In vitro, in vivo and in silico anti-hyperglycemic inhibition by sinigrin

OBJECTIVE@#To evaluate the anti-hyperglycemic potential of sinigrin using in vitro, in silico and in vivo streptozotocin (STZ) induced hyperglycemic zebrafish model.@*METHODS@#The in vitro enzyme inhibition assay was carried out to determine the IC value against α-glucosidase and α-amylase, in silico molecular docking was performed against both enzymes with PyRx tool and simulations were performed using GROMACS tool. Hyperglycemia was induced in zebrafishes using three intraperitoneal injections on alternating days for 1 week at 350 mg/kg of STZ. Hyperglycemic fishes were treated intraperitoneally with 50, 100 and 150 mg of sinigrin/kg of body weight for 24 h and glucose levels were measured.@*RESULTS@#The sinigrin showed very strong inhibition against α-glucosidase and α-amylase with 0.248 and 0.00124 μM while reference drug acarbose showed IC value of 73.0700 and 0.0017 μM against α-glucosidase and α-amylase, respectively. Kinetic analysis revealed that sinigrin has the mixed type mode of inhibition against α-glucosidase. Molecular docking results revealed its strong binding affinity with α-glucosidase (-10.00 kcal/mol) and α-amylase (-8.10 kcal/mol). Simulations graphs confirmed its stability against both enzymes. Furthermore, in hyperglycemic zebrafishes most significant (P < 0.001) reduction of glucose was occurred at 150 mg/kg, moderate significant reduction of glucose was observed at 100 mg/kg and no any significant reduction of glucose was measured at 50 mg/kg.@*CONCLUSIONS@#It can be evident from the present results that sinigrin has potent anti-hyperglycemic activity and it may prove to be effective treatment for the hyperglycemia.

In vitro, in vivo and in silico anti-hyperglycemic inhibition by sinigrin

Objective To evaluate the anti-hyperglycemic potential of sinigrin using in vitro, in silico and in vivo streptozotocin (STZ) induced hyperglycemic zebrafish model. Methods The in vitro enzyme inhibition assay was carried out to determine the IC

Withaferin A-Caused Production of Intracellular Reactive Oxygen Species Modulates Apoptosis via PI3K/Akt and JNKinase in Rabbit Articular Chondrocytes

Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.

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 (IkappaB-alpha) was rescued by resveratrol treatment, and the SN50 peptide-mediated inhibition of NF-kappa B (NF-kappaB) activity potently blocked SNP-induced caspase-3 activation and apoptosis. Our results suggest that resveratrol inhibits NO-induced apoptosis through the NF-kappaB pathway in articular chondrocytes.

Snapshot of degenerative aging of porcine intervertebral disc: a model to unravel the molecular mechanisms

Larger animal models, such as porcine, have been validated as appropriate models of the human disc with respect to biomechanics and biochemistry. They are advantageous for research as the models are relatively straightforward to prepare and easily obtainable for research to perform surgical techniques. The intention of this study was to quantitatively analyze gene expression for collagen and proteoglycan components of the extracellular matrix and for collagenase (MMP-1) in porcine discs of varying ages (Newborn; 2-3weeks, Mature; 6-9 month, Older; 2-3 years). In this study, we observed that the cell number and GAG (glycosaminoglycan) formation dramatically decreased with aging. Also, gene expression in the annulus fibrosus (AF) and nucleus pulposus (NP) cells changed with aging. The level of MMP-1 mRNA increased with age and both type I, II collagens decreased with age. The level of aggrecan mRNA was highest in the mature group and decreased significantly with aging. In the mature group, MMP-1 expression was minimal compared to the newborn group. In AF cells, type II collagen was expressed at a high level in the mature group with a higher level of aggrecan, when aged NP showed a decrease in type II collagen. The model of IVD degeneration in the porcine disc shows many changes in gene expression with age that have been previously documented for human and may serve as a model for studying changes in IVD metabolism with age. We concluded that the porcine model is excellent to test hypotheses related to disc degeneration while permitting time-course study in biologically active systems.

Endoplasmic reticulum stress (ER-stress) by 2-deoxy-D-glucose (2DG) reduces cyclooxygenase-2 (COX-2) expression and N-glycosylation and induces a loss of COX-2 activity via a Src kinase-dependent pathway in rabbit articular chondrocytes

Endoplasmic reticulum (ER) stress regulates a wide range of cellular responses including apoptosis, proliferation, inflammation, and differentiation in mammalian cells. In this study, we observed the role of 2-deoxy-D-glucose (2DG) on inflammation of chondrocytes. 2DG is well known as an inducer of ER stress, via inhibition of glycolysis and glycosylation. Treatment of 2DG in chondrocytes considerably induced ER stress in a dose- and time-dependent manner, which was demonstrated by a reduction of glucose regulated protein of 94 kDa (grp94), an ER stress-inducible protein, as determined by a Western blot analysis. In addition, induction of ER stress by 2DG led to the expression of COX-2 protein with an apparent molecular mass of 66-70kDa as compared with the normally expressed 72-74 kDa protein. The suppression of ER stress with salubrinal (Salub), a selective inhibitor of eif2-alpha dephosphorylation, successfully prevented grp94 induction and efficiently recovered 2DG-modified COX-2 molecular mass and COX-2 activity might be associated with COX-2 N-glycosylation. Also, treatment of 2DG increased phosphorylation of Src in chondrocytes. The inhibition of the Src signaling pathway with PP2 (Src tyrosine kinase inhibitor) suppressed grp94 expression and restored COX-2 expression, N-glycosylation, and PGE2 production, as determined by a Western blot analysis and PGE2 assay. Taken together, our results indicate that the ER stress induced by 2DG results in a decrease of the transcription level, the molecular mass, and the activity of COX-2 in rabbit articular chondrocytes via a Src kinase-dependent pathway.

2-Deoxy-D-glucose regulates dedifferentiation through beta-catenin pathway in rabbit articular chondrocytes

2-deoxy-D-glucose (2DG) is known as a synthetic inhibitor of glucose. 2DG regulates various cellular responses including proliferation, apoptosis and differentiation by regulation of glucose metabolism in cancer cells. However, the effects of 2DG in normal cells, including chondrocytes, are not clear yet. We examined the effects of 2DG on dedifferentiation with a focus on the beta-catenin pathway in rabbit articular chondrocytes. The rabbit articular chondrocytes were treated with 5 mM 2DG for the indicated time periods or with various concentrations of 2DG for 24 h, and the expression of type II collagen, c-jun and beta-catenin was determined by Western blot, RT-PCR, immunofluorescence staining and immunohistochemical staining and reduction of sulfated proteoglycan synthesis detected by Alcain blue staining. Luciferase assay using a TCF (T cell factor)/LEF (lymphoid enhancer factor) reporter construct was used to demonstrate the transcriptional activity of beta-catenin. We found that 2DG treatment caused a decrease of type II collagen expression. 2DG induced dedifferentiation was dependent on activation of beta-catenin, as the 2DG stimulated accumulation of beta-catenin, which is characterized by translocation of beta-catenin into the nucleus determined by immunofluorescence staining and luciferase assay. Inhibition of beta-catenin degradation by inhibition of glycogen synthase kinase 3-beta with lithium chloride (LiCl) or inhibition of proteasome with z-Leu-Leu-Leu-CHO (MG132) accelerated the decrease of type II collagen expression in the chondrocytes. 2DG regulated the post-translational level of beta-catenin whereas the transcriptional level of beta-catenin was not altered. These results collectively showed that 2DG regulates dedifferentiation via beta-catenin pathway in rabbit articular chondrocytes.

Curcumin inhibits cellular condensation and alters microfilament organization during chondrogenic differentiation of limb bud mesenchymal cells

Curcumin is a well known natural polyphenol product isolated from the rhizome of the plant Curcuma longa, anti-inflammatory agent for arthritis by inhibiting synthesis of inflammatory prostaglandins. However, the mechanisms by which curcumin regulates the functions of chondroprogenitor, such as proliferation, precartilage condensation, cytoskeletal organization or overall chondrogenic behavior, are largely unknown. In the present report, we investigated the effects and signaling mechanism of curcumin on the regulation of chondrogenesis. Treating chick limb bud mesenchymal cells with curcumin suppressed chondrogenesis by stimulating apoptotic cell death. It also inhibited reorganization of the actin cytoskeleton into a cortical pattern concomitant with rounding of chondrogenic competent cells and down-regulation of integrin beta1 and focal adhesion kinase (FAK) phosphorylation. Curcumin suppressed the phosphorylation of Akt leading to Akt inactivation. Activation of Akt by introducing a myristoylated, constitutively active form of Akt reversed the inhibitory actions of curcumin during chondrogenesis. In summary, for the first time, we describe biological properties of curcumin during chondrogenic differentiation of chick limb bud mesenchymal cells. Curcumin suppressed chondrogenesis by stimulating apoptotic cell death and down-regulating integrin-mediated reorganization of actin cytoskeleton via modulation of Akt signaling.

A MELAS syndrome family harboring two mutations in mitochondrial genome

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.

Ectopic expression of cyclooxygenase-2-induced dedifferentiation in articular chondrocytes

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.

15-Deoxy-delta 12,14-ProstaglandinJ2 Regulates Dedifferentiation through Peroxisome Proliferator-Activated Receptor-gamma-Dependent Pathway but Not COX-2 Expression in Articular Chondrocytes

Peroxisome proliferator-activated receptors-gamma (PPAR-gamma) is critical for phenotype determination at early differentiation stages of mesenchymal cells, whereas its physiological role is unclear. Therefore, we investigated the role of 15-deoxy-delta 12,14-prostaglandinJ2 (15d-PGJ2), the natural receptor ligand for PPAR-gamma, on dedifferentiation and inflammatory responses, such as COX-2 expression and PGE2 production, in articular chondrocytes. Our data indicate that the 15d-PGJ2 caused a loss of differentiated chondrocyte phenotype as demonstrated by inhibition of type II collagen and proteoglycan synthesis. 15d-PGJ2 also induced COX-2 expression and PGE2 production. The 15d-PGJ2-induced dedifferentiation effect seems to be dependent on PPAR-gamma activation, as the PPRE luciferase activity increased and PPAR-gamma antagonist, BADGE, abolished type II collagen expression. However, BADGE did not block 15d-PGJ2-induced COX-2 expression. Collectively, our findings suggest that PPAR-gamma-dependent and -independent mechanisms of 15d-PGJ2-induced dedifferentiation and inflammatory responses in articular chondrocytes, respectively. Additionally, these data suggest that targeted modulation of the PPAR-gamma pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.

ERK-1/-2 and p38 Kinase Oppositely Regulate 15-deoxy-delta(12,14)-prostaglandinJ2-Induced PPAR-gamma Activation That Mediates Dedifferentiation But Not Cyclooxygenase-2 Expression in Articular Chondrocytes

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. In this study, we investigated the effects of 15d-PGJ2, a high-affinity ligand of PPAR-gamma, on dedifferentiation and on inflammatory responses such as COX-2 expression and PGE2 production in rabbit articular chondrocytes with a focus on ERK-1/-2, p38 kinase, and PPAR-gamma activation. We report here that 15d-PGJ2 induced dedifferentiation and/or COX-2 expression and subsequent PGE2 production. 15d-PGJ2 treatment stimulated activation of ERK-1/-2, p38 kinase, and PPAR-gamma. Inhibition of ERK-1/-2 with PD98059 recovered 15d-PGJ2-induced dedifferentiation and enhanced PPAR-gamma activation, whereas inhibition of p38 kinase with SB203580 potentiated dedifferentiation and partially blocked PPAR-gamma activation. Inhibition of ERK-1/-2 and p38 kinase abolished 15d-PGJ2-induced COX-2 expression and subsequent PGE2 production. Our findings collectively suggest that ERK-1/-2 and p38 kinase oppositely regulate 15d-PGJ2-induced dedifferentiation through a PPAR-gamma-dependent mechanism, whereas COX-2 expression and PGE2 production is regulated by ERK-1/-2 through a PPAR-gamma-independent mechanism but not p38 kinase in articular chondrocytes. Additionally, these data suggest that targeted modulation of the PPAR-gamma and mitogen-activated protein kinase pathway may offer a novel approach for therapeutic inhibition of joint tissue degradation.

Src Kinase Regulates Nitric Oxide-induced Dedifferentiation and Cyclooxygenase-2 Expression in Articular Chondrocytes via p38 Kinase-dependent Pathway

BACKGROUND: Nitric oxide (NO) in articular chondrocytes regulates dedifferentiation and inflammatory responses by modulating MAP kinases. In this study, we investigated whether the Src kinase in chondrocytes regulates NO-induced dedifferentiation and cyclooxygenase-2 (COX-2) expression. METHODS: Primary chondrocytes were treated with various concentrations of SNP for 24 h. The COX-2 and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin E(2) (PGE(2)) was determined by using a PGE(2) assay kit. Expression and distribution of p-Caveolin and COX-2 in rabbit articular chondrocytes and cartilage explants were determined by immunohistochemical staining and immunocytochemical staining, respectively. RESULTS: SNP treatment stimulated Src kinase activation in a dose-dependent manner in articular chondrocytes. The Src kinase inhibitors PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine], a significantly blocked SNP-induced p38 kinase and caveolin-1 activation in a dose-dependent manner. Therefore, to determine whether Src kinase activation is associated with dedifferentiation and/or COX-2 expression and PGE(2) production. As expected, PP2 potentiated SNP-stimulated dedifferentiation, but completely blocked both COX-2 expression and PGE2 production. And also, levels of p-Caveolin and COX-2 protein expression were increased in SNP-treated primary chondrocytes and osteoarthritic and rheumatoid arthritic cartilage, suggesting that p-Caveolin may play a role in the inflammatory responses of arthritic cartilage. CONCLUSION: Our previously studies indicated that NO caused dedifferentiation and COX-2 expression is regulated by p38 kinase through caveolin-1 (1). Therefore, our results collectively suggest that Src kinase regulates NO-induced dedifferentiation and COX-2 expression in chondrocytes via p38 kinase in association with caveolin-1.

p38 Kinase Regulates Nitric Oxide-induced Dedifferentiation and Cyclooxygenase-2 Expression of Articular Chondrocytes

BACKGROUND: Caveolin, a family of integral membrane proteins are a principal component of caveolae membranes. In this study, we investigated the effect of p38 kinase on differentiation and on inflammatory responses in sodium nitroprusside (SNP)- treated chondrocytes. METHODS: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. SNP was used as a nitric oxide (NO) donor. In this experiments measuring SNP dose response, primary chondrocytes were treated with various concentrations of SNP for 24 h. The time course of the SNP response was determined by incubating cells with 1 mM SNP for the indicated time period (0~24 h). The cyclooxygenase-2 (COX-2) and type II collagen expression levels were determined by immunoblot analysis, and prostaglandin E2 (PGE2) assay was used to measure the COX-2 activity. The tyrosine phosphorylation of caveolin-1 was determined by immunoblot analysis and immunostaining. RESULTS: SNP treatment stimulated tyrosine phosphorylation of caveolin-1 and activation of p38 kinase. SNP additionally caused dedifferentiation and inflammatory response. We showed previously that SNP treatment stimulated activation of p38 kinase and ERK-1/-2. Inhibition of p38 kinase with SB203580 reduced caveolin-1 tyrosine phosphorylation and COX-2 expression but enhanced dedifferentiation, whereas inhibition of ERK with PD98059 did not affect caveolin-1 tyrosine phosphorylation levels, suggesting that ERK at least is not related to dedifferentiation and COX-2 expression through caveolin-1 tyrosine phosphorylation. CONCLUSION: Our results indicate that SNP in articular chondrocytes stimulates dedifferentiation and inflammatory response via p38 kinase signaling in association with caveolin-1 phosphorylation.

Ectopic Expression of Caveolin-1 Induces COX-2 Expression in Rabbit Articular Chondrocytes via MAP Kinase Pathway

BACKGROUND: Caveolin-1 is a principal component of caveolae membranes in vivo. Although expression of caveolae structure and expression of caveolin family, caveolin-1, -2 and -3, was known in chondrocytes, the functional role of caveolae and caveolins in chondrocytes remains unknown. In this study, we investigated the role of caveolin-1 in articular chondrocytes. METHODS: Rabbit articular chondrocytes were prepared from cartilage slices of 2-week-old New Zealand white rabbits by enzymatic digestion. Caveolin-1 cDNA was transfected to articular chondrocytes using LipofectaminePLUS. The cyclooxygenase-2 (COX-2) expression levels were determined by immunoblot analysis, immunostaining, immunohistochemistry, and prostaglandin E2 (PGE2) assay was used to measure the COX-2 activity. RESULTS: Ectopic expression of caveolin-1 induced COX-2 expression and activity, as indicated by immunoblot analysis and PGE2 assay. And also, overexpression of caveolin-1 stimulated activation of p38 kinase and ERK-1/ -2. Inhibition of p38 kinase and ERK-1/-2 with SB203580 and PD98059, respectively, led to a dose-dependent decrease COX-2 expression and PGE2 production in caveolin-1-transfected cells. CONCLUSION: Taken together, our data suggest that ectopic expression of caveolin-1 contributes to the expression and activity of COX-2 in articular chondrocytes through MAP kinase pathway.

Whole blood and Plasma Vitamin C Concentrations of Elementary School Children in Chinju

PURPOSE: Recommended dietary allowance of vitamin C was determined on the basis of preventing the scurvy without considerations of the important function of the vitamin C as a first line antioxidant. So we measured the whole blood and plasma vitamin C concentrations of the contemporay healthy elementary school children in Chinju for the establishment of the optimal daily vitamin C requirment in the elementary school children. METHODS: Whole blood and plasma vitamin C concentrations were measured by the 2,4-dinitrophenylhydrazine method in 338 children from the 1st to the 6th grade of one elementary school in Chinju. RESULTS: Whole blood and plasma vitamin C concentrations were 1.36+/-0.34mg/dL and 1.07+/-0.33mg/dL respectively. There existed an close relationship between whole blood and plasma vitamin C concentrations (r=0.77, p=0.0001). Whole blood vitamin C concentration decreased as the age became older (r=-0.22 p=0.0001), but plasma vitamin C concentration did not change. There were no sex differences in the whole blood and plasma vitamin C concentrations except in the 3rd grade (p<0.05). Twenty-three of 338 elementary school children (6.8%) had the plasma vitamin C concentration less than 0.6mg/dL. CONCLUSIONS: We produced the blood and plasma vitamin C concentrations of the contemporay elementary school children in Chinju. These values were not satisfactory in consideration of the importance of the childhood health.

Exchange Transfusion Using Percutaneous Femoral Vein Catheterization in Neonates: Comparison with Umbilical Vein Catheterization

Traditionally umbilical vein has been used for exchange transfusion in neonates. This method is relatively safe and effective but with a few complications. So via percutaneous femoral vein catheters we tried exchange transfusions in 30 patients with neonatal hyperbilirubinemia admitted to GNUH from September 1990 to August 1992. Femoral vein catheterization succeeded in 2~3 trials, In all cases high bilirubin level was lowered by half at the end of exchange transfusion. Transient microscopic hematuria by bladder puncture occurred in one neonate. Exchange transfusion via femoral vein catheter is a sage, easy and effective method.