Combination of human platelet lysate and 3D gelatin scaffolds to enhance osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells.

Bone disorders are major health issues requiring specialized care; however, the traditional bone grafting method had several limitations. Thus, bone tissue engineering has become a potential alternative. In therapeutic treatments, using fetal bovine serum (FBS) as a culture supplement may result in the risk of contamination and host immunological response; therefore, human platelet lysate (hPL) has been considered a viable alternative source. This study attempted to compare the effectiveness and safety of different culture supplements, either FBS or hPL, on the osteoblastic differentiation potential of mesenchymal stem cells derived from human amniotic fluid (hAF-MSCs) under a three-dimensional gelatin scaffold. The results indicate that hAF-MSCs have the potential to be used in clinical applications as they meet the criteria for mesenchymal stem cells based on their morphology, the expression of a particular surface antigen, their proliferation ability, and their capacity for multipotent differentiation. After evaluation by MTT and Alamar blue proliferation assay, 10% of hPL was selected. The osteogenic differentiation of hAF-MSCs under three-dimensional gelatin scaffold using osteogenic-induced media supplemented with hPL was achievable and markedly stimulated osteoblast differentiation. Moreover, the expressions of osteoblastogenic related genes, including OCN, ALP, and COL1A1, exhibited the highest degree of expression under hPL-supplemented circumstances when compared with the control and the FBS-supplemented group. The induced cells under hPL-supplemented conditions also presented the highest ALP activity level and the greatest degree of calcium accumulation. These outcomes would indicate that hPL is a suitable substitute for animal derived serum. Importantly, osteogenic differentiation of human amniotic fluid derived mesenchymal stem cells using hPL-supplemented media and three-dimensional scaffolds may open the door to developing an alternative construct for repairing bone defects.

Bromelain Extract Exerts Antiarthritic Effects via Chondroprotection and the Suppression of TNF-α-Induced NF-κB and MAPK Signaling.

Bromelain, a mixture of proteases in pineapple rhizome, has beneficial biological properties. Following absorption, the compound remains biologically active in mammalian blood and tissues. Bromelain has multiple clinical and therapeutic applications because of its anti-arthritic activities. Anti-inflammation is one of the putative therapeutic effects of bromelain on osteoarthritis (OA) and rheumatoid arthritis (RA), but the molecular mechanisms in cartilage and synovial fibroblast has not been reported. Thus, in this study, interleukin (IL)-1ß/oncostatin M-induced porcine cartilage and TNF-α-induced synovial fibroblast were used as the inflamed OA and RA models, respectively. The results demonstrated the chondroprotective effects of bromelain on cartilage degradation and the downregulation of inflammatory cytokine (tumor necrosis factor (TNF)-α, IL-1ß, IL-6, IL-8) expression in TNF-α-induced synovial fibroblasts by suppressing NF-κB and MAPK signaling. The evidence from this study supported and explained the anti-inflammatory and analgesic effects of bromelain on arthritis in animal models and clinical studies.

Anti-Inflammatory Effect of Pineapple Rhizome Bromelain through Downregulation of the NF-κB- and MAPKs-Signaling Pathways in Lipopolysaccharide (LPS)-Stimulated RAW264.7 Cells.

Bromelain is a mixture of proteolytic enzymes derived from pineapple (Ananas comosus) fruit and stem possessing several beneficial properties, particularly anti-inflammatory activity. However, the molecular mechanisms underlying the anti-inflammatory effects of bromelain are unclear. This study investigated the anti-inflammatory effects and inhibitory molecular mechanisms of crude and purified rhizome bromelains on lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. RAW264.7 cells were pre-treated with various concentrations of crude bromelain (CB) or purified bromelain (PB), and then treated with LPS. The production levels of pro-inflammatory cytokines and mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α were determined by Griess and ELISA assays. The expressions of inducible nitric oxide synthetase (iNOS), cyclooxygenase (COX)-2, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs)-signaling pathway-related proteins were examined by western blot analysis. The pre-treatment of bromelain dose-dependently reduced LPS-induced pro-inflammatory cytokines and mediators, which correlated with downregulation of iNOS and COX-2 expressions. The inhibitory potency of PB was stronger than that of CB. PB also suppressed phosphorylated NF-κB (p65), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha, extracellular signal-regulated kinases, c-Jun amino-terminal kinases, and p38 proteins in LPS-treated cells. PB then exhibited potent anti-inflammatory effects on LPS-induced inflammatory responses in RAW264.7 cells by inhibiting the NF-κB and MAPKs-signaling pathways.

Sesamin Promotes Neurite Outgrowth under Insufficient Nerve Growth Factor Condition in PC12 Cells through ERK1/2 Pathway and SIRT1 Modulation.

The promotion of neurogenesis can be a promising strategy to improve and restore neuronal function in neurodegenerative diseases. Nerve growth factor (NGF) plays a key role in neurite outgrowth and synaptic formation during brain repair stage. Nowadays, there are several studies on the developing methods to enhance the endogenous NGF activity for treatment and restore the neuronal function. In this study, the potentiating effect of sesamin, a major lignan in sesame seeds (Sesamum indicum) and oil, on NGF-induced neurogenesis and its involved mechanisms were firstly reported. Sesamin effectively enhanced the PC12 neuron-like cell differentiation and neurite length under insufficient conditions of NGF. The neuronal markers including synaptophysin and growth-associated protein-43 along with the synaptic connections were significantly increased in combination treatment between sesamin and NGF. Moreover, sesamin also increased the level of phospho-ERK1/2 and SIRT1 protein, an important regulatory protein of the neurogenesis process. The neurogenesis was blocked by the specific SIRT1 inhibitor, JGB1741, suggesting that the neuritogenic effect of sesamin was associated with SIRT1 protein modulation. Taken together, the potentiating effect of sesamin on NGF-induced neurogenesis in this finding could be used for alternative treatment in neurodegenerative diseases, including Alzheimer's disease.

Effects of sesamin on primary human synovial fibroblasts and SW982 cell line induced by tumor necrosis factor-alpha as a synovitis-like model.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease that causes chronic synovitis, cartilage degradation and bone deformities. Synovitis is the term for inflammation of the synovial membrane, an early stage of RA. The pathogenesis of the disease occurs through cytokine induction. The major cytokine that increases the severity of RA is TNF-α. Thus, inhibition of the TNF-α cascade is an effective way to diminish the progression of the disease. We are interested in investigating the difference between primary human synovial fibroblast (hSF) cells and SW982 as synovitis models induced by TNF-α and in monitoring their responses to sesamin as an anti-inflammatory phytochemical. METHOD: The designed experiments were performed in hSF cells or the SW982 cell line treated with 10 ng/ml TNF-α with or without 0.25, 0.5 or 1 µM sesamin. Subsequently, pro-inflammatory cytokine genes and proteins were measured in parallel with a study of associated signalling transduction involved in inflammatory processes, including NF-κB and MAPK pathways. RESULTS: The results demonstrated that although hSF and SW982 cells responded to TNF-α induction in the same fashion, they reacted at different levels. TNF-α could induce IL-6, IL-8 and IL-1ß in both cell types, but the levels in SW982 cells were much higher than in hSF cells. This characteristic was due to the different induction of MAPKs in each cell type. Both cell types reacted to sesamin in almost the same fashion. However, hSF cells were more sensitive to sesamin than SW982 cells in terms of the anti-RA effect. CONCLUSIONS: The responses of TNF-α-induced hSF and SW982 were different at the signal transduction level. However, the two cell types showed almost the same reaction to sesamin treatment in terms of the end point of the response.

Effects of sesamin on chondroitin sulfate proteoglycan synthesis induced by interleukin-1beta in human chondrocytes.

BACKGROUND: Numerous studies have reported on the health benefits of sesamin, a major lignin found in sesame (S. indicum) seeds. Recently, sesamin was shown to have the ability to promote chondroitin sulfate proteoglycan synthesis in normal human chondrocytes. This study assesses the anti-inflammatory effect of sesamin on proteoglycans production in 3D chondrocyte cultures. METHODS: To evaluate the effects of sesamin on IL-1ß-treated human articular chondrocytes (HAC) pellets, the pellets were pre-treated with IL-1ß then cultured in the presence of various concentrations of sesamin for 21 days. During that period, the expression of IL-1ß, glycosaminoglycans (GAGs) content and Chondroitin sulfate proteoglycans (CSPGs) synthesis genes (ACAN, XT-1, XT-2, CHSY1 and ChPF) was measured. The GAGs accumulation in the extracellular matrix was determined on day 21 by histological analysis. RESULTS: There was clear evidence that sesamin upregulated expression of all the CSPGs synthesis genes, in contrast to the down-regulation of IL-1ß expression both in genes and in protein levels. The level of release and matrix accumulation of GAGs in IL-1ß pre-treated HAC pellets in the presence of sesamin was recovered. These results correlate with the histological examination which showed that sesamin enhanced matrix CSPGs accumulation. CONCLUSIONS: Sesamin enhances CSPGs synthesis, suppresses IL-1ß expression and ameliorates IL-1ß induced inflammation in human chondrocytes. Sesamin could have therapeutic benefits for treating inflammation in osteoarthritis.

Hepcidin suppression in ß-thalassemia is associated with the down-regulation of atonal homolog 8.

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in ß-thalassemia. We found that inhibition of hepcidin expression in ß-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in ß-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with ß-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the ß-thalassemia patient sera. In conclusion, hepcidin suppression in ß-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in ß-thalassemia.

Ex vivo model exhibits protective effects of sesamin against destruction of cartilage induced with a combination of tumor necrosis factor-alpha and oncostatin M.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease associated with chronic inflammatory arthritis. TNF-α and OSM are pro-inflammatory cytokines that play a key role in RA progression. Thus, reducing the effects of both cytokines is practical in order to relieve the progression of the disease. This current study is interested in sesamin, an active compound in sesame seeds. Sesamin has been shown to be a chondroprotective agent in osteoarthritis models. Here, we have evaluated a porcine cartilage explant as a cartilage degradation model related to RA induced by TNF-α and/or OSM in order to investigate the effects of sesamin on TNF-α and OSM in the cartilage degradation model. METHODS: A porcine cartilage explant was induced with a combination of TNF-α and OSM (test group) or IL-1ß and OSM (control group) followed by a co-treatment of sesamin over a long-term period (35 days). After which, the tested explants were analyzed for indications of both the remaining and the degradation aspects using glycosaminoglycan and collagen as an indicator. RESULTS: The combination of TNF-α and OSM promoted cartilage degradation more than either TNF-α or OSM alone and was comparable with the combination of IL-1ß and OSM. Sesamin could be offering protection against cartilage degradation by reducing GAGs and collagen turnover in the generated model. CONCLUSIONS: Sesamin might be a promising agent as an alternative treatment for RA patients. Furthermore, the generated model revealed itself to be an impressive test model for the analysis of phytochemical substances against the cartilage degradation model for RA. The model could be used to test for the prevention of cartilage degradation in other biological agents induced with TNF-α and OSM as well.

Effect of sesamin against cytokine production from influenza type A H1N1-induced peripheral blood mononuclear cells: computational and experimental studies.

In 2009, swine flu (H1N1) had spread significantly to levels that threatened pandemic influenza. There have been many treatments that have arisen for patients since the WHO first reported the disease. Although some progress in controlling influenza has taken place during the last few years, the disease is not yet under control. The development of new and less expensive anti-influenza drugs is still needed. Here, we show that sesamin from the seeds of the Thai medicinal plant Sesamum indicum has anti-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) induced by 2009 influenza virus type A H1N1. In this study, the combinatorial screening method combined with the computational approach was applied to investigate the new molecular binding structures of sesamin against the 2009 influenza virus type A H1N1 (p09N1) crystallized structure. Experimental methods were applied to propose the mechanisms of sesamin against cytokine production from H1N1-induced human PBMC model. The molecular dynamics simulation of sesamin binding with the p09N1 crystallized structure showed new molecular binding structures at ARG118, ILE222, ARG224, and TYR406, and it has been proposed that sesamin could potentially be used to produce anti-H1N1 compounds. Furthermore, the mechanisms of sesamin against cytokine production from influenza type A H1N1-induced PBMCs by ELISA and signaling transduction showed that sesamin exhibits the ability to inhibit proinflammatory cytokines, IL-1ß and TNF-α, and to enhance the activity of the immune cell cytokine IL-2 via downregulating the phosphorylated JNK, p38, and ERK1/2 MAPK signaling pathways. This information might very well be useful in the prevention and treatment of immune-induced inflammatory disorders.

Sesamin stimulates osteoblast differentiation through p38 and ERK1/2 MAPK signaling pathways.

BACKGROUND: Osteoporosis is a worldwide health problem predominantly affecting post-menopausal women. Therapies aimed at increasing bone mass in osteoporetic patients lag behind comparable investigation of therapeutic strategies focusing on the bone resorption process. Sesamin, a major lignan compound found in Sesamun indicum Linn., has a variety of pharmacological effects, though its activity on bone cell function is unclear. Herein we examine the effect of this lignan on osteoblast differentiation and function. METHOD: Cell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 h of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 h of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stained with Alizarin Red S. MAPK signaling pathway activation was observed by using western blotting. RESULTS: Sesamin promoted the gene expression of COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19. CONCLUSIONS: The data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the p38 and ERK MAPK signaling pathway and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts. Therefore, sesamin may be a promising phytochemical that could be developed for supplementation of osteoporotic therapy.

Polyoxypregnane glycoside from Dregea volubilis extract inhibits IL-1ß-induced expression of matrix metalloproteinase via activation of NF-κB in human chondrocytes.

Interleukin-1ß (IL-1ß) induces the expression of matrix metalloproteinases (MMPs) implicated in cartilage and joint degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). Polyoxypregnane glycoside (PPG), active compound was identified from Dregea volubilis extract by chemical analysis, shown to exert chondroprotective effects in cartilage explant models. However, no studies have been undertaken for the molecular investigation of whether PPG constituents protect the human articular chondrocyte (HAC). In the present studies, HAC was co-treated with IL-1ß and PPG. The expression of MMPs, type II collagen, phosphorylation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathway were determined by Western immunoblotting. PPG (6.25-25 µM) decreased the IL-1ß-induced HA release from chondrocyte to culture medium. The mode of action of PPG was likely mediated through inhibiting expression of MMP-1, -3 and -13 in the medium, which was associated with the inhibition of mRNA expression. PPG had no effect on IL-1ß-induced phosphorylation of MAPK pathway. Conversely, PPG decreased phosphorylation of IκB kinase and IκBα degradation. Taken together, these results indicate that PPG may inhibit cartilage degradation in OA and may also be used as nutritional supplement for maintaining joint integrity and function.

Alpinia galanga extracts downregulate interleukin-1ß-induced matrix metalloproteinases expression in human synovial fibroblasts.

Alpinia galanga has been used as alternative medicine for anti-rheumatic activities. However, the precise action of the extract on arthritic diseases is not yet fully understood. In this study, we investigated the effects of A. galanga extracts on the expression of genes involved in catabolic activities in an interleukin-1ß (IL-1ß)-induced human synovial fibroblast as an inflammatory model. Confluent primary human synovial fibroblasts were treated for 24 h with A. galanga hexane extracts in the presence of recombinant human IL-1ß. MMPs in the culture medium were monitored by gelatin zymography. Total RNA was isolated from the cell lysate and analyzed via semi-quantitative RT-PCR. After treatment with A. galanga extracts, MMP-2 activity in the culture medium was significantly reduced. In addition, MMP-1, MMP-3, MMP-13, and Cox-2 expression were downregulated. These data suggest that the decrease of gene expression and production of MMPs in synovial fibroblasts against inflammatory stimuli could be due to the effects of the A. galanga extracts. Therefore, A. galanga extracts might be a promising therapeutic agent for arthritis.

The effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chondrocytes.

Osteoarthritis (OA) is the most common form of arthritis and affects millions of people worldwide. Patients have traditionally been treated with non-steroidal anti-inflammatory drugs (NSAIDs), but these are associated with significant side effects. Purification of the acetone extract of Alpinia galanga afforded p-hydroxycinnamaldehyde, as identified by nuclear magnetic resonance and mass spectrometry analyses. By exploiting the cartilage explant culture, p-hydroxycinnamaldehyde suppressed loss of uronic acid, resulting in release of hyaluronan (HA), sulfated glycosaminoglycans (s-GAGs) and matrix metalloproteinases (MMPs). p-Hydroxycinnamaldehyde and interleukin-1beta (IL-1beta), when incubated in primary human chondrocytes, also reduced release of HA, s-GAG and MMP-2. The results demonstrated: (a) that expression levels of the catabolic genes MMP-3 and MMP-13 were suppressed and (b) mRNA expression levels of anabolic genes of collagen II, SOX9 and aggrecan were increased. This study shows that p-hydroxycinnaldehyde from A. galanga Linn. is a potential therapeutic agent for treatment of OA.

Raised serum chondroitin sulfate epitope level in ovarian epithelial cancer.

OBJECTIVE: To determine the value of serum chondroitin sulfate epitope WF6 and hyaluronan (HA) levels as a biomarker for early detection of ovarian epithelial cancer and other gynecological disorders. METHOD: Serum WF6 CS epitope and HA were measured in 91 patients with ovarian epithelial cancer, 39 patients with non-cancer gynecological disorders and 30 healthy women. Serum chondroitin sulfate (CS) WF6 epitope was determined by a competitive immunoassay with the monoclonal antibodies WF6, which specifically recognizes an epitope in native CS chains. In addition, serum HA concentration was measured by an ELISA-based assay with a biotinylated affinity HA-binding proteins. RESULTS: The serum concentration of CS (WF6) epitope was highly increased in epithelial types of ovarian cancer and at all stages of development (p < 0.005). Serum HA in ovarian cancer patients was significantly higher than normal controls (p < 0.05). CONCLUSION: These results reflect changes in ECM metabolism in progressive ovarian cancer, which cause an increase in serum CS epitopes and HA. Therefore, serum CS epitopes may provide useful biomarkers for cancers and other disorders of the ovary. Measurement of serum HA provided complementary information, which may be useful as a discriminator between benign ovarian disorders and malignant ovarian diseases.