The combination of resveratrol and Bletilla striata polysaccharide decreases inflammatory markers of early osteoarthritis knee and the preliminary results on LPS-induced OA rats.

Osteoarthritis (OA) of the knee is characterized by progressive deterioration and loss of articular cartilage with associatedstructural and performance changes in the entire joint, and current treatments for OA only aim to relieve symptoms, rather than to prevent or reverse disease progression. Recently, treatments targeting "early osteoarthritis" (EOA) have attracted attention. However, during EOA stage, chondrocytes may change behaviors to express pro-inflammatory cytokines and free radicals, which would cause detrimental effects to the synovial cavity and further cartilage wear. In this study, we combined resveratrol (Res) and Bletilla striata polysaccharide (BSP) as anti-inflammatories and antioxidants to diffuse free radicals and to alleviate inflammation from the synovial cavity both short term and long term. The current study introduced a new method for harvesting BSP from as-received Bletilla striata to achieve high yields, shortened extraction times, and maintained structure/functions. In addition, it combined Res and home-extracted BSP (Res-BSP) to alleviate oxidative stress and inflammation in a Lipopolysaccharide (LPS)-induced OA model. The gene expressions of inflammatory genes iNOs, IL-1ß, IL-6, and MMP-13 were upregulated 5.7-fold, 6.5-fold, 8.6-fold, and 4.5-fold, respectively on OA-like chondrocytes and the gene expressions were significantly downregulated to 3.3-fold, 2.1-fold, 4.9-fold, and 0.1-fold, respectively, once OA-like chondrocytes were treated with Res-BSP (p < 0.05, compared with OA-like chondrocytes). The gene expressions of chondrogenic genes TGFß1, SOX9, and type II collagen were downregulated by 0.8-fold, 2.2-fold, and 0.8-fold, respectively, based on the control group as a baseline. While it was significantly upregulated by 3.4-fold, 0.32-fold, and 0.4-fold, respectively, once OA-like chondrocytes were treated with Res-BSP. (p < 0.05, compared with OA-like chondrocytes). Finally, we elucidated the role of Res-BSP in EOA in suppressing COX-2 and activating p-Smad 2/3 and p-Erk1/2. We believe that the combination of Res and BSP has great potential as an alternative therapeutic strategy for EOA treatment in future.

Safety and Efficacy of Kartigen® in Treating Cartilage Defects: A Randomized, Controlled, Phase I Trial.

Here, we aimed to investigate the safety and preliminary efficacy of Kartigen®, a matrix with autologous bone marrow mesenchymal stem cell-derived chondrocyte precursors embedded in atelocollagen. As a surgical graft, Kartigen® was implanted onto the cartilage defects at the weight-bearing site of the medial femoral condyle of the knee. Fifteen patients were enrolled and stratified into two groups, undergoing either Kartigen® implantation (n = 10) or microfracture (control group, n = 5). The primary endpoint was to evaluate the safety of Kartigen® by monitoring the occurrence of adverse events through physician queries, physical examinations, laboratory tests, and radiological analyses for 2 years. There were no infections, inflammations, adhesions, loose body, or tumor formations in the Kartigen®-implanted knees. The preliminary efficacy was assessed using the International Knee Documentation Committee (IKDC) score, visual analog scale, and second-look arthroscopy. The postoperative IKDC scores of the Kartigen® group significantly improved in the 16th week (IKDC = 62.1 ± 12.8, p = 0.025), kept increasing in the first year (IKDC = 78.2 ± 15.4, p < 0.005), and remained satisfactory in the second year (IKDC = 73.6 ± 13.8, p < 0.005), compared to the preoperative condition (IKDC = 47.1 ± 17.0), while the postoperative IKDC scores of the control group also achieved significant improvement in the 28th week (IKDC = 68.5 ± 6.1, p = 0.032) versus preoperative state (IKDC = 54.0 ± 9.1). However, the IKDC scores decreased in the first year (IKDC = 63.5 ± 11.6) as well as in the second year (IKDC = 52.6 ± 16.4). Thirteen patients underwent second-look arthroscopy and biopsy one year after the operation. The Kartigen® group exhibited integration between Kartigen® and host tissue with a smooth appearance at the recipient site, whereas the microfracture group showed fibrillated surfaces. The histological and immunohistochemical analyses of biopsy specimens demonstrated the columnar structure of articular cartilage and existence of collagen type II and glycosaminoglycan mimic hyaline cartilage. This study indicates that Kartigen® is safe and effective in treating cartilage defects.

MicroRNA detection using lateral flow nucleic acid strips with gold nanoparticles.

In this study, the tested microRNA and the detection probe perfectly match with the capture probe instead of the traditional sandwich methods in which the tested oligonucleotide matches with the detection and capture probes. To avoid non-specific signals, mung-bean nuclease, a single-strand-specific nuclease, catalyzes the degradation of the capture probe if there is no tested miRNA in the samples. The gold nanoparticles conjugate the thiol-DNA as the detection probe and the biotin-single strand DNA serves as the capture probe. The avidin-biotin-Au-sample complex is captured by the anti-avidin antibody immobilized on a flow strip. The detection and quantification of the gold nanoparticle signal indicate the existence and quantity of the target miRNA. One fmol and five amol of the synthetic microRNA were detected without and with the silver enhancement, respectively. This highly sensitive and specific assay takes about 70 min after the RNA purification and preparation. It is simple, convenient, fast, and suitable for point-of-care.

Mediation of the inhibitory effect of thyroid hormone on proliferation of hepatoma cells by transforming growth factor-beta.

Thyroid hormone (triiodothyronine, T3) regulates growth, development and differentiation. To examine the influence of T3 on hepatoma cell growth, thyroid receptor (TR)alpha1 or TRbeta1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-beta neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-beta was studied to delineate the repression mechanism. TGF-beta was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-beta repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-beta neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.

Indirect regulation of human dehydroepiandrosterone sulfotransferase family 1A member 2 by thyroid hormones.

Thyroid hormone, T(3), regulates cell metabolism, differentiation, and development. cDNA microarrays were performed to study the mechanism of target gene regulation after T(3) treatment in a thyroid hormone receptor-alpha (TRalpha)-overexpressing hepatoma cell line (HepG2-TRalpha). The differentially expressed target genes are several metabolic enzymes, including dehydroepiandrosterone-sulfotransferase family 1A member 2 (SULT2A1). Enzyme SULT2A1 was elevated roughly 5-fold at the protein level and 9-fold increase at the mRNA level after 48 h T(3) treatment in HepG2-TRalpha cells. Cycloheximide inhibited T(3)-induced SULT2A1 expression, suggesting that regulation was indirect. SULT2A1 has been reported to be regulated by the two transcription factors, steroidogenic factor 1 (SF1) and GATA, in the human adrenal gland. T(3) induced a 2.5- to 3.5-fold elevation of SF1 at the protein level and a 6.2-fold increase at the RNA level in HepG2-TRalpha cells. About seven SF1 binding sites exist on the SULT2A1 gene. To identify and localize the critical SF1 binding site, series of deletion mutants of SULT2A1 promoter fragments in pGL2 plasmid were constructed. The promoter activity of the SULT2A1 gene was enhanced about 2.8- to 7.1-fold by T(3). The -228 SF1 binding site was identified as the most critical site because deleting this region reduced T(3)-induced expression. Transcription factor SF1 application enhanced the -228 but not -117 reporter plasmid activities. SULT2A1 and SF1 up-regulation at protein and RNA levels in thyroidectomized rats occurred after T(3) application. In summary, this work demonstrated that the SULT2A1 gene was mediated by SF1 and indirectly regulated by T(3). Further study is required to elucidate the physiological importance of SULT2A1 induction mediated by T(3).

Regulation of fibronectin by thyroid hormone receptors.

Thyroid hormones regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors and associated pathways. We investigated the triiodothyronine (T3) modulation of gene expression, in human hepatocellular carcinoma cell lines, via a PCR-based cDNA subtraction method. Here we present further data on one of the T3-upregulated genes, fibronectin (FN). We demonstrate that the induction of FN protein expression by T3 in TRalpha1 and TRbeta1 over-expressing cells was time and dose-dependent at the mRNA and protein levels. Blockade of protein synthesis by cycloheximide almost completely inhibited the concomitant induction of FN mRNA by T3, indicating that T3 indirectly regulates FN. Furthermore, nuclear-run on and FN promoter assay clearly can specifically increase the number of FN transcriptional demonstrated that the presence of T3 initiations. In addition, we further confirmed that the up-regulation of FN by T3 was mediated, at least in part, by transforming growth factor-beta (TGF-beta), because the induction of FN was blocked in a dose-dependent manner by the addition of TGF-beta neutralizing antibody. In an effort to elucidate the we demonstrated the involvement of the signaling pathways involved in the activation of FN by T3, mitogen activated protein kinase/c-Jun N-terminal kinase/p38 MAPK (MAPK/JNK/p38) pathway. Although T3 induces the expression of TGF-beta, neither wild-type nor dominant-negative Smad3 or Smad4 over-expression affected the activation of FN by T3. Thus, we demonstrate that T3 regulates FN gene expression indirectly at the transcriptional level, with the participation of the MAPK/JNK/p38 pathway and the TGF-beta signaling pathway but independent of Smad3/4.

Thyroid hormone receptor-dependent transcriptional regulation of fibrinogen and coagulation proteins.

Thyroid hormone (T(3)) regulates growth, development, and differentiation. These activities are mediated by the nuclear thyroid hormone receptors (TRs), which belong to the steroid/TR superfamily of ligand-dependent transcription factors. The effect of T(3) treatment on target gene regulation was investigated in a TRalpha-overexpressing hepatoma cell line (HepG2-TRalpha), by performing cDNA microarrays. We demonstrate that 148 of the 7597 genes represented were up-regulated by T(3), including fibrinogen and several other components of the coagulation factor system. To confirm the microarray results, fibrinogen and a small number of the blood clotting components were further investigated using quantitative RT-PCR. The T(3)-induction ratios observed with quantitative RT-PCR for factors such as thrombin (8-fold), coagulation factor X (4.9-fold), and hepatoglobin (30-fold) were similar to those observed by the cDNA microarray analysis. Further investigation, using HepG2-TRalpha (cell lines, revealed a 2- to 3-fold induction of fibrinogen transcription after 24 h of T(3) treatment. In addition, T(3) treatment increased the level of fibrinogen protein expression 2.5- to 6-fold at 48 h. The protein synthesis inhibitor, cycloheximide, did not inhibit the induction of fibrinogen by T(3), indicating that this regulation was direct. Furthermore, transcription run-on experiments indicate that the induction of fibrinogen by T(3) is regulated largely at the level of transcription. Similar observations were made on the regulation of fibrinogen by T(3) using rats that received surgical thyroidectomy (TX) as an in vivo model. These results suggest that T(3) plays an important role in the process of blood coagulation and inflammation and may contribute to the understanding of the association between thyroid diseases and the misregulation of the inflammatory and clotting profile evident in the circulatory system of these patients.

P53 is a regulator of the metastasis suppressor gene Nm23-H1.

p53, a tumor suppressor gene involved in the G1 cell cycle checkpoint, is also the most frequently mutated gene in human cancer. In addition, p53 modifies the ability of tumor cells to metastasize. The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase, was previously identified in cells with low metastatic potential. Although p53 and Nm23-H1 proteins play an important part in regulating the progression of cancer, any functional relationship between these two proteins is currently unknown. Here we report an association between p53 levels and expression of the Nm23-H1 gene. Our data indicate that wild-type (wt) p53 upregulated the expression of Nm23-H1 at protein and mRNA levels in MCF-7 and J7B cells. This capacity of wt p53 to regulate expression of Nm23-H1 was not only dependent on the endogenous but also the exogenous origin of p53, and could not be reproduced with mutant p53. Subsequently, the invasive ability of MCF-7 and J7B cells was suppressed upon induction of the Nm23-H1 protein by p53. In contrast, increased levels of p53 downregulated the expression of Nm23-H1 at the protein and mRNA levels in RKO and H1299 cells and, as a consequence, increased the invasive ability of both cell types. Thus, our results implicated the differential regulation of Nm23-H1 by p53 in different cell types as an important component in the molecular mechanisms of tumor metastasis.