Human Osteoarthritis Chondrocytes Research Articles

The inhibition of PLCγ1 protects chondrocytes against osteoarthritis, implicating its binding to Akt.

Previous studies have addressed the involvement of phosphoinositide-specifc phospholipase γ1 (PLCγ1) and protein kinase B (PKB/Akt) in osteoarthritis (OA) pathogenesis, but it is not ascertained the possibility of them to be potential targets for OA therapy. Here, through local intra-articular injection of PLCγ or Akt inhibitor in a rat OA model induced by anterior cruciate ligament transaction plus medial meniscus resection, the architecture of chondrocyte and matrix organization of articular cartilage were observed using histopathological assays and Aggrecan, Col2, PLCγ1, and Akt levels were detected using immunohistochemistry assays. By treatment of Akt or PLCγ inhibitor and transfection of different PLCγ1- or Akt-expressing vectors in rat OA model chondrocytes, Aggrecan, Col2, PLCγ1, p-PLCγ1, Akt, and p-Akt levels were detected using western blotting analysis. The binding between PLCγ1 and Akt was assessed with co-immunoprecipitation assays in human OA chondrocytes. These results showed that PLCγ inhibition protected chondrocytes against OA, but Akt inhibition did not dramatically aggravate OA progression. There were mutual antagonism and binding between PLCγ1 and Akt that could be regulated by their phosphorylation levels. Consequently, the data reveal that the inhibition of PLCγ1 may provide an attractive therapeutic target for OA therapy, implicating its binding to Akt.

Osteoarthritic human chondrocytes proliferate in 3D co-culture with mesenchymal stem cells in suspension bioreactors.

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage-generating cells. Previous studies in static culture have shown that hACs co-cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co-culture of hMSCs and OA hACs under serum-free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125-ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75-fold increase over 16days. Whereas the apparent growth rate was lower than that achieved during co-culture as a 2D monolayer in static culture flasks, bioreactor co-culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum-free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell-based cartilage repair.

Paeoniflorin inhibits IL‑1β‑induced expression of inflammatory mediators in human osteoarthritic chondrocyte.

Interleukin (IL)-1β serves an important role in the promotion of the growth of osteoarthritis (OA) lesions. Paeoniflorin (PF) has been identified to exert anti‑inflammatory and anti‑arthritic effects. However, it is uncertain whether PF may affect the expression levels of inflammatory mediators in OA chondrocytes. Therefore, the objective of the present study was to determine the effects of PF on the expression levels of inflammatory mediators in IL‑1β‑stimulated human OA chondrocytes. The results of the present study determined that PF inhibited the production of nitric oxide and prostaglandin E2 induced by IL‑1β, and the expression of inducible nitric oxide synthase and cyclooxygenase‑2 in chondrocytes. Additionally, PF significantly inhibited the IL‑1β‑stimulated production of metalloproteinase‑3 (MMP‑3) and MMP‑13 in chondrocytes. PF inhibited the expression of nuclear factor‑κB (NF‑κB), p65 and NF‑κB inhibitor α degradation was induced by IL‑1β in chondrocytes. The results of the present study suggest that PF may inhibit IL‑1β‑induced expression ofinflammatorymediators in human OA chondrocytes by suppressing the activation of the NF‑κB signaling pathway. Therefore, PF may be a potential agent in the future treatment of OA.

Could Oxidative Stress Regulate the Expression of MicroRNA-146a and MicroRNA-34a in Human Osteoarthritic Chondrocyte Cultures?

Oxidative stress and the overproduction of reactive oxygen species (ROS) play an important role in the pathogenesis of osteoarthritis (OA). Accumulating evidence has demonstrated the involvement of microRNAs (miRNAs) dysregulation in disease development and progression. In this study, we evaluated the effect of oxidative stress on miR-146a and miR-34a expression levels in human OA chondrocytes cultures stimulated by H2O2. Mitochondrial ROS production and cell apoptosis were detected by flow cytometry. The antioxidant enzymes SOD-2, CAT, GPx, the transcriptional factor NRF2 and the selected miRNAs were analyzed by qRT-PCR. The H2O2-induced oxidative stress was confirmed by a significant increase in superoxide anion production and of the apoptotic ratio. Furthermore, H2O2 significantly up-regulated the expression levels of SOD-2, CAT, GPx and NRF2, and modulated miR-146a and miR-34a gene expression. The same analyses were carried out after pre-treatment with taurine, a known antioxidant substance, which, in our experience, counteracted the H2O2-induced effect. In conclusion, the induction of oxidative stress affected cell apoptosis and the expression of the enzymes involved in the oxidant/antioxidant balance. Moreover, we demonstrated for the first time the modification of miR-146a and miR-34a in OA chondrocytes subjected to H2O2 stimulus and we confirmed the antioxidant effect of taurine.

Thrombospondin-1 Partly Mediates the Cartilage Protective Effect of Adipose-Derived Mesenchymal Stem Cells in Osteoarthritis.

Assuming that mesenchymal stem cells (MSCs) respond to the osteoarthritic joint environment to exert a chondroprotective effect, we aimed at investigating the molecular response setup by MSCs after priming by osteoarthritic chondrocytes in cocultures. We used primary human osteoarthritic chondrocytes and adipose stem cells (ASCs) in mono- and cocultures and performed a high-throughput secretome analysis. Among secreted proteins differentially induced in cocultures, we identified thrombospondin-1 (THBS1) as a potential candidate that could be involved in the chondroprotective effect of ASCs. Secretome analysis revealed significant induction of THBS1 in ASCs/chondrocytes cocultures at mRNA and protein levels. We showed that THBS1 was upregulated at late stages of MSC differentiation toward chondrocytes and that recombinant THBS1 (rTHBS1) exerted a prochondrogenic effect on MSC indicating a role of THBS1 during chondrogenesis. However, compared to control ASCs, siTHBS1-transfected ASCs did not decrease the expression of hypertrophic and inflammatory markers in osteoarthritic chondrocytes, suggesting that THBS1 was not involved in the reversion of osteoarthritic phenotype. Nevertheless, downregulation of THBS1 in ASCs reduced their immunosuppressive activity, which was consistent with the anti-inflammatory role of rTHBS1 on T lymphocytes. THBS1 function was then evaluated in the collagenase-induced OA model by comparing siTHBS1-transfected and control ASCs. The protective effect of ASCs evaluated by histological and histomorphological analysis of cartilage and bone was not seen with siTHBS1-transfected ASCs. Our data suggest that THBS1 did not exert a direct protective effect on chondrocytes but might reduce inflammation, subsequently explaining the therapeutic effect of ASCs in OA.

Phlpp inhibitors block pain and cartilage degradation associated with osteoarthritis.

Phlpp protein phosphatases are abnormally abundant within human osteoarthritic articular chondrocytes and may contribute to the development of osteoarthritis. Mice lacking Phlpp1 were previously shown to be resistant to post-traumatic osteoarthritis. Here a small molecule with therapeutic properties that inhibits Phlpp1 and Phlpp2 was tested for its ability to slow post-traumatic OA in mice and to stimulate anabolic pathways in human articular cartilage from OA joints. PTOA was induced in male C57Bl/6 mice by surgically destabilizing the meniscus. Seven weeks after surgery, mice received a single intra-articular injection of the Phlpp inhibitor NSC117079 or saline. Mechanical allodynia was measured with von Frey assays, mobility was tracked in an open field system, and cartilage damage was assessed histologically. A single intra-articular injection of the Phlpp inhibitor NSC117079 attenuated mechanical allodynia and slowed articular cartilage degradation in joints with a destabilized meniscus. Animals treated with the Phlpp inhibitor 7 weeks after injury maintained normal activity levels, while those in the control group traveled shorter distances and were less active 3 months after the joint injury. NSC117079 also increased production of cartilage extracellular matrix components (glycosaminoglycans and aggrecan) in over 90% of human articular cartilage explants from OA patients and increased phosphorylation of Phlpp1 substrates (AKT2, ERK1/2, and PKC) in human articular chondrocytes. Our results indicate that Phlpp inhibitor NSC117079 is a novel osteoarthritis disease modifying drug candidate that may have palliative affects. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1487-1497, 2018.

Different roles of Akt and mechanistic target of rapamycin in serum‑dependent chondroprotection of human osteoarthritic chondrocytes.

Despite various animal serums being used widely to culture chondrocytes, the regulatory mechanism of serum on chondrocyte activities has not been elucidated. In the present study, human osteoarthritis (OA) chondrocytes were used to perform invitro investigations on the effect of different concentrations of bovine fetal serum on extracellular matrix synthesis, cell proliferation and autophagy using the Cell Counting Kit‑8 analysis, a laser‑scanning confocal microscope, and western blot analysis. The results demonstrated that 5% serum exerted a chondroprotective effect more than the other concentrations of serum, as it simultaneously promoted cell proliferation, autophagy, and ECM synthesis in human OA chondrocytes. Furthermore, the decreased mechanistic target of rapamycin (mTOR) and increased Akt were observed in 5% serum‑treated OA chondrocytes. Either mTOR or Akt inhibitor influenced the effect of 5% serum on cell proliferation and autophagy in human OA chondrocytes, which was associated with LC‑3B or B‑cell lymphoma-2 (Bcl‑2) signal molecules. Consistent with previous studies, the present study proposes that 5% serum promotes cell proliferation via the Akt/Bcl‑2 axis and induces autophagy via the mTOR/LC‑3B axis in human OA chondrocytes. Furthermore, the different roles of Akt and mTOR in the cell processes of human OA chondrocytes require consideration for preclinical and clinical therapy of OA.

Expression of miR-206 in Human Knee Articular Chondrocytes and Effects of miR-206 on Proliferation and Apoptosis of Articular Chondrocytes

BackgroundIncreasing evidence has demonstrated that microRNAs regulate the development of cartilage and osteogenesis. Whether miR-206 participates in the development of human articular cartilage remains largely unknown. This study aimed to investigate the role of miR-206 in human chondrocytes. MethodsExpression of miR-206 was initially assessed in human osteoarthritis (OA) tissues and articular chondrocytes through quantitative real-time polymerase chain reaction. The effects of miR-206 on proliferation and apoptosis of human chondrocytes were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining assay. Then, the effects of miR-206 on type II collagen alpha 1 (Col2a1), aggrecan, runt-related transcription factor 2 (RUNX2) and matrix metalloproteinase13 (MMP13) were examined with quantitative real-time polymerase chain reaction and Western blot analysis. ResultsMiR-206 was significantly increased in human OA tissues and chondrocytes. MiR-206 significantly inhibited the proliferation of chondrocytes, but promoted apoptosis. Expression of Col2a1 and aggrecan were dramatically decreased, and the expression of RUNX2 and MMP13 were significantly increased when miR-206 was overexpressed. ConclusionsMiR-206 may participate in cartilage degradation in OA. Manipulation of the expression of miR-206 in human chondrocytes may be a novel therapeutic strategy for the treatment of OA.

Pleiotropic Roles of Nrf2 as Regulators of Chondrocyte Apoptosis, Oxidative Stress, Inflammatory Response and Catabolic and Anabolic Pathways in Osteoarthritis

Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we investigated the regulation of oxidative stress, inflammatory response, apoptosis, catabolic and anabolic factors by Nrf2 in IL-1β-stimulated human OA chondrocytes. Ectopic expression of Nrf2 in OA chondrocytes suppressed the generation of ROS while Nrf2 knockdown significantly increased the ROS levels in IL-1β-stimulated OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1β-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction, suppression of pro-apoptotic proteins Bax, Bad and Bid expression and enhanced expression of anti-apoptotic proteins Bcl2, Bcl-xl and Mcl-1 in OA chondrocytes. To examine the role of Nrf2 in IL-1β-induced inflammatory response, we determined the mRNA expression and protein levels of several cytokines and chemokines using PCR array and multiplex immunoassay. Our data demonstrate that Nrf2 activation inhibited the IL-1β-mediated expression of several inflammatory mediators and Gene Ontology analysis revealed significant enrichment of MAPK signaling, TNF signaling, NF-κβ signaling and cytokine receptor signaling pathways. Additionally, Nrf2 activation inhibited the expression matrix degrading proteases-MMP-13, 3, 9, and ADAMTS-4 and enhanced the expression of OA related anabolic factors-COL2A1, ACAN, FRZB and GDF5 and SOX-5, 8 and 9 in IL-1β-stimulated OA chondrocytes. Importantly, we also discovered that Nrf2 mediated suppression of catabolic factors was dependent on ERK1/2 activation by Nrf2. Our data demonstrate that Nrf2 regulates multiple pathways involved in chondrocyte apoptosis, inflammation and matrix degradation via activation of ERK1/2/ELK1-P70S6K-P90RSK axis. Our findings collectively support the development of therapies targeting Nrf2 for the management of OA.

Hyperosmolar Potassium (K+) Treatment Suppresses Osteoarthritic Chondrocyte Catabolic and Inflammatory Protein Production in a 3-Dimensional In Vitro Model.

The main goal of this study was to provide a proof-of-concept demonstrating that hyperosmolar K+ solutions can limit production of catabolic and inflammatory mediators in human osteoarthritic chondrocytes (OACs). A 3-dimensional in vitro model with poly(ethylene glycol) diacrylate (PEGDA) hydrogels was used. Catabolic and pro-inflammatory protein production from encapsulated OACs was assessed following culture for 1 or 7 days in the presence or absence of 80 mM K+ gluconate, 80 mM sodium (Na+) gluconate, or 160 mM sucrose, each added to culture media (final osmolarity ~490 mOsm). Relative to untreated controls, OACs treated with hyperosmolar (80 mM Na+ gluconate or 160 mM sucrose) solutions produced lower levels of catabolic and inflammatory mediators in a marker- and time-dependent manner (i.e., MMP-9 after 1 day; MCP-1 after 7 days ( P ≤ 0.015)). In contrast, OAC treatment with 80 mM K+ gluconate reduced catabolic and inflammatory mediators to a greater extent (both the number of markers and degree of suppression) relative to untreated, Na+ gluconate, or sucrose controls (i.e., MMP-3, -9, -13, TIMP-1, MCP-1, and IL-8 after 1 day; MMP-1, -3, -9, -13, TIMP-1, MCP-1, and IL-8 after 7 days ( P ≤ 0.029). Hyperosmolar K+ solutions are capable of attenuating protein production of catabolic and inflammatory OA markers, providing the proof-of-concept needed for further development of a K+-based intra-articular injection for OA treatment. Moreover, K+ performed significantly better than Na+- or sucrose-based solutions, supporting the application of K+ toward improving irrigation solutions for joint surgery.

A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes

Background/ObjectiveOsteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β. MethodsThe hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. ResultsBME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1β induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. ConclusionThe above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1β induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis.

The use of mechano growth factor to prevent cartilage degeneration in knee osteoarthritis.

Previous studies have attached more importance to growth factors in treating cartilage degeneration and osteoarthritis (OA). Here, the capability of mechano growth factor-C24E (MGF) to prevent osteoarthritic cartilage degeneration was evaluated in vitro and in vivo. Using in vitro cultured human OA chondrocytes treated with 10-60-ng/ml MGF for 12hr, we detected the cell proliferation, migration, and anabolism of OA chondrocytes. The unfolded protein response and the protein characteristic of OA pathology, such as transforming growth factor β, SMAD family member 3, and hypoxia-inducible factor 2α of OA chondrocytes, were also detected by western blotting. Furthermore, protein kinase RNA-like endoplasmic reticulum kinase was knocked down via small interfering RNA to illuminate the potential mechanism of MGF's treatment of OA. In a rabbit knee joint OA model, cartilage degeneration was inhibited after 2weeks of treatment with 0.1-10-μg/ml MGF. This study demonstrated that MGF treatment can inhibit the pathological apoptosis of OA chondrocytes and promote the proliferation, migration, and matrix synthesis of the chondrocytes. The results also demonstrate that the degeneration of OA cartilage can be delayed by MGF treatment partially via unfolded protein response regulated by protein kinase RNA-like endoplasmic reticulum kinase and suggest a potential therapeutic application of MGF for OA treatment.

MiR-204 enhances the progression of osteoarthritis by suppressing the production of IL-1β.

Recent studies suggest that cytokines and microRNAs play a key role in the destruction of cartilage matrix in osteoarthritis (OA) tissues. In the current study, we focused on miR-204, which has never been explored in OA. We found that the level of miR-204 was markedly reduced in the OA cartilage tissues compared with that of normal control. Real time PCR analysis demonstrated that the level of miR-204 was markedly decreased after IL-1β treatment for 3, 6, 12 h in the normal chondrocytes and OA chondrocytes, respectively. Furthermore, overexpression of miR-204 markedly suppressed the protein levels of IL-1β, COX-2 and IL6 in human OA chondrocytes and chondrogenic SW1353 cells. Dual luciferase reporter assay demonstrated that miR-204 significantly suppressed the relative luciferase activity of pmirGLO-IL-1β-3'UTR, indicating that IL-1β was a target gene of miR-204. More importantly, treatment with IL-1β significantly enhanced the protein levels of IL-1β, COX-2 and IL6. However, overexpression of miR-204 could partially abolish such effects. In conclusion, our data demonstrate that reduced miR-204 expression enhances the destruction of the cartilage tissues among OA patients mainly through targeting IL-1β.

Trehalose ameliorates oxidative stress-mediated mitochondrial dysfunction and ER stress via selective autophagy stimulation and autophagic flux restoration in osteoarthritis development

Oxidative stress-related apoptosis and autophagy play crucial roles in the development of osteoarthritis (OA), a progressive cartilage degenerative disease with multifactorial etiologies. Here, we determined autophagic flux changes and apoptosis in human OA and tert-Butyl hydroperoxide (TBHP)-treated chondrocytes. In addition, we explored the potential protective effects of trehalose, a novel Mammalian Target of Rapamycin (mTOR)-independent autophagic inducer, in TBHP-treated mouse chondrocytes and a destabilized medial meniscus (DMM) mouse OA model. We found aberrant p62 accumulation and increased apoptosis in human OA cartilage and chondrocytes. Consistently, p62 and cleaved caspase-3 levels increased in mouse chondrocytes under oxidative stress. Furthermore, trehalose restored oxidative stress-induced autophagic flux disruption and targeted autophagy selectively by activating BCL2 interacting protein 3 (BNIP3) and Phosphoglycerate mutase family member 5 (PGAM5). Trehalose could ameliorate oxidative stress-mediated mitochondrial membrane potential collapse, ATP level decrease, dynamin-related protein 1 (drp-1) translocation into the mitochondria, and the upregulation of proteins involved in mitochondria and endoplasmic reticulum (ER) stress-related apoptosis pathway. In addition, trehalose suppressed the cleavage of caspase 3 and poly(ADP-ribose) polymerase (PARP) and prevented DNA damage under oxidative stress. However, the anti-apoptotic effects of trehalose in TBHP-treated chondrocytes were partially abolished by autophagic flux inhibitor chloroquine and BNIP3- siRNA. The protective effect of trehalose was also found in mouse OA model. Taken together, these results indicate that trehalose has anti-apoptotic effects through the suppression of oxidative stress-induced mitochondrial injury and ER stress which is dependent on the promotion of autophagic flux and the induction of selective autophagy. Thus, trehalose is a promising therapeutic agent for OA.

Saikosaponin A inhibits IL-1β-induced inflammatory mediators in human osteoarthritis chondrocytes by activating LXRα

Saikosaponin a (SSa), one of the main active components of Bupleurum falcatum, has been reported to have anti-inflammatory effect. In the present study, we investigated the anti-inflammatory effect of SSa on IL-1β-stimulated human osteoarthritis chondrocytes. The cells were pretreated with SSa 12 h before IL-1β treatment. The production of PGE2 and NO were detected by ELISA and Griess method. The levels of MMP1, MMP3, and MMP13 were measured by ELISA and qRT-PCR. The expression of NF-κB and LXRα were tested by western blot analysis. The results showed that SSa inhibited IL-1β-induced PGE2 and NO production in a concentration-dependent manner. SSa also suppressed IL-1β-induced MMP1, MMP3, and MMP13 production. Furthermore, SSa significantly attenuated IL-1β-induced phosphorylation levels of NF-κB p65 and IκBα. SSa also up-regulated the expression of LXRα. The inhibition of SSa on PGE2, NO, MMP1, MMP3, and MMP13 production were reversed by LXRα siRNA or GGPP, the inhibitor of LXRα. In conclusion, our results demonstrated that SSa inhibited inflammatory responses in human chondrocytes in vitro. SSa might be a potential therapeutic drug for osteoarthritis.

Baicalin suppresses IL-1β-induced expression of inflammatory cytokines via blocking NF-κB in human osteoarthritis chondrocytes and shows protective effect in mice osteoarthritis models

Osteoarthritis (OA) is a degenerative joint disease with an inflammatory component that drives the degradation of cartilage extracellular matrix. Baicalin, a predominant flavonoid isolated from the dry root of Scutellaria baicalensis Georgi, has been reported to have anti-inflammatory effects. However, the anti-inflammatory effects of baicalin on OA have not been reported. Our study aimed to investigate the effect of baicalin on OA both in vitro and in vivo. In vitro, human OA chondrocytes were pretreated with baicalin (10, 50, 100μM) for 2h and subsequently stimulated with IL-1β for 24h. Production of NO and PGE2 were evaluated by the Griess reaction and ELISAs. The mRNA expression of COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5, aggrecan and collagen-II were measured by real-time PCR. The protein expression of COX-2, iNOS, MMP-3, MMP-13, ADAMTS-5, p65, p-p65, IκBα and p-IκBα was detected by Western blot. The protein expression of collagen-II was evaluated by immunofluorescence. Luciferase activity assay was used to assess the relative activity of NF-kB. In vivo, the severity of OA was determined by histological analysis. We found that baicalin significantly inhibited the IL-1β-induced production of NO and PGE2, expression of COX-2, iNOS, MMP-3, MMP-13 and ADAMTS-5 and degradation of aggrecan and collagen-II. Furthermore, baicalin dramatically suppressed IL-1β-stimulated NF-κB activation. In vivo, treatment of baicalin not only prevented the destruction of cartilage but also relieved synovitis in mice OA models. Taken together, these results suggest that baicalin may be a potential agent in the treatment of OA.

Fluoxetine ameliorates cartilage degradation in osteoarthritis by inhibiting Wnt/β-catenin signaling.

Abnormal activation of the Wnt/β-catenin signaling is implicated in the osteoarthritis (OA) pathology. We searched for a pre-approved drug that suppresses abnormally activated Wnt/β-catenin signaling and has a potency to reduce joint pathology in OA. We introduced the TOPFlash reporter plasmid into HCS-2/8 human chondrosarcoma cells to estimate the Wnt/β-catenin activity in the presence of 10 μM each compound in a panel of pre-approved drugs. We found that fluoxetine, an antidepressant in the class of selective serotonin reuptake inhibitors (SSRI), down-regulated Wnt/β-catenin signaling in human chondrosarcoma cells. Fluoxetine inhibited both Wnt3A- and LiCl-induced loss of proteoglycans in chondrogenically differentiated ATDC5 cells. Fluoxetine increased expression of Sox9 (the chondrogenic master regulator), and decreased expressions of Axin2 (a marker for Wnt/β-catenin signaling) and Mmp13 (matrix metalloproteinase 13). Fluoxetine suppressed a LiCl-induced increase of total β-catenin and a LiCl-induced decrease of phosphorylated β-catenin in a dose-dependent manner. An in vitro protein-binding assay showed that fluoxetine enhanced binding of β-catenin with Axin1, which is a scaffold protein forming the degradation complex for β-catenin. Fluoxetine suppressed LiCl-induced β-catenin accumulation in human OA chondrocytes. Intraarticular injection of fluoxetine in a rat OA model ameliorated OA progression and suppressed β-catenin accumulation.

RAAV-mediated overexpression of TGF-β via vector delivery in polymeric micelles stimulates the biological and reparative activities of human articular chondrocytes in vitro and in a human osteochondral defect model.

Recombinant adeno-associated virus (rAAV) vectors are clinically adapted vectors to durably treat human osteoarthritis (OA). Controlled delivery of rAAV vectors via polymeric micelles was reported to enhance the temporal and spatial presentation of the vectors into their targets. Here, we tested the feasibility of delivering rAAV vectors via poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) (poloxamer and poloxamine) polymeric micelles as a means to overexpress the therapeutic factor transforming growth factor-beta (TGF-β) in human OA chondrocytes and in experimental human osteochondral defects. Application of rAAV-human transforming growth factor-beta using such micelles increased the levels of TGF-β transgene expression compared with free vector treatment. Overexpression of TGF-β with these systems resulted in higher proteoglycan deposition and increased cell numbers in OA chondrocytes. In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted. Delivery of therapeutic rAAV vectors via PEO-PPO-PEO micelles may provide potential tools to remodel human OA cartilage.

REAC regenerative treatment efficacy in experimental chondral lesions: a pilot study on ovine animal model.

Radioelectric asymmetric conveyor (REAC) technology is a platform designed to optimize cell polarity. Cell polarity is a universal biological phenomenon that is implicated in cell differentiation, proliferation, morphogenesis, aging, and rejuvenation. In this work, we investigate a timing and administration protocol for tissue optimization regenerative treatment type C, in order to treat aging-related chondral damage or injuries and gain insights into regenerative processes of articular cartilage in humans. The chondral lesion produced in this study in an animal model (6 knee joints of 4 adult sheep) was 6 mm in diameter and about 2 mm deep. These lesions, which did not involve subchondral bone, tend to increase in size and depth and are not completely repaired with normal hyaline articular cartilage since adult articular cartilage is avascular and has a very slow turnover at the cellular and molecular level. Moreover, the hydration of articular cartilage is reduced with aging and with decreased mitotic activity, synthesis, and population size of chondrocytes. Six months posttreatment, lesions appeared filled, though not completely, with newly generated tissue of the light opalescent color of healthy articular cartilage, which otherwise covered the underlying subchondral bone. The newly formed tissue surface appeared to be quite regular. Nearly complete regeneration of subchondral bone occurred, with little vascularization and ossification nuclei almost absent. The results of this study confirm previous data obtained in vitro on the regenerative effects of REAC technology on human normal and osteoarthritic chondrocytes exposed to IL-1β. The present findings indicate that REAC tissue optimization-regenerative treatment type C is a promising therapeutic tool among the other REAC regenerative treatment protocols for the treatment of cartilage lesions.

TRPA1 expression is downregulated by dexamethasone and aurothiomalate in human chondrocytes: TRPA1 as a novel factor and drug target in arthritis

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