Sauchinone prevents IL‐1β‐induced inflammatory response in human chondrocytes

Sauchinone is one of the active lignan isolated from Saururus chinensis, which has been considered to possess various pharmacological activities, such as antitumor, hepatoprotective, antioxidant, and anti‐inflammatory effects. However, the functional roles of sauchinone in interleukin‐1 beta (IL‐1β)‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Thus, in this study, we investigated the anti‐inflammatory effects of sauchinone in IL‐1β‐stimulated chondrocytes. Our results demonstrated that sauchinone significantly attenuated NO and PGE2 production, as well as inhibited iNOS and COX‐2 expression in IL‐1β‐stimulated OA chondrocytes. In addition, sauchinone efficiently inhibited IL‐1β‐induced MMP‐3 and MMP‐13 release in human OA chondrocytes. Furthermore, sauchinone significantly attenuated the activation of NF‐κB in human OA chondrocytes. In conclusion, we showed for the first time that sauchinone inhibited inflammatory response in IL‐1β‐stimulated human chondrocytes probably through inhibiting the activation of NF‐κB signaling pathway. These data suggest that sauchinone may be a potential agent in the treatment of OA.     

Gypenoside inhibits interleukin‐1β‐induced inflammatory response in human osteoarthritis chondrocytes

Gypenoside (GP), the main active ingredient of Gynostemma pentaphyllum, possesses a variety of pharmacological capacities including anti‐inflammation, anti‐oxidation, and anti‐tumor. However, the effects of GP on IL‐1β‐stimulated human osteoarthritis (OA) chondrocytes are still unknown. Therefore, this study aimed to investigate the anti‐inflammatory effects of GP on IL‐1β‐stimulated human OA chondrocytes and explore the possible mechanism. Our results showed that GP dose‐dependently inhibited IL‐1β‐induced NO and PGE2 production in human OA chondrocytes. In addition, treatment of GP inhibited the expression of MMP3 and MMP13, which was increased by IL‐1β. Finally, we found that pretreatment of GP obviously suppressed NF‐κB activation in IL‐1β‐stimulated human OA chondrocytes. Taken together, the results demonstrated that GP has chondro‐protective effects, at least in part, through inhibiting the activation of NF‐κB signaling pathway in human OA chondrocytes. Thus, these findings suggest that GP may be considered as an alternative therapeutic agent for the management of OA patients.     

Prostaglandin E2 exerts catabolic effects in osteoarthritis cartilage: evidence for signaling via the EP4 receptor

Elevated levels of PGE(2) have been reported in synovial fluid and cartilage from patients with osteoarthritis (OA). However, the functions of PGE(2) in cartilage metabolism have not previously been studied in detail. To do so, we cultured cartilage explants, obtained from patients undergoing knee replacement surgery for advanced OA, with PGE(2) (0.1-10 muM). PGE(2) inhibited proteoglycan synthesis in a dose-dependent manner (maximum 25% inhibition (p < 0.01)). PGE(2) also induced collagen degradation, in a manner inhibitable by the matrix metalloproteinase (MMP) inhibitor ilomastat. PGE(2) inhibited spontaneous MMP-1, but augmented MMP-13 secretion by OA cartilage explant cultures. PCR analysis of OA chondrocytes treated with PGE(2) with or without IL-1 revealed that IL-1-induced MMP-13 expression was augmented by PGE(2) and significantly inhibited by the cycolooygenase 2 selective inhibitor celecoxib. Conversely, MMP-1 expression was inhibited by PGE(2), while celecoxib enhanced both spontaneous and IL-1-induced expression. IL-1 induction of aggrecanase 5 (ADAMTS-5), but not ADAMTS-4, was also enhanced by PGE(2) (10 muM) and reversed by celecoxib (2 muM). Quantitative PCR screening of nondiseased and end-stage human knee OA articular cartilage specimens revealed that the PGE(2) receptor EP4 was up-regulated in OA cartilage. Moreover, blocking the EP4 receptor (EP4 antagonist, AH23848) mimicked celecoxib by inhibiting MMP-13, ADAMST-5 expression, and proteoglycan degradation. These results suggest that PGE(2) inhibits proteoglycan synthesis and stimulates matrix degradation in OA chondrocytes via the EP4 receptor. Targeting EP4, rather than cyclooxygenase 2, could represent a future strategy for OA disease modification.     

Rosmarinic acid down‐regulates NO and PGE2 expression via MAPK pathway in rat chondrocytes

Rosmarinic acid (RosA) is a water‐soluble polyphenol, which can be isolated from many herbs such as orthosiphon diffuses and rosmarinus officinalis. Previous studies have shown that RosA possesses various biological properties. In this study, we investigate the anti‐osteoarthritic effects of RosA in rat articular chondrocytes. Chondrocytes were pre‐treated with RosA, followed by the stimulation of IL‐1β. Real‐time PCR and Western blot were performed to detect the expression of matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13. Nitric oxide and PGE₂ production were measured by Griess reagent and enzyme‐linked immunosorbent assay (ELISA). The expression of mitogen‐activated protein kinase (MAPK) and nuclear factor‐κB (NF‐κB) was also investigated by Western blot analysis. We found that RosA down‐regulated the MMPs expression as well as nitric oxide and PGE₂ production in IL‐1β‐induced chondrocytes. In addition, RosA inhibited p38 and JNK phosphorylation as well as p65 translocation. The results suggest that RosA may be considered a possible agent in the treatment of OA.     

Cirsium japonicum var. maackii and apigenin block Hif‐2α‐induced osteoarthritic cartilage destruction

Although Hif‐2α is a master regulator of catabolic factor expression in osteoarthritis development, Hif‐2α inhibitors remain undeveloped. The aim of this study was to determine whether Cirsium japonicum var. maackii (CJM) extract and one of its constituents, apigenin, could attenuate the Hif‐2α‐induced cartilage destruction implicated in osteoarthritis progression. In vitro and in vivo studies demonstrated that CJM reduced the IL‐1β‐, IL‐6, IL‐17‐ and TNF‐α‐induced up‐regulation of MMP3, MMP13, ADAMTS4, ADAMTS5 and COX‐2 and blocked osteoarthritis development in a destabilization of the medial meniscus mouse model. Activation of Hif‐2α, which directly up‐regulates MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression, is inhibited by CJM extract. Although cirsimarin, cirsimaritin and apigenin are components of CJM and can reduce inflammation, only apigenin effectively reduced Hif‐2α expression and inhibited Hif‐2α‐induced MMP3, MMP13, ADAMTS4, IL‐6 and COX‐2 expression in articular chondrocytes. IL‐1β induction of JNK phosphorylation and IκB degradation, representing a critical pathway for Hif‐2α expression, was completely blocked by apigenin in a concentration‐dependent manner. Collectively, these effects indicate that CJM and one of its most potent constituents, apigenin, can lead to the development of therapeutic agents for blocking osteoarthritis development as novel Hif‐2α inhibitors.     

The pro‐inflammatory effect of NR4A3 in osteoarthritis

NR4A3 is a member of nuclear receptor subfamily 4, which is an important regulator of cellular function and inflammation. In this study, high expression of NR4A3 in human osteoarthritis (OA) cartilage was firstly observed. To explore the relationship between NR4A3 and OA, we used a lentivirus overexpression system to simulate its high expression and study its role in OA. Additionally, siRNA‐mediated knockdown of NR4A3 was used to confirm the findings of overexpression experiments. The results showed the stimulatory effect of IL‐1β on cartilage matrix‐degrading enzyme expression such as MMP‐3, 9, INOS and COX‐2 was enhanced in NR4A3‐overexpressed chondrocytes and decreased in NR4A3‐knockdown chondrocytes at both mRNA and protein levels, while IL‐1β‐induced chondrocyte‐specific gene (collagen 2 and SOX‐9) degradation was only regulated by NR4A3 at protein level. Furthermore, overexpression of NR4A3 would also enhance EBSS‐induced chondrocytes apoptosis, while knockdown of NR4A3 decreased apoptotic level after EBSS treatment. A pathway study indicated that IL‐1β‐induced NF‐κB activation was enhanced by NR4A3 overexpression and reduced by NR4A3 knockdown. We suggest that NR4A3 plays a pro‐inflammatory role in the development of OA, and we also speculate that NR4A3 mainly regulates cartilage matrix‐degrading gene expression under inflammatory conditions via the NF‐κB pathway.     

Berberine ameliorates cartilage degeneration in interleukin‐1β‐stimulated rat chondrocytes and in a rat model of osteoarthritis via Akt signalling

Berberine, a plant alkaloid used in Chinese medicine, has broad cell‐protective functions in a variety of cell lines. Chondrocyte apoptosis contributes to the pathogenesis of cartilage degeneration in osteoarthritis (OA). However, little is known about the effect and underlying mechanism of berberine on OA chondrocytes. Here, we assessed the effects of berberine on cartilage degeneration in interleukin‐1β (IL‐1β)‐stimulated rat chondrocytes and in a rat model of OA. The results of an MTT assay and western blotting analysis showed that berberine attenuated the inhibitory effect of IL‐1β on the cell viability and proliferating cell nuclear antigen expression in rat chondrocytes. Furthermore, berberine activated Akt, which triggered p70S6K/S6 pathway and up‐regulated the levels of aggrecan and Col II expression in IL‐1β‐stimulated rat chondrocytes. In addition, berberine increased the level of proteoglycans in cartilage matrix and the thickness of articular cartilage, with the elevated levels of Col II, p‐Akt and p‐S6 expression in a rat OA model, as demonstrated by histopathological and immunohistochemistry techniques. The data thus strongly suggest that berberine may ameliorate cartilage degeneration from OA by promoting cell survival and matrix production of chondrocytes, which was partly attributed to the activation of Akt in IL‐1β‐stimulated articular chondrocytes and in a rat OA model. The resultant chondroprotective effects indicate that berberine merits consideration as a therapeutic agent in OA.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Chondroprotective and anti‐inflammatory effects of amurensin H by regulating TLR4/Syk/NF‐κB signals

The low‐grade, chronic inflammation initiated by TLR4‐triggered innate immune responses has a central role on early osteoarthritis. Amurensin H is a resveratrol dimer with anti‐inflammatory and anti‐apoptotic effects, while its effects on TLR‐4 signals to inhibit osteoarthritis are still unclear. In the present study, treatment with amurensin H for 2 weeks in monosodium iodoacetate‐induced mice significantly slows down cartilage degeneration and inflammation using macroscopic evaluation, haematoxylin and eosin (HE) staining and micro‐magnetic resonance imaging. In IL‐1β‐stimulated rat chondrocytes, amurensin H suppresses the production of inflammatory mediators including nitric oxide, IL‐6, IL‐17, PGE2 and TNF‐α using Greiss and ELISA assay. Amurensin H inhibits matrix degradation via decreasing levels of MMP‐9 and MMP‐13 using Western blot assay, promotes synthesis of type II collagen and glycosaminoglycan using immunostaining and safranin O staining, respectively. Amurensin H inhibits intracellular and mitochondrial reactive oxygen species (ROS) generation, and mitochondrial membrane depolarization using DCFH‐DA, MitoSOX Red and JC‐1 assay as well. IL‐1β stimulates TLR4 activation and Syk phosphorylation in chondrocytes, while amurensin H inhibits TLR4/Syk signals and downstream p65 phosphorylation and translocation in a time and dose‐dependent manner. Together, these results suggest that amurensin H exerts chondroprotective effects by attenuating oxidative stress, inflammation and matrix degradation via the TLR4/Syk/NF‐κB pathway.     

Seomae mugwort and jaceosidin attenuate osteoarthritic cartilage damage by blocking IκB degradation in mice

Seomae mugwort, a Korean native variety of Artemisia argyi, exhibits physiological effects against various diseases. However, its effects on osteoarthritis (OA) are unclear. In this study, a Seomae mugwort extract prevented cartilage destruction in an OA mouse model. In vitro and ex vivo analyses revealed that the extract suppressed MMP3, MMP13, ADAMTS4 and ADAMTS5 expression induced by IL‐1β, IL‐6 and TNF‐α and inhibited the loss of extracellular sulphated proteoglycans. In vivo analysis revealed that oral administration of the extract suppressed DMM‐induced cartilage destruction. We identified jaceosidin in Seomae mugwort and showed that this compound decreased MMP3, MMP13, ADAMTS4 and ADAMTS5 expression levels, similar to the action of the Seomae mugwort extract in cultured chondrocytes. Interestingly, jaceosidin and eupatilin combined had similar effects to Seomae mugwort in the DMM‐induced OA model. Induction of IκB degradation by IL‐1β was blocked by the extract and jaceosidin, whereas JNK phosphorylation was only suppressed by the extract. These results suggest that the Seomae mugwort extract and jaceosidin can attenuate cartilage destruction by suppressing MMPs, ADAMTS4/5 and the nuclear factor‐κB signalling pathway by blocking IκB degradation. Thus, the findings support the potential application of Seomae mugwort, and particularly jaceosidin, as natural therapeutics for OA.     

NOV/CCN3 induces cartilage protection by inhibiting PI3K/AKT/mTOR pathway

Osteoarthritis (OA), an age‐related degenerative joint disease, is pathologically characterized by articular cartilage degeneration and synovial inflammation. Nephroblastoma overexpressed (NOV or CCN3), a matricellular protein, is a primary member of the CCN family (Cyr61, Ctgf, NOV) of proteins and is involved in various inflammatory disorders. Previous studies reported that CCN3 might play a therapeutic role in OA. However, the underlying mechanism remains unclear. In this study, we confirmed the expression of CCN3 was decreased in human and rat OA articular cartilage. Recombinant CCN3 ameliorated the IL‐1β‐induced matrix catabolism, as demonstrated by MMP1, MMP3, MMP13, ADAMTS5 and iNOS expression, in vitro. In addition, the degradation of cartilage matrix such as collagen 2 and aggrecan could be reversed by CCN3. Furthermore, we found CCN3 promoted autophagy as Atg5, Beclin1 and LC3‐II expression were increased. High‐mobility group box 1 was negatively correlated with CCN3 in IL‐1β‐induced osteoarthritis responses, and HMGB1 is involved in the protective effect of CCN3 in OA. Moreover, CCN3 overexpression decreased the expression of HMGB1 and reversed the IL‐1β induced MMPs production. Additionally, recombinant CCN3 or CCN3 overexpression attenuated the activation of PI3K/AKT/mTOR pathway induced by IL‐1β. Our study presents new mechanisms of CCN3 in osteoarthritis and indicates that CCN3 can serve as a novel potential therapeutic target for osteoarthritis.     

The protective effect of sinapic acid in osteoarthritis: In vitro and in vivo studies

The anti‐inflammatory effect of sinapic acid (SA) has been reported in several studies. However, whether SA has the same effect on osteoarthritis (OA) has yet to be clearly elucidated. We designed a series of in vitro and in vivo procedures to verify the above conjecture. Compared with controls, SA‐pretreated human chondrocytes showed lower levels of interleukin (IL)‐1β‐induced IL‐6, prostaglandin E2 (PGE2), nitric oxide (NO) and tumour necrosis factor‐α (TNF‐α) in vitro. Meanwhile, SA could also reverse the degradation of type II collage and aggrecan, as well as the overproduction of matrix metalloproteinase‐9 (MMP‐9) and matrix metalloproteinase‐13 (MMP‐13), inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)‐2 and a disintegrin and metalloproteinase thrombospondin motifs (ADAMTS)‐5. Furthermore, activation of nuclear factor κB (NF‐κB), which was induced by IL‐1β, was also inhibited by SA through the pathway of nuclear factor‐erythroid 2‐related factor‐2 (Nrf2)/heme oxygenase 1. In vivo, SA could delay the progress of mice OA models. We propose that SA may be applied as a potential therapeutic drug in OA treatment.     

Hydrogen sulphide decreases IL‐1β‐induced activation of fibroblast‐like synoviocytes from patients with osteoarthritis

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H₂S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H₂S on fibroblast‐like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro‐inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL‐1β and treated with the H₂S donor NaHS. Cellular responses were analysed by ELISA, quantitative real‐time PCR, phospho‐MAPkinase array and Western blotting. Treatment‐induced effects on cellular structure and synovial architecture were investigated in three‐dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL‐1β‐induced secretion of IL‐6, IL‐8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo‐proteinases MMP‐2 and MMP‐14. IL‐1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL‐1β‐induced activation of several MAPK whereas it increased phosphorylation of pro‐survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer‐like structure; stimulation with IL‐1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H₂S partially antagonizes IL‐1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA.     

Differential responses of chondrocytes from normal and osteoarthritic human articular cartilage to mechanical stimulation

Mechanical stimulation is critically important for the maintenance of normal articular cartilage integrity. Molecular events regulating responses of chondrocytes to mechanical forces are beginning to be defined. Chondrocytes from normal human knee joint articular cartilage show increased levels of aggrecan mRNA following 0.33 Hz mechanical stimulation whilst at the same time relative levels of MMP3 mRNA are decreased. This anabolic response, associated with membrane hyperpolarisation, is activated via an integrin-dependent interleukin (IL)-4 autocrine/paracrine loop. Work in our laboratory suggests that this chondroprotective response may be aberrant in osteoarthritis (OA). Chondrocytes from OA cartilage show no changes in aggrecan or MMP3 mRNA following 0.33 Hz mechanical stimulation. alpha5beta1 integrin is the mechanoreceptor in both normal and OA chondrocytes but downstream signalling pathways differ. OA chondrocytes show membrane depolarisation following 0.33 Hz mechanical stimulation consequent to activation of an IL1beta autocrine/paracrine loop. IL4 signalling in OA chondrocytes is preferentially through the type I (IL4alpha/cgamma) receptor rather than via the type II (IL4alpha/IL13R) receptor. Altered mechanotransduction and signalling in OA may contribute to changes in chondrocyte behaviour leading to increased cartilage breakdown and disease progression.     

Prostaglandin E2 enhances interleukin-8 production via EP4 receptor in human pulmonary microvascular endothelial cells

Prostaglandin E(2) (PGE(2)) is a bioactive prostanoid implicated in the inflammatory processes of acute lung injury/acute respiratory distress syndrome. This study investigated whether PGE(2) can induce production of interleukin (IL)-8, the major chemokine for neutrophil activation, from human pulmonary microvascular endothelial cells (HPMVECs). PGE(2) significantly enhanced IL-8 protein production with increases in IL-8 mRNA expression and intracellular cAMP levels. HPMVECs expressed only EP4 receptor mRNA. The PGE(2) effects were mimicked by a selective EP4 receptor agonist, ONO-AE1-329, and inhibited by a selective EP4 receptor antagonist, ONO-AE3-208, or a protein kinase A inhibitor, Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt. The specific agonist for EP1, EP2, or EP3 receptor did not induce IL-8 production. PGE(2)-induced IL-8 production was accompanied by p38 phosphorylation and was significantly inhibited by a p38 inhibitor, SB-203580, but not by an ERK1/2 inhibitor, U-0126, or a JNK inhibitor, SP-600125. Additionally, PGE(2) increased cyclooxygenase-2 expression with no change in constitutive cyclooxygenase-1 expression, suggesting possible involvement of an autocrine or paracrine manner. In conclusion, PGE(2) enhances IL-8 production via EP4 receptor coupled to G(s) protein in HPMVECs. Activation of the cAMP/protein kinase A pathway, followed by p38 activation, is essential for these mechanisms. Because neutrophils play a critical role in the inflammation of acute lung injury/acute respiratory distress syndrome, IL-8 released from the pulmonary microvasculature in response to PGE(2) may contribute to pathophysiology of this disease.     

Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well‐known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVβ3 and αVβ5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), matrix metalloproteinase‐3 (MMP‐3), and MMP‐13 induced by excessive mechanical stress. In addition, the protein expression of IL1‐β and MMP‐13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress‐induced integrin signaling in chondrocytes by using western blotting. The levels of p‐FAK, p‐ERK, p‐JNK, and p‐p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVβ3 and αVβ5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL‐1β, TNF‐α, MMP‐3, and MMP‐13 through phosphorylation of FAK and MAPKs.     

Cyclin D1 Gene Silencing Promotes IL‐1β‐Induced Apoptosis in Rat Chondrocytes

This study investigated the effects of cyclin D1 gene silencing on cell proliferation and apoptosis of interleukin‐1β (IL‐1β)‐induced osteoarthritis (OA) chondrocytes. Chondrocytes from healthy sprague‐dawley rats were divided into blank, OA model (chondrocytes underwent IL‐1β inducement), OA trial (chondrocytes underwent IL‐1β inducement with cyclin D1‐shRNA treatment), and negative control (NC; chondrocytes underwent IL‐1β inducement and control‐shRNA treatment) groups. Cell proliferation was assessed by CCK‐8 assay, and cell cycle and apoptosis by flow cytometry. qRT‐PCR and Western blotting were performed to detect cyclin D1 and apoptosis‐related factors expression levels. Chondrocyte proliferation increased after 72–96 h after incubation. The OA trial group exhibited reduced cell proliferation at 48, 72, and 96 h after treatment. The OA model, OA trial, and NC groups all contained more cells arrested in G1 phase and had higher apoptosis rates than the blank group. Additionally, the OA trial group contained more cells arrested in G1 phase, with increased apoptosis rates compared to the OA model and NC groups. The OA model group had lowest expression of cyclin D1 whereas the blank group contained the highest among the four groups. qRT‐PCR also showed that the OA model, OA trial, and NC groups all had increased expression levels of Bax and reduced expression levels of Bcl‐2 and P53 compared to the blank group, whereby by the OA group had the most significant change. The combined evidence in our study shows that cyclin D1 gene silencing suppresses proliferation and induces apoptosis of rat chondrocytes in IL‐1β‐induced OA. J. Cell. Biochem. 119: 290–299, 2018. © 2017 Wiley Periodicals, Inc.     

Acacetin inhibits expression of matrix metalloproteinases via a MAPK‐dependent mechanism in fibroblast‐like synoviocytes

It is well known that rheumatoid arthritis (RA) is an autoimmune joint disease in which fibroblast‐like synoviocytes (FLSs) play a pivotal role. In this study, we investigated the anti‐arthritic properties of acacetin in FLSs. The expression of matrix metalloproteinase (MMP)‐1, MMP‐3 and MMP‐13 were investigated by quantitative RT‐PCR and western blot at gene and protein levels. At the same time, the phosphorylation of mitogen‐activated protein kinases (MAPK) was investigated. The DNA‐binding activity of NF‐κB was investigated by electrophoretic mobility shift assay. We found that acacetin inhibits p38 and JNK phosphorylation and reduces MMP‐1, MMP‐3 and MMP‐13 expression in interleukin‐1β‐induced FLSs. Our results suggest that acacetin has antiarthritic effects in FLSs. Thus, acacetin should be further studied for the treatment of arthritis.     

Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE(2), the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE(2) and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-gamma. The inhibitory effect of PGE(2) but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE(2) in 3T3-L1 adipocyte differentiation.