Nepetin inhibits osteoclastogenesis by inhibiting RANKL‐induced activation of NF‐κB and MAPK signalling pathway,and autophagy

Aseptic prosthetic loosening due to wear particle–induced inflammatory osteolysis is the main cause of failure for artificial joint replacement. The inflammatory response and the production of pro‐osteoclastic factors lead to elevation of osteoclast formation and excessive activity results in extensive bone destruction around the bone‐implant interface. Here we showed that Nepetin, a natural bioactive flavonoid with proven anti‐inflammatory and anti‐proliferative properties, potently inhibited RANKL‐induced osteoclast differentiation, formation and bone resorption in vitro, and protected mice against the deleterious effects of titanium particle–induced calvarial osteolysis in vivo. Mechanistically, Nepetin attenuated RANKL‐induced activation of NF‐κB and MAPK signalling pathways and TRAF6‐dependent ubiquitination of Beclin 1 which is necessary for the induction of autophagy. In brief, our study demonstrates the potential therapeutic application of Nepetin against osteoclast‐mediated osteolytic diseases.     

Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis

Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal‐cysteine‐protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL‐mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non‐eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL‐mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non‐eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL‐stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL‐stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL‐primed osteoclastogenesis was observed in male and female CatB‐knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL‐primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.     

NLRP3 regulates alveolar bone loss in ligature‐induced periodontitis by promoting osteoclastic differentiation

ObjectivesNLRP3 inflammasome is a critical part of the innate immune system and plays an important role in a variety of inflammatory diseases. However, the effects of NLRP3 inflammasome on periodontitis have not been fully studied.Materials and methodsWe used ligature‐induced periodontitis models of NLRP3 knockout mice (NLRP3^KO) and their wildtype (WT) littermates to compare their alveolar bone phenotypes. We further used Lysm‐Cre/Rosa^nTnG mouse to trace the changes of Lysm‐Cre⁺ osteoclast precursors in ligature‐induced periodontitis with or without MCC950 treatment. At last, we explored MCC950 as a potential drug for the treatment of periodontitis in vivo and in vitro.ResultsHere, we showed that the number of osteoclast precursors, osteoclast differentiation and alveolar bone loss were reduced in NLRP3^KO mice compared with WT littermates, by using ligature‐induced periodontitis model. Next, MCC950, a specific inhibitor of the NLRP3 inflammasome, was used to inhibit osteoclast precursors differentiation into osteoclast. Further, we used Lysm‐Cre/Rosa^nTnG mice to demonstrate that MCC950 decreases the number of Lysm‐Cre⁺ osteoclast precursors in ligature‐induced periodontitis. At last, treatment with MCC950 significantly suppressed alveolar bone loss with reduced IL‐1β activation and osteoclast differentiation in ligature‐induced periodontitis.ConclusionOur findings reveal that NLRP3 regulates alveolar bone loss in ligature‐induced periodontitis by promoting osteoclastic differentiation.     

F‐actin dynamics in midgut cells enables virus persistence in vector insects

Hemipteran insects that transmit plant viruses in a persistent circulative manner acquire, retain and transmit viruses for their entire life. The mechanism enabling this persistence has remained unclear for many years. Here, we determined how wheat dwarf virus (WDV) persists in its leafhopper vector Psammotettix alienus. We found that WDV caused the up‐regulation of actin‐depolymerizing factor (ADF) at the mRNA and protein levels in the midgut cells of leafhoppers after experiencing a WDV acquisition access period (AAP) of 6, 12 or 24 h. Experimental inhibition of F‐actin depolymerization by jasplakinolide and dsRNA injection led to lower virus accumulation levels and transmission efficiencies, suggesting that depolymerization of F‐actin regulated by ADF is essential for WDV invasion of midgut cells. Exogenous viral capsid protein (CP) inhibited ADF depolymerization of actin filaments in vitro and in Spodoptera frugiperda 9 (Sf9) cells because the CP competed with actin to bind ADF and then blocked actin filament disassembly. Interestingly, virions colocalized with ADF after a 24‐h AAP, just as actin polymerization occurred, indicating that the binding of CP with ADF affects the ability of ADF to depolymerize F‐actin, inhibiting WDV entry. Similarly, the luteovirus barley yellow dwarf virus also induced F‐actin depolymerization and then polymerization in the gut cells of its vector Schizaphis graminum. Thus, F‐actin dynamics are altered by nonpropagative viruses in midgut cells to enable virus persistence in vector insects.     

MgIG exerts therapeutic effects on crizotinib‐induced hepatotoxicity by limiting ROS‐mediated autophagy and pyroptosis

Crizotinib (CRIZO) has been widely employed to treat non‐small‐cell lung cancer. However, hepatic inflammatory injury is the major toxicity of CRIZO, which limits its clinical application, and the underlying mechanism of CRIZO‐induced hepatotoxicity has not been fully explored. Herein, we used cell counting kit‐8 assay and flow cytometry to detect CRIZO‐induced cytotoxicity on human hepatocytes (HL‐7702). CRIZO significantly reduced the survival rate of hepatocytes in a dose‐dependent manner. Furthermore, the reactive oxygen species (ROS) assay kit showed that CRIZO treatment strongly increased the level of ROS. In addition, CRIZO treatment caused the appearance of balloon‐like bubbles and autophagosomes in HL‐7702 cells. Subsequently, Western blotting, quantitative real‐time PCR and ELISA assays revealed that ROS‐mediated pyroptosis and autophagy contributed to CRIZO‐induced hepatic injury. Based on the role of ROS in CRIZO‐induced hepatotoxicity, magnesium isoglycyrrhizinate (MgIG) was used as an intervention drug. MgIG activated the Nrf2/HO‐1 signalling pathway and reduced ROS level. Additionally, MgIG suppressed hepatic inflammation by inhibiting NF‐κB activity, thereby reducing CRIZO‐induced hepatotoxicity. In conclusion, CRIZO promoted autophagy activation and pyroptosis via the accumulation of ROS in HL‐7702 cells. MgIG exerts therapeutic effects on CRIZO‐induced hepatotoxicity by decreasing the level of ROS.     

Nur77 Prevents Osteoporosis by Inhibiting the NF‐κB Signalling Pathway and Osteoclast Differentiation

Inflammation is a major risk factor for osteoporosis, and reducing inflammatory levels is important for the prevention of osteoporosis. Although nuclear receptor 77 (Nur77) protects against inflammation in a variety of diseases, its role in osteoporosis is unknown. Therefore, the main purpose of this study was to investigate the osteoprotective and anti‐inflammatory effects of Nur77. The microCT and haematoxylin and eosin staining results indicated that knockout of Nur77 accelerated femoral bone loss in mice. The enzyme‐linked immunosorbent assay (ELISA) results showed that knockout of Nur77 increased the serum levels of hsCRP and IL‐6. The expression levels of NF‐κB, IL‐6, TNF‐α and osteoclastogenesis factors (TRAP, NFATC1, Car2, Ctsk) in the femurs of Nur77 knockout mice were increased significantly. Furthermore, in vitro, shNur77 promoted the differentiation of RAW264.7 cells into osteoclasts by activating NF‐κB, which was confirmed by PDTC treatment. Mechanistically, Nur77 inhibited osteoclast differentiation by inducing IκB‐α and suppressing IKK‐β. In RAW264.7 cells, overexpression of Nur77 alleviated inflammation induced by siIκB‐α, while siIKK‐β alleviated inflammation induced by shNur77. Consistent with the in vivo studies, we found that compared with control group, older adults with high serum hsCRP levels were more likely to suffer from osteoporosis (OR = 1.76, p < 0.001). Our data suggest that Nur77 suppresses osteoclast differentiation by inhibiting the NF‐κB signalling pathway, strongly supporting the notion that Nur77 has the potential to prevent and treat osteoporosis.     

Novel chloroquine derivative suppresses melanoma cell growth by DNA damage through increasing ROS levels

Melanoma is a fatal cancer with a significant feature of resistance to traditional chemotherapeutic drugs and radiotherapy. A mutation in the kinase BRAF is observed in more than 66% of metastatic melanoma cases. Therefore, there is an urgent need to develop new BRAF‐mutant melanoma inhibitors. High‐dose chloroquine has been reported to have antitumour effects, but it often induces dose‐limiting toxicity. In this study, a series of chloroquine derivatives were synthesized, and lj‐2‐66 had the best activity and was selected for further investigation. Furthermore, the anti‐BRAF‐mutant melanoma effect and mechanism of this compound were explored. CCK‐8 and colony formation assays indicated that lj‐2‐66 significantly inhibited the proliferation of BRAF‐mutant melanoma cells. Flow cytometry revealed that lj‐2‐66 induced G2/M arrest in melanoma cells and promoted apoptosis. Furthermore, lj‐2‐66 increased the level of ROS in melanoma cells and induced DNA damage. Interestingly, lj‐2‐66 also played a similar role in BRAF inhibitor‐resistant melanoma cells. In summary, we found a novel chloroquine derivative, lj‐2‐66, that increased the level of ROS in melanoma cells and induced DNA damage, thus leading to G2/M arrest and apoptosis. These findings indicated that lj‐2‐66 may become a potential therapeutic drug for melanoma harbouring BRAF mutations.     

MST1/2 inhibitor XMU‐MP‐1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system

The Hippo signalling pathway has been considered as potential therapeutic target in self‐renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20‐like kinase 1/2 (MST1/2) as the core serine‐threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU‐MP‐1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU‐MP‐1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU‐MP‐1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.     

Ferulic acid (FA) protects human retinal pigment epithelial cells from H2O2‐induced oxidative injuries

The aim of present study is to investigate whether Ferulic acid (FA), a natural polyphenol antioxidant, was able to protect ARPE‑19 cells from hydrogen peroxide (H₂O₂)‑induced damage, and elucidate the underlying mechanisms. Our results revealed that FA pre‐treatment for 24 hours can reverse cell loss of H₂O₂‐induced ARPE‐19 cells via the promotion of cell proliferation and prevention of apoptosis, as evidenced by 5‐ethynyl‐2′‐deoxyuridine (EdU) incorporation and terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, respectively. Moreover, the addition of FA (5 mM) can decrease Bax and cleaved caspase‐3 protein expression, but increase Bcl‐2 protein expression in ARPE‐19 cells. Furthermore, H₂O₂‐induced oxidative stress in ARPE‐19 cells was significantly alleviated by FA, illustrated by reduced levels of ROS and MDA. In addition, the attenuated antioxidant enzymes activities of (SOD, CAT and GPX) and GSH level were reversed almost to the normal base level by the pre‐addition of FA for 24 hours. In all assays, FA itself did not exert any effect on the change of the above parameters. These novel findings indicated that FA effectively protected human ARPE‐19 cells from H₂O₂‐induced oxidative damage through its pro‐proliferation, anti‐apoptosis and antioxidant activity, suggesting that FA has a therapeutic potential in the prevention and treatment of AMD.     

Periplaneta Americana extract inhibits osteoclastic differentiation in vitro

ObjectivesPeriplaneta americana extract (PAE) is proven to be promising in treating fever, wound healing, liver fibrosis, and cardiovascular disease. However, the role of PAE in skeletal disorders remains unclear. This study investigated whether PAE regulates osteoclastic differentiation in vitro via the culture using RAW264.7 cells and bone marrow derived macrophages (BMDMs).Materials and MethodsRAW264.7 cells and BMDMs were cultured and induced for osteoclastic differentiation supplementing with different concentrations of PAE (0, 0.1, 1, and 10 mg/mL). Cell counting kit‐8 (CCK‐8) assay was used to detect the cytotoxicity and cell proliferation. TRAP staining, actin ring staining, real‐time quantitative PCR (RT‐qPCR), and bone resorption activity test were performed to detect osteoclastic differentiation. RT‐qPCR and enzyme‐linked immunosorbent assay (ELISA) were conducted to assay the expression and secretion of inflammatory factors. RNA sequencing (RNA‐seq) and western blot analysis were carried out to uncover the underlying mechanism.ResultsCCK‐8 results showed that 10 mg/mL and a lower concentration of PAE did not affect cell proliferation. RT‐qPCR analysis verified that PAE down‐regulated the osteoclastic genes Nfatc1, Ctsk, and Acp5 in macrophages. Moreover, PAE restrained the differentiation, formation, and function of osteoclasts. Besides, RT‐qPCR and ELISA assays showed that PAE decreased inflammatory genes expression and reduced the secretion of inflammatory factors, including IL1β, IL6, and TNFα. Subsequent RNA‐seq analysis identified possible genes and signaling pathways of PAE‐mediated osteoclastogenesis suppression.ConclusionsOur study indicates that PAE has inhibitive effects on osteoclastogenesis and may be a potential therapeutic alternative for bone diseases.

Periplaneta americana extract (PAE), the animal medicine material extracted from the insects Periplaneta americana, is proven to possess a variety of pharmacological functions. However, the role of PAE in skeletal disorders remains unclear. In this study, we found that PAE decreased osteoclast genes expression Nfatc1, Ctsk, and Acp5 in macrophages. Besides, PAE restrained the differentiation, formation, and function of osteoclasts. Moreover, PAE suppressed the LPS‐induced inflammation. Subsequent RNA‐seq analysis identified the signaling pathways of PAE‐mediated osteoclastogenesis suppression. Our study indicated that PAE has inhibitive effects on osteoclastic differentiation and may be a potential therapeutic Chinese medicine for bone diseases.     

Adverse PFAS effects on mouse oocyte in vitro maturation are associated with carbon‐chain length and inclusion of a sulfonate group

ObjectivesPer‐ and polyfluoroalkyl substances (PFAS) are man‐made chemicals that are widely used in various products. PFAS are characterized by their fluorinated carbon chains that make them hard to degrade and bioaccumulate in human and animals. Toxicological studies have shown PFAS toxic effects: cytotoxicity, immunotoxicity, neurotoxicity, and reproductive toxicity. However, it is still unclear how the structures of PFAS, such as carbon‐chain length and functional groups, determine their reproductive toxicity.Methods and ResultsBy using a mouse‐oocyte‐in‐vitro‐maturation (IVM) system, we found the toxicity of two major categories of PFAS, perfluoroalkyl carboxylic acid (PFCA) and perfluoroalkyl sulfonic acid (PFSA), is elevated with increasing carbon‐chain length and the inclusion of the sulfonate group. Specifically, at 600 μM, perfluorohexanesulfonic acid (PFHxS) and perfluorooctanesulfonic acid (PFOS) reduced the rates of both germinal‐vesicle breakdown (GVBD) and polar‐body extrusion (PBE) as well as enlarged polar bodies. However, the shorter PFSA, perfluorobutanesulfonic acid (PFBS), and all PFCA did not show similar adverse cytotoxicity. Further, we found that 600 μM PFHxS and PFOS exposure induced excess reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP). Cytoskeleton analysis revealed that PFHxS and PFOS exposure induced chromosome misalignment, abnormal F‐actin organization, elongated spindle formation, and symmetric division in the treated oocytes. These meiotic defects compromised oocyte developmental competence after parthenogenetic activation.ConclusionsOur study provides new information on the structure‐toxicity relationship of PFAS.

Reproductive toxicity of PFAS is determined by their chemical structures. An inverted U‐shaped relationship is shown between toxic effects of PFAS and carbon‐chain length; carbon‐chain length around 10 is the most toxic. PFSA has higher and unique toxicity effects on mouse oocyte maturation compared to PFCA because PFSA has an extra sulfonate group.     

Topical astilbin ameliorates imiquimod‐induced psoriasis‐like skin lesions in SKH‐1 mice via suppression dendritic cell‐Th17 inflammation axis

Astilbin, an essential component of Rhizoma smilacis glabrae, exerts significant antioxidant and anti‐inflammatory effects against various autoimmune diseases. We have previously reported that astilbin decreases proliferation and improves differentiation of HaCaT keratinocytes in a psoriatic model. The present study was designed to evaluate the potential therapeutic effects of topical administration of astilbin on an imiquimod (IMQ)‐induced psoriasis‐like murine model and to reveal their underlying mechanisms. Topical administration of astilbin at a lower dose alleviated IMQ‐induced psoriasis‐like skin lesions by inducing the differentiation of epidermal keratinocytes in mice, and the therapeutic effect was even better than that of calcipotriol. Moreover, the inflammatory skin disorder was relieved by astilbin treatment characterized by a reduction in both IL‐17‐producing T cell accumulation and psoriasis‐specific cytokine expression in skin lesions. Furthermore, we found that astilbin inhibited R837‐induced maturation and activation of bone marrow‐derived dendritic cells and decreased the expression of pro‐inflammatory cytokines by downregulating myeloid differentiation factor 88. Our findings provide the convincing evidence that lower doses of astilbin might attenuate psoriasis by interfering with the abnormal activation and differentiation of keratinocytes and accumulation of IL‐17‐producing T cells in skin lesions. Our results strongly support the pre‐clinical application of astilbin for psoriasis treatment.     

Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.     

Sirtuin 1 ameliorates defenestration in hepatic sinusoidal endothelial cells during liver fibrosis via inhibiting stress‐induced premature senescence

ObjectivePremature senescence is related to progerin and involves in endothelial dysfunction and liver diseases. Activating sirtuin 1 (SIRT1) ameliorates liver fibrosis. However, the mechanisms of premature senescence in defenestration of hepatic sinusoidal endothelial cells (HSECs) and how SIRT1 affects HSECs fenestrae remain elusive.MethodsWe employed the CCl₄‐induced liver fibrogenesis rat models and cultured primary HSECs in vitro, administered with the SIRT1‐adenovirus vector, the activator of SIRT1 and knockdown NOX2. We measured the activity of senescence‐associated β‐galactosidase (SA‐β‐gal) in HSECs. Meanwhile, the protein expression of SIRT1, NOX2, progerin, Lamin A/C, Ac p53 K381 and total p53 was detected by Western blot, co‐immunoprecipitation and immunofluorescence.ResultsIn vivo, premature senescence was triggered by oxidative stress during CCl₄‐induced HSECs defenestration and liver fibrogenesis, whereas overexpressing SIRT1 with adenovirus vector lessened premature senescence to relieve CCl₄‐induced HSECs defenestration and liver fibrosis. In vitro, HSECs fenestrae disappeared, with emerging progerin‐associated premature senescence; these effects were aggravated by H₂O₂. Nevertheless, knockdown of NOX2, activation of SIRT1 with resveratrol and SIRT1‐adenovirus vector inhibited progerin‐associated premature senescence to maintain fenestrae through deacetylating p53. Furthermore, more Ac p53 K381 and progerin co‐localized with the abnormal accumulation of actin filament (F‐actin) in the nuclear envelope of H₂O₂‐treated HSECs; in contrast, these effects were rescued by overexpressing SIRT1.ConclusionSIRT1‐mediated deacetylation maintains HSECs fenestrae and attenuates liver fibrogenesis through inhibiting oxidative stress‐induced premature senescence.     

Tumour necrosis factor‐α promotes BMHSC differentiation by increasing P2X7 receptor in oestrogen‐deficient osteoporosis

The exact mechanism of tumour necrosis factor α (TNF‐α) promoting osteoclast differentiation is not completely clear. A variety of P2 purine receptor subtypes have been confirmed to be widely involved in bone metabolism. Thus, the purpose of this study was to explore whether P2 receptor is involved in the differentiation of osteoclasts. Mouse bone marrow haematopoietic stem cells (BMHSCs) were co‐cultured with TNF‐α to explore the effect of TNF‐α on osteoclast differentiation and bone resorption capacity in vitro, and changes in the P2 receptor were detected at the same time. The P2 receptor was silenced and overexpressed to explore the effect on differentiation of BMHSCs into osteoclasts. In an in vivo experiment, the animal model of PMOP was established in ovariectomized mice, and anti‐TNF‐α intervention was used to detect the ability of BMHCs to differentiate into osteoclasts as well as the expression of the P2 receptor. It was confirmed in vitro that TNF‐α at a concentration of 20 ng/mL up‐regulated the P2X7 receptor of BMHSCs through the PI3k/Akt signalling pathway, promoted BMHSCs to differentiate into a large number of osteoclasts and enhanced bone resorption. In vivo experiments showed that more P2X7 receptor positive osteoclasts were produced in postmenopausal osteoporotic mice. Anti‐TNF‐α could significantly delay the progression of PMOP by inhibiting the production of osteoclasts. Overall, our results revealed a novel function of the P2X7 receptor and suggested that suppressing the P2X7 receptor may be an effective strategy to delay bone formation in oestrogen deficiency‐induced osteoporosis.     

Osteocytic cells exposed to titanium particles increase sclerostin expression and inhibit osteoblastic cell differentiation mostly via direct cell‐to‐cell contact

The mechanism underlying induction of periprosthetic osteolysis by wear particles remains unclear. In this study, cultured MLO‐Y4 osteocytic cells were exposed to different concentrations of titanium (Ti) particles. The results showed that Ti particles increased expression of the osteocytic marker SOST/sclerostin in a dose‐dependent manner, accelerated apoptosis of MLO‐Y4 cells, increased the expression of IL‐6, TNF‐α and connexin 43. SOST silence alleviated the increase of MLO‐Y4 cells apoptosis, decreased the expression of IL‐6, TNF‐α and connexin 43 caused by Ti particles. The different co‐culture systems of MLO‐Y4 cells with MC3T3‐E1 osteoblastic cells were further used to observe the effects of osteocytic cells'' changes induced by Ti particles on osteoblastic cells. MLO‐Y4 cells treated with Ti particles inhibited dramatically differentiation of MC3T3‐E1 cells mostly through direct cell‐to‐cell contact. SOST silence attenuated the inhibition effects of Ti‐induced MLO‐Y4 on MC3T3‐E1 osteoblastic differentiation, which ALP level and mineralization of MC3T3‐E1 cells increased and the expression of ALP, OCN and Runx2 increased compared to the Ti‐treated group. Taken together, Ti particles had negative effects on MLO‐Y4 cells and the impact of Ti particles on osteocytic cells was extensive, which may further inhibit osteoblastic differentiation mostly through intercellular contact directly. SOST/sclerostin plays an important role in the process of mutual cell interaction. These findings may help to understand the effect of osteocytes in wear particle‐induced osteolysis.     

NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic β‐cells for protection

Sustained hyperglycaemia and hyperlipidaemia incur endoplasmic reticulum stress (ER stress) and reactive oxygen species (ROS) overproduction in pancreatic β‐cells. ER stress or ROS causes c‐Jun N‐terminal kinase (JNK) activation, and the activated JNK triggers apoptosis in different cells. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an inducible multi‐stress response factor. The aim of this study was to explore the role of NR4A1 in counteracting JNK activation induced by ER stress or ROS and the related mechanism. qPCR, Western blotting, dual‐luciferase reporter and ChIP assays were applied to detect gene expression or regulation by NR4A1. Immunofluorescence was used to detect a specific protein expression in β‐cells. Our data showed that NR4A1 reduced the phosphorylated JNK (p‐JNK) in MIN6 cells encountering ER stress or ROS and reduced MKK4 protein in a proteasome‐dependent manner. We found that NR4A1 increased the expression of cbl‐b (an E3 ligase); knocking down cbl‐b expression increased MKK4 and p‐JNK levels under ER stress or ROS conditions. We elucidated that NR4A1 enhanced the transactivation of cbl‐b promoter by physical association. We further confirmed that cbl‐b expression in β‐cells was reduced in NR4A1‐knockout mice compared with WT mice. NR4A1 down‐regulates JNK activation by ER stress or ROS in β‐cells via enhancing cbl‐b expression.