Effect of Alpinia galanga extract on cartilage degradation and on gene expression in human chondrocyte and synovial fibroblast metabolism

We investigated the effects of A. galanga extract on metabolism and gene expression involved in the interleukin-1β (IL-1β) response of human chondrocyte and synovial fibroblast. A. galanga extract inhibited IL-1β enhanced matrix breakdown of the cartilage explants in a dose-dependent manner. It suppressed uronic acid loss from the tissue and decreased the release of sulfated GAG and hyaluronan into the medium. MMP-2 and MMP-9 activity in the culture medium of chondrosarcomas and synovial fibroblasts were significantly reduced in the presence of A. galanga extract, which also suppressed the production of MMP-1,-3 and-13. The A. galanga extract also significantly increased type II collagen, SOX9 and aggrecan gene expression, suggesting an ability to enhance anabolic activity. At a high dose of A. galanga extract there was a down-regulation of aggrecan gene expression. Comparison with Diacerein® showed its general anti-inflammatory potential to be similar. The A. galanga extract was shown to inhibit IL-1β-stimulated cartilage matrix degradation in both systems. Additionally, the extract showed the potential to up-regulate certain chondrocyte anabolic genes. It may, therefore, offer some cartilage protective and anti-inflammatory properties as a therapeutic agent in arthritis.     

Two phenolic constituents from Alpinia galanga rhizomes

Chemical investigation of the chloroform extract of the rhizomes of Alpinia galanga yielded p-hydroxycinnamaldehyde and [di-(p-hydroxy-cis-styryl)] methane. The former is isolated for the first time in nature and the latter is a new chemical component. These compounds were characterized from spectral studies and chemical reactions.     

Caesalpinia sappan extract inhibits IL1β-mediated overexpression of matrix metalloproteinases in human chondrocytes

Exacerbated production of matrix metalloproteinases (MMPs) is a key event in the progression of osteoarthritis (OA) and represents a promising target for the management of OA with nutraceuticals. In this study, we sought to determine the MMP-inhibitory activity of an ethanolic Caesalpinia sappan extract (CSE) in human OA chondrocytes. Thus, human articular chondrocytes isolated from OA cartilage and SW1353 chondrocytes were stimulated with Interleukin-1beta (IL1β), without or with pretreatment with CSE. Following viability assays, the production of MMP-2 and MMP-13 was assessed using ELISA, whereas mRNA levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13 and TIMP-1, TIMP-2, TIMP-3 were quantified using RT-qPCR assays. Chondrocytes were co-transfected with a MMP-13 luciferase reporter construct and NF-kB p50 and p65 expression vectors in the presence or absence of CSE. In addition, the direct effect of CSE on the proteolytic activities of MMP-2 was evaluated using gelatin zymography. We found that CSE significantly suppressed IL1β-mediated upregulation of MMP-13 mRNA and protein levels via abrogation of the NF-kB(p65/p50)-driven MMP-13 promoter activation. We further observed that the levels of IL1β-induced MMP-1, MMP-3, MMP-7, and MMP-9 mRNA, but not TIMP mRNA levels, were down-regulated in chondrocytes in response to CSE. Zymographic results suggested that CSE did not directly interfere with the proteolytic activity of MMP-2. In summary, this study provides evidence for the MMP-inhibitory potential of CSE or CSE-derived compounds in human OA chondrocytes. The data indicate that the mechanism of this inhibition might, at least in part, involve targeting of NF-kB-mediated promoter activation.     

Association of IL-17A and IL-17F single nucleotide polymorphisms with susceptibility to osteoarthritis in a Korean population

The damage incurred in osteoarthritis (OA) is mediated by a variety of cytokines, growth factors and inflammatory mediators. The importance of the interleukin-17 (IL-17) family in inflammatory and autoimmune disease is becoming increasingly apparent. Microsatellite association mapping reveals a primary osteoarthritis susceptibility locus on chromosome 6p12.3-q13. IL-17A and IL-17F genes that resided on chromosome 6p12.3-q13 are believed to play an important role in the primary OA susceptibility. We investigated the allele and genotype of IL-17A G-197A and IL-17F T7488C in 302 OA patients and 300 healthy subjects as controls. We employed a PCR-SSCP assay to identify the genotypes IL-17A G-197A and IL-17F T7488C. For IL-17A G-197A, there were significant differences in frequencies of genotype and allele of IL-17A G-197A between OA patients and controls (both p < 0.0001). For IL-17F T7488C, there were no significant differences in the allele frequency and genotype distribution for IL-17F T7488C between OA patients and controls (p = 0.938 and p = 0.1735, respectively). In conclusion, current study showed that polymorphism of IL-17A G-197A may be closely associated with susceptibility to the development of OA in the Korean population. However, there was no relationship between IL-17F T7488C polymorphism and OA susceptibility.     

MicroRNA-33a regulates cholesterol synthesis and cholesterol efflux-related genes in osteoarthritic chondrocytes

IntroductionSeveral studies have shown that osteoarthritis (OA) is strongly associated with metabolism-related disorders, highlighting OA as the fifth component of the metabolic syndrome (MetS). On the basis of our previous findings on dysregulation of cholesterol homeostasis in OA, we were prompted to investigate whether microRNA-33a (miR-33a), one of the master regulators of cholesterol and fatty acid metabolism, plays a key role in OA pathogenesis.MethodsArticular cartilage samples were obtained from 14 patients with primary OA undergoing total knee replacement surgery. Normal cartilage was obtained from nine individuals undergoing fracture repair surgery. Bioinformatics analysis was used to identify miR-33a target genes. miR-33a and sterol regulatory element-binding protein 2 (SREBP-2) expression levels were investigated using real-time PCR, and their expression was also assessed after treatment with transforming growth factor-β1 (TGF-β1) in cultured chondrocytes. Akt phosphorylation after treatment with both TGF-β1 and miR-33a inhibitor or TGF-β1 and miR-33a mimic was assessed by Western blot analysis. Furthermore, we evaluated the effect of miR-33a mimic and miR-33a inhibitor on Smad7, a negative regulator of TGF-β signaling, on cholesterol efflux-related genes, ATP-binding cassette transporter A1 (ABCA1), apolipoprotein A1 (ApoA1) and liver X receptors (LXRα and LXRβ), as well as on matrix metalloproteinase-13 (MMP-13), using real-time PCR.ResultsWe found that the expression of miR-33a and its host gene SREBP-2 was significantly elevated in OA chondrocytes compared with normal chondrocytes. Treatment of cultured chondrocytes with TGF-β1 resulted in increased expression of both miR-33a and SREBP-2, as well as in rapid induction of Akt phosphorylation, whereas TGF-β-induced Akt phosphorylation was enhanced by miR-33a and suppressed by inhibition of miR-33a, as a possible consequence of Smad7 regulation by miR-33a. Moreover, treatment of normal chondrocytes with miR-33a resulted in significantly reduced ABCA1 and ApoA1 mRNA expression levels and significantly elevated MMP-13 expression levels, promoting the OA phenotype, whereas miR-33a’s suppressive effect was reversed using its inhibitor.ConclusionsOur findings suggest, for the first time to our knowledge, that miR-33a regulates cholesterol synthesis through the TGF-β1/Akt/SREBP-2 pathway, as well as cholesterol efflux-related genes ABCA1 and ApoA1, in OA chondrocytes, pointing to its identification as a novel target for ameliorating the OA phenotype.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Hyaluronic Acid Suppresses the Expression of Metalloproteinases in Osteoarthritic Cartilage Stimulated Simultaneously by Interleukin 1β and Mechanical Load

Purpose

In patients with osteoarthritis (OA), intraarticular injection of hyaluronic acid (HA) frequently results in reduced pain and improved function for prolonged periods of time, i.e. more than 6 months. However, the mechanisms underlying these effects are not fully understood. Our underlying hypothesis is that HA modifies the enzymatic breakdown of joint tissues.

Methods

To test this hypothesis, we examined osteochondral cylinders from 12 OA patients. In a bioreactor, these samples were stimulated by interleukin 1β (Il1ß) (2 ng/ml) plus mechanical load (2.0 Mpa at 0.5 Hz horizontal and 0.1 Hz vertical rotation), thus the experimental setup recapitulated both catabolic and anabolic clues of the OA joint.

Results

Upon addition of HA at either 1 or 3 mg/ml, we observed a significant suppression of expression of metalloproteinase (MMP)-13. A more detailed analysis based on the Kellgren and Lawrence (K&L) OA grade, showed a much greater degree of suppression of MMP-13 expression in grade IV as compared to grade II OA. In contrast to the observed MMP-13 suppression, treatment with HA resulted in a suppression of MMP-1 expression only at 1 mg/ml HA, while MMP-2 expression was not significantly affected by either HA concentration.

Conclusion

Together, these data suggest that under concurrent catabolic and anabolic stimulation, HA exhibits a pronounced suppressive effect on MMP-13. In the long-run these findings may benefit the development of treatment strategies aimed at blocking tissue degradation in OA patients.     

Gene polymorphism analysis of Yersinia enterocolitica outer membrane protein A and putative outer membrane protein A family protein

Background

Yersinia enterocolitica outer membrane protein A (OmpA) is one of the major outer membrane proteins with high immunogenicity. We performed the polymorphism analysis for the outer membrane protein A and putative outer membrane protein A (p-ompA) family protein gene of 318 Y. enterocolitica strains.

Results

The data showed all the pathogenic strains and biotype 1A strains harboring ystB gene carried both ompA and p-ompA genes; parts of the biotype 1A strains not harboring ystB gene carried either ompA or p-ompA gene. In non-pathogenic strains (biotype 1A), distribution of the two genes and ystB were highly correlated, showing genetic polymorphism. The pathogenic and non-pathogenic, highly and weakly pathogenic strains were divided into different groups based on sequence analysis of two genes. Although the variations of the sequences, the translated proteins and predicted secondary or tertiary structures of OmpA and P-OmpA were similar.

Conclusions

OmpA and p-ompA gene were highly conserved for pathogenic Y. enterocolitica. The distributions of two genes were correlated with ystB for biotype 1A strains. The polymorphism analysis results of the two genes probably due to different bio-serotypes of the strains, and reflected the dissemination of different bio-serotype clones of Y. enterocolitica.     

Structural characterization and protective effect against murine sepsis of fucogalactans from Agaricus bisporus and Lactarius rufus

Fucogalactans from edible Agaricus bisporus (RFP-Ab) and wild Lactarius rufus (RFP-Lr) mushrooms were obtained on aqueous extraction followed by purification. RFP-Ab had M_w 43.8 × 10⁴ g mol⁻¹ and RFP-Lr M_w 1.4 × 10⁴ g mol⁻¹. RFP-Lr had a (1 → 6)-linked α-d-Galp main-chain partially substituted at O-2 by nonreducing end-units of α-l-Fucp (29%). While RFP-Ab had a similar main chain, it was partially substituted at O-2 by nonreducing end-units of α-l-Fucp (2.8%) and β-d-Galp (14.5%), and partially methylated at HO-3. Both RFP-Lr and RFP-Ab were tested in mice against polymicrobial sepsis. Lethality rate, myeloperoxidase (MPO) activity and cytokine levels were determined. It was observed a reduction in late mortality rate by 62.5% and 50%, respectively, prevention of neutrophil accumulation in ileum and decreasing in TNF-α and IL-1β serum levels.     

Chondroprotective and anti-inflammatory effects of sesamin

Osteoarthritis (OA) is a major disability of elderly people. Sesamin is the main compound in Sesamun indicum Linn., and it has an anti-inflammatory effect by specifically inhibiting Δ5-desaturase in polyunsaturated fatty acid biosynthesis. The chondroprotective effects of sesamin were thus studied in a porcine cartilage explant induced with interleukin-1beta (IL-1β) and in a papain-induced osteoarthritis rat model. With the porcine cartilage explant, IL-1β induced release of sulfated-glycosaminoglycan (s-GAG) and hydroxyproline release, and this induction was significantly inhibited by sesamin. This ability to inhibit these processes might be due to its ability to decrease expression of MMP-1, -3 and -13, which can degrade both PGs and type II collagen, both at the mRNA and protein levels. Interestingly, activation of MMP-3 might also be inhibited by sesamin. Moreover, in human articular chondrocytes (HACs), some pathways of IL-1β signal transduction were inhibited by sesamin: p38 and JNK. In the papain-induced OA rat model, sesamin treatment reversed the following pathological changes in OA cartilage: reduced disorganization of chondrocytes in cartilage, increased cartilage thickness, and decreased type II collagen and PGs loss. Sesamin alone might increase formation of type II collagen and PGs in the cartilage tissue of control rats. These results demonstrate that sesamin efficiently suppressed the pathological processes in an OA model. Thus, sesamin could be a potential therapeutic strategy for treatment of OA.