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.

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.

Glucosamine sulfate suppresses the expression of matrix metalloproteinase-3 in osteosarcoma cells in vitro.

BACKGROUND: Glucosamine, a common dietary supplement, has a possible anti-sarcoma effect. However, an understanding of the underlying mechanism of such an effect is limited. For this study we hypothesized that glucosamine suppresses the basal level of matrix metalloproteinase expression in human osteosarcoma cell lines. METHODS: We examined the osteosarcoma cell lines, MG-63 and SaOS-2. Cells were exposed to 0, 10, 50 and 100 µg/ml glucosamine sulfate for 48 h and treatment toxicity was determined through measurement of cell viability and proliferation. Relative gene expression of matrix metalloproteinase (MMP)-2, -3 and -9 was quantified by real-time polymerase chain reaction. Protein levels of MMP-2 and -9 were assessed by ELISA. RESULTS: Administration of 10, 50 or 100 µg/ml glucosamine sulfate had no effect on the cell viability of MG-63 and SaOS-2 cells. A significant reduction of MMP expression in both cell lines was observed only for MMP-3, while a decrease in MMP-9 was seen in SaOS-2 cells. The expression of MMP-2 was not significantly affected in either cell line. Protein level of MMP-3 was reduced in both cell lines upon stimulation with 10 µg/ml glucosamine sulfate whereas for MMP-9 a decrease could only be observed in SaOS-2 cells. CONCLUSION: In this study, we found a pronounced suppressive effect of glucosamine sulfate particularly on MMP-3 and also MMP-9 mRNA and protein levels in osteosarcoma cell lines in vitro. The data warrants further investigations into the potential anti-tumor efficacy of glucosamine sulfate in osteosarcoma.

[Effect of Warm-needle-moxibustion on Behavior Reactions and TNF-α and MMP-3 Contents in Knee Cartilage of Rabbits with Knee Osteoarthritis].

OBJECTIVE: To observe the effect of warm-needle-moxibustion on behavior changes and the levels of tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-3 (MMP-3) in the knee cartilage tissue of rabbits with knee osteoarthritis (KOA) so as to explore its mechanisms in relieving KOA. METHODS: Thirty male Japanese white rabbits were randomly divided into normal, model and treatment groups (n=10 in each group). The KOA model was established by using immobilization me-thod. The internal and external "Dubi" (ST 35), "Xuehai" (SP 10) and "Yanglingquan "(GB 34) were punctured with filiform needles first, followed by attaching an ignited moxa column to the handle of each inserted needles. The treatment was conducted for 20 min every time, once daily for 4 weeks. The behavioral scores were assessed according to the pain severity, gait, joint motion range and articular swelling severity in reference to the modified Lequesne's methods. The TNF-α and MMP-3 contents in the knee joint cartilage tissue were assayed by radioimmunoassay, and pathological changes of the articular cartilage tissue were observed under microscope after sectioning and H.E. staining. RESULTS: The Lequesne total score, and the cartilage TNF-α and MMP-3 contents were significantly increased in the model group than in the normal group (P<0.05), and obviously down-regulated after warm-needle-moxibustion intervention (P<0.05). Under light microscope, the inflammatory infiltration of the articular cartilage and the disordered arrangement of chondrocytes in KOA rabbits were improved in the treatment group. CONCLUSIONS: Warm-needle-moxibustion intervention can improve the motor function of rabbits with KOA, which may be related to its effects in reducing the level of TNF-α and MMP-3 in articular cartilage tissue.

Effects of electrical stimulation on periodontal tissue remodeling in rats.

BACKGROUND AND OBJECTIVE: Electric current is used to promote wound healing. However, it is unclear whether electrical stimulation contributes to gingival tissue remodeling. This study examined the effects of electrical stimulation on gingival tissue remodeling in a rat periodontitis model. MATERIAL AND METHODS: Male Wistar rats (n = 28, 8 wks of age) were divided into four groups of seven rats each. The control group did not receive any treatment for 6 wks. In the other groups, periodontitis was ligature-induced for 4 wks. After 4 wks, the rats with periodontitis were given daily electrical stimulation of 0, 50 or 100 µA for 2 wks. RESULTS: The periodontitis group stimulated with 0 µA showed a higher density of polymorphonuclear leukocytes and a lower density of collagen in gingival tissue compared with the control group (p < 0.05). The two remaining groups treated with 50 or 100 µA of electrical stimulation exhibited a lower density of polymorphonuclear leukocytes (p < 0.05) and a higher density of collagen than the group stimulated with 0 µA (p < 0.05). They also showed higher expression of fibroblast growth factor-2 than the group treated with 0 µA of electrical stimulation (p < 0.05). CONCLUSION: Electric stimulation may offer a novel approach to promote gingival tissue remodeling in periodontal lesions.

Effects of cranberry components on human aggressive periodontitis gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis (AgP) causes rapid periodontal breakdown involving AgP gingival fibroblast production of cytokines [i.e. interleukin (IL)-6, a bone metabolism regulator], and matrix metalloproteinase (MMP)-3. Lipopolysaccharide upregulates fibroblast IL-6 and MMP-3, via transcription factors (i.e. NF-κB). Cranberry (Vaccinium macrocarpon) inhibits lipopolysaccharide-stimulated macrophage and normal gingival fibroblast activities, but little is known of its effects on AgP fibroblasts. Objectives of this study are to use AgP fibroblasts, to determine cytotoxicity of cranberry components or periodontopathogen (Fusobacterium nucleatum, Porphyromonas gingivalis) lipopolysaccharide ± cranberry components, and effects of cranberry components on lipopolysaccharide-stimulated NF-κB activation and IL-6 and MMP-3 production. MATERIAL AND METHODS: AgP fibroblasts were incubated ≤ 6 d with high molecular weight non-dialyzable material (NDM) (derived from cranberry juice (1-500 µg/mL) or lipopolysaccharide (1 µg/mL) ± NDM. Membrane damage and viability were assessed by enzyme activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Secreted IL-6 and MMP-3 were measured by ELISA. NF-κB p65 was measured via binding to an oligonucleotide containing the NF-κB consensus site. Data were analyzed using analysis of variance and Scheffe's F procedure for post hoc comparisons. RESULTS: Short-term exposure to NDM, or lipopolysaccharide ± NDM caused no membrane damage. NDM (≤ 100 µg/mL) or lipopolysaccharide ± NDM had no effect on viability ≤ 7 d exposure. NDM (50 µg/mL) inhibited lipopolysaccharide-stimulated p65 (P ≤ 0.003) and constitutive or lipopolysaccharide-stimulated MMP-3 (P ≤ 0.02). NDM increased AgP fibroblast constitutive or lipopolysaccharide-stimulated IL-6 (P ≤ 0.0001), but inhibited normal human gingival fibroblast IL-6 (P ≤ 0.01). CONCLUSION: Lack of toxicity of low NDM concentrations, and its inhibition of NF-κB and MMP-3, suggest that cranberry components may regulate AgP fibroblast inflammatory responses. Distinct effects of NDM on AgP and gingival fibroblast production of IL-6 (which can have both positive and negative effects on bone metabolism) may reflect phenotypic differences in IL-6 regulation in the two cell types.

[Effect of acupuncture on expression of matrix metalloproteinase and tissue inhibitor in cartilage of rats with knee osteoarthritis].

OBJECTIVE: To explore the curative mechanism of acupuncture treatment on osteoarthritis (OA). METHODS: Forty cases of female SD rats were randomly divided into a normal group, a model group, an acupuncture group and a medication group, 10 cases in each group. OA animal model was established by using the method of heel tendon resection for unilateral hind limb. The acupuncture group was treated with electroacupuncture at "Xiqian"(ST 35) and "Housanli"(ST 36), and the medication group with inunction of Diclofenac cream, and the normal group and the model group without any treatment. The expression of matrix metalloproteinase-1, 3 (MMP-1, MMP-3) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the cartilage were observed by immunohistochemistry. RESULTS: There were significant differences among four groups. The expressions of MMP-1, MMP-3 and TIMP-1 in the model, acupuncture and medication groups were all significantly stronger than those in the normal group (all P < 0.01). The expressions of MMP-1 and MMP-3 in the acupuncture and medication groups were down regulated and TIMP-1 expression up-regulated with significant differences as compared with the model group (all P < 0.01), and the expressions of MMP-1 and MMP-3 in acupuncture group were significantly lower, while TIMP-1 expression significantly higher than that in the medication group (all P < 0.01). CONCLUSION: Acupuncture can down-regulate the expression of MMP-1 and MMP-3 and up-regulate the expression of TIMP1, which is superior to that of Diclofenac cream, showing that acupuncture has a certain protective effect on cartilage from OA.

Statin therapy alone and in combination with an acyl-CoA:cholesterol O-acyltransferase inhibitor on experimental atherosclerosis.

The ability to modify the enzymatic processes involved in promoting atherosclerotic plaque disruption and to serially monitor atherosclerotic evolution could provide novel information in the management of patients with atherosclerosis. We studied the effects of a statin (atorvastatin) and its combination with an acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor (avasimibe) on atherosclerotic regression and plaque stability as measured by matrix metalloproteinase 1 and 3 (MMP-1 and MMP-3) levels. Watanabe Heritable Hyperlipidemic (WHHL) rabbits treated with atorvastatin alone experienced an attenuated increase in atherosclerotic burden versus controls as determined by MR imaging. The mean vessel wall area (VWA) prior to drug therapy was 5.57 +/- 0.01 mm2. The VWA increased to 6.71 +/- 0.03 and 7.16 +/- 0.03 mm2, respectively, in atorvastatin-treated and control groups (p < 0.0001 for both). The combination of atorvastatin and avasimibe induced a significant regression of the previously established atherosclerotic lesions, with the VWA decreasing to 4.54 +/- 0.04 mm2 (p = 0.009). Atorvastatin alone induced a nonsignificant reduction in the percent staining of MMP-1 in atherosclerotic lesions, but the combination treatment with avasimibe led to a significant reduction versus controls (p = 0.005). However, a reduction in MMP-3 staining was significant for rabbits treated with both atorvastatin alone (p = 0.007) and in combination with avasimibe (p = 0.04) versus controls. In this animal model, the addition of avasimibe to atorvastatin has beneficial effects on both atherosclerotic plaque regression and stabilization.

Simvastatin inhibits cigarette smoking-induced emphysema and pulmonary hypertension in rat lungs.

RATIONALE: In cigarette smoking-induced chronic obstructive pulmonary disease, structural and functional derangements are characterized by parenchymal destruction and pulmonary hypertension. Statins are 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase inhibitors that have been used as lipid-lowering agents. These drugs also have additional pharmacologic properties, including antiinflammation, scavenging reactive oxygen species, restoring endothelial function, and antithrombogenesis, all of which can counteract the harmful effects of cigarette smoking. OBJECTIVE: We performed assays to determine whether simvastatin could attenuate lung damage induced by chronic cigarette smoking in rats. METHODS: In Sprague-Dawley rats exposed to cigarette smoke for 16 weeks, morphologic changes in the lungs and pulmonary arterial pressure were examined. MAIN RESULTS: Simvastatin inhibited lung parenchymal destruction and development of pulmonary hypertension, and also inhibited peribronchial and perivascular infiltration of inflammatory cells and induction of matrix metalloproteinase-9 activity in lung tissue. Simvastatin additionally prevented pulmonary vascular remodeling and the changes in endothelial nitric oxide synthase expression induced by smoking. In human lung microvascular endothelial cells, simvastatin increased expression of endothelial nitric oxide synthase mRNA. CONCLUSIONS: Simvastatin ameliorated the structural and functional derangements of the lungs caused by cigarette smoking, partly by suppressing inflammation and matrix metalloproteinase-9 induction and preventing pulmonary vascular abnormality. These findings indicate that statins may play a role in the treatment of cigarette smoking-induced chronic obstructive pulmonary disease.

The in vitro effects of dehydroepiandrosterone on human osteoarthritic chondrocytes.

OBJECTIVE: To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on human osteoarthritic chondrocytes. DESIGN: Chondrocytes isolated from human osteoarthritic knee cartilage were three-dimensionally cultured in alginate beads, except for cell proliferation experiment. Cells were treated with DHEA in the presence or absence of IL-1beta. The effects on chondrocytes were analyzed using a 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay (for chondrocyte proliferation), a dimethylmethylene blue (DMB) assay (for glycosaminoglycan (GAG) synthesis), and an indole assay (for DNA amount). Gene expressions of type I and II collagen, metalloproteinase-1 and -3 (MMP-1 and -3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) as well as the IL-1beta-induced gene expressions of MMP-1 and -3 were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The protein synthesis of MMP-1 and -3 and TIMP-1 was determined by Western blotting. RESULTS: The treatment of chondrocytes with DHEA did not affect chondrocyte proliferation or GAG synthesis up to 100 micro M of concentration. The gene expression of type II collagen increased in a dose-dependent manner, while that of type I decreased. DHEA suppressed the expression of MMP-1 significantly at concentrations exceeding 50 micro M. The gene expression of MMP-3 was also suppressed, but this was without statistical significance. The expression of TIMP-1 was significantly increased by DHEA at concentrations exceeding 10 micro M. The effects of DHEA on the gene expressions of MMP-1 and -3 were more prominent in the presence of IL-1beta, in which DHEA suppressed not only MMP-1, but also MMP-3 at the lower concentrations, 10 and 50 micro M, respectively. Western blotting results were in agreement with RT-PCR, which indicates that DHEA acts at the gene transcription level. CONCLUSIONS: Our study demonstrates that DHEA has no toxic effect on chondrocytes up to 100 micro M of concentration and has an ability to modulate the imbalance between MMPs and TIMP-1 during OA at the transcription level, which suggest that it has a protective role against articular cartilage loss.

Affinity NMR.

Because binding ligands are directly detected and identified, the diffusion-based NMR method and NOE pumping approach promise to greatly simplify deconvolution in drug screening. An additional advantage of these techniques is that low-affinity ligands, which might be missed by high-throughput screening, can be detected and could serve as synthetic precursors for higher affinity ligands. The biggest challenge to NMR methodology lies in its sensitivity. Compared with other techniques, such as MS (25), NMR methods for screening mixtures are limited by their relative insensitivity. Because of issues such as solubility, stability, and mass limitation, it is not in general judicious to simply increase the concentration of the mixture. Improvements in hardware and software are necessary to extend the applicability of the affinity NMR method to the screening of larger and more complex mixtures. A boost in sensitivity and screening capacity of NMR technique is possible by the implementation of microcoil (26) and flow probe techniques. An upsurge in the capabilities of mixture analysis could be achieved with a combination of independent and complementary techniques (e.g., HPLC, MS) (27). As a unique, nondestructive, and versatile tool, NMR will continue on its fast track of development in the support of drug discovery.