Tocopheryl Phosphate Inhibits Rheumatoid Arthritis-Related Gene Expression In Vitro and Ameliorates Arthritic Symptoms in Mice.

Anti-rheumatoid arthritis (RA) effects of α-tocopherol (α-T) have been shown in human patients in a double-blind trial. However, the effects of α-T and its derivatives on fibroblast-like synoviocytes (FLS) during the pathogenesis of RA remain unclear. In the present study, we compared the expression levels of genes related to RA progression in FLS treated with α-T, succinic ester of α-T (TS), and phosphate ester of α-T (TP), as determined via RT-PCR. The mRNA levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-3, and MMP-13 were reduced by treatment with TP without cytotoxicity, while α-T and TS did not show such effects. Furthermore, intraperitoneal injection of TP ameliorated the edema of the foot and joint and improved the arthritis score in laminarin-induced RA model mice. Therefore, TP exerted anti-RA effects through by inhibiting RA-related gene expression.

Bone marrow mesenchymal stem cell-derived exosomes inhibit chondrocyte apoptosis and the expression of MMPs by regulating Drp1-mediated mitophagy.

Osteoarthritis (OA) is a joint degenerative disease commonly seen in the elderly. Bone marrow mesenchymal stem cell-exosomes (BMSC-exosomes) are closely associated with the progression of OA. Here, we investigated whether BMSC-exosomes can affect OA development by regulating mitophagy. Primary rat chondrocytes were treated with advanced glycation end products (AGEs) to induce cell damage. The results of flow cytometry showed that AGEs treatment significantly promoted apoptosis of chondrocytes. AGEs treatment also enhanced the expression of matrix metalloproteinases (MMPs), MMP-3 and MMP-13, and dynamin-related protein 1 (Drp1) in chondrocytes. To investigate the impact of BMSC-exosomes on chondrocytes, chondrocytes were treated with BMSC-exosomes. AGEs-mediated increase of apoptosis and up-regulation of MMP-3, MMP-13, and Drp1 in chondrocytes were abrogated by BMSC-exosomes. Western blot analysis of autophagy-related proteins and Mito-Keima assay revealed that BMSC-exosome treatment elevated the expression of autophagy-related proteins, LC3-II/LC3-I and Beclin-1, and promoted mitophagy in the AGEs-treated chondrocytes. Moreover, Drp1 overexpression repressed the expression of LC3-II/LC3-I and Beclin-1, and enhanced apoptosis and the expression of MMP-3 and MMP-13 in AGEs-treated chondrocytes. BMSC-exosomes reversed the impact of Drp1 overexpression on AGEs-treated chondrocytes. In conclusion, this work demonstrates that BMSC-exosomes inhibit chondrocyte apoptosis and the expression of MMPs, which attributes to regulate Drp1-mediated mitophagy. Thus, BMSC-exosomes may be a potential treatment for OA.

Lysophosphatidic Acid Augments the Gene Expression and Production of Matrix Metalloproteinases-1 and -3 in Human Synovial Fibroblasts in Vitro.

Rheumatoid arthritis (RA) is an inflammatory disease with joint dysfunction following cartilage degradation. The level of lysophosphatidic acid (LPA) has been reported to be augmented in human synovial fluid from patients with RA. However, it remains to be elucidated whether LPA participates in cartilage destruction. In the present study, we have demonstrated that the production of promatrix metalloproteinases (proMMPs)-1 and -3 was augmented along with an increase of extracellular signal-regulated kinase (ERK)1/2 phosphorylation through LPA receptor 1 (LPAR1) in human synovial fibroblasts. These results suggest that LPA transcriptionally increases MMP production by the activation of an LPAR1/ERK1/2 signal pathway in human synovial fibroblasts. Thus, LPA is likely to be a pathological candidate for cartilage degradation in RA.

Identification of miR-515-3p and its targets, vimentin and MMP3, as a key regulatory mechanism in esophageal cancer metastasis: functional and clinical significance.

Metastasis is the main factor of treatment failure in cancer patients, but the underlying mechanism remains to be elucidated and effective new treatment strategies are urgently needed. This study aims to explore novel key metastasis-related microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). By comparing miRNA profiles of the highly metastatic ESCC cell sublines, we established through serial in vivo selection with the parental cells, we found that the expression level of miR-515-3p was lower in ESCC tumor tissues than adjacent normal tissues, further decreased in metastatic tumors, and moreover, markedly associated with advanced stage, metastasis and patient survival. The in vitro and in vivo assays suggested that miR-515-3p could increase the expression of the epithelial markers as well as decrease the expression of the mesenchymal markers, and more importantly, suppress invasion and metastasis of ESCC cells. Mechanistically, we revealed that miR-515-3p directly regulated vimentin and matrix metalloproteinase-3 (MMP3) expression by binding to the coding sequence and 3'untranslated region, respectively. In addition, the data from whole-genome methylation sequencing and methylation-specific PCR indicated that the CpG island within miR-515-3p promoter was markedly hypermethylated in ESCC cell lines and ESCC tumor tissues, which may lead to deregulation of miR-515-3p expression in ESCC. Furthermore, our preclinical experiment provides solid evidence that systemic delivery of miR-515-3p oligonucleotide obviously suppressed the metastasis of ESCC cells in nude mice. Taken together, this study demonstrates that miR-515-3p suppresses tumor metastasis and thus represents a promising prognostic biomarker and therapeutic strategy in ESCC.

7­MEGA™ 500 regulates the expression of COX­2, MMP­3 and type 1 procollagen in UVB­irradiated human keratinocytes and dermal fibroblasts.

AlaskOmega® Omega 7 500, also known as Omega­7 fatty acid or 7­MEGA™, is a highly concentrated palmitoleic acid (C16:1). Little is known about how 7­MEGA regulates skin inflammation and wrinkle formation in cultured skin cells. The present study aimed to investigate the effects of 7­MEGA on the expression of cyclooxygenase­2 (COX­2), matrix metallopeptidase (MMP)­1/3 and type 1 procollagen, which are markers of skin inflammation and wrinkle formation, in ultraviolet B (UVB)­irradiated human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). No toxicity was observed upon treatment of HDFs and HaCaT cells with 0.5­2.5 µl/ml 7­MEGA. The exposure of HaCaT cells to 10 mJ/cm2 UVB for 6 h resulted in increased protein and/or mRNA expression of COX­2 and MMP­3. Treatment of HaCaT cells with 2.5 µl/ml 7­MEGA suppressed the UVB­induced expression of COX­2 and MMP­3 in these cells. In addition, treatment with 2.5 µl/ml 7­MEGA attenuated the UVB­induced expression and phosphorylation levels of c­Fos and c­Jun, two components of the activator protein­1 (AP­1) transcription factor, in HaCaT cells. Exposure of HDFs to 60 mJ/cm2 UVB for 6 h significantly decreased the expression of type 1 procollagen protein, whereas treatment with 2.5 µl/ml 7­MEGA partially reversed the effects of UVB on the expression of type 1 procollagen protein. These results demonstrated for the first time that 7­MEGA regulated the expression of COX­2, MMP­3 and type 1 procollagen in UVB­irradiated skin cells. The present study suggested that 7­MEGA may serve as a novel agent against UVB­induced skin inflammation and damage.

Danshen Attenuates Intervertebral Disc Degeneration via Antioxidation in SD Rats.

OBJECTIVE: To investigate the effects of Danshen on the imaging and histological parameters, expression levels of ECM-associated proteins and inflammatory factors, and antioxidative activity in the degenerated intervertebral disc (IVD) of SD rats. METHODS: Sixty male rats were randomly divided into three groups (control, IDD, and Danshen IDD). Percutaneous needle puncture in Co8-9 intervertebral disc was conducted in all rats of the IDD and Danshen IDD groups to induce intervertebral disc degeneration (IDD). After operation, animals of the Danshen IDD group were administrated with Danshen granules (3 g/kg body weight ) by gavage once a day. Four weeks later, the coccygeal vertebrae were harvested and used for imaging (disc height and MR signal), histological, immunohistochemical, and biochemical [water content, glycosaminoglycans (GAG), superoxide dismutase (SOD2), glutathione (GSH), and malondialdehyde (MDA)] analyses. RESULTS: The puncture induced significant decreased IVD space and MR T2 signal at both 2 and 4 weeks, which were attenuated by Danshen treatment. The disc degeneration in the IDD group (HE and Safranin O-Fast Green histological staining was markedly more serious compared with that in the control group. Four weeks of Danshen treatment significantly alleviated this degeneration compared with the IDD group. Needle puncture resulted in the upregulation of IL-1ß and TNF-α, MMP-3, and downregulation of COL2 and aggrecan in the IDD group. However, this change was significantly weakened by Danshen treatment. Significantly lower water and GAG content, as well as the SOD2 and GSH levels, in the IDD group were found compared with those in the control group. However, the above parameters of the Danshen IDD group were significantly higher than those of the IDD group. Danshen treatment significantly decreased the content of MDA which was increased by needle puncture in the IDD group. CONCLUSION: Danshen can attenuate intervertebral disc degeneration in SD rats by suppressing the oxidation reaction.

Leonurine Hydrochloride Suppresses Inflammatory Responses and Ameliorates Cartilage Degradation in Osteoarthritis via NF-κB Signaling Pathway.

Leonurine hydrochloride (LH) has been reported to exhibit a number of biological properties such as suppression of inflammation. This study aimed to examine whether the progression of osteoarthritis (OA) could be delayed by the administration of LH in an OA model. Rat chondrocytes were treated with LH under the condition of TNF-α-induced inflammation. After that, real-time PCR and Western blotting were conducted to evaluate relative gene/protein expression levels. For the in vivo study, rats were randomly allocated to a control group (anterior cruciate ligament transection (ACLT) surgery, treatment with saline) and LH group (ACLT surgery, treatment with LH). Articular cartilage degeneration was assessed by histological evaluation. It was found that LH significantly suppressed the expression of MMP-1, MMP-3, MMP-13, IL-6, and ADAMTS-5 in cells via the NF-κB signaling pathway. In addition, it is revealed that intra-articular injection of LH significantly ameliorated cartilage degeneration in a rat OA model. Taken together, these results indicate that LH attenuates progression of OA by inhibition of inflammation via the NF-κB signaling pathway and represents a potential preventive therapy for OA.

Heparanase-Regulated Syndecan-1 Shedding Facilitates Herpes Simplex Virus 1 Egress.

Herpes simplex virus 1 (HSV-1) can infect virtually all cell types in vitro An important reason lies in its ability to exploit heparan sulfate (HS) for attachment to cells. HS is a ubiquitous glycosaminoglycan located on the cell surface and tethered to proteoglycans such as syndecan-1. Previously, we have shown that heparanase (HPSE) facilitates the release of viral particles by cleaving HS. Here, we demonstrate that HPSE is a master regulator where, in addition to directly enabling viral release via HS removal, it also facilitates cleavage of HS-containing ectodomains of syndecan-1, thereby further enhancing HSV-1 egress from infected cells. Syndecan-1 cleavage is mediated by upregulation of matrix metalloproteases (MMPs) that accompanies higher HPSE expression in infected cells. By overexpressing HPSE, we have identified MMP-3 and MMP-7 as important sheddases of syndecan-1 shedding in corneal epithelial cells, which are natural targets of HSV-1 infection. MMP-3 and MMP-7 were also naturally upregulated during HSV-1 infection. Altogether, this paper shows a new connection between HSV-1 release and syndecan-1 shedding, a phenomenon that is regulated by HPSE and executed by the MMPs. Our results also identify new molecular markers for HSV-1 infection and new targets for future interventions.IMPORTANCE HSV-1 is a common cause of recurrent viral infections in humans. The virus can cause a range of mucosal pathologies. Efficient viral egress from infected cells is an important step for HSV-1 transmission and virus-associated pathologies. Host mechanisms that contribute to HSV-1 egress from infected cells are poorly understood. Syndecan-1 is a common heparan sulfate proteoglycan expressed by many natural target cells. Despite its known connection with heparanase, a recently identified mediator of HSV-1 release, syndecan-1 has not been previously investigated in HSV-1 release. In this study, we demonstrate that the shedding of syndecan-1 by MMP-3 and MMP-7 supports viral egress. We show that the mechanism behind the activation of these MMPs is mediated by heparanase, which is upregulated upon HSV-1 infection. Our study elucidates a new connection between HSV-1 egress, heparanase, and matrix metallopeptidases; identifies new molecular markers of infection; and provides potential new targets for therapeutic interventions.

Dexmedetomidine exerts dual effects on human annulus fibrosus chondrocytes depending on the oxidative stress status.

Dexmedetomidine (Dex) is an anesthetic widely used in lumbar discectomy, but its effect on chondrocytes remains unclear. Dex is speculated to promote cartilage degeneration by activating α-2 adrenergic receptor. However, the antioxidative and anti-inflammatory effects of Dex implied the potential chondrocyte protective effect under stress conditions. The present study aimed to determine the effect of Dex on chondrocytes under non-stress and stress conditions. Chondrocytes were isolated from human annulus fibrosus (AF) tissues and oxidative stress was induced by treatment with 1 mM hydrogen peroxide (H2O2). Chondrocytes were treated with Dex alone or in combination with H2O2 Treatment with Dex alone decreased mRNA expression of COL2A1 and increased that of MMP-3 and MMP-13, thus contributing to cartilage degeneration. However, Dex prevented H2O2-induced death and degeneration of chondrocytes partly by enhancing antioxidant capacity. Mechanistically, Dex attenuated H2O2-mediated activation of NF-κB and NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), both of which play key roles in inflammation and inflammatory damage. Dex inactivated NLRP3 through the suppression of NF-κB and JNK signals. Co-treatment with Dex and H2O2 increased protein level of XIAP (X-linked inhibitor-of-apoptosis, an anti-apoptosis protein), compared with H2O2 treatment alone. H2O2 treatment increased the expression of neural precursor cell expressed developmentally down-regulated protein 4 (NEDD4) that is a ubiquitin ligase targeting XIAP. However, Dex decreased the amount of NEDD4 adhering to XIAP, thus protecting XIAP protein from NEDD4-mediated ubiquitination and degradation. Given that surgery inevitably causes oxidative stress and inflammation, the protective effect of Dex on chondrocytes during oxidative stress is noteworthy and warrants further study.

Vitexin alleviates interleukin-1ß-induced inflammatory responses in chondrocytes from osteoarthritis patients: Involvement of HIF-1α pathway.

It has been reported that vitexin has anti-inflammatory effects in osteoarthritis (OA) rats. However, the effects of vitexin on interleukins-1ß (IL-1ß)-stimulated OA patient-derived chondrocytes have not been reported. The purpose of this study was to investigate the anti-inflammatory effects of vitexin on IL-1ß-stimulated human osteoarthritis chondrocytes and to reveal the involvement of hypoxia-inducible factor 1α (HIF-1α) pathway. Enzyme-linked immunosorbent assay, quantitative real-time PCR and Western blotting assays were employed. ELISA results demonstrated that the proinflammatory cytokine levels of interleukins-6 (IL-6) and tumour necrosis factor α (TNF-α) in the serum and synovial fluid and HIF-1α level in the synovial fluid were significantly elevated in OA patients compared to normal healthy subjects. Moreover, the Western blotting results indicated that the protein expression of HIF-1α was significantly higher in the cartilage tissues of OA patients. OA patient-derived chondrocytes were stimulated by IL-1ß and treated with different concentration of vitexin for 24 hours. Vitexin showed no cytotoxicity and increased the survival of chondrocytes under IL-1ß stimulation. Vitexin suppressed IL-1ß-induced production of NO and prostaglandin E2 (PGE2 ) in chondrocytes culture. The treatment of vitexin significantly inhibited IL-1ß-induced expressions of proinflammatory cytokine levels of IL-6, TNF-α, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13. Furthermore, Western blotting results demonstrated that HIF-1α is involved in vitexin's protective effects on IL-1ß-stimulated injuries in OA patient-derived chondrocytes. Our study demonstrates that vitexin alleviates IL-1ß-induced inflammatory responses in chondrocytes from osteoarthritis patients, which may be attributed partly to the inhibition of HIF-1α pathway.

Synergistic Role of Oxidative Stress and Blood-Brain Barrier Permeability as Injury Mechanisms in the Acute Pathophysiology of Blast-induced Neurotrauma.

Blast-induced traumatic brain injury (bTBI) has been recognized as the common mode of neurotrauma amongst military and civilian personnel due to an increased insurgent activity domestically and abroad. Previous studies from our laboratory have identified enhanced blood-brain barrier (BBB) permeability as a significant, sub-acute (four hours post-blast) pathological change in bTBI. We also found that NADPH oxidase (NOX)-mediated oxidative stress occurs at the same time post-blast when the BBB permeability changes. We therefore hypothesized that oxidative stress is a major causative factor in the BBB breakdown in the sub-acute stages. This work therefore examined the role of NOX1 and its downstream effects on BBB permeability in the frontal cortex (a region previously shown to be the most vulnerable) immediately and four hours post-blast exposure. Rats were injured by primary blast waves in a compressed gas-driven shock tube at 180 kPa and the BBB integrity was assessed by extravasation of Evans blue and changes in tight junction proteins (TJPs) as well as translocation of macromolecules from blood to brain and vice versa. NOX1 abundance was also assessed in neurovascular endothelial cells. Blast injury resulted in increased extravasation and reduced levels of TJPs in tissues consistent with our previous observations. NOX1 levels were significantly increased in endothelial cells followed by increased superoxide production within 4 hours of blast. Blast injury also increased the levels/activation of matrix metalloproteinase 3 and 9. To test the role of oxidative stress, rats were administered apocynin, which is known to inhibit the assembly of NOX subunits and arrests its function. We found apocynin completely inhibited dye extravasation as well as restored TJP levels to that of controls and reduced matrix metalloproteinase activation in the sub-acute stages following blast. Together these data strongly suggest that NOX-mediated oxidative stress contributes to enhanced BBB permeability in bTBI through a pathway involving increased matrix metalloproteinase activation.

Sudachitin Inhibits Matrix Metalloproteinase-1 and -3 Production in Tumor Necrosis Factor-α-Stimulated Human Periodontal Ligament Cells.

Sudachitin, a polymethoxylated flavonoid found in the skin of Citrus sudachi, is a biologically active substance. The aim of this study was to examine whether sudachitin could be used to inhibit the expression of matrix metalloproteinase (MMP)-1 and MMP-3, which are involved in the destruction of periodontal tissues in periodontal lesions, in tumor necrosis factor (TNF)-α-stimulated human periodontal ligament cells (HPDLC). Sudachitin suppressed TNF-α-induced MMP-1 and MMP-3 production in HPDLC. On the other hand, it enhanced tissue inhibitor of metalloproteinase (TIMP)-1 expression. The level of Akt phosphorylation in the TNF-α-stimulated HPDLC was decreased by sudachitin treatment. Moreover, an Akt inhibitor reduced MMP-1 and MMP-3 production and increased TIMP-1 production. These findings indicate that sudachitin reduces MMP-1 and MMP-3 production in TNF-α-stimulated HPDLC by inhibiting the Akt pathway.

Curcumin Inhibits Proliferation of Synovial Cells by Downregulating Expression of Matrix Metalloproteinase-3 in Osteoarthritis.

OBJECTIVE: To investigate the association between curcumin and the differentially expressed genes (DEG) in synovial tissues of osteoarthritis. METHODS: Microarray analysis was used to screen for the DEG in osteoarthritis synovial cells. Curcumin-related genes were identified through the drug-gene interaction network STITCH (http://stitch.embl.de/cgi/input.pl). Expression levels of fibronectin 1 (FN1) and collagen III protein were measured by western blot. MTT assay was used to examine the effects of different concentrations of curcumin on cell viability. Western blot and quantitative real-time polymerase chain reaction were used to validate the different expression levels of matrix metalloproteinase-3 (MMP3). Clone formation assay, flow cytometry, and the TUNEL method were conducted for detecting the cell proliferation and apoptosis rate. RESULTS: In the two chips of GSE1919 and GSE55235, the average expression of MMP3 in the osteoarthritis group was 63.7% and 12.9% higher than that of the healthy control, respectively. The results of western blot also showed that the average expression of MMP3 in 30 osteoarthritis patients was 132% higher than that of the healthy group, which confirmed that MMP3 was highly expressed in osteoarthritis group. The results of MTT showed that at 72 h, the cell viability of 40 µmol/L curcumin was the lowest and 79.6% lower than for the 0 µmol/L group, so the final curcumin concentration of 40 µmol/L was selected for subsequent experiments. Western blot results further showed that the expression of MMP3 was 44% lower in the untreated groups compared with the curcumin group, and the expressions of FN1 and collagen III were increased by 112% and 84%, respectively, which indicated that curcumin inhibited MMP3 expression and decreased osteoarthritis synovial cell activity. Cloning formation experiments showed that cell numbers increased by 75% and 20.5% in untreated and curcumin groups, and compared with the untreated group, the cells in the curcumin group decreased by 30.8%. Flow cytometry showed that the apoptotic rate in the curcumin group increased by 85.1% compared with the untreated group, but for a single group, MMP3 decreased the apoptotic rate by 53.9% and 46.7%, respectively. CONCLUSIONS: MMP3 was highly expressed in osteoarthritis synovial cells. Curcumin could reduce cell viability, inhibit cell proliferation, increase cell apoptosis, and eventually alleviate inflammation of osteoarthritis by inhibiting the expression of MMP3.

Effect of Static Compression Loads on Intervertebral Disc: An in Vivo Bent Rat Tail Model.

OBJECTIVE: To evaluate how well different magnitudes of compression-induced degenerative changes using a bent rat tail model simulated human lumbar lordosis. It has been shown that compression plays an important role in intervertebral disc degeneration (IDD). METHODS: Sprague-Dawley rats (n = 25) were instrumented with a special compressive apparatus that was used to bend the intervertebral disc between the 8th and the 10th caudal vertebral bodies using two Kirschner wires inserted percutaneously into the middle of two tail vertebrae. Then, rats were divided into five different static compression loads (control, sham, 1.8 N, 4.5 N, and 7.2 N). The degeneration of the discs was evaluated by magnetic resonance imaging (MRI), histology, gene expression of anabolism and catabolism after 2 weeks. We used the signal characteristics of the disc in T2-weighted MRI to reflect the changes caused by degeneration as this is the most relevant and clinically recognized way to assess IDD. Pfirrmann classification was used to classify disc images. The tail discs from C8-9 and C9-10 with their two adjacent half vertebrae were carefully cut out and decalcified. Then the sections were paraffin-embedded and cut into 5-µm sections by histotome. Finally, they were stained with Safranin O-Fast Green and hematoxylin, and hematoxylin and eosin, respectively. Images were taken using a microscope and staining and compression-induced changes were assessed by a Masuda's grading scale. The relative expression levels of mRNA encoding rat anabolic genes and catabolic genes were evaluated by real-time reverse transcription (RT)-polymerase chain reaction (PCR). The mRNA expression fold change of the target gene was calculated using the 2-ΔΔCt method in the loaded and unloaded disc. RESULTS: As the loading magnitude increased, static compression produced a significantly progressive decrease in nucleus intensity on T2-weighted MRI, a decrease of aggrecan and Type II collagen, an increase in Matrix metallopeptidase-3 (MMP-3) and MMP-13 expressions, and a histomorphological degeneration. The sham group had a score of 1.4 ± 0.3, the 1.8 N group had a score of 2.4 ± 0.3, the 4.5 N group had a score of 3.2 ± 0.3, and the 7.2 N group had a score of 4.4 ± 0.3, which was based on the Pfirrmann classification score, in which the control group had a score of 1. These results demonstrated that the sham group was not significantly different from the control group. Histological analysis showed that in the loaded disc, the size of the nucleus was reduced and that the annular layer was disorganized. Based on the Masuda grading scale, scores were as follows: for the control group, 3.8 ± 0.35; sham, 4.2 ± 0.35; 1.8 N, 5.4 ± 0.35; 4.5 N, 7.6 ± 0.35; and 7.2 N, 10 ± 0.35. The gene expression was divided into the following: anabolic genes (aggrecan, collagen type1-α1, and collagen type2-α1) and catabolic genes (MMP-3 and MMP-13). Aggrecan and collagen type 2 were, respectively, downregulated from 0.42 ± 0.04 to 0.21 ± 0.04 and from 0.93 ± 0.06 to 0.17 ± 0.06 as the magnitude of compression increased, whereas collagen type 1 was significantly upregulated, from 2.49 ± 0.19 to 4.40 ± 0.19, when compared with the control group (from 1.8 to 7.2 N, P < 0.05). Catabolic genes MMP-3 and MMP-13 were significantly upregulated in all experimental groups (P < 0.05, MMP-3: from 1.46 ± 0.18 to 3.44 ± 0.18; MMP-13: from 1.19 ± 0.12 to 2.82 ± 0.13); however, MMP-13 exhibited no significant changes but tended to be upregulated when compared with the 1.8 N group with the 4.5 N group. CONCLUSIONS: Different stresses led to different processes of degenerative changes, the concave disc degenerating more severely as stress gradually increased.

Celastrol inhibits colorectal cancer cell proliferation and migration through suppression of MMP3 and MMP7 by the PI3K/AKT signaling pathway.

Colorectal cancer (CRC) is one of the most frequent malignant tumors. Signaling by the PI3K/AKT pathway is crucial for CRC development and progression, including proliferation and migration. Celastrol has an anticancer effect, but its mechanism needs to be determined. Here, we showed that celastrol suppressed CRC cell proliferation and migration. Celastrol treatment also decreased the PI3K/AKT pathway components, and MMP3 and MMP7 expression levels. In addition, knockdown of AKT, not mTOR, inhibited MMP3 and MMP7 expression levels and AKT silencing promoted the celastrol-induced effects on CRC cell proliferation and migration. Taken together, these findings indicated that the celastrol-induced antitumor effects were mediated through MMP3 and MMP7 by the PI3K/AKT signaling pathway.

Synergistic effect of l-ascorbic acid and hyaluronic acid on the expressions of matrix metalloproteinase-3 and -9 in human chondrocytes.

Proinflammatory cytokines and reactive oxygen species (ROS) are known to be involved in the initiation and progression of osteoarthritis (OA). New evidence clarifying the correlation between ROS and inflammation has indicated that oxidative stress can up-regulate inflammatory cytokines. l-Ascorbic acid (AA), an antioxidant, has been shown to have anti-inflammatory effects and improve matrix deposition in chondrocytes. The purpose of this study was to examine the effects of hyaluronic acid (HA; 100 µg/mL) supplemented with AA (50 µg/mL) on human normal and interleukin-1 beta-stimulated (IL-1ß, 10 ng/mL) chondrocytes. HA, AA, and HA + AA treatment did not change cell morphology, viability, proliferation, and glycosaminoglycan production in normal chondrocytes. HA, AA, and HA + AA, by contrast, partially restored viability and morphology of hypertrophic chondrocytes, and HA and HA + AA further decreased the cytotoxicity of IL-1ß. Real-time PCR revealed that AA and HA + AA had no substantial effects on unstimulated chondrocytes, except for down-regulation of matrix metalloproteinase (MMP)-9 mRNA levels. For IL-1ß-stimulated chondrocytes, significant down-regulation of IL-1ß, tumor necrosis factor-alpha (TNF-α), MMP-3, and MMP-9 mRNA expression was found when cells were cultured in HA-supplemented media. Moreover, HA + AA supplementation further significantly decreased MMP-3 and MMP-9 mRNA expression. The protein production of MMP-3 was decreased, with a significant difference between the HA + AA group and HA group. The antioxidant capacity and superoxide dismutases activity were also partially restored in stimulated chondrocytes. HA supplemented with AA modulates MMPs expression and antioxidant fuction in chondrocytes. AA may enhance the anticatabolic effects of HA on OA chondrocytes. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1809-1817, 2018.

The inhibitory effect of Aconiti Sinomontani Radix extracts on the proliferation and migration of human synovial fibroblast cell line SW982.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconiti Sinomontani Radix is frequently used in the treatment of Bi syndrome in traditional Chinese medicine. Several reports indicate that Aconiti Sinomontani Radix has therapeutic effects for rheumatoid arthritis (RA). However, the cellular mode of action is still unclear. To investigate the effect of alkaloid extracts of Aconiti Sinomontani Radix on proliferation and migration of human synovial sarcoma SW982 cells as well as the molecular mechanism underlying. MATERIALS AND METHODS: SW982 cells were examined for proliferation by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method. Wound scratch assays were performed to assess the migrated rate of SW982 cells. Quantitative real-time PCR was used to measure the mRNA expression levels of Wnt5a, Runx2, MMP3, and Bmp2. Western blotting was used to measure the phosphorylated levels of JNK and NF-κB as well as the expression of MMP3. RESULTS: The alkaloid extract from Aconiti Sinomontani Radix (MQA) and MQB, which removed lappaconitine from MQA significantly inhibited the proliferation of SW982 in a dose-dependent manner. The proliferation inhibitory effect of MQB was more potent. Incubation with 10µg/ml MQB for 12, 24, and 36h inhibited the migration of SW982 cells by 83%, 58%, and 42%, respectively. Treatment with different concentrations of MQB for 24h inhibited mRNA expression of Wnt5a, Runx2, and MMP3, but Bmp2 mRNA expression was elevated by MQB. Further, MQB inhibited phosphorylation of JNK and NF-κB p65 as well as MMP3 expression by Western blotting analysis. CONCLUSION: The results showed that MQB inhibited proliferation and migration of SW982 cells possibly through suppressing Wnt5a-mediated JNK and NF-κB pathways. These results indicated that MQB might be an active extract of Aconiti Sinomontani Radix for targeting fibroblast-like synoviocytes (FLS) and be potential for RA therapy.

BMAL1 Deficiency Contributes to Mandibular Dysplasia by Upregulating MMP3.

Skeletal mandibular hypoplasia (SMH), one of the common types of craniofacial deformities, seriously affects appearance, chewing, pronunciation, and breathing. Moreover, SMH is prone to inducing obstructive sleep apnea syndrome. We found that brain and muscle ARNT-like 1 (BMAL1), the core component of the molecular circadian oscillator, was significantly decreased in mandibles of juvenile SMH patients. Accordingly, SMH was observed in circadian-rhythm-disrupted or BMAL1-deficient mice. RNA sequencing and protein chip analyses suggested that matrix metallopeptidase 3 (MMP3) is the potential target of BMAL1. Interestingly, in juvenile SMH patients, we observed that MMP3 was obviously increased. Consistently, MMP3 was upregulated during the whole growth period of 3-10 weeks in Bmal1-/- mice. Given these findings, we set out to characterize the underlying mechanism and found BMAL1 deficiency enhanced Mmp3 transcription through activating p65 phosphorylation. Together, our results provide insight into the mechanism by which BMAL1 is implicated in the pathogenesis of SMH.

Interleukin-17 regulates matrix metalloproteinase activity in human pulmonary tuberculosis.

Tuberculosis (TB) is characterized by extensive pulmonary matrix breakdown. Interleukin-17 (IL-17) is key in host defence in TB but its role in TB-driven tissue damage is unknown. We investigated the hypothesis that respiratory stromal cell matrix metalloproteinase (MMP) production in TB is regulated by T-helper 17 (TH -17) cytokines. Biopsies of patients with pulmonary TB were analysed by immunohistochemistry (IHC), and patient bronchoalveolar lavage fluid (BALF) MMP and cytokine concentrations were measured by Luminex assays. Primary human airway epithelial cells were stimulated with conditioned medium from human monocytes infected with Mycobacterium tuberculosis (Mtb) and TH -17 cytokines. MMP secretion, activity, and gene expression were determined by ELISA, Luminex assay, zymography, RT-qPCR, and dual luciferase reporter assays. Signalling pathways were examined using phospho-western analysis and siRNA. IL-17 is expressed in TB patient granulomas and MMP-3 is expressed in adjacent pulmonary epithelial cells. IL-17 had a divergent, concentration-dependent effect on MMP secretion, increasing epithelial secretion of MMP-3 (p < 0.001) over 72 h, whilst decreasing that of MMP-9 (p < 0.0001); mRNA levels were similarly affected. Both IL-17 and IL-22 increased fibroblast Mtb-dependent MMP-3 secretion but IL-22 did not modulate epithelial MMP-3 expression. Both IL-17 and IL-22, but not IL-23, were significantly up-regulated in BALF from TB patients. IL-17-driven MMP-3 was dependent on p38 MAP kinase and the PI3K p110α subunit. In summary, IL-17 drives airway stromal cell-derived MMP-3, a mediator of tissue destruction in TB, alone and with monocyte-dependent networks in TB. This is regulated by p38 MAP kinase and PI3K pathways. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Exploration about changes of IL-10, NF-κB and MMP-3 in a rat model of cervical spondylosis.

OBJECTIVES: To investigate the relationship and mechanism between IL-10, NF-κB and MMP-3 in cervical degenerative disease induced by unbalanced dynamic and static forces in rats. METHODS: Sixty Sprague Dawley rats were randomized into test (n=45) and control (n=15) groups, which were randomly subdivided into three groups corresponding to one-month, three-month and six-month post-operation. Test group included 10, 15, 20 rats at corresponding postoperative stage and control group had five rats at each time point. By excising cervicodorsal muscles and ligaments of rats to establish unbalanced dynamic and static rat model in test group. The expression of IL-10, NF-κB and MMP-3 in the intervertebral disc tissue samples of both test and control group rats were detected by immunohistochemistry at one-month, three-month and six-month post-operation. The results were analyzed and compared among groups. RESULTS: Compared with the control group, the positive expression of IL-10 in test group was significantly higher at three-month (P<0.05). In the same model group, IL-10 was highest at one-month. Compared with the control group, NF-kB in test group was higher at one-month, three-month and six-month. In the same model group, NF-kB was the highest at one-month, followed by the time at three-month and six-month. And, compared with the control group, MMP-3 was significantly higher in test group at one-month (P<0.05). CONCLUSION: Cervical degeneration may accompanied with the changes of IL-10, NF-κB and MMP-3.