 and cyclooxygenase-2) and matrix metalloproteinases (MMP-1, -2, -9, and -13) were also counteracted by fullerol and LN. CONCLUSION: Our data demonstrate that LN and fullerol individually, and in combination, promote matrix production and have anti-inflammatory and anti-catabolic effects on AF cells.

Oral Polyphosphate Suppresses Bacterial Collagenase Production and Prevents Anastomotic Leak Due to Serratia marcescens and Pseudomonas aeruginosa.

OBJECTIVE: The objective of this study was to determine the effect of polyphosphate on intestinal bacterial collagenase production and anastomotic leak in mice undergoing colon surgery. BACKGROUND: We have previously shown that anastomotic leak can be caused by intestinal pathogens that produce collagenase. Because bacteria harbor sensory systems to detect the extracellular concentration of phosphate which controls their virulence, we tested whether local phosphate administration in the form of polyphosphate could attenuate pathogen virulence and prevent leak without affecting bacterial growth. METHODS: Groups of mice underwent a colorectal anastomosis which was then exposed to collagenolytic strains of either Serratia marcescens or Pseudomonas aeruginosa via enema. Mice were then randomly assigned to drink water or water supplemented with a 6-mer of polyphosphate (PPi-6). All mice were sacrificed on postoperative day 10 and anastomoses assessed for leakage, the presence of collagenolytic bacteria, and anastomotic PPi-6 concentration. RESULTS: PPi-6 markedly attenuated collagenase and biofilm production, and also swimming and swarming motility in both S. marcescens and P. aeruginosa while supporting their normal growth. Mice drinking PPi-6 demonstrated increased levels of PPi-6 and decreased colonization of S. marcescens and P. aeruginosa, and collagenase activity at anastomotic tissues. PPi-6 prevented anastomotic abscess formation and leak in mice after anastomotic exposure to S. marcescens and P. aeruginosa. CONCLUSIONS: Polyphosphate administration may be an alternative approach to prevent anastomotic leak induced by collagenolytic bacteria with the advantage of preserving the intestinal microbiome and its colonization resistance.

Inflammatory cytokine of IL-1ß is involved in T-2 toxin-triggered chondrocyte injury and metabolism imbalance by the activation of Wnt/ß-catenin signaling.

Mycotoxin T-2 exerts a causative role in Kashin-Beck disease (KBD) suffering chondrocyte apoptosis and cartilage matrix homeostasis disruption. Recent research corroborated the aberrant levels of pro-inflammatory cytokine IL-1ß in KBD patients and mycotoxin environment. In the present study, we investigated the relevance of IL-1ß in T-2 toxin-evoked chondrocyte cytotoxic injury and aberrant catabolism. High levels of IL-1ß were detected in serum and cartilages from KBD patients and in T-2-stimulated chondrocytes. Moreover, knockdown of IL-1ß antagonized the adverse effects of T-2 on cytotoxic injury by enhancing cell viability and inhibiting apoptosis. However, exogenous supplementation of IL-1ß further aggravated cell damage in response to T-2. Additionally, cessation of IL-1ß rescued T-2-elicited tilt of matrix homeostasis toward catabolism by elevating the transcription of collagen II and aggrecan, promoting release of sulphated glycosaminoglycans (sGAG) and TIMP1, and suppressing matrix metalloproteinases production including MMP-1, MMP-3 and MMP-13. Conversely, IL-1ß stimulation deteriorated T-2-induced disruption of matrix metabolism balance toward catabolism. Mechanistic analysis found the high activation of Wnt/ß-catenin in KBD patients and chondrocytes upon T-2. Furthermore, this activation was mitigated after IL-1ß inhibition, but further enhanced following IL-1ß precondition. Importantly, blocking this pathway by transfection with ß-catenin alleviated the adverse roles of IL-1ß on cytotoxic injury and metabolism disorders under T-2 conditioning. Together, this study elucidates a new insight into how T-2 deteriorates the pathological progression of KBD by regulating inflammation-related pathways, indicating a promising anti-inflammation strategy for KBD therapy.

Collagen-derived N-acetylated proline-glycine-proline upregulates the expression of pro-inflammatory cytokines and extracellular matrix proteases in nucleus pulposus cells via the NF-κB and MAPK signaling pathways.

N-acetylated proline-glycine-proline (N-Ac-PGP) is a chemokine involved in inflammatory diseases and is found to accumulate in degenerative discs. N-Ac-PGP has been demonstrated to have a pro-inflammatory effect on human cartilage endplate stem cells. However, the effect of N-Ac-PGP on human intervertebral disc cells, especially nucleus pulposus (NP) cells, remains unknown. The purpose of this study was to investigate the effect of N-Ac-PGP on the expression of pro-inflammatory factors and extracellular matrix (ECM) proteases in NP cells and the molecular mechanism underlying this effect. Therefore, Milliplex assays were used to detect the levels of various inflammatory cytokines in conditioned culture medium of NP cells treated with N-Ac-PGP, including interleukin-1ß (IL-1ß), IL-6, IL-17, tumor necrosis factor-α (TNF-α) and C-C motif ligand 2 (CCL2). RT-qPCR was also used to determine the expression of pro-inflammatory cytokines and ECM proteases in the NP cells treated with N-Ac-PGP. Moreover, the role of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways in mediating the effect of N-Ac-PGP on the phenotype of NP cells was investigated using specific signaling inhibitors. Milliplex assays showed that NP cells treated with N-Ac-PGP (10 and 100 µg/ml) secreted higher levels of IL-1ß, IL-6, IL-17, TNF-α and CCL2 compared with the control. RT-qPCR assays showed that NP cells treated with N-Ac-PGP (100 µg/ml) had markedly upregulated expression of matrix metalloproteinase 3 (MMP3), MMP13, a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4), ADAMTS5, IL-6, CCL-2, CCL-5 and C-X-C motif chemokine ligand 10 (CXCL10). Moreover, N-Ac-PGP was shown to activate the MAPK and NF-κB signaling pathways in NP cells. MAPK and NF-κB signaling inhibitors suppressed the upregulation of proteases and pro-inflammatory cytokines in NP cells treated with N-Ac-PGP. In conclusion, N-Ac-PGP induces the expression of pro-inflammatory cytokines and matrix catabolic enzymes in NP cells via the NF-κB and MAPK signaling pathways. N-Ac-PGP is a novel therapeutic target for intervertebral disc degeneration.

Collagenolytic enzymes produced by fungi: a systematic review

Abstract Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.

Inhibition of collagen synthesis by IWR-1 in normal and keloid-derived skin fibroblasts.

AIMS: Keloid is a benign tumor that is characterized by the hyperproliferation of dermal fibroblasts and excessive deposition of extracellular matrix (ECM) especially the collagen. Aberrant activation of Wnt/ß-catenin signaling is implicated in the pathogenesis of keloid. In this study, we investigated the effects of IWR-1, a small molecule inhibitor for Wnt/ß-catenin signaling via the inhibition of tankyrase, on production of collagen and matrix metalloproteinase (MMP) in dermal fibroblasts. MAIN METHODS: We cultured human normal skin- and keloid-derived fibroblasts, then treated with IWR-1. The effects of IWR-1 on collagen and MMP production were determined by Western blot, ELISA and zymography. KEY FINDINGS: IWR-1 significantly suppressed the proliferation and migration of both the normal and keloid fibroblasts. IWR-1 also inhibited the production and secretion of type I collagen from the fibroblasts. In addition, IWR-1 significantly increased the expression of MMPs, such as MMP-1, MMP-3 and MMP-13, along with the increase of gelatinase activity. These results suggest that inhibitory effect of IWR-1 on collagen production may be related with the increased MMP activity. SIGNIFICANCE: This study provides the possible action mechanism of IWR-1 on regulation of collagen expression, on which to base further investigation for preventing skin fibrotic diseases such as keloid.

Collagenolytic enzymes produced by fungi: a systematic review.

Specific proteases capable of degrading native triple helical or denatured collagen have been required for many years and have a large spectrum of applications. There are few complete reports that fully uncover production, characterization and purification of fungi collagenases. In this review, authors searched through four scientific on line data bases using the following keywords (collagenolytic OR collagenase) AND (fungi OR fungus OR fungal) AND (production OR synthesis OR synthesize) AND (characterization). Scientific criteria were adopted in this review to classify found articles by score (from 0 to 10). After exclusion criteria, 21 articles were selected. None obtained the maximum of 10 points defined by the methodology, which indicates a deficiency in studies dealing simultaneously with production, characterization and purification of collagenase by fungi. Among microorganisms studied the non-pathogenic fungi Penicillium aurantiogriseum and Rhizoctonia solani stood out in volumetric and specific collagenase activity. The only article found that made sequencing of a true collagenase showed 100% homology with several metalloproteinases fungi. A clear gap in literature about collagenase production by fungi was verified, which prevents further development in the area and increases the need for further studies, particularly full characterization of fungal collagenases with high specificity to collagen.

Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during maturation.

Although participation of matrix metalloproteinases (MMPs) in reproductive tract remodeling has been strongly suggested in mammalian species, the role of MMPs in the avian oviduct has received little attention. To gain a better understanding of the potential role of the MMP system in avian oviduct development, mRNA and protein expression, localization of selected MMPs and their tissue inhibitors (TIMPs), and gelatinolytic activity in the oviduct of growing chickens were examined. The oviducts were collected from Hy-Line Brown hens before (10, 12, 14 and 16 weeks of age) and after (week 17) the onset of egg laying. The MMP-2, -7, -9 and TIMP-2 and -3 genes were found to be differentially expressed in all examined oviductal sections: the infundibulum, magnum, isthmus and shell gland on both mRNA (by real time polymerase chain reaction) and protein (by western blotting and immunohistochemistry) levels. In the course of oviduct development, the relative expression of all genes decreased in most sections. Protein level of MMP-9 was diminished, while MMP-7 and TIMP-3 were elevated in the oviduct of growing birds. MMP-2 and TIMP-2 protein levels remained constant, with a slight increase in MMP-2 concentration just before reaching maturity. The relative activity of MMP-2 and -9 (assessed by gelatin zymography) was higher (P < 0.05, P < 0.01) in immature birds compared with adults. Immunohistochemistry demonstrated cell- and tissue-specific localization of MMPs and TIMPs in the wall of the chicken oviduct. We concluded that changes in the expression of examined MMPs and their inhibitors, as well as alterations in MMP activity occurring simultaneously with changes in the morphology of the chicken oviduct, suggest the involvement of the MMP system in the proper development and functioning of this organ. Mechanisms regulating the expression and activity of MMPs require further clarification.

Gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, Gentiana macrophylla Pall have been prescribed for the treatment of pain and inflammatory conditions. In addition, it is a common Tibetan medicinal herb used for the treatment of tonsillitis, urticaria, and rheumatoid arthritis (RA), while the flowers of G. macrophylla Pall have been traditionally treated as an anti-inflammatory agent to clear heat in Mongolian medicine. The secoiridoid glycosides and their derivatives are the primary active components of G. macrophylla and have been demonstrated to be effective as anti-inflammatory agents. MATERIALS AND METHODS: Solvent extraction and D101 macroporous resin columns were employed to concentratethe gentiopicroside. Gentiopicroside cytotoxicity was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; the toxicity of gentiopicroside in chondrocytes was reconfirmed using Hoechst staining. Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were utilized to explore the protective effects and mechanisms of gentiopicroside prevents interleukin-1 beta induced inflammation response in rat articular chondrocyte. RESULTS: The MTT assay demonstrated that 50, 500, and 1,500 µg/mL of gentiopicroside exhibited no significant toxicity to chondrocytes (P>0.05) after 24h. Using immunohistochemistry, ELISA, RT-PCR, Western blot method to explore the protective effect and mechanism of gentiopicroside on chondrocytes induced by IL-1ß. The results showed some pathways of IL-1ß signal transduction were inhibited by gentiopicroside in rat chondrocytes: p38, ERK and JNK. Meanwhile, gentiopicroside showed inhibition in the IL-1ß-induced release of MMPs while increasing Collagen type II expression. CONCLUSIONS: The current study demonstrated that gentiopicroside exhibited a potent protective effect on IL-1ß induced inflammation response in rat articular chondrocyte. Thus, gentiopicroside could be a potential therapeutic strategy for treatment of OA.