Identification of anti-photoaging components of Olea europaea leaves based on spectrum-effect relationship.

In this study, to identify bioactive components of Olea europaea leaves extract (OLE), chemometrics analyses including bivariate correlation analysis and partial least squares regression were used to establish the relationships between the chromatograms and anti-photoaging effect of OLE samples. Firstly, the fingerprint of olive leaves extract was determined by high-performance liquid chromatography (HPLC). Photoaging models of HaCaT cells were established by UVB irradiation. The photoaging resistance of OLE was evaluated by cell viability using the MTT assay. Chemometrics analyses showed that compounds 14, 19, 20, 24, 26, and 28 might be the major anti-photoaging components of OLE. Furthermore, after separation by HSCCC and NMR identification, compound 19 is luteoloside and compound 24 is oleuropein. Oleuropein and luteoloside were docked with collagenase (MMP-1), stromelysin (MMP-3), and gelatinase (MMP-9), respectively. The results showed that oleuropein and luteoloside inhibited their activity by directly interacting with MMP-1, MMP-3, and MMP-9, thereby exhibiting anti-photoaging activity. The current bioassay and spectrum-effect relationships are proper for associating sample quality with the active ingredient, and our finding would provide foundation and further understanding of the quality evaluation and quality control of Olea europaea.

Effects of metformin on human gingival fibroblasts: an in vitro study.

OBJECTIVE: To investigate the effects of metformin (MF) treatment on the matrix metalloproteinases (MMPs) and proinflammatory cytokines production from lipopolysaccharide (LPS) - stimulated human gingival fibroblasts (HGFs). METHODS: HGFs were obtained from subcultures of biopsies from clinically healthy gingival tissues of patients undergoing oral surgeries. Cell cytotoxicity assay was used to determine the effect of different concentrations of MF on viability of HGFs. HGFs were then incubated and treated with different concentrations of MF and Porphyromonas gingivais (Pg) LPS. MMP-1, MMP-2, MMP-8, MMP-9, IL-1ß, and IL-8 expression analysis was performed using xMAP technology (Luminex 200, Luminex, Austin, TX, USA). Student's t-test for a single sample was used to compare the mean values of the study groups with the control value. A p-value of <0.05 and 95% confidence intervals were used to report the statistical significance and precision of mean values. RESULTS: Concentrations of 0.5, 1- and 2-mM MF had a minimal non-significant cytotoxic effect on the HGFs and caused statistically significant reduction of MMP-1, MMP-2, MMP-8 and IL-8 expressed by the LPS-stimulated HGFs. CONCLUSION: The results of the present study confirm that MF suppresses MMP-1, MMP-2, MMP-8 and IL-8 in LPS-stimulated HGFs suggesting an anti-inflammatory effect of MF and potential adjunct therapeutic role in the treatment of periodontal diseases.

Protective effects of Capsicum fruits and their constituents on damage in TNF-α-stimulated human dermal fibroblasts.

BACKGROUND: Antioxidant and anti-inflammatory effects of natural products on skin cells have been proved to be effective in improving skin damage. Capsicum species contain capsaicinoids that have antioxidant and anti-inflammatory properties, and various subspecies are cultivated. In this study, the effects of four Capsicum fruits and major constituents on oxidative stress and inflammatory reactions were measured using human dermal fibroblasts (HDFs) to verify their effects on skin damage. RESULTS: The inhibitory effects of nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E2 (PGE2 ) by cucumber hot pepper, red pepper (RDP), Shishito pepper (SSP), and Cheongyang pepper were determined in HDFs. RDP and SSP inhibited the production of NO, ROS, and PGE2 in tumor necrosis factor-alpha-stimulated HDFs. Additionally, SSP seeds restored tumor necrosis factor-alpha-induced increase in matrix metalloproteinase-1 and decreased procollagen I α1 (COLIA1). In high-performance liquid chromatography analysis of the capsaicinoids capsaicin (CAP) and dihydrocapsaicin (DHC), CAP was detected at a higher level than DHC in the peel and seeds of all four types of Capsicum fruits, and the total amount of capsaicinoids was the highest in SSP. CAP and DHC, which are major constituents of Capsicum fruits, also inhibited NO, ROS, and PGE2 and restored matrix metalloproteinase-1 and procollagen I α1. CONCLUSION: RDP and SSP were shown to have a significant protective effect on skin damage, including oxidative stress, inflammatory reactions, and reduction of collagens. Capsaicinoids CAP and DHC were proved as active constituents. This research may provide basic data for developing Capsicum fruits as ingredients to improve skin damage, such as inflammation and skin aging. © 2022 Society of Chemical Industry.

Genetic bases for the metabolism of the DMS precursor S-methylmethionine by Saccharomyces cerevisiae.

Dimethyl sulfide (DMS) is a sulfur containing volatile that enhances general fruity aroma and imparts aromatic notes in wine. The most important precursor of DMS is S-methylmethionine (SMM), which is synthesized by grapes and can be metabolized by the yeast S. cerevisiae during wine fermentation. Precursor molecules left after fermentation are chemically converted to DMS during wine maturation, meaning that wine DMS levels are determined by the amount of remaining precursors at bottling. To elucidate SMM metabolism in yeast we performed quantitative trait locus (QTL) mapping using a population of 130 F2-segregants obtained from a cross between two wine yeast strains, and we detected one major QTL explaining almost 30% of trait variation. Within the QTL, gene YLL058W and SMM transporter gene MMP1 were found to influence SMM metabolism, from which MMP1 has the bigger impact. We identified and characterized a variant coding for a truncated transporter with superior SMM preserving attributes. A population analysis with 85 yeast strains from different origins revealed a significant association of the variant to flor strains and minor occurrence in cheese and wine strains. These results will help selecting and improving S. cerevisiae strains for the production of wine and other fermented foods containing DMS such as cheese or beer.

Maternal omega-3 polyunsaturated fatty acids supplementation in pregnancy decreases MMP-1 levels in breastmilk: a cross-sectional study.

INTRODUCTION: Anti-inflammatory properties of fish-oil are well known and suggested during pregnancy. MMP-1 is involved in inflammation and tissue remodelling. There have been studies focused on anti-inflammatory effect of maternal omega use on human milk while little is known about the effect of omega use on breastmilk proteases. Leptin is an important hormone that influences MMP levels in various tissues and exerts its metabolic effects. In our study we assessed the levels of MMP-1, TIMP-1, leptin, IL-6 and FA's including PUFA in breastmilk from women who used omega-3. MATERIALS AND METHODS: Our study was a cross-sectional study included 67(Group 1, n = 32, omega user; Group 2 n = 35, non-user)lactating women and their infan MMP-1, TIMP-1, leptin, IL-6 and FA's were evaluated in breastmilk of both groups. MMP-1, TIMP-1, IL-6 and leptin were measured by enzyme-linked immunoabsorbent assay (ELISA) method. Breastmilk fatty acids were measured by gas chromatography flame ionisation detector (GC-FID). RESULTS: Matrix metalloproteinase-1 (MMP-1) levels in breastmilk were significantly lower in breastmilk from omega users (mean ± SD, 0.455 ± 0.1) than non-users (mean ± SD, 0.677 ± 0.289) (p=.0001). MMP-1 and omega 6:3 ratio were positively correlated (r: 0.301, p=.01). MMP levels were correlated with IL-6 (Pearson's r: 0.411, p<.001). MMP-1 and leptin levels were positively correlated (r: .388, p=.001). CONCLUSION: MMP-1 levels in breastmilk, may be modified by maternal omega use in pregnancy which may help to redirect extracellular matrix remodelling and metabolic programming in early infancy.

Spatial expression of metallothionein, matrix metalloproteinase-1 and Ki-67 in human epidermal wounds treated with zinc and determined by quantitative immunohistochemistry: A randomised double-blind trial.

Reepithelialisation is fundamental to wound healing, but our current understanding largely relies on cellular and animal studies. The aim of the present randomised double-blind three-arm controlled trial was to correlate genuine epidermal wound healing with key proteins and topical zinc treatment in humans. Sixty wounds were produced using deroofed suction blisters in 30 healthy volunteers and randomised to topical zinc sulphate (n = 20), placebo (n = 20), or control (n = 20) treatment for 4 days. All wounds with perilesional skin were processed for automatic immunostaining of paraffin tissue sections with monoclonal antibodies against Ki-67, metallothionein (MT) and matrix metalloproteinase (MMP)-1. Protein expression was quantified by automated digital image analysis. Epidermal Ki-67 and MT labelling indices were increased in keratinocytes in the neoepidermis (∼1.1 mm) and at the wound edge (0.5 mm) compared to normal skin. Increased MMP-1 immunostaining was restricted to the neoepidermis. MT was robustly upregulated in the upper dermis of the wounds. Zinc treatment enhanced MMP-1 expression beneath the neoepidermis via paracrine mechanisms and MT under the neoepidermis and in the nonepithelialised wound bed via direct actions of zinc as indicated by the induction of MT2A mRNA but not MMP-1 mRNA in cultured normal human dermal fibroblasts by zinc sulphate. The present human study demonstrates that quantitative immunohistochemistry can identify proteins involved in reepithelialisation and actions of external compounds. Increased dermal MT expression may contribute to the anti-inflammatory activities of zinc and increased MMP-1 levels to promote keratinocyte migration.

Novel histopathological classification of meningiomas based on dural invasion.

AIMS: Histological invasion into the adjacent brain parenchyma is frequently investigated in meningioma because it is an important morphological criterion for grade II meningioma according to the 2016 WHO classification. However, few studies have focused on dural invasion of meningiomas. Herein, we propose a novel histopathological classification based on dural invasion of meningiomas. METHODS: Forty-nine cases with WHO grade I meningiomas who underwent Simpson grade I removal were collected. After the meningeal layer (ML) and periosteal layer (PL) of dura mater were visualised by Masson's trichrome stain, we evaluated the depth (to the ML and PL) and the patterns (1, expanding; 2, infiltrating) of dural invasion of meningiomas using serial paraffin sections. Invasion-associated markers, including Ki-67, matrix metalloproteinase (MMP)-1, MMP-9 and MMP-13, aquaporin 1 and Na-K-2Cl cotransporter, were quantitatively analysed by immunohistochemistry. RESULTS: Thirty-five cases (71.4%) showed the dural invasion. In 27 of these 35 cases (77.1%), dural invasion was localised in ML. Type 1 (expanding type) and type 2 (infiltrating type) invasions were observed in 23 and 12 cases, respectively. The recurrence rate in cases with type 2 invasion was significantly higher than that in cases with type 1 invasion. The percentage of MMP-1-positive tumour cells was also significantly higher in cases with dural invasion than those without, suggesting involvement of MMP-1 in dural invasion. CONCLUSIONS: We quantitatively evaluated the depth and patterns of dural invasion in meningiomas. The patterns of dural invasion were associated with meningioma recurrence.

CXCL17-mediated downregulation of type I collagen via MMP1 and miR-29 in skin fibroblasts possibly contributes to the fibrosis in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by excessive deposition of collagen in the skin and internal organs. Recent studies have shown that chemokine (C-X-C motif) ligands (CXCLs) are involved in the pathogenesis of SSc. OBJECTIVE: Our aim was to examine the anti-fibrotic potential of CXCL17, a newly discovered chemokine, in cultured skin fibroblasts and in a bleomycin-induced SSc mouse model. Moreover, we examined serum level of CXCL17 in patients with SSc. METHODS: Type I collagen expression was evaluated in SSc skin and cultured fibroblasts treated with CXCL17 using immunoblotting and quantitative reverse transcription-PCR. Serum CXCL17 levels were determined using enzyme-linked immunosorbent assay in 63 patients with SSc and 17 healthy subjects. A bleomycin-induced SSc mouse model was used to evaluate the effect of CXCL17 on skin fibrosis. RESULTS: CXCL17 reduced the expression of type I collagen in healthy control fibroblasts. CXCL17 also induced matrix metalloproteinase 1 (MMP1) and miR-29 expression in fibroblasts, indicating that CXCL17 regulates type I collagen expression in part via post-transcriptional mechanisms through MMP1 and miR-29. We found that local injection of CXCL17 attenuated bleomycin-induced skin fibrosis in mice. CXCL17 levels in SSc skin were lower than those in healthy controls, in contrast to the high serum CXCL17 levels in patients with SSc. The low expression of CXCL17 in SSc skin possibly affects type I collagen accumulation in this disease. CONCLUSION: Our data indicate that understanding CXCL17 signaling may lead to a better therapeutic approach for SSc.

Anti-skin aging properties of protocatechuic acid in vitro and in vivo.

BACKGROUND: Protocatechuic acid has reported containing antioxidant effects. However, information on its other biological activities such as anti-wrinkle properties is limited AIMS: The objective of this study was to evaluate an antioxidant, collagen synthesis, MMP-1 inhibition (in vitro), and anti-wrinkle (in vivo) effects of protocatechuic acid (PCA) as a potent ingredient for wrinkle-care cosmetic. METHODS: Antioxidant effect was evaluated based on its scavenging activity for free radicals (DPPH, ABTS+). To evaluate the anti-skin aging potency of PCA, levels of MMP-1 and type I procollagen were measured using an ELISA kit in cultured human dermal fibroblasts. To further investigate if PCA could increase collagen synthesis, full-thickness human skin explants were immunostained with an anti-collagen I antibody. In an in vivo study, 22 female subjects were enrolled in a placebo-controlled trial. Facial wrinkle, especially crow's feet around eyes, was treated with lotion-containing 0.02% PCA for 8 weeks and compared with the placebo. RESULTS: In in vitro study, PCA showed high antioxidant activ ity. PCA also showed potential to induce the synthesis of type I collagen in human dermal fibroblast and skin explants. It inhibited MMP-1 secretion from UVA-irradiated human dermal fibroblast. An in vivo study, treatment with lotion-containing 0.02% PCA for 8 weeks significantly reduced the percentage of all skin wrinkle parameters. CONCLUSION: Based on the results of in vitro assays and in vivo skin testing in human subjects, PCA shows potential in anti-wrinkle or anti-skin aging treatments.

Identification of peptide inhibitors of matrix metalloproteinase 1 using an in-house assay system for the enzyme.

Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in the degradation of extracellular matrix proteins. As one of the isoforms, MMP-1 breaks down collagen, and its activity is known to be important in wound healing. Its timely and adequate level of expression is pivotal because MMP-1 is also involved in the damage or aging of skins as well as in certain types of cancers. Thus, both assaying the MMP-1 activity and developing its inhibitors are of great importance. We here developed an in-house assay system that gave us the high degree of freedom in screening peptide inhibitors of MMP-1. The assay system utilized a circularly permutated fusion of ß-lactamase and its inhibitory protein through an MMP-1-sensitive linker so that the activity of MMP-1 could be translated into that of ß-lactamase. As a proof of concept, we applied the developed assay system to initial screens of MMP-1 inhibitors and successfully identified one lead peptide that inhibited the collagenase activity of the enzyme.

The effect of colchicine on alveolar bone loss in ligature-induced periodontitis.

Colchicine is widely used in the treatment of several inflammatory diseases due to its anti-inflammatory effect, but effects on bone metabolism are unclear. The aim of this study was to evaluate the effects of systemically-administered colchicine on healthy periodontium and experimentally-induced periodontitis. In total, 42 male Wistar rats were included in this study. A non-ligated group constituting the negative control group (Control, C, n = 6) and a ligature-only group forming the positive control group (LO, n = 12) were created separately. Twelve rats were treated with 0.4 mg/kg colchicine and another 12 with 1 mg/kg colchicine. In the colchicine-administered groups, right mandibles constituted the ligated groups (1 mgC-L or 0.4 mgC-L) and left mandibles formed the corresponding non-ligated controls (1mgC or 0.4mgC). Silk ligatures were placed at the gingival margin of the lower first molars. The animals were euthanized at different time-points of healing (11 or 30 days). Alveolar bone loss was clinically measured and TRAP+ osteoclasts, osteoblastic activity, and MMP-1 expression were examined histologically. There was no increase in alveolar bone loss with either colchicine dose in healthy periodontium (p > 0.05) and the highest level of alveolar bone loss, TRAP+ osteoclast number, and MMP-1 expression were measured in the LO group (p < 0.05). The 0.4 mgC-L group showed less alveolar bone loss at 11 days (p < 0.05), but greater loss at 30 days. The 1 mgC-L group showed higher osteoblast number than the other ligated groups (p < 0.05) at both time-points. In summary, colchicine did not increase alveolar bone loss in healthy periodontium and also may tend to reduce periodontitis progression. However, further extensive study is necessary to understand the mechanism of colchicine action on alveolar bone loss in periodontitis.

The Effect of Oral Administration of PUFAs on the Matrix Metalloproteinase Expression in Gastric Adenocarcinoma Patients Undergoing Chemotherapy.

OBJECTIVE: Gastric cancer is the third-leading cause of cancer-related mortality and the fifth most common cancer globally. Polyunsaturated fatty acids (PUFAs) are considered as functional ingredients that improve the efficacy of chemotherapeutic drugs. The aim of this study is to investigate the effect of PUFAs administration on matrix metalloproteinases (MMPs). METHODS: This study was designed as a randomized, double-blind trial. Thirty-four newly diagnosed patients with gastric cancer were randomly divided into two groups: control group (n = 17) and case group (n =17). Both groups received the same dose (75 mg/m2) of cisplatin. Control group received cisplatin plus placebo and the case group received cisplatin plus PUFAs [3600 mg/day, for three courses (each course included 3 weeks)]. The mRNA and protein expression of MMPs determined by real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. RESULTS: The relative gene expression of MMP-1 and MMP-9 was significantly lower in case group than control. The protein expression of MMP-1 and MMP-9 was significantly lower in case group than control. CONCLUSION: According to the results of this study, PUFAs reduced the expression of MMPs in gastric cancer cells. It seems that PUFAs may have an inhibitory effect on invasion and metastasis of gastric cancer cells.

MMP-1/TIMP-1 expressions in rectal submucosa of females with obstructed defecation syndrome associated with internal rectal prolapse.

OBJECTIVE: To explore the MMP-1/TIMP-1 expressions in rectal submucosa of females with obstructed defecation syndrome (ODS) associated with internal rectal prolapse (IRP). METHODS: Fifty-six female patients with ODS associated with IRP were enrolled as Case group, and 43 female hemorrhoids of stages III-IV without constipation and IRP were enrolled as Control group. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were performed to test the expressions of MMP-1/TIMP-1 in the rectal submucosa. Western blotting was used to examine protein expressions of MMP-1/TIMP-1 and pro-inflammatory cytokines (IL-6 and TNF-α) in the rectal submucosa. EVG staining was conducted to detect collagen and elastic fibers in rectal submucosa. RESULTS: The increased expression of MMP-1 was negatively linked to the decreased TIMP-1 level in the rectal submucosa of patients with ODS associated with IRP. Besides, the expressions of IL-6 and TNF-α were increased in the Case group as compared with the Control group. Additionally, ODS severity and the pro-inflammatory cytokines was positively linked to MMP-1, but negatively related to TIMP-1 in Case group. EVG staining showed that the area ratios of collagen and elastic fibers were lower in Case group than Control group. Through Pearson's correlation analysis, the area ratios of collagen and elastic fibers were positively associated with MMP-1 expression, but negatively correlated with TIMP-1 expression in rectal submucosa of patients with ODS associated with IRP. CONCLUSION: Elevated MMP-1 and reduced TIMP-1 were found in ODS associated with IRP, which was related to the ODS severity, inflammation and contents of collagen and elastic fibers.

The effect of colchicine on alveolar bone loss in ligature-induced periodontitis

Abstract Colchicine is widely used in the treatment of several inflammatory diseases due to its anti-inflammatory effect, but effects on bone metabolism are unclear. The aim of this study was to evaluate the effects of systemically-administered colchicine on healthy periodontium and experimentally-induced periodontitis. In total, 42 male Wistar rats were included in this study. A non-ligated group constituting the negative control group (Control, C, n = 6) and a ligature-only group forming the positive control group (LO, n = 12) were created separately. Twelve rats were treated with 0.4 mg/kg colchicine and another 12 with 1 mg/kg colchicine. In the colchicine-administered groups, right mandibles constituted the ligated groups (1 mgC-L or 0.4 mgC-L) and left mandibles formed the corresponding non-ligated controls (1mgC or 0.4mgC). Silk ligatures were placed at the gingival margin of the lower first molars. The animals were euthanized at different time-points of healing (11 or 30 days). Alveolar bone loss was clinically measured and TRAP+ osteoclasts, osteoblastic activity, and MMP-1 expression were examined histologically. There was no increase in alveolar bone loss with either colchicine dose in healthy periodontium (p > 0.05) and the highest level of alveolar bone loss, TRAP+ osteoclast number, and MMP-1 expression were measured in the LO group (p < 0.05). The 0.4 mgC-L group showed less alveolar bone loss at 11 days (p < 0.05), but greater loss at 30 days. The 1 mgC-L group showed higher osteoblast number than the other ligated groups (p < 0.05) at both time-points. In summary, colchicine did not increase alveolar bone loss in healthy periodontium and also may tend to reduce periodontitis progression. However, further extensive study is necessary to understand the mechanism of colchicine action on alveolar bone loss in periodontitis.

Black rice (Oryza sativa L.) extract modulates ultraviolet-induced expression of matrix metalloproteinases and procollagen in a skin cell model.

Exposure of the skin to ultraviolet (UV) radiation causes extracellular matrix (ECM) collapse in the dermis, owing to an increase in matrix metalloproteinase (MMP) production in both the epidermis and dermis, and a decrease in type I collagen expression in the dermis. Recently, black rice (Oryza sativa L.) was reported to have a wide range of pharmacological effects in various settings. However, the effects of black rice extract (BRE) on UV­irradiated skin cells have not yet been characterized. BRE treatment did not affect cell morphology and viability of HaCaT and human dermal fibroblasts (HDF). We demonstrated that BRE downregulated basal and UV­induced MMP­1 expression in HaCaT cells. Furthermore, BRE significantly increased type I procollagen expression, and decreased MMP­1 and MMP­3 expression in UV­irradiated HDF. The underlying mechanisms of these results involve a decrease in p38 and c­Jun N­terminal kinase activity, and suppression of UV­induced activation of activator protein­1 (AP­1). BRE reduced UV­induced reactive oxygen species production in HaCaT cells in a dose­dependent manner. Indeed, mass spectrometry revealed that BRE contained antioxidative flavonoid components such as cyanidin­3­O­ß­D­glycoside and taxifolin­7­O­glucoside. These findings suggest that BRE attenuates UV­induced ECM damage by modulating mitogen­activated protein kinase and AP­1 signaling, and could be used as an active ingredient for preventing photoaging of the skin.

Molecular Biological Effects of Weightlessness and Hypergravity on Intervertebral Disc Degeneration.

INTRODUCTION: The rate of intervertebral disc degeneration (IVDD) is influenced by environmental factors. Extracellular matrix (ECM) destruction and apoptosis of intervertebral disc cells are major characteristics of IVDD. ECM degradation is closely linked to up-regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMP). This study aimed to elucidate the molecular biological changes during IVDD under conditions of weightlessness and hypergravity. METHODS: A total of 120 rabbits were divided randomly into four groups: control group, weightlessness group, hypergravity group, and mixed (hypergravity + weightlessness) group. Tail-suspension was used to simulate a weightless environment, and an animal centrifuge (+7 Gz three times for 60 s) was used to mimic hypergravity conditions. After exposure to the above conditions for 30, 60, and 90 d, respectively, 10 rabbits were selected from each group for immunohistochemical determination of MMP-1, MMP-3, and TIMP-1 expression. TUNEL staining was also carried out to detect apoptotic cells in each group at each time point. RESULTS: MMP-1, MMP-3, and TIMP-1 were rarely expressed in the control group, but were positively expressed in the other three groups. The strongest expression was in the mixed group at every time point, followed by the hypergravity group, and then the weightlessness group. Cell apoptosis index followed a similar trend to MMPs and TIMP-1 expression. DISCUSSION: The results suggested that weightlessness and hypergravity may both aggravate IVDD over time, with hypergravity having a particularly marked effect.Wu D, Zheng C, Wu J, Huang R, Chen X, Zhang T, Zhang L. Molecular biological effects of weightlessness and hypergravity on intervertebral disc degeneration. Aerosp Med Hum Perform. 2017; 88(12):1123-1128.

An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.

Collagen zymography is an SDS-PAGE-based method for detecting both the proenzyme and active forms of collagenases. Although collagen zymography is used for assessment of the matrix metalloproteinases MMP-1 and MMP-13, it can be difficult to detect these collagenases due to technical issues. Moreover, it remains unclear whether the collagenase activity of MMP-8 can be detected by this method. Here, we present an improved collagen zymography method that allows quantification of the activities of MMP-1, MMP-8, and MMP-13. Activities of recombinant collagenases could be detected in collagen zymogram gels copolymerized with 0.3 mg/mL type I collagen extracted from rat tail tendon. This improved method is sensitive enough to detect the activity of as little as 1 ng of collagenase. We generated standard curves for the three collagenases to quantify the collagenolytic activity levels of unknown samples. To validate our improved method, we investigated MMP-1 activity levels in human thyroid cancer (8505C) and normal thyroid (Nthy-ori-3-1) cell lines, finding that the proenzyme and active MMP-1 levels were greater in 8505C cells than in Nthy-ori-3-1 cells. Taken together, our data show that collagen zymography can be used in both molecular and clinical investigations to evaluate collagenase activities in various pathological conditions.

Compounds isolated from Eriobotrya deflexa leaves protect against ultraviolet radiation B-induced photoaging in human fibroblasts.

Ultraviolet (UV) irradiation leads to skin photoaging because of the upregulation of matrix metalloproteinase (MMP)-1 and downregulation of type I collagen and tissue inhibitor of metalloproteinase (TIMP)-1. Eriobotrya deflexa (Hemsl.) Nakai (Rosaceae) is a flowering plant endemic to Taiwan, and its leaves have been used as an expectorant and in antitussive folk remedy. Our previous studies have demonstrated that an E. deflexa leaf extract functions as a free radical scavenger. The current evaluated the antiphotoaging effect of partitioned fractions and specific compounds from the leaves of E. deflexa by using bioguided isolation, compound identification, and biological activity testing with UVB-irradiated human fibroblasts (WS-1 cells). E. deflexa leaves were extracted with 95% ethanol and then partitioned using a sequential treatment of n-hexane, ethyl acetate, and n-butanol (n-BuOH). The bioactive n-BuOH fraction was used for isolation and purification through chromatography. The compounds were identified by analyzing their physical and spectroscopic properties. We identified eight compounds from this fraction; of these compounds, 3-O-α-l-rhamnopyranosyl-(1‴→6″)-ß-d-galactopyranoside (1), hyperin (2), afzelin (5), and cryptochlorogenic acid methyl ester (7) were isolated from E. deflexa for the first time, and they exhibited MMP-1 inhibition activity. The IC50 values were 96.5, 89.5, 93.4, and 92.8µM for 1, 2, 5, and 7, respectively. These compounds also enhanced the expression of procollagen type I, and TIMP-1 and hyperin (2) were found to be most effective with IC50 values of 56.7 and 70.3µM, respectively. Hyperin (2) could reduce intracellular reactive oxygen species production in UVB-irradiated WS-1 cells, with the corresponding IC50 value being 80.7µM. Liquid chromatography triple-quadrupole mass spectrometry was used for the quantitative and chemical fingerprint analysis of active compounds. Quercetin 3-O-α-l-rhamnopyranosyl-(1‴→6″)-ß-d-galactopyranoside (1), hyperin (2), afzelin (5), and cryptochlorogenic acid methyl ester (7) constituted 24.2±3.9, 5.5±1.0, 3.4±0.3, and 67.1±8.1mg/g of dry weight in the active n-BuOH fraction, respectively. Our results demonstrate that the extract and the isolated active compounds from E. deflexa leaves possess the potential for protection against skin photoaging.

Local wound healing biomarkers for real-time assessment of periodontal regeneration: pilot study.

BACKGROUND AND OBJECTIVES: Within the same surgical procedure, a great variability on achievement of clinical outcomes exists and may be associated to different molecular factors related to tissue healing. The aim of the present study was to assess the distribution of clinical success separately in regenerative therapy (REG) and open flap debridement (OFD) to evaluate if factors related with healing of epithelium, connective tissue and bone may be associated to the clinical outcome within each surgical procedure. MATERIAL AND METHODS: Sixteen patients underwent periodontal REG and nine patients underwent OFD. Periodontal wound fluid was collected at baseline, 3-5, 7, 14 and 21 d after surgery, and expression of wound healing proteins was assessed. Pocket depth and clinical attachment level were taken at baseline and at 6 mo of follow-up. Percentage pocket depth reduction and percentage clinical attachment level gain were computed. Patients were regarded as better or worse responders depending on their percentage pocket depth reduction or percentage clinical attachment level gain. RESULTS: Higher percentage of better responders was observed in the REG group (68.7%) compared to the OFD group (22.2%). At 21 d, no difference in the profile of most of the proteins emerged, with two exceptions, both regarding REG treatment. Bone morphogenetic protein-7 tended to increase in better responders and to decrease in worse responders. Matrix metalloproteinase-1 increased in worse responders and remained substantially unchanged in better responders. CONCLUSION: Local expression of matrix metalloproteinase-1 and bone morphogenetic protein-7 during wound healing is associated with the clinical performance of periodontal regenerative surgery. The use of local biomarkers offers the potential for real-time assessment of the periodontal healing process.

MMP-1 and MMP-8 expression in giant-cell fibroma and inflammatory fibrous hyperplasia.

The aim of this study is to compare the immunoexpression of metalloproteinases 1 and 8 in giant-cell fibroma, inflammatory fibrous hyperplasia and normal mucosa. Twenty-two cases of giant-cell fibroma, inflammatory fibrous hyperplasia and oral mucosa (control) each were subjected to immunohistochemistry using anti-metalloproteinase-1 and anti-metalloproteinase-8 antibodies. Eight images of each case were captured and analysed through the a) application of a count grid to count the number of positive neutrophils, macrophages, lymphocytes, plasma cells, fibroblasts and blood vessels to obtain the percentage of staining and b) semi-automated segmentation quantifying the stained area in square micrometres. Statistical tests included ANOVA Two-way, Kruskal Wallis and Games-Howell, with a significance level of 5%. An increased percentage of metalloproteinase-1-immunopositive blood vessels were observed in giant-cell fibroma (26.6±22.4; p=0.02) and inflammatory fibrous hyperplasia (34.3±31.5; p=0.01) compared with the control group (19.6±9.2). No significant differences in inflammatory cells, fibroblasts and total area of metalloproteinase-1 and -8 were noted among the three groups. Metalloproteinase-1 apparently acts within the pathogenesis of giant-cell fibroma and inflammatory fibrous hyperplasia.