Resveratrol-mediated inhibition of cyclooxygenase-2 in melanocytes suppresses melanogenesis through extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase/Akt signalling.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene), has been reported to exert a variety of important pharmacological effects including anti-inflammatory, anticancer, and direct inhibition of tyrosinase. This study aimed to examine the expression of melanogenic molecules following down-regulation of cyclooxygenase (COX)-2 expression by resveratrol and the related signal transduction pathways in mouse B16F10 melanoma cells and zebrafish larvae. We report that resveratrol suppressed COX-2 in melanocytes and decreased the expressions of tyrosinase and microphthalmia-associated transcription factor (MITF). Furthermore, inhibition of COX-2 with NS398 enhanced resveratrol-reduced tyrosinase and MITF expression. Resveratrol also induced phosphorylation of extracellular signal-regulated 1/2 (ERK1/2) and phosphoinositide-3 (PI-3)-kinase/Akt. Inhibition of ERK1/2 or PI-3K/Akt by PD98059 and LY294002 restored the decreased tyrosinase activity and MITF expression via resveratrol-mediated down-regulation of COX-2. Additionally, resveratrol inhibited body pigmentation in zebrafish. These results indicated that resveratrol inhibited melanogenesis by down-regulating COX-2 via ERK1/2 and PI-3K/Akt pathways in B16F10 cells.

A nano sensor for sensitive and selective detection of Cu2+ based on fluorescein: Cell imaging and drinking water analysis.

A fluorescein-based nano probe was designed and synthesized for ultra-sensitive detection of Cu2+ in aqueous solution. The formation of fluorescent organic nanoparticles confirmed by using particle size analysis and scanning electron microscopy. UV-Vis. absorption and fluorescence spectroscopy displays excellent photophysical properties of prepared nanoparticles as compared to parent molecule i.e. N-(3',6'-dihydroxy-3-oxo-3,3a-dihydrospiro[isoindole-1,9'-xanthene]-2(7aH)-yl)-1-naphthamide (FNH) in acetone. A series of 18 metal ion was examined with FNH nanoparticles (FNHNPs) to examine the change in fluorescence response. Pleasingly, only copper ion (Cu2+) shows selective and sensitive fluorescence enhancement effect, which discussed on chelation-enhanced fluorescence phenomenon. Other competing metal ions does not affect the FNHNPs fluorescence enhancement induced by Cu2+ ion. The excited state complexation through chelation-enhanced fluorescence of FNHNPs was further supported by UV-Vis. absorption and fluorescence decay titration of FNHNPs with and without the addition of Cu2+. The present investigation approach serves extremely low detection limit of 1.62 ng/mL (0.024 µM) for Cu2+ in aqueous solution. In addition, benefit of present study includes practical application for the quantitative estimation of Cu2+ in drinking water sample and intracellular cell imaging for Cu2+.

Rosmarinic acid induces rabbit articular chondrocyte differentiation by decreases matrix metalloproteinase-13 and inflammation by upregulating cyclooxygenase-2 expression.

BACKGROUND: Matrix metalloproteinases (MMPs) are known to play an important role in the degradation of the extracellular matrix and the pathological progression of osteoarthritis (OA). The natural polyphenolic compound rosmarinic acid (Ros. A) has been shown to suppress the inhibitory activity of matrix metalloproteinases (MMPs). However, the effects of Ros. A on OA have not been investigated. METHODS: In the current study, primary articular chondrocytes were cultured from rabbit articular cartilage and treated with Ros. A. Phenotypic characterization was performed by western blotting to assess specific markers, prostaglandin E2 (PGE2) assays, and alcian blue staining to measure sulfated-proteoglycan production. RESULTS: We report that in rabbit articular chondrocytes, Ros. A increased type II collagen, sulfated-proteoglycan, cyclooxygenase-2 (COX-2), and PGE2 production in a dose- and time-dependent manner. Furthermore, Ros. A suppressed the expression of MMP-13. In addition, treatment with Ros A activated extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling pathways. Inhibition of MMP-13 enhanced Ros. A-induced type II collagen expression and sulfated-proteoglycan synthesis but COX-2 and PGE2 production were unchanged. Ros. A-mediated up-regulation of ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which prevented induction of the associated inflammatory response. Inhibition of p38 kinase with SB203580 enhanced the increase in type II collagen expression via Ros. A-mediated down-regulation of MMP-13. CONCLUSIONS: Results suggest that ERK-1/2 regulates Ros. A-induced inflammation and that p38 regulates differentiation by inhibiting MMP-13 in rabbit articular chondrocytes.

Isolation, characterization, and in silico, in vitro and in vivo antiulcer studies of isoimperatorin crystallized from Ostericum koreanum.

CONTEXT: Ostericum koreanum (Maxim.) Kitagawa (Apiaceae) roots are traditionally used as an analgesic and antiulcer agent. However, the antiulcer potential of isoimperatorin isolated from O. koreanum has not yet been explored. AIM: To evaluate the antiulcer activity of isoimperatorin isolated from the roots of O. koreanum. MATERIALS AND METHODS: Isoimperatorin was isolated as cubic crystals by repeated column chromatography of the ethyl acetate fraction and structure was verified with 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS-FAB). The crystals obtained were analyzed with the single crystal X-ray method. The MTT assay was used to determine its cytotoxicity against chondrocytes at different concentrations (0.0-737.74 µM, 24 h). The in vivo antiulcer activity of isoimperatorin (40 mg/kg) was determined against ethanol-, indomethacin- and pyloric ligation-induced ulcers in Sprague-Dawley rats. Furthermore, the effect of isoimperatorin (0.0-737.74 µM, 24 h) on the expression of type II collagen in chondrocytes was determined using western blot method. The in vitro urease inhibitory activity of isoimperatorin (0-80 µM) and molecular docking was also performed against urease. RESULTS AND DISCUSSION: Isoimperatorin demonstrated significant inhibitory activity (IC50 36.43 µM) against urease as compared to the standard drug thiourea (IC50 33.57 µM) without cytotoxic effects. It provided 70.9%, 67.65% and 54.25% protection in ulcer models induced by ethanol, indomethacin and pyloric ligation, respectively. Isoimperatorin showed the highest expression level of type II collagen at 368.87 µM. The docking results confirmed strong binding affinity with the target protein. CONCLUSION: Isoimperatorin may be used to develop antiulcer drugs with decreased side effects.

PEP-1-SIRT2-induced matrix metalloproteinase-1 and -13 modulates type II collagen expression via ERK signaling in rabbit articular chondrocytes.

Matrix metalloproteinases (MMPs) are critical for the degradation of the extracellular matrix (ECM), which includes cartilage-specific collagen types I, II and XI. We previously found that PEP-1-sirtuin (SIRT)2 could induce dedifferentiation of articular chondrocytes; however, the underlying mechanisms remains unclear. We addressed this in the present study by examining the association between PEP-1-SIRT2 and the expression of MMP-1 and MMP-13 and type II collagen in rabbit articular chondrocytes. We found that PEP-1-SIRT2 increased MMP-1 and -13 expression in a dose- and time-dependent manner, as determined by western blotting. A similar trend in MMP-1 and -13 levels was observed in cultures during expansion to four passages. Pharmacological inhibition of MMP-1 and -13 blocked the PEP-1-SIRT2-induced decrease in type II collagen level. Phosphorylation of extracellular regulated kinase (ERK) was increased by PEP-1-SIRT2; however, treatment with the mitogen-activated protein kinase inhibitor PD98059 suppressed PEP-1-SIRT2-induced MMP-1 and -13 expression and dedifferentiation while restoring type II collagen expression in passage 2 cells. These results suggest that PEP-1-SIRT2 promotes MMP-induced dedifferentiation via ERK signaling in articular chondrocytes.

Applications of Chondrocyte-Based Cartilage Engineering: An Overview.

Chondrocytes are the exclusive cells residing in cartilage and maintain the functionality of cartilage tissue. Series of biocomponents such as different growth factors, cytokines, and transcriptional factors regulate the mesenchymal stem cells (MSCs) differentiation to chondrocytes. The number of chondrocytes and dedifferentiation are the key limitations in subsequent clinical application of the chondrocytes. Different culture methods are being developed to overcome such issues. Using tissue engineering and cell based approaches, chondrocytes offer prominent therapeutic option specifically in orthopedics for cartilage repair and to treat ailments such as tracheal defects, facial reconstruction, and urinary incontinence. Matrix-assisted autologous chondrocyte transplantation/implantation is an improved version of traditional autologous chondrocyte transplantation (ACT) method. An increasing number of studies show the clinical significance of this technique for the chondral lesions treatment. Literature survey was carried out to address clinical and functional findings by using various ACT procedures. The current study was conducted to study the pharmacological significance and biomedical application of chondrocytes. Furthermore, it is inferred from the present study that long term follow-up studies are required to evaluate the potential of these methods and specific positive outcomes.

PEP-1-SIRT2 causes dedifferentiation and COX-2 expression via the MAPK pathways in rabbit articular chondrocytes.

SIRT2 is a member of the mammalian sirtuin protein family, primarily found in the cytoplasm. It regulates numerous cellular processes including aging, DNA repair, cell cycle, and survival under stress conditions. However, the biological function and mechanism of the SIRT2 protein was not well understood in normal cells such as primary chondrocytes. In this study, we examined the effects of SIRT2 on differentiation and inflammation in rabbit articular chondrocytes by using a cell-permeative PEP-1-SIRT2 protein. Our results indicate that PEP-1-SIRT2-induced a loss of type II collagen and decreased sulfate proteoglycan levels in a dose- and time-dependent manner, as examined by Western blotting, alcian blue staining, and immunohistochemistry. Furthermore, PEP-1-SIRT2 caused an inflammatory response by inducing the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). In addition, after treatment with PEP-1-SIRT2, phosphorylation of both p38 and ERK was observed. Inhibition of ERK with PD98059 (PD) suppressed PEP-1-SIRT2-induced dedifferentiation and COX-2 expression. Reduction in PEP-1-SIRT2-induced inflammatory response was observed upon inhibition of p38 by SB203580 (SB). The same pattern was demonstrated in PEP-1-SIRT2-induced dedifferentiation and inflammatory response during culture with serial passages. During expansion to four passages, levels of type II collagen decreased, whereas levels of COX-2 and SIRT2 increased and activated ERK and p38. Furthermore, PEP-1-SIRT2 enhances dedifferentiation through the ERK pathway and inflammatory response through the ERK and p38 pathways in rabbit chondrocytes in vitro. These findings suggest that PEP-1-SIRT2 induces dedifferentiation via the ERK pathway and inflammation through the p38 and ERK pathways in rabbit articular chondrocytes.

Resveratrol regulates type II collagen and COX-2 expression via the ERK, p38 and Akt signaling pathways in rabbit articular chondrocytes.

Resveratrol, a naturally occurring polyphenolic phytoalexin antioxidant compound present in grapes and red wine, has been reported to induce various biochemical responses. It has been shown to possess anti-aging, anti-inflammatory and anti-proliferative activities in several cell types. However, the effects of resveratrol in normal cells, including chondrocytes, have not yet been clearly elucidated. The aim of the present study was to evaluate the effects of resveratrol on differentiation and inflammation in rabbit articular chondrocytes and to investigate the underlying mechanism of action. Rabbit articular chondrocytes were treated with 20 µM resveratrol for different time periods or with various concentrations of resveratrol for 24 h. It was observed that the expression levels of type II collagen and sulfated proteoglycan, as determined by western blot analysis and Alcian blue staining, respectively, increased following treatment with resveratrol in a concentration-dependent manner at concentrations up to 20 µM and then decreased at higher concentrations. The expression levels of cyclooxygenase (COX-2) and prostaglandin E2 (PGE2) began to increase at 10 min after the addition of resveratrol, reached peak levels at 3 h and decreased from the peak level thereafter, as determined by western blot analysis and PGE2 assay, respectively. It was also demonstrated that resveratrol caused phosphorylation of mitogen-activated protein kinase proteins [extracellular signal-regulated kinases (ERK), p38 and c-Jun N-terminal kinases (JNK)] and Akt in rabbit articular chondrocytes. The inhibition of ERK, p38 kinase, phosphoinositide 3-kinase (PI3K) and Akt with PD98059, SB203580, LY294002 and triciribine, respectively, suppressed resveratrol-induced type II collagen and COX-2 expression. However, inhibition of JNK with SP600125 produced no clear changes in the expression levels of type II collagen and COX-2. The results suggest that resveratrol in articular chondrocytes stimulates differentiation and inflammation via the ERK, p38 and Akt signaling pathways.

Induction of G2/M Arrest by Berberine via Activation of PI3K/Akt and p38 in Human Chondrosarcoma Cell Line.

Berberine is a clinically important natural isoquinoline alkaloid found in many medicinal herbs. Berberine has been shown to have many pharmacological effects including antimicrobial, antitumor, and anti-inflammatory activities. However, the effects and mechanism of action of berberine have not been studied in chondrosarcoma. Therefore, the effects of berberine on proliferation in a human chondrosarcoma cell line (HTB-94) were investigated. Berberine inhibited cell proliferation in a concentration-dependent manner. We also determined that inhibition of cell proliferation by berberine occurred via G2/M phase arrest in HTB-94 cells. Berberine induced cell cycle arrest at the G2/M phase by upregulation of p53 and p21 expression and suppressed cyclin B1, cyclin-dependent kinase 1 (cdc2), cdc25c, and phosphorylated retinoblastoma tumor-suppressor protein (pRb) expression. In addition, berberine stimulated phosphorylation of protein kinase B (Akt) and p38 kinase. Inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt with LY294002 (LY) and p38 kinase with SB203580 (SB), respectively, decreased berberine-induced p53 and p21 expression and restored cell proliferation and expression of cyclin B1, cdc2, cdc25c, and pRb cell cycle progression proteins. These results suggest that berberine-induced inhibition of cell proliferation by cell cycle arrest at the G2/M phases was regulated through PI3K/Akt and p38 kinase pathways in HTB-94 chondrosarcoma cells.

Resveratrol Inhibits Nitric Oxide-Induced Apoptosis via the NF-Kappa B Pathway in Rabbit Articular Chondrocytes.

Resveratrol (trans-3,4'-trihydroxystillbene), a naturally occurring polyphenolic antioxidant found in grapes and red wine, elicits diverse biochemical responses and demonstrates anti-aging, anti-inflammatory, and anti-proliferative effects in several cell types. Previously, resveratrol was shown to regulate differentiation and inflammation in rabbit articular chondrocytes, while the direct production of nitric oxide (NO) in these cells by treatment with the NO donor sodium nitroprusside (SNP) led to apoptosis. In this study, the effect of resveratrol on NO-induced apoptosis in rabbit articular chondrocytes was investigated. Resveratrol dramatically reduced NO-induced apoptosis in chondrocytes, as determined by phase-contrast microscopy, the MTT assay, FACS analysis, and DAPI staining. Treatment with resveratrol inhibited the SNP-induced expression of p53 and p21 and reduced the expression of procaspase-3 in chondrocytes, as detected by western blot analysis. SNP-induced degradation of I-kappa B alpha (IκB-α) was rescued by resveratrol treatment, and the SN50 peptide-mediated inhibition of NF-kappa B (NF-κB) activity potently blocked SNP-induced caspase-3 activation and apoptosis. Our results suggest that resveratrol inhibits NO-induced apoptosis through the NF-κB pathway in articular chondrocytes.