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.     

Promotion of the prehierarchical follicle growth by postovulatory follicles involving PGE2–EP2 signaling in chickens

The postovulatory follicle (POF) in birds is an enigmatic structure, the function of which remains largely unknown. Previous studies on chickens have shown that removal of POFs leads to the postponement of oviposition and the disturbance of broody behavior. One suggestion is that POFs may secrete some crucial hormones or cytokines to act on reproductive organs. However, such secretions and their specific target organs remain to be identified. Here, we investigate the putative functions of POFs in promoting the development of prehierarchical follicles in chickens and explore the possible signaling mechanisms controlling these processes. Results show that POFs express steroidogenic acute regulatory protein (STAR), cholesterol side‐chain cleavage enzyme (CYP11A1), cyclooxygenase 1 (COX1), and COX2 in granulosa cells (GCs), and, most notably, that POF1 produces more prostaglandin E2 (PGE₂) or prostaglandin F2α than do the F1 follicle or the other POFs. Using coculture systems, we also found that POF1 or GCs from POF1 (POF1‐GCs) significantly promote the proliferation of theca externa cells of small white follicles (SWFs, one phase of the prehierarchical follicle). Treatment with PGE₂ significantly facilitates theca externa cell proliferation in SWFs. This POF‐stimulating effect on SWF growth was prevented by treatment with indomethacin (COX inhibitor) or TG6‐10‐1 (PGE₂ type 2 receptor [EP2] antagonist). Therefore, POF1 may secrete PGE₂ to stimulate the progression of SWF by PGE₂–EP2 signaling. These results indicate that POF1 may serve as a transient supplementary endocrine gland in the chicken ovary that stimulates the development of the prehierarchical follicles through PGE₂–EP2 signaling.     

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.     

Prostaglandin E2 downregulates TNF-alpha-induced production of matrix metalloproteinase-1 in HCS-2/8 chondrocytes by inhibiting Raf-1/MEK/ERK cascade through EP4 prostanoid receptor activation

Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-alpha (TNF-alpha). In response to TNF-alpha, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1, and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38 mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-alpha-induced MMP-1 release. A serine/threonine kinase, Akt was not activated in TNF-alpha-stimulated HCS-2/8 cells. TNF-alpha stimulated the production of PGE(2) in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE(2) potently inhibited TNF-alpha-induced both MMP-1 production and activation of ERK1/2. The effects of PGE(2) were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE(2) signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-alpha-induced MMP-1 production. Furthermore, the suppression of MMP-1 production by exogenously added PGE(2) was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-alpha, and that the produced PGE(2) downregulates the expression of MMP-1 by blockage of TNF-alpha-induced Raf-1 activation through EP4-PGE(2) receptor activation.     

Simvastatin abolishes nitric oxide‐ and reactive oxygen species‐induced cyclooxygenase‐2 expression by blocking the nuclear factor κB pathway in rabbit articular chondrocytes

Nitric oxide (NO) and reactive oxygen species (ROS) have been shown to be linked with numerous diseases, including osteoarthritis (OA). Our study aimed to examine the effect of simvastatin on NO‐ or ROS‐induced cyclooxygenase‐2 (COX‐2) expression in OA. Simvastatin has attracted considerable attention since the discovery of its pharmacological effects on different pathogenic processes, including inflammation. Here, we report that simvastatin treatment blocked sodium nitroprusside (SNP)‐ and interleukin 1 beta (IL‐1β)‐induced COX‐2 production. In addition, simvastatin attenuated SNP‐induced NO production and IL‐1β‐induced ROS generation. Treatment with simvastatin prevented SNP‐ and IL‐1β‐induced nuclear factor kappa B (NF‐κB) activity. Inhibiting NO production and ROS generation using N‐acetylcysteine (NAC) and NG‐monomethyl‐ l ‐arginine ( l ‐NMMA), respectively, accelerated the influence of simvastatin on NF‐κB activity. In addition, NAC blocked SNP and simvastatin‐mediated COX‐2 production and NF‐κB activity but did not alter IL‐1β and simvastatin‐mediated COX‐2 expression. l ‐NMMA treatment also abolished IL‐1β‐mediated COX‐2 expression and NF‐κB activation, whereas SNP and simvastatin‐mediated COX‐2 expression were not altered compared with the levels in the SNP and simvastatin‐treated cells. Our findings suggested that simvastatin blocks COX‐2 expression by inhibiting SNP‐induced NO production and IL‐1β‐induced ROS generation by blocking the NF‐κB pathway.     

Long noncoding RNA HNF1A‐AS1 regulates proliferation and apoptosis of glioma through activation of the JNK signaling pathway via miR‐363‐3p/MAP2K4

Long noncoding RNAs (lncRNAs) have been proven to exert important functions in the various biological processes of human cancers. It has been reported that lncRNA HNF1 homeobox A antisense RNA 1 (HNF1A‐AS1) was abnormally expressed and played a role in the initiation and development of various human cancers. In this study, we confirmed that the expression level of HNF1A‐AS1 was increased in glioma tissues and cells. Knockdown of HNF1A‐AS1 inhibited cell proliferation and promoted cell apoptosis in glioma. Then, we disclosed the downregulation of miR‐363‐3p in glioma tissues and cell lines. The interaction between HNF1A‐AS1 and miR‐363‐3p was identified in glioma cells. Furthermore, an inverse correlation between HNF1A‐AS1 and miR‐363‐3p was observed in glioma tissues. Afterwards, we recognized that MAP2K4 was a direct target of miR‐363‐3p. The expression of MAP2K4 was negatively correlated with miR‐363‐3p while positively related to HNF1A‐AS1 in glioma tissues. We also found the regulatory effect of HNF1A‐AS1 on the MAP2K4‐dependent JNK signaling pathway. All findings indicated that HNF1A‐AS1 induces the upregulation of MAP2K4 to activate the JNK signaling pathway to promote glioma cell growth by acting as a miR‐363‐3p sponge.     

Renoprotective effect of berberine via regulating the PGE2‐EP1‐Gαq‐Ca2+ signalling pathway in glomerular mesangial cells of diabetic rats

G‐protein coupled receptor‐mediated pathogenesis is of great importance in the development of diabetic complications, but the detailed mechanisms have not yet been clarified. Therefore, we aimed to explore the roles of the prostaglandin E2 receptor 1 (EP1)‐mediated signalling pathway and develop a corresponding treatment for diabetic nephropathy (DN). To create the DN model, rats fed a high‐fat and high‐glucose diet were injected with a single dose of streptozotocin (35 mg/kg, i.p.). Then, rats were either treated or not with berberine (100 mg/kg per day, i.g., 8 weeks). Cells were isolated from the renal cortex and cultured in high‐sugar medium with 20% foetal bovine serum. Prostaglandin E₂ (PGE₂) levels were determined by ELISA, and cells were identified by fluorescence immunoassay. We measured the biochemical characteristics and observed morphological changes by periodic‐acid‐Schiff staining. The expression of the EP1 receptor and the roles of GRK2 and β‐arrestin2 were identified using western blotting and flow cytometry. Downstream proteins were detected by western blot, while molecular changes were assessed by ELISA and laser confocal scanning microscopy. Berberine not only improved the majority of biochemical and renal functional parameters but also improved the histopathological alterations. A significant increase in PGE₂ level, EP1 membrane expression and Gαq expression, and concentration of Ca²⁺ were observed, accompanied by increased GRK2 and β‐arrestin2 levels soon afterwards. Berberine decreased the abnormal concentration of Ca²⁺, the increased levels of PGE₂, the high expression of EP1 and Gαq and suppressed the proliferation of mesangial cells. The EP1 receptor, a critical therapeutic target of the signalling pathway, contributed to mesangial cell abnormalities, which are linked to renal injury in DN. The observed renoprotective effects of berberine via regulating the PGE₂‐EP1‐Gαq‐Ca²⁺ signalling pathway indicating that berberine could be a promising anti‐DN medicine in the future.     

Targeting EP4 downstream c‐Jun through ERK1/2‐mediated reduction of DNMT1 reveals novel mechanism of solamargine‐inhibited growth of lung cancer cells

Lung cancer is the most common cancer and the leading cause of cancer deaths worldwide. We previously showed that solamargine, one natural phytochemicals from traditional plants, inhibited the growth of lung cancer cells through inhibition of prostaglandin E2 (PGE₂) receptor EP4. However, the potential downstream effectors of EP4 involving in the anti‐lung cancer effects of solamargine still remained to be determined. In this study, we further verified that solamargine inhibited growth of non‐small‐cell lung cancer (NSCLC) cells in multiple cell lines. Mechanistically, solamargine increased phosphorylation of ERK1/2. Moreover, solamargine inhibited the protein expression of DNA methyltransferase 1 (DNMT1) and c‐Jun, which were abrogated in cells treated with MEK/ERK1/2 inhibitor (PD98059) and transfected with exogenously expressed DNMT1 gene, respectively. Interestingly, overexpressed DNMT1 gene antagonized the effect of solamargine on c‐Jun protein expression. Intriguingly, overexpressed c‐Jun blocked solamargine‐inhibited lung cancer cell growth, and feedback resisted the solamargine‐induced phosphorylation of ERK1/2. A nude mouse xenograft model implanted with lung cancer cells in vivo confirmed the results in vitro. Collectively, our results show that solamargine inhibits the growth of human lung cancer cells through reduction of EP4 protein expression, followed by increasing ERK1/2 phosphorylation. This results in decrease in DNMT1 and c‐Jun protein expressions. The inter‐correlations between EP4, DNMT1 and c‐Jun and feedback regulation of ERK1/2 by c‐Jun contribute to the overall responses of solamargine in this process. This study uncovers an additional novel mechanism by which solamargine inhibits growth of human lung cancer cells.     

P2X4 receptors mediate PGE2 release by tissue‐resident macrophages and initiate inflammatory pain

Prostaglandin E2 (PGE2) is a key mediator of inflammation and contributes to pain hypersensitivity by promoting sensory neurons hyperexcitability. PGE2 synthesis results from activation of a multi‐step enzymatic cascade that includes cyclooxygenases (COXs), the main targets of non‐steroidal anti‐inflammatory drugs (NSAIDs). Although NSAIDs are widely prescribed to reduce inflammatory symptoms such as swelling and pain, associated harmful side effects restrict their long‐term use. Therefore, finding new drugs that limit PG production represents an important therapeutic issue. In response to peripheral inflammatory challenges, mice lacking the ATP‐gated P2X4 channel (P2X4R) do not develop pain hypersensitivity and show a complete absence of inflammatory PGE2 in tissue exudates. In resting conditions, tissue‐resident macrophages constitutively express P2X4R. Stimulating P2X4R in macrophages triggers calcium influx and p38 MAPK phosphorylation, resulting in cytosolic PLA2 (cPLA2) activation and COX‐dependent release of PGE2. In naive animals, pain hypersensitivity was elicited by transfer into the paw of ATP‐primed macrophages from wild type, but not P2X4R‐deficient mice. Thus, P2X4Rs are specifically involved in inflammatory‐mediated PGE2 production and might therefore represent useful therapeutic targets.     

miR‐370 and miR‐373 regulate the pathogenesis of osteoarthritis by modulating one‐carbon metabolism via SHMT‐2 and MECP‐2, respectively

The aim of this study was to determine the mechanism underlying the association between one‐carbon metabolism and DNA methylation during chronic degenerative joint disorder, osteoarthritis (OA). Articular chondrocytes were isolated from human OA cartilage and normal cartilage biopsied, and the degree of cartilage degradation was determined by safranin O staining. We found that the expression levels of SHMT‐2 and MECP‐2 were increased in OA chondrocytes, and 3′UTR reporter assays showed that SHMT‐2 and MECP‐2 are the direct targets of miR‐370 and miR‐373, respectively, in human articular chondrocytes. Our experiments showed that miR‐370 and miR‐373 levels were significantly lower in OA chondrocytes compared to normal chondrocytes. Overexpression of miR‐370 or miR‐373, or knockdown of SHMT‐2 or MECP‐2 reduced both MMP‐13 expression and apoptotic cell death in cultured OA chondrocytes. In vivo, we found that introduction of miR‐370 or miR‐373 into the cartilage of mice that had undergone destabilization of the medial meniscus (DMM) surgery significantly reduced the cartilage destruction in this model, whereas introduction of SHMT‐2 or MECP‐2 increased the severity of cartilage destruction. Together, these results show that miR‐370 and miR‐373 contribute to the pathogenesis of OA and act as negative regulators of SHMT‐2 and MECP‐2, respectively.     

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.     

4‐octyl Itaconate inhibits lipopolysaccharide (LPS)‐induced osteoarthritis via activating Nrf2 signalling pathway

Small molecule drug intervention for chondrocytes is a valuable method for the treatment of osteoarthritis (OA). The 4‐octyl itaconate (OI) is a cellular derivative of itaconate with sound cell permeability and transformation rate. We attempted to confirm the protective role of OI in chondrocytes and its regulatory mechanism. We used lipopolysaccharide (LPS) to induce chondrocyte inflammation injury. After the OI treatment, the secretion and mRNA expression of Il‐6, Il‐10, Mcp‐1 and Tnf‐α were detected by ELISA and qPCR. The protective effect of OI on articular cartilage was further verified in surgical destabilization of the medial meniscus model of OA. Cell death and apoptosis were evaluated based on CCK8, LDH, Typan blue staining, Annexin V and TUNEL analyses. The small interfering RNAs were used to knockout the Nrf2 gene of chondrocytes to verify the OI‐mediated Nrf2 signalling pathway. The results revealed that OI protects cells from LPS‐induced inflammatory injury and attenuates cell death and apoptosis induced by LPS. Similar protective effects were also observed on articular cartilage in mice. The OI activated Nrf2 signalling pathway and promoted the stable expression and translocation of Nrf2 into the nucleus. When the Nrf2 signalling pathway was blocked, the protective effect of OI was significantly counteracted in chondrocytes and a mouse arthritis model. Both itaconate and its derivative (i.e., OI) showed important medical effects in the treatment of OA.