Protective effects of stem cells from human exfoliated deciduous teeth derived conditioned medium on osteoarthritic chondrocytes.
ABSTRACT: Treatment of osteoarthritis (OA) is still a major clinical challenge due to the limited inherent healing capacity of cartilage. Recent studies utilising stem cells suggest that the therapeutic benefits of these cells are mediated through the paracrine mechanism of bioactive molecules. The present study evaluates the regenerative effect of stem cells from human exfoliated deciduous teeth (SHED) conditioned medium (CM) on OA chondrocytes. The CM was collected after the SHED were cultured in serum-free medium (SFM) for 48 or 72 h and the cells were characterised by the expression of MSC and pluripotency markers. Chondrocytes were stimulated with interleukin-1? and treated with the CM. Subsequently, the expression of aggrecan, collagen type 2 (COL 2), matrix metalloproteinase-13 (MMP-13), nuclear factor-kB (NF-kB) and the level of inflammatory and anti-inflammatory markers were evaluated. SHED expressed mesenchymal stromal cell surface proteins but were negative for haematopoietic markers. SHED also showed protein expression of NANOG, OCT4 and SOX2 with differential subcellular localisation. Treatment of OA chondrocytes with CM enhanced anti-inflammation compared to control cells treated with SFM. Furthermore, the expression of MMP-13 and NF-kB was significantly downregulated in stimulated chondrocytes incubated in CM. The study also revealed that CM increased the expression of aggrecan and COL 2 in OA chondrocytes compared to SFM control. Both CM regenerate extracellular matrix proteins and mitigate increased MMP-13 expression through inhibition of NF-kB in OA chondrocytes due to the presence of bioactive molecules. The study underscores the potential of CM for OA treatment.
Project description: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 (IL1?), 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 IL1?-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 IL1?-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.
Project description:OBJECTIVE:The aggrecanase ADAMTS-5 and the collagenase matrix metalloproteinase 13 (MMP-13) are constitutively secreted by chondrocytes in normal cartilage, but rapidly endocytosed via the cell surface endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP-1) and subsequently degraded. This endocytic system is impaired in osteoarthritic (OA) cartilage due to increased ectodomain shedding of LRP-1. The aim of this study was to identify the LRP-1 sheddase(s) in human cartilage and to test whether inhibition of LRP-1 shedding prevents cartilage degradation in OA. METHODS:Cell-associated LRP-1 and soluble LRP-1 (sLRP-1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. LRP-1 sheddases were identified by proteinase inhibitor profiling and gene silencing with small interfering RNAs. Specific monoclonal antibodies were used to selectively inhibit the sheddases. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively. RESULTS:Shedding of LRP-1 was increased in OA cartilage compared with normal tissue. Shed sLRP-1 bound to ADAMTS-5 and MMP-13 and prevented their endocytosis without interfering with their proteolytic activities. Two membrane-bound metalloproteinases, ADAM-17 and MMP-14, were identified as the LRP-1 sheddases in cartilage. Inhibition of their activities restored the endocytic capacity of chondrocytes and reduced degradation of aggrecan and collagen in OA cartilage. CONCLUSION:Shedding of LRP-1 is a key link to OA progression. Local inhibition of LRP-1 sheddase activities of ADAM-17 and MMP-14 is a unique way to reverse matrix degradation in OA cartilage and could be effective as a therapeutic approach.
Project description:The issue of whether ERK activation determines matrix synthesis or degradation in osteoarthritis (OA) pathogenesis currently remains controversial. Our previous study shows that PLC?1 and mTOR are involved in the matrix metabolism of OA cartilage. Investigating the interplays of PLC?1, mTOR and ERK in matrix degradation of OA will facilitate future attempts to manipulate ERK in OA prevention and therapy. Here, cultured human normal chondrocytes and OA chondrocytes were treated with different inhibitors or transfected with expression vectors, respectively. The levels of ERK, p-ERK, PLC?1, p-PLC?1, mTOR, p-mTOR and MMP-13 were then evaluated by Western blotting analysis. The results manifested that the expression level of ERK in human OA chondrocytes was lower than that in human normal articular chondrocytes, and the up-regulation of ERK could promote matrix synthesis, including the decrease in MMP-13 level and the increase in Aggrecan level in human OA chondrocytes. Furthermore, the PLC?1/ERK axis and a mutual inhibition of mTOR and ERK were observed in human OA chondrocytes. Interestingly, activated ERK had no inhibitory effect on MMP-13 expression in PLC?1-transformed OA chondrocytes. Combined with our previous study, the non-effective state of ERK activation by PLC?1 on MMP-13 may be partly attributed to the inhibition of the PLC?1/mTOR axis on the PLC?1/ERK axis. Therefore, the study indicates that the mutual inhibition of ERK and mTOR is involved in PLC?1-mediated MMP-13 expression in human OA chondrocytes, with important implication for the understanding of OA pathogenesis as well as for its prevention and therapy.
Project description:Osteoarthritis (OA) is characterized by cartilage degradation in the affected joints. Pomegranate fruit extract (PFE) inhibits cartilage degradation in vitro. The aim of this study was to determine whether oral consumption of PFE inhibits disease progression in rabbits with surgically induced OA.OA was surgically induced in the tibiofemoral joints of adult New Zealand White rabbits. In one group, animals were fed PFE in water for 8 wk postsurgery. In the second group, animals were fed PFE for 2 wk before surgery and for 8 wk postsurgery. Histologic assessment and scoring of the cartilage was per Osteoarthritis Research Society International guidelines. Gene expression and matrix metalloproteinases (MMP) activity were determined using quantitative reverse transcriptase polymerase chain reaction and fluorometric assay, respectively. Interleukin (IL)-1 ?, MMP-13, IL-6, prostaglandin (PG)E2, and type II collagen (COL2A1) levels in synovial fluid/plasma/culture media were quantified using enzyme-linked immunosorbent assay. Expression of active caspase-3 and poly (ADP-ribose) polymerase p85 was determined by immunohistochemistry. Effect of PFE and inhibitors of MMP-13, mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-?B was studied in IL-1 ?-stimulated rabbit articular chondrocytes.Safranin-O-staining and chondrocyte cluster formation was significantly reduced in the anterior cruciate ligament transaction plus PFE fed groups. Expression of MMP-3, MMP-9, and MMP-13 mRNA was higher in the cartilage of rabbits given water alone but was significantly lower in the animals fed PFE. PFE-fed rabbits had lower IL-6, MMP-13, and PGE2 levels in the synovial fluid and plasma, respectively, and showed higher expression of aggrecan and COL2A1 mRNA. Significantly higher numbers of chondrocytes were positive for markers of apoptosis in the joints of rabbits with OA given water only compared with those in the PFE-fed groups. PFE pretreatment significantly reduced IL-1 ? induced IL-6 and MMPs expression in rabbit articular chondrocytes. These effects were also mimicked using MMP-13, MAPK, and NF-?B inhibitors in IL-1 ?-stimulated rabbit chondrocytes. In an in vitro activity assay, PFE blocked the activity of MMP-13. Like MAPK and NF-?B inhibitors, PFE was also effective in inhibiting IL-1 ?-induced PGE2 production in rabbit chondrocytes. PFE also reversed the inhibitory effect of IL-1? on COL2A1 mRNA and protein expression in IL-1 ?-stimulated rabbit chondrocytes.The present data highlight the chondroprotective effects of PFE oral consumption in a model of posttraumatic OA and suggest that PFE-derived compounds may have potential value in the management of OA.
Project description:OBJECTIVE:Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. METHODS:Chondrocytes were stimulated with interleukin-1beta (IL-1beta) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)-based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription-PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-kappaB was evaluated using specific inhibitors. RESULTS:In IL-1beta-stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3'-untranslated region (3'-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA and inhibited the IL-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b. CONCLUSION:Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.
Project description:Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action.We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes' transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-β (TGF-β) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-β alone can induce SREBP-2, we treated normal chondrocytes with TGF-β and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-β activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels.We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-β induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment.
Project description:INTRODUCTION: Accumulation of advanced glycation end products (AGEs) in joints contributes to the pathogenesis of cartilage damage in osteoarthritis (OA). We aim to explore the potential chondroprotective effects of resveratrol on AGEs-stimulated porcine chondrocytes and cartilage explants. METHODS: Chondrocytes were isolated from pig joints. Activation of the I?B kinase (IKK)-I?B?-nuclear factor-kappaB (NF-?B) and c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)-activator protein-1 (AP-1) pathways was assessed by electrophoretic mobility shift assay (EMSA), Western blot and transfection assay. The levels of inducible nitric oxide synthase (iNOS)-NO and cyclooxygenase-2 (COX-2)-prostaglandin E2 (PGE2) were measured by Western blot, Griess reaction or ELISA. The expression and enzyme activity of matrix metalloproteinase-13 (MMP-13) were determined by real time RT/PCR and gelatin zymography, respectively. RESULTS: We show that AGEs-induced expression of iNOS and COX-2 and production of NO and PGE2 were suppressed by resveratrol. Such effects of resveratrol were likely mediated through inhibiting IKK-I?B?-NF-?B and JNK/ERK-AP-1 signaling pathways induced by AGEs. By targeting these critical signaling pathways, resveratrol decreased AGEs-stimulated expression and activity of MMP-13 and prevented AGEs-mediated destruction of collagen II. Histochemistry analysis further confirms that resveratrol could prevent AGEs-induced degradation of proteoglycan and aggrecan in cartilage explants. CONCLUSIONS: The present study reveals not only the effects and mechanisms regarding how resveratrol may protect cartilage from AGEs-mediated damage but also the potential therapeutic benefit of resveratrol in the treatment of OA.
Project description:Osteoarthritis (OA) is a chronic degenerative disease wherein the articular cartilage exhibits inflammation and degradation. Scutellarin (SCU) is a flavonoid glycoside with a range of pharmacological activities, as shown in previous studies demonstrating its anti-inflammatory activity. How SCU impacts the progression of OA, however, has not been explored to date. Herein, we assessed the impact of SCU on murine chondrocytes in an OA model system. In in vitro assays, we measured chondrocyte expression of key OA-associated factors such as matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) via qRT-PCR and Western blotting, the expression of interleukin 6 (IL-6), tumor necrosis factor-? (TNF-?), and prostaglandin E2 (PGE2) were detected by qRT-PCR. Our results showed that the downregulation of MMP-13, ADAMTS-5, COX-2, and iNOS expression by SCU and the overproduction of IL-6, TNF-?, and PGE2 induced by IL-1? were all inhibited by SCU in a concentration-dependent manner. Moreover, SCU was able to reverse aggrecan and collagen II degradation and nuclear factor-?B (NF-?B) and nuclear factor erythroid-derived 2-like 2 (Nrf2) signaling pathway activation both in vivo and in vitro. We further used a destabilization of the medial meniscus (DMM) murine model of OA to explore the therapeutic benefits of SCU in vivo. Together, our findings suggest SCU to be a potentially valuable therapeutic agent useful for treating OA.
Project description:It has been suggested that microRNA-9 (miR-9) is associated with the development of knee osteoarthritis (OA). This study was aimed to investigate the association between the mechanism of miR-9 targeting nuclear factor kappa-B1 (NF-?B1) and the proliferation and apoptosis of knee OA chondrocytes.Cartilage samples were collected from 25 patients with knee OA and 10 traumatic amputees, and another 15 OA rat models, together with 15 rats without knee OA lesions were also established. MiR-9 expressions in both knee OA cartilage and normal cartilage samples were detected using quantitative real-time PCR. The expressions of related genes (NF-?B1, IL-6, and MMP-13) in the two groups were also detected. Dual luciferase reporter gene assay was employed to examine the effect of miR-9 on the luciferase activity of NF-?B1 3'UTR. Knee OA chondrocytes were transfected with miR-9 mimics, miR-9 inhibitor, and NF-?B1 siRNA, respectively, and changes in cellular proliferation and apoptosis were detected via MTT assay and flow cytometric analysis, respectively. Western blotting assay was used to detect the expressions of NF-?B1, interleukin-6 (IL-6), and matrix metalloproteinase-13 (MMP-13).According to results from human OA samples and rat OA models, miR-9 was significantly downregulated in knee OA cartilage tissues compared with normal cartilage tissues (P?<?0.01). The expressions of NF-?B1, IL-6, and MMP-13 in knee OA cartilage tissues were significantly higher than those in normal cartilage tissues (P?<?0.01). Dual luciferase reporter gene assay showed that miR-9 could bind to the 3'UTR of NF-?B1 and significantly inhibit the luciferase activity by 37% (P?<?0.01). Upregulation of miR-9 or downregulation of NF-?B1 could promote cell proliferation and suppress cell apoptosis.Conclusively, downregulated miR-9 can facilitate proliferation and antiapoptosis of knee OA chondrocytes by directly binding to NF-kB1, implying that stimulating miR-9 expressions might assist in treatment of knee OA.
Project description:Recent studies confer to IL-36? pro-inflammatory properties. However, little is known about the expression and function of IL-36? in cartilage. This study sought to analyze the expression of IL-36? in healthy and OA cartilage. Next, we determined the effects of recombinant IL-36? on catabolism and inflammation in chondrocytes. For completeness, part of the signaling pathway elicited by IL-36? was also explored. IL-36? expression was evaluated by immunohistochemistry and RT-qPCR. Expression of MMP-13, NOS2 and COX-2 was also determined in OA articular chondrocytes treated with recombinant IL-36?. I?B-? and P-p38 was explored by western blot. We observed a low constitutive expression of IL-36? in healthy human chondrocytes. However, OA chondrocytes likely expressed more IL-36? than healthy chondrocytes. In addition, immune cells infiltrated into the joint and PBMCs express higher levels of IL-36? in comparison to chondrocytes. OA chondrocytes, treated with IL-36?, showed significant increase in the expression of MMP-13, NOS2 and COX-2. Finally, IL-36? stimulated cells showed NF?B and p38 MAPK activated pathways. IL-36? acts as a pro-inflammatory cytokine at cartilage level, by increasing the expression of markers of inflammation and cartilage catabolism. Like other members of IL-1 family, IL-36? acts through the activation of NF?B and p38 MAPK pathway.