Ginkgo biloba leaf extract that has been identified as a natural platelet-activating factor receptor (PAFR) antagonist. We investigated the effect of ginkgolide B on high glucose-induced TLR4 activation in human umbilical vein endothelial cells (HUVECs).  Ginkgolide B is a  Protein expression was analyzed by immunoblotting. Small-interfering RNA (siRNA) was used to knock down PAFR and TLR4 expression.  Ginkgolide B suppressed the expression of TLR4 and MyD88 that was induced by high glucose. Ginkgolide B also reduced the levels of platelet endothelial cell adhesion molecule-1, interleukin-6, and monocyte chemotactic protein 1. Further, we examined the association between PAFR and TLR4 by coimmunoprecipitation. The result showed that high glucose treatment caused the binding of PAFR and TLR4, whereas ginkgolide B abolished this binding. The functional analysis indicated that PAFR siRNA treatment reduced TLR4 expression, and TLR4 siRNA treatment decreased PAFR expression in high glucose-treated HUVECs, further supporting the coimmunoprecipitation data. Ginkgolide B inhibited the phosphorylation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) and p38 mitogen-activated protein kinase (MAPK).  Ginkgolide B exerted protective effects by inhibiting the TLR4-mediated inflammatory response in high glucose-treated endothelial cells. The mechanism of action of ginkgolide B might be associated with inhibition of the JAK2/STAT3 and p38 MAPK phosphorylation. Diabetes is the most important risk factor for cardiovascular disease, which is the leading cause of morbidity and mortality in patients with diabetes. Macrovascular and microvascular damage is the main pathological characteristic of diabetic complications. However, the molecular mechanism of vascular inflammatory injury in diabetes remains unclear. Growing evidence demonstrates that toll-like receptors (TLRs) of the innate immune system are involved in the pathological process of diabetes –3. Toll-β-arrestin-dependent extracellular signal-regulated kinase (ERK) signaling pathway [Platelet-activating factor (PAF) is a potent phospholipid inflammatory mediator. By binding PAF receptor (PAFR), PAF elicits inflammatory responses in various cell types. A recent study reported that the activity of PAF-acetylhydrolase  increased in hypercholesterolemic minipigs . Platele pathway . Agrawal pathway . Moreove pathway . Our pre pathway . These r705 and dimerizes and translocates to the nucleus to regulate gene expression [705 to induce dimerization and increase STAT3 DNA-binding activity [727 is mediated by various serine kinases, such as MAPKs, cyclin-dependent kinases, and protein kinase C, and this modification increases the transcriptional activity of STAT3 by facilitating protein-protein interactions with transcriptional coactivators [Signal transducer and activator of transcription (STAT) proteins are a family of latent transcription factors that are activated by cytokines and growth factors. Mammals possess seven STAT proteins; of these, STAT3 is a multifunctional member that is involved in the acute-phase response, development, cell growth and differentiation, and immunity. STAT3 is phosphorylated by Janus family kinase (JAK) at Tyrpression . The actactivity . The photivators . A recentivators . HoweverGinkgo biloba leaf extract that has been identified as a natural PAFR antagonist [Ginkgolide B is a tagonist . Our pretagonist , 25. HowAccording to the Declaration of Helsinki, the umbilical cords were donated by cesarean section patients, from whom we received written informed consent. The study was approved by the Ethics Committee of the Beijing Institute of Geriatrics (number 201420).β-actin antibodies were purchased from Santa Cruz Biotechnology . Polyclonal anti-p38 MAPK, polyclonal anti-phosphorylated p38 MAPK, monoclonal anti-ERK, and monoclonal anti-phosphorylated ERK antibodies were purchased from Cell Signaling Technologies . Dylight 594 goat anti-mouse antibodies were purchased from Zhongshan Jinqiao Biotechnology .Ginkgolide B (95% purity) was purchased from Daguanyuan Company . Monoclonal anti-TLR4 antibody was purchased from Abcam . Polyclonal anti-PAFR antibody was purchased from Cayman Chemical . Monoclonal polyclonal anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) antibodies, polyclonal anti-phosphorylated STAT3 antibodies, anti-STAT3 antibodies, and monoclonal anti-μg/ml streptomycin, and 20 ng/ml endothelial growth factor  in an incubator at 37°C and 5% CO2. HUVECs at passage 3 were used in the present study.Primary HUVECs were collected from human umbilical veins by digestion in 0.1% collagenase I in M199 medium  for 15 min. The solution was collected and centrifuged at 1000 rotations per minute (rpm) for 10 min. The cells were cultured in M199 medium that contained 10% fetal bovine serum , 2 mM glutamine, 100 U/ml penicillin, 100 μM phenylmethylsulfonyl fluoride (PMSF), and 100 μg/ml leupeptin) and then centrifuged at 15000 ×g at 4°C for 5 min. Protein samples were boiled in sodium dodecyl sulfate (SDS) loading buffer for 5 min, run on SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane. Primary antibody incubations were performed overnight at 4°C. Horseradish peroxidase-conjugated secondary antibody was applied for 1 h at room temperature and developed using Super Signal developing reagent . Blot densitometry was then performed, and the bands were analyzed using a Gene Genius Bio Imaging System.HUVECs were incubated with various concentrations of ginkgolide B  for 1 h and then exposed to 30 mM glucose for 8 h. Cell lysis was performed in lysis buffer , 5 mM ethylene glycol tetraacetic acid (EGTA), 1 β-actin. Nonimmune isotype immunoglobulin G was used for immunoprecipitation as a negative control.For immunoprecipitation, the cells were incubated with or without ginkgolide B for 1 h, and then high glucose (30 mM) was added for 8 h. HUVEC lysates were incubated with antibodies against PAFR or TLR4 overnight at 4°C, followed by incubation with protein A/G Sepharose beads for 2 h at 4°C. The samples were centrifuged at 800 ×g for 5 min to collect protein A/G beads, which were washed four times and boiled in SDS buffer. Eluates from the beads were separated by 8% SDS-PAGE and immunoblotted for PAFR, TLR4, and TLR4 mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). Cells were treated with various doses of ginkgolide B for 1 h, and high glucose was added for another 8 h. Total RNAs were isolated for HUVEC culturing using Trizol reagent . RT-PCR was performed using SYBR Premix Ex Taq mix . The primers were the following: forward (5′-CCGCTTCCTGGTCTTATCAT) and reverse (5′-TCTGCTGCAACTCATTTCAT). After amplification, portions of the PCR mixtures were electrophoresed on a 2% agarose gel and visualized by a Bio-Rad Gel Doc 2000 device .For the detection of interleukin-6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) in the supernatant, cells were plated in 24-well plates. The cells were then treated with and without ginkgolide B for 1 h, and then high glucose (30 mM) was added for 8 h. After centrifugation at 1000 rpm for 10 min, the supernatant was collected to measure IL-6 and MCP-1. The levels of secreted IL-6 and MCP-1 were determined by an enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer's instructions.HUVECs were cultured on six-well culture plates. The cells were pretreated with ginkgolide B for 1 h and then with high glucose (30 mM) for 8 h. The cells were fixed with 4% paraformaldehyde for 15 min and washed twice. The cells were then incubated with antibody against TLR4 or STAT3 for 1.5 h. After washing three times, Dylight-conjugated antibody was added to the cells for 1.5 h at room temperature. The nucleus was stained by DAPI for 5 min at room temperature, and staining was observed under a fluorescence microscope.HUVECs were cotransfected with Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) that contained PAFR-selective small-interfering RNA (siRNA) or TLR4 siRNA for 48 h. The siRNA PAFR sequence was 5′-GGCCAUUAAUGAUGCACAU(dTdT)-3′ . PAFR sit-test. All of the calculations were performed using SPSS 18.0 software . Values of p < 0.05 were considered statistically significant.Quantitative data are presented as mean ± SEM. Significant differences between two groups were analyzed by two-tail unpaired Student's TLR4 plays an important role in diabetes and diabetic complications. To determine the effects of ginkgolide B on the TLR4-mediated inflammatory response in high glucose-treated HUVECs, we investigated the effects of ginkgolide B on the expression of TLR4 and MyD88, which is a downstream molecule of the TLR4 signaling pathway. The cells were incubated with various doses of ginkgolide B  for 1 h and then exposed to 30 mM glucose for 8 h. Semiquantitative RT-PCR was performed to determine TLR4 mRNA expression. As shown in Figures To evaluate the effects of ginkgolide B on inflammatory protein expression in high glucose-treated HUVECs, PECAM-1 expression was detected by Western blot. As shown in Our previous studies found that high glucose treatment increased PAFR expression in HUVECs. We hypothesized that PAFR might be associated with TLR4 under condition of high glucose stimulation. We performed coimmunoprecipitation (Co-IP) experiments using anti-TLR4 antibody and anti-PAFR antibody. The results showed that anti-TLR4 antibody immunoprecipitated TLR4 and PAFR , and antTo further determine the interaction between PAFR and TLR4 in high glucose-treated HUVECs, we conducted a functional analysis using siRNA to knock down the PAFR and TLR4 genes. HUVECs were transfected with siRNA against PAFR and a negative control siRNA for 48 h, and a high glucose concentration (30 mM) was added to the cells for another 8 h. As shown in κB activation and the inflammatory response [705. As shown in Figures μM) attenuated STAT3 phosphorylation that was induced by high glucose, and 1 μM CLI-095 completely inhibited STAT3 phosphorylation. To determine the inhibitory effect of ginkgolide B on STAT3 phosphorylation is associated with TLR4 activation, we then investigated whether combined CLI-095 and ginkgolide B synergistically inhibits STAT3 phosphorylation. As shown in μM CLI-095 alone only partially suppressed STAT3 phosphorylation, whereas the combination of both treatments completely attenuated STAT3 phosphorylation.TLR4 signaling is linked to nuclear transcription factor-response , 28. HowμM) dose dependently inhibited STAT3 phosphorylation that was induced by LPS. Additionally, we also evaluated whether combined stattic and ginkgolide B synergistically inhibits STAT3 phosphorylation. As shown in μM) or ginkgolide B (0.4 mg/ml) alone could not completely suppress STAT3 phosphorylation; however, the combination of both completely attenuated STAT3 phosphorylation induced by LPS.To further confirm the suppressive effect of ginkgolide B on JAK2/STAT3 phosphorylation is associated with TLR4 signal, we used the TLR4-specific agonist LPS. As shown in Figures μM) could inhibit p38 MAPK phosphorylation in high glucose-treated HUVECs (data not shown). As shown in Figures μM) dose dependently decreased TLR4 and PECAM-1 expression in glucose-treated HUVECs and 1 μM SB203580 completely inhibited their expression.The MAPK family has garnered significant attention because of its vast implications in signaling and crosstalk with other signaling networks. To explore the underlying mechanisms of action of ginkgolide B on TLR4 activation, we investigated the effect of ginkgolide B on p38 MAPK phosphorylation that was induced by high glucose. As shown in α) in peripheral blood mononuclear cells [Immune dysfunction plays a key role in chronic inflammation, including diabetes and atherosclerosis , 30. Earar cells . In the −/− animals. Both TLR4 and PAFR may influence lung inflammation that is induced by lipoteichoic acid  either by sensing LTA directly or through the recognition and signaling of endogenous mediators that are induced by LTA [Ginkgolide B is a PAF receptor antagonist. We evaluated whether the action of ginkgolide B is associated with PAFR blockade in high glucose-stimulated HUVECs. Immunoprecipitation revealed that high glucose stimulation increased TLR4 and PAFR expression and enhanced the binding between PAFR and TLR4. Ginkgolide B attenuated these effects. This suggests an interaction between PAFR and TLR4 in high glucose-stimulated HUVECs. We also performed a functional analysis using siRNA. PARF knockdown with PAFR siRNA reduced TLR4 expression, and knockdown with TLR4 siRNA decreased PAFR expression in nonhigh glucose-treated cells and high glucose-treated cells. Our findings demonstrated an interaction between TLR4 and PAFR activation in high glucose-treated HUVECs. The inhibition of high glucose-induced TLR4 activation by ginkgolide B appears to be associated with PAFR suppression. These results are consistent with a previous study that showed that the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype in PAFRd by LTA .Growing evidence indicates that the JAK2/STAT3 pathway plays an important role in tumors and inflammation. Some studies have shown that STAT3 is involved in the IL-6-mediated inflammatory response . We founGinkgolide B suppressed the expression of the TLR4-mediated inflammatory response and inhibited the binding of PAFR and TLR4 in high glucose-treated HUVECs. The mechanism of action of ginkgolide B appears to be associated with blockade of the JAK2/STAT3 pathway and p38MAPK phosphorylation. Our study provided evidence that PAFR inhibition might be considered as a therapeutic strategy for diabetic vascular inflammation.


However, the detailed mechanisms of TNF-α induced cPLA2 expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA2 mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE2 secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE2 ELISA kit. These TNF-α-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS , NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47phox, TRAF2, NIK, IKKα, IKKβ, or p65. TNF-α markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47phox. In addition, TNF-α also stimulated p47phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47phox complex formation in HPAEpiCs, which were attenuated by a TNF-α neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated IKKα/β and NF-κB p65 phosphorylation, NF-κB (p65) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-α-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKα/β pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA2 by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway.Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A Moreover, our previous findings also provided insights into the correlation between COX-2 and cPLA2 expression in ATPγS-stimulated vascular smooth muscle cells (VSMCs) with similar molecular mechanisms and functional coupling to amplify the occurrence of vascular inflammation . Therefore, up-regulation of cPLA2 may play a key role in local and systemic inflammation in airway diseases. However, the molecular mechanisms by which TNF-α induces cPLA2 expression and PGE2 synthesis in HPAEpiCs are not completely understood.The occurrence and exacerbation of lung diseases, including chronic obstructive pulmonary disease (COPD) and asthma, is dependent on the severity of lung inflammation . Eicosanthmatics . Phosphose of AA . The conof AA  are products of normal cellular metabolism acting as second messengers . However2 gene activity in various cell types , anti-p47phox (sc-14015), anti-Gαs (sc-823), anti-TRAF2 (sc-7346), anti-ASK1 (sc-5294), anti-TNFR1 (sc-52739), anti-NIK (sc7211), anti-IKKα (sc7218), anti-IKKβ (sc8014), anti-p65 (sc-7151) and anti-phospho-serine (sc-81515) antibodies were from Santa Cruz . An anti-GAPDH antibody (#MCA-1D4) was from Encor . Human TNF-α neutralizing antibody (#7321), anti-phospho-tyrosine (#9411), anti-phospho-ASK1 (#3765), anti-phospho-p65 (#3031), and anti-phospho-IKKα/β (#2697) antibodies were from Cell Signaling . N-Acetyl cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (APO), and Bay11-7082 were from Biomol . Dihydroethidium (DHE) and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) were from Molecular Probes . SDS-PAGE supplies were from MDBio Inc . All other reagents were from Sigma .Recombinant human TNF-α was from R&D System . Anti-cPLAHuman pulmonary alveolar epithelial cells (HPAEpiCs) were ordered from ScienCell Research Lab . The passages 4–7 were used throughout this study. HPAEpiCs were cultured in DMEM/F12 medium containing 10% FBS, as previously described g at 4°C for 1 h to prepare the whole cell lysate, as previously described  antibody for 24 h, and then incubated with an anti-mouse horseradish peroxidase antibody (1:2000) for 1 h. ECL reagents and UVP BioSpectrum 500 Imaging System  were used to detect and capture the immunoreactive bands. The image densitometry of each immunoreactive band was analyzed and quantified by the UN-SCAN-IT gel software .Serum-starved HPAEpiCs were incubated with TNF-α at 37°C for the various time points. At the end of treatment, the cells were harvested and centrifuged at 45000 × escribed . The den2 and GAPDH genes were used, as previously described  and primers specific for cPLAescribed . The rea2O2 levels were determined by measuring fluorescence of 2′,7′-dichloro fluorescein diacetate (DCF-DA) and the The intracellular H6 cells/ml) were cultured in 6-well plates and then incubated with TNF-α after growth to confluence and serum-starved. At the end of incubation, cells were harvested and centrifuged at 400 × g for 10 min at 4°C. The cell pellet was kept on ice after re-suspended by 35 μl/per well of ice-cold RPMI-1640 medium. The complex including NADPH (1 μM) or lucigenin (20 μM), 5 μl of cell suspension (0.2 × 105 cells) and a final 200 μl volume of pre-warmed (37°C) RPMI-1640 medium were prepared and the chemiluminescence was immediate recorded with an Appliskan luminometer (Thermo®) in out-of-coincidence mode. Appropriate blanks and controls were used. There was no background chemiluminescence of lucigenin in NADPH or NADH alone group (30–40 counts per min), as previously described HPAEpiCs  as cPLA2-luc plasmid. The activity of cPLA2-luc was detected using a luciferase assay system , as previously described Human cPLA2 were detected by a PGE2 enzyme immunoassay kit (Cayman) according to the manufacturer’s instructions, as previously described The serum-starved cells were treated with TNF-α for the different time points. At the end of treatment, culture media were collected and stored at -80°C. The concentrations of PGEphox, TRAF2, NIK, IKKα, IKKβ, and p65 siRNA together with scramble siRNA were purchased from Sigma . LipofectamineTM RNAiMAX reagents were used to prepare siRNA liposome complexes (100 nM of siRNAs) according to the manufacturer’s instructions, as previously described All human ASK1, p47phox, anti-TNFR1, anti-TRAF2, or anti-ASK1 antibody at 4°C for 24 h, and then 10 μl of 50% protein A-agarose beads was added and mixed at 4°C for 24 h. The immunoprecipitates were collected and washed thrice with a lysis buffer without Triton X-100. 5x Laemmli buffer was added and subjected to electrophoresis on SDS-PAGE, and then blotted using an anti-phospho-tyrosine, anti-phospho-serine, anti-p47phox, anti-TRAF2, anti-TNFR1, or anti-ASK1 antibody, as previously described  and the supernatant (cytosolic fraction).The cell lysates were sonicated for 5 s at output 1.5 using a sonicator  and then centrifuged at 6800 × t-test or by one-way analysis of variance (ANOVA) followed with Tukey’s post hoc test at p < 0.05 level of significance.Data were showed as the mean or mean ± SEM of five individual experiments and estimated using GraphPad Prism Program . All the data were analyzed by paired two-tailed Student’s 2 expression. Here we reported that TNF-α-induced cPLA2 protein levels were significantly reduced by pretreatment with a ROS scavenger (NAC) or the inhibitors of NADPH oxidase (APO and DPI) . In addition, pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated cPLA2 mRNA expression and promoter activity . The p47phox protein, one of cytosolic subunits of NADPH oxidase, contributed to acute NADPH oxidase activation via being phosphorylated and binding to p22phox . To confirm this cPLA2 expression is mediated through TNF-α-dependent induction, HPAEpiCs were pretreated with a human TNF-α neutralizing antibody followed by TNF-α treatment. We found that TNF-α neutralizing antibody significantly blocked the cPLA2 induction in a dose-dependent manner . These results indicated that TNF-α induces cPLA2 expression via NADPH oxidase and ROS in HPAEpiCs.Reactive oxygen species play both deleterious and beneficial roles. Accumulation of ROS and cellular oxidative stress trigger expression of inflammatory genes and result in tissue damages and various diseases . Thus, w p22phox . Thus, w2 expression is due to activation of NADPH oxidase and ROS generation. Here, we found that TNF-α markedly induced superoxide and hydrogen peroxide production, determined by using DHE or DCF under a fluorescence microscope . On the other hand, we also observed that TNF-α time-dependently induces NADPH oxidase activation , which was reduced by TNFR1 neutralizing antibody, APO, or DPI . These results suggested that TNF-α induced ROS generation via NADPH oxidase activation. The p47phox is phosphorylated on several serine residues within the polybasic region of the protein, and these multiple phosphorylation events induce conformational changes that permit p47phox to interact with the cytoplasmic tail of p22phox and to initiate the formation of the active NADPH oxidase complex . Finally, we investigated whether TNF-α-induced superoxide and hydrogen peroxide production are mediated through NADPH oxidase activation. Here, we observed that TNF-α-induced superoxide and hydrogen peroxide production were inhibited by TNFR1 neutralizing antibody, APO, DPI and NAC . These results suggested that TNF-α induces ROS generation via p47phox translocation and NADPH oxidase activation in HPAEpiCs.TNF-α may stimulate ROS production by several sources, such as mitochondria, but recent studies have strongly suggested that a major source of ROS is a phagocyte-type NADPH oxidase. Several reports also demonstrate that TNF-α triggers several signal transduction pathways to activate the NOX activity and enhances intracellular ROS generation leading to expression of inflammatory genes . Therefo complex . Previoual cells . Thus, w2 expression. Here, we demonstrated that TNF-α induced cPLA2 expression via TRAF2 by transfection with TRAF2 siRNA . We found that transfection with TRAF2 siRNA reduced TRAF2 protein expression by about 50% and significantly attenuated the TNF-α-induced cPLA2 expression from 5.4-fold to 2.2-fold. In addition, we also showed that TNF-α stimulated TRAF2 and TNFR1 complex formation and its downstream components . Therefore, we further studied whether TNF-α promoted the association of TNFR1, TRAF2, ASK1, and p47phox in HPAEpiCs. Data in Figure 3B showed that TNF-α time-dependently induced TNFR1, TRAF2, ASK1, and p47phox complex formation. Importantly, this interaction was blocked by pretreatment with TNF-α neutralizing antibody.Both tissue injury-related proinflammatory and programmed cell death pathways are activated by TNF-α via binding to TNFR1 . TRAF2 p2 expression from 4.9-fold to 1.9-fold . ASK1 plays a pivotal role in ROS generation. In resting cells, endogenous ASK1 constitutively associated with thioredoxin (Trx) forming an inactive high-molecular-mass complex, known as ASK1 signalosome. Once cellular ROS increased, Trx dissociated from ASK1 signalosome and resulted in full activation of higher-molecular-mass complex by the recruitment of TRAF2 and TRAF6 . Consistently, this interaction was also blocked by pretreatment with a TNF-α neutralizing antibody. To confirm whether ASK1 phosphorylation is necessary for TNF-α-induced cPLA2 expression, activation of the ASK1 was assayed by Western blot using an antibody specific for the phosphorylated form of ASK1. We found that TNF-α significantly stimulated ASK1 phosphorylation in a time-dependent manner . These results suggested that TNF-α stimulates TNFR1, TRAF2, ASK1, and p47phox complex formation leading to NADPH oxidase activation and ROS generation in HPAEpiCs.Various cell stresses, including ROS, TNF-α, lipopolysaccharide (LPS), and ER stress, activate ASK1 and resulting in apoptosis, differentiation and inflammation . Howevernd TRAF6 . Thus, wphox are involved in TNF-α-induced ROS generation. As shown in Figure 4A, transfection with TRAF2, ASK1, or p47phox siRNA markedly inhibited TNF-α-induced superoxide and hydrogen peroxide production. In addition, these siRNAs also inhibited the TNF-α-induced NADPH oxidase activation . Moreover, our previous findings also provided the correlation between COX-2 and cPLA2 expression in ATPγS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vascular inflammation . These results suggested that TNF-α induces ROS generation and PGE2 release via TRAF2, ASK1, and p47phox in HPAEpiCs.Next, we investigated whether TRAF2, ASK1 and p47ammation . Therefolization . In this2 expression via NIK, IKKα, and IKKβ by transfection with respective siRNAs. Transfection with NIK, IKKα, or IKKβ siRNAs knocked down their own protein levels and subsequently attenuated the TNF-α-induced cPLA2 expression . To ascertain whether NF-κB participated in TNF-α-induced cPLA2 expression, a selective pharmacological inhibitor of NF-κB, Bay11-7082, was used for this purpose. Pretreatment with Bay11-7082 significantly reduced the TNF-α-induced cPLA2 protein, mRNA, and promoter activity . Moreover, p65 siRNA was used to confirm the role of NF-κB in TNF-α-mediated effects. In addition, transfection with p65 siRNA also reduced TNF-α-induced cPLA2 expression  and PGE2 secretion  in these cells. The non-canonical NF-κB pathway is activated through particular TNF-α receptors that bind to the TRAF2 which leads to translocation of NF-κB into nucleus and induction of target gene expression  together with enhancing NF-κB promoter activity . These results suggested that TNF-α-induced cPLA2 expression is mediated through NIK, IKKα/β, and NF-κB in these cells.NF-κB is mainly involved in stress-induced immune and inflammatory responses. Activities of NF-κB are regulated by various inflammatory cytokines such as TNF-α . The sigpression . TherefoFigure 6A, TNF-α markedly stimulated NF-κB p65 phosphorylation which was inhibited by NAC, DPI, APO or Bay11-7082. In addition, pretreatment with NAC, DPI, or APO also inhibited TNF-α-induced NF-κB promoter activity and NF-κB p65 translocation in HPAEpiCs . On the other hand, we also observed that TNF-α significantly stimulated IKKα/β phosphorylation in a time-dependent manner, which was reduced by NAC, DPI or APO . Finally, we investigated whether TNF-α stimulates NF-κB p65 phosphorylation via an NIK/IKKα/β pathway. As shown in Figure 6E, transfection with NIK siRNA attenuated the TNF-α-induced IKKα/β and NF-κB p65 activation. In addition, transfection with siRNA of IKKα or IKKβ markedly inhibited TNF-α-induced NF-κB p65 phosphorylation . These results suggested that TNF-α stimulates NF-κB p65 activation through an NIK/IKKα/β-dependent cascade in HPAEpiCs.In this study, we investigated whether TNF-α stimulates NF-κB p65 activation via an NADPH oxidase/ROS-dependent pathway. As shown in 2 by mesenchymal cells in several extra-pulmonary sites contributes to the production of PGE2 which functions as biologically active lipid mediators in inflammatory responses , ROS (NAC), or NF-κB (Bay11-7082) or transfection with siRNA of ASK1, TRAF2, p47phox, NIK, IKKα, IKKβ, or p65 attenuated TNF-α-induced cPLA2 expression and PGE2 production in HPAEpiCs. Our studies confirmed that activation of NADPH oxidase, ROS, NIK, IKKα/β, and NF-κB via association of TNFR1, TRAF2, ASK1, and p47phox may be essential for cPLA2 expression in TNF-α-stimulated HPAEpiCs. cPLA2 induction and TNF-α-induced complex formation of TNFR1, TRAF2, ASK1, and p47phox were blocked by pretreatment with TNF-α neutralizing antibody. These results suggested that up-regulation of cPLA2 by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway.Various degrees of inflammation and tissue remodeling are characteristics of different pulmonary disorders including asthma and COPD. Expression of cPLAesponses . TNF-α hammation , but fewammation . HoweverSeveral reports indicate that TNF-α may regulate inflammatory protein expression via activating various downstream protein kinases . Effectsphox) and p22phox is anchored in plasma membrane which assembles with the regulatory subunits  distributed in cytoplasm and leading to ROS generation , the inhibitors of NADPH oxidase (DPI and APO) or transfection with p47phox siRNA significantly attenuated TNF-α-induced cPLA2 expression and PGE2 synthesis. DPI or APO pretreatment reduced TNF-α-stimulated ROS generation. These results revealed that TNF-α increased cPLA2/PGE2 expression via NADPH oxidase-dependent ROS generation. p47phox is phosphorylated on several serine residues within the polybasic region of the protein, and these multiple phosphorylation events induce conformational changes that permit p47phox to interact with the cytoplasmic tail of p22phox and to initiate the formation of the active oxidase /MKK7-JNK and MKK3/6-p38 pathways. ASK1 can be activated by various types of stresses, such as ROS, TNF-α, and ER stress, and exerts pivotal roles in regulating cell apoptosis, differentiation, and inflammation . Therefond TRAF6 . Indeed,tivation , in this2 expression. Various extracellular stimuli such as TNF-α and IL-1β, viruses and environmental particulates (PM10s), and oxidative stress regulate the activation of NF-κB  to regulate the levels of PGEs (2 synthesis induced by LPS (2 expression in ATPγS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vascular inflammation (2 treatment also induced cPLA2 expression in VSMCs, which exerted as a positive feedback regulator in this response (2 could be a good index of AA release that is more sensitive than [3H]AA mobilization (2 may further induce cPLA2 expression to amplify the occurrence of pulmonary inflammation. In this study, although the effect of PGE2 on COX-2 expression was not investigated, we tested the effect of TNF-α on PGE2 synthesis as a parameter of cPLA2 activity which may be an important issue for further study in HPAEpiCs.COX-2 and cPLA species . cPLA2 htive PGs . On the  of PGEs . In our d by LPS . Moreoveammation . PGE2 trresponse . Therefolization  and PGE2Figure 7 addresses a model for the molecular mechanisms of TNF-α-stimulated cPLA2 expression and PGE2 release in HPAEpiCs. To our knowledge, this is the first study to indicate that in HPAEpiCs, TNF-α-regulated activation of TNFR1/TRAF2/ASK1/p47phox-dependent NADPH oxidase is required for the expression of cPLA2. Finally, activation of the ROS/NIK/IKKα/β/NF-κB pathway leads to cPLA2 gene activation and expression. It is an important link for TNF-α-regulated cPLA2 expression in the pathogenesis of lung inflammatory diseases. Therefore, uncovering the signaling components in TNF-α-mediated cPLA2 expression in HPAEpiCs is important to develop new therapeutic strategies in pulmonary diseases.According to the literature reports and our results, C-CL, W-NL, R-LC, C-yW, L-DH, and C-MY substantially contributed to the conception or design of the work, the acquisition, analysis, and interpretation of data for the work. C-CL, W-NL, R-LC, C-yW, L-DH, and C-MY drafted the work and revised it critically for important intellectual content. C-CL, W-NL, R-LC, CyW, L-DH, and C-MY finally approved the version to be published. C-CL, W-NL, R-LC, C-yW, L-DH, and C-MY agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer HH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi—a process termed ‘cell shedding’. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes.Certain members of the microbiota genus  They Bifidobacterium, has been linked to inflammatory bowel disease (IBD) [There is a growing body of evidence suggesting that the microbiota influences intestinal epithelial cell (IEC) function, including gene expression, cell division and energy balance –11. Thesse (IBD) –15, undeThe epithelium of the small intestine represents the first line of defence against entry of bacteria into host tissues. Cell division in the crypt, under physiological conditions, is counterbalanced by cell shedding from the villi to maintain homeostasis and integrity of the crypt/villus axis. When the epithelial cell is shed, a discontinuity in the villus epithelial monolayer is created, which potentially compromises the epithelial barrier. In health, epithelial barrier function is maintained , owing tTNF-α is a key cytokine in IBD. We and others have shown that TNF-α induces apoptosis of villus tip epithelial cells causing excessive shedding, leading to breakdown of the epithelial barrier and microulceration ,20. DelaBifidobacterium, may play a role in protecting against the cell shedding response by modulating IEC function. To determine the contribution of bifidobacteria in cell shedding, we employed a well characterized in vivo mouse model in which pathological cell shedding is induced by intraperitoneal (IP) administration of lipopolysaccharide (LPS), driving mononuclear cell expression of TNF-α and subsequent caspase-3-positive shedding cells [B. breve UCC2003) positively modulates the small intestinal cell shedding response via host MyD88- and bacterial EPS-dependent interactions which serve to significantly reduce apoptotic signalling in the epithelial compartment. These data identify a previously unknown mechanism by which Bifidobacterium protects its host against pathological cell shedding. These findings may thus have important implications for the future design of therapeutic strategies in the context of intestinal diseases.Given reports of beneficial effects of certain members of the gut microbiota in IBD and potential roles of microbial dysbiosis in these diseases, we hypothesized that certain health-promoting microbiota members, including ng cells . Our res2.2.1.C57 BL/6 Jax mice (6–10 weeks) were obtained from Charles River. Vil-cre MyD88 transgenic mice (i.e. Cre recombinase expression causes truncation and resulting non-function of the MyD88 protein in IECs) were obtained from the Wellcome Trust Sanger Institute (kind gift from S. Clare).2.2.Bifidobacterium breve strains UCC2003, UCC2003del and UCC2003inv were used for animal inoculations. These strains and corresponding culturing conditions have been previously described in detail [l-cysteine before being reconstituted in sterile PBS at a final concentration of approximately 1 × 1010 bacteria ml−1. 0.1 ml of inoculum was then administered to mice by oral gavage in 3 × 24 h doses followed by plating of faecal pellets on RCA containing 50 mg l−1 mupirocin to confirm stable colonization. Control mice received oral gavage of PBS only.n detail . In brie2.3.B. breve or PBS control, mice received an IP injection of 1.25 mg kg−1 LPS from Escherichia coli 0111:B4 (Sigma) or sterile saline (control) and mice were sacrificed 1.5 h post-challenge with LPS. Proximal small intestine was collected in 10% neutral buffered formalin saline (Sigma) and fixed for 24 h followed by paraffin embedding. Samples of proximal small intestine were also collected into RNA Later (Qiagen) for transcriptome analysis or frozen on dry ice for subsequent ELISA analysis. In some cases, proximal small intestine was also collected into Hanks buffered saline solution (HBSS) for isolation of IECs.Twenty-four hours after the last doses of 2.4.Sections (5 µm) of paraffin-embedded small intestinal tissue were sectioned and used for immunohistochemistry. Following de-parafinization and rehydration, tissue sections were treated with 1% hydrogen peroxide in methanol to block endogenous peroxidases. Subsequently, slides were treated using heat-induced antigen retrieval in 0.01 M citrate acid buffer (pH 6) followed by incubation with a rabbit polyclonal anti-active caspase-3 (CC3) antibody (AF835: R&D Systems). Visualization of caspase-3 positivity was via a peroxidase-labelled anti-rabbit EnVision secondary antibody (Dako) and 3,3′-diaminobenzidine followed by counterstaining with haematoxylin. For macrophage staining, an antibody against F4/80 antigen (ab6640: Abcam) was employed using biotinylated anti-rat (BA-9401) and avidin–biotin reagent .2.5.core, WinCrypts [IECs were counted on a cell positional basis from villus tip (cell position (CP) 1) down towards the crypts under 400× magnification. Twenty well-orientated hemi-villi were counted per mouse and analysed using the SinCrypts  and PRIS2.6.Samples fixed in RNAlater solution were processed through RNeasy plus mini spin columns to isolate total RNA (Qiagen). In brief, samples were homogenized using a rotor stator hand held homogenizer in buffer RLT before processing through a QIAshredder column and subsequently RNeasy mini-spin columns. Purified RNA was eluted into RNAase free water. Reverse transcription was performed using the Quantitect reverse transcription kit (Qiagen) and cDNA used for real-time (RT-)PCR analysis. For RT-PCR, transcripts were amplified using Quantifast SYBR green mastermix (Qiagen) and Quantitect primer assays for TNF-α, TNF-R1 and F4/80 (EMR1). Expression of the housekeeping gene hypoxanthine–guanine phosphoribosyltransferase (HPRT) 5′-GACCAGTCAACAGGGGACAT-3′ (sense) and 5′-AGGTTTCTACCAGTTCCAGC-3′ (antisense)  was also2.7.2 pieces and washed in a solution containing 0.154 M NaCl and 1 mM DTT, and subsequently a solution containing 1.5 mM KCl, 96 mM NaCl, 27 mM tri-sodium citrate, 8 mM NaH2PO4 and 5.6 mM Na2HPO4, pH 7.3. IECs were then isolated by incubation in PBS containing 1.5 mM EDTA and 0.5 mM DTT, shaking at 200 r.p.m. and at 37°C. Purity of epithelial preparations was confirmed by histological analysis of stripped intestinal mucosa and by FACS analysis of isolated cells. For FACS analysis, 5 × 106 cells were stained with anti-mouse CD45-A700 (Biolegend) on ice for 30 min. After two washes in HBSS containing 0.01 BSA, 2 mM EDTA, 20 mM HEPES and 0.01% NaN3, propidium iodide was added (Biolegend) and samples analysed on a Sony FCS SH-800 flow cytometer. Data were analysed using FlowJo (TreeStar).IECs were isolated using a modification of the Weiser methodology . In brie2.8.Frozen proximal small intestinal samples were homogenized in extraction buffer containing protease inhibitors (Roche), cleared by centrifugation and analysed using a commercial ELISA kit for TNF-α (eBioscience) as per manufacturer's protocol. Measurement of TNF-α immunoreactivity was at 450 nm, using a Fluostar Optima plate reader (BMG Labtech).2.9.iji [Isolated IECs were lysed in CelLytic MT reagent (Sigma) before centrifugation at 10 000 rpm for 10 min to pellet cellular debris. Supernatants were mixed with 2 × Laemmli sample buffer before being separated by sodium dodecyl sulfate (SDS)–PAGE with 3–14% acrylamide gel and transferred to Hybond-P PVDF membrane  and blocking with 5% Marvel milk in with tris(hydroxymethyl)aminomethane (Tris). (Tris)-buffered saline containing Tween 20 (TTBS) immunostaining was performed with 1/1000 anti-TNF-R1 antibody (Abcam) and 1/5000 goat anti-Rabbit IgG HRP conjugate (Millipore) on a reduced gel. Macrophage expression was analysed similarly using antibody against F4/80 antigen (Abcam) at 1 : 1000 and goat anti-rat IgG-HRP , on a non-reduced gel. Washes were in TTBS. For detection, Immobilon Western chemiluminescent HRP substrate (Millipore) was applied to the membrane as recommended by the manufacturer and signal was detected, using a FluorChem E imaging system (Protein Simple). Band densities were quantified using Fiji .2.10.Real-rime ready Custom Panel 480–96+ PCR arrays were obtained (Roche) and quantitative PCR analysis performed. RNA was extracted from whole small intestinal tissue preserved in RNAlater reagent (Sigma), using RNeasy plus mini kits (Qiagen). Reverse transcription was performed, using Transcriptor First Strand cDNA Synthesis Kit followed by analysis of targets using LightCycler 480 Probes Master on a LightCycler 480 platform . Standard protocols as per manufacturer recommendations were followed. CT values of target genes were normalized to expression of the housekeeping gene HPRT and fold change versus control samples calculated using the delta/delta CT method .2.11.rism v. 5 software (GraphPad Software). A p-value of less than 0.05 was used as significant in all cases.Experimental results were plotted and analysed for statistical significance with P3.3.1.a). Recent studies have demonstrated that following IP injection of mice with LPS isolated from Escherichia coli 0111:B4, a potent cell shedding response is induced, similar to that observed in relapsing IBD patients [−1 LPS, not only at the villus tip, but also along the shoulders and sides of the villus (figure 1b). Effects of LPS on the cell shedding response were found to be dose-dependent, in agreement with previous observations [Caspase-3 is activated in IECs during their extrusion from the tips of small intestinal villi ,28. Simifigure 1patients . In agrervations   in 3 × 24 h doses orally to establish stable colonization [B. breve UCC2003 and induction of cell shedding with LPS, mice showed a marked reduction in the levels of CC3-positive shedding cells compared with LPS-treated control mice receiving PBS gavage . Cell count analysis confirmed significant reduction (p < 0.01) in cell shedding at the majority of positions along the length of the villus in B. breve UCC2003-treated mice (c). Thus, B. breve appears to modulate epithelial integrity/survival during periods of inflammatory insult.Various members of the microbiota are known to promote a healthy gut , althougnization . ColonizS gavage a,b. Cellted mice c. Thus, B. breve UCC2003 versus control treated mice (C57 BL/6), but overall, no notable differences (but expected increase in Actinobacteria in the B. breve UCC2003 group) in microbiota class abundance between the treatment groups . Bifidobacterial colonization takes place along the gastrointestinal (GI) tract including the small/large intestine and caecum. RNAscope analysis showed that B. breve UCC2003 was found in intimate contact with the IECs of the small intestine in colonized C57 BL/6 mice . Together, these data suggest that colonization with B. breve does not produce significant shifts in the overall gut microbiota community structure and that the observed protective effects after colonization are more likely to be related to direct effects of B. breve, possibly through interactions with the IECs.Previous studies have indicated that bifidobacteria may modulate the composition of other microbiota members, and within the context of IBD, studies have linked microbiota disturbances with active disease. Thus, to determine if bifidobacterial colonization impacts the gut microbiota, we analysed the community composition using a 16S rRNA-based sequencing approach. We found minor changes to the community structure in 3.3.B. breve have been described to possess immunomodulatory properties [B. breve, was caused by reduced expression of TNF-α from macrophages, we isolated RNA and protein from whole small intestine of control and B. breve UCC2003-treated C57 BL/6 mice following LPS-mediated induction of cell shedding. As shown in figure 3a, no significant difference (p > 0.05) in levels of TNF-α protein was observed between groups, and this was confirmed at the transcriptional level (data not shown). We also found no significant changes (p > 0.05) in expression of TNF-α in the plasma of B. breve UCC2003-treated versus control mice following LPS-induced cell shedding (figure 3b), nor any significant difference (p > 0.05) in the numbers/levels of F4/80+ macrophages infiltrating the small intestine (figure 3c–f). Together, these data suggest that modulation of the reduced cell shedding response is independent of TNF-α induction. Because the microbiota may be able to interact directly with IECs, we postulated that B. breve modulates a signalling pathway downstream of the TNF-α ligand. To test whether expression of TNF-R1 was altered in the epithelium following dosing with B. breve UCC2003, IECs were isolated from whole small intestinal tissue using a modified Weiser methodology [figure 3g,h). Subsequent quantitative RT-PCR and western blot analysis of isolated IEC populations showed no significant changes (p > 0.05) to expression of the TNF-R1 transcript or protein following exposure to B. breve UCC2003 (figure 3i–k), suggesting that there is no impairment of signalling at the level of the receptor.LPS-induced cell shedding is caused by the release of TNF-α from lamina propria tissue-resident macrophages, which binds to TNF-receptor 1 (TNF-R1), on IECs , therebyoperties . Thus, thodology , after w3.4.B. breve elicits its protective effects via epithelial TLR signalling pathways.IECs sample microbe-associated molecular patterns (MAMPS) of the intestinal luminal contents using a variety of receptors including members of the nucleotide-binding oligomerization domain (NOD) family, the C-type lectin receptor (CLR) family and the Toll-like receptor (TLR) superfamily. MyD88 is a critical adaptor protein in signalling downstream of the majority of the TLR family members . We thus−/− villin-cre mice (i.e. IEC MyD88 KO mice) colonized with B. breve UCC2003, showed similar rates (p > 0.05) of LPS-induced cell shedding to PBS gavaged IEC MyD88−/− mice. In comparison, control mice (i.e. C57 BL/6 MyD88+/+ villin-cre) showed the expected protection (p < 0.01) against cell shedding in the presence of B. breve UCC2003 (a–d). Furthermore, RT-PCR analysis of IEC homogenates showed increased expression (p < 0.001) of TLR2 in B. breve UCC2003-colonized mice when compared with control mice . Taken together, these data indicate that functional MyD88 signalling, potentially via TLR2 is required for modulating the protective effect of B. breve against cell shedding outcomes.C57 BL/6 MyD88 UCC2003 a–d. Furt3.5.eps gene clusters represent a relatively conserved feature of bifidobacterial genomes, including those of the species B. breve [B. breve UCC2003-EPSdel) that expresses neither EPS1 nor EPS2 [B. breve EPS-positive or EPS-negative strains followed by challenge with LPS . Strikingly, when colonized with the B. breve UCC2003-EPSdel, no significant protection (p > 0.05) against cell shedding was observed in control (i.e. PBS) versus colonized mice .Recently, a number of functions modulated by bifidobacteria have been shown to be mediated through surface-associated EPS including resistance to gut infection . InteresB. breve . In ordenor EPS2 . Mice wezed mice a,b.FiguBifidobacterium breve UCC2003 controls EPS biosynthesis via a bidirectional gene cluster which results in expression of either EPS1 (B. breve UCC2003) or EPS2 (B. breve UCC2003-EPSInv) [B. breve UCC2003-EPSInv. Colonization with EPS2 expressing B. breve (i.e. B. breve UCC2003-EPSInv) also failed to show any significant protection (p > 0.05) against LPS-induced cell shedding, suggesting considerable variation in the protective response dependent upon EPS genetic and chemical structure and organization . All strains are directly compared in electronic supplementary material, figure S4.-EPSInv) . Thus, tnization c,d. All in vivo for optimal translation to human patients.Together, these studies emphasize the striking strain variant specificity that is observed with regard to the individual protective effects of these bacteria following LPS-induced cell shedding. This is probably regulated by the specific molecules produced by each strain, including the EPS. This highlights the critical need to fully genetically characterize ‘probiotic’ strains of bacteria to enable a detailed dissection of their functional effects 3.6.B. breve UCC2003 and the influence of EPS, whole small intestinal samples from control (i.e. PBS) and colonized (EPS-positive B. breve UCC2003 and EPS-negative B. breve UCC2003-del) mice following challenge with LPS were analysed using a custom RT-PCR array  showed significant increases (more than twofold and p < 0.01) in IL-6 and Tnfrs15 when compared with control and LPS-challenged mice. Moreover, numerous other apoptotic and inflammatory genes were significantly upregulated  including Bad, Cycs , casp4, Fas, Traf5 and Tnfrs9. In contrast, in EPS-positive-colonized mice (i.e. B. breve UCC2003), our analysis showed only subtle changes to the expression of the majority of the targets when compared with PBS-treated control mice challenged with LPS . In addition, while significant elevation (more than twofold and p < 0.05) in IL-6 and Tnfrs15 was observed following colonization with B. breve UCC2003, Tnfrs15 expression was markedly decreased versus B. breve UCC2003-EPSdel-colonized mice (threefold versus 16-fold increase). These data suggest that signalling via EPS may downregulate inflammatory and apoptotic networks, which would otherwise lead to elevated cell shedding.In order to gain further insights into the changes taking place in the small intestine following colonization with 4.B. breve significantly reduces pathological/apoptotic epithelial cell shedding, through a previously unknown mechanism involving bifidobacterial EPS-MyD88 signalling.We report that colonization of mice with Clostridium, Bacteroides, Faecalibacterium and (of particular interest here) Bifidobacterium [a priori administration of B. breve UCC2003  conferred a significant level of protection, which manifested as significantly reduced caspase-3 positivity within the villus epithelium (a–c). Previous studies have highlighted that bifidobacterial supplementation may also modulate the wider microbiota in mouse models [B. breve-colonized mice (as indicated by taxa abundance), there are no notable differences , suggesting limited effects on overall microbiota profiles . These data therefore suggests a more direct link between bifidobacteria and maintenance of epithelial integrity in the prevention of intestinal inflammation.The gut microbiota appears central to maintaining epithelial barrier integrity and, importantly, disturbances in the microbiota appear pivotal in IBD pathogenesis. Indeed, IBD patients (paediatric and adult cohorts) have been shown to possess a reduced overall microbiota diversity and reductions in specific genera including acterium ,15,35. Pacterium . Followiithelium a–c. Preve models . HoweverBifidobacterium predominantly colonizes the colon of infants and adults, as determined from faecal or mucosal scrapings, or biopsy samples [Bifidobacterium colonization in the lower SI . Notably, select studies using ileostomy effluents and illeum biopsies have indicated bifidobacteria  are present in this area of the infant and adult GI tract [Previous studies have indicated that  samples ,39; howeGI tract ,41. TherB. breve UCC2003-treated or control (i.e. PBS) mice (a–f), suggesting that the protection conferred by Bifidobacterium strains is TNF-α independent. Previous studies have indicated that colonization of B. breve UCC2003 during homeostatic conditions does not induce differences in splenic TNF-α-positive macrophage numbers when compared with non-colonized controls [i–k), it appears that macrophages, TNF-α production and TNF-R1 signalling are not involved in modulating this protective response and suggests that B. breve UCC2003 acts preferentially from the luminal side through interactions with IECs. We cannot exclude the potential for EPS to block signalling via TNF-R1. However, TNF-R1 expression appears to be restricted to the basolateral surface of epithelial cells and thus B. breve would not be expected to have direct access to this cellular compartment for direct inhibition via binding [B. breve UCC2003 and B. breve UCC2003-del-colonized mice, which suggests EPS does not play a key role via TNF-R1.As previously mentioned, studies have shown that this experimental model of LPS-induced cell shedding is driven by an induction in expression of TNF-α from the intestinal mucosa ,42. One BS) mice a–f, suggcontrols . Coupled binding . Further−/− villin-cre) showed no protection against cell shedding after colonization of B. breve UCC2003 ; this was in stark contrast to MyD88-positive control animals, which again showed significant protection against LPS-induced cell shedding . Furthermore, we observed significant increases in IEC TLR2 expression in B. breve UCC2003-colonized mice (e). Interestingly, previous work has indicated that TLR2 may enhance ZO-1 associated intestinal epithelial barrier integrity [B. bifidum has been shown to reduce apoptosis in vitro (necrotizing enterocolitis IEC-6 cell model), as also indicated by reduced CC3-positive cells [B. breve UCC2003 may regulate epithelial integrity in response to LPS-induced cell shedding (as marked by caspase-3) via these central MyD88 signalling mechanisms, potentially downstream of TLR2.To delineate these protective luminal bifidobacterial–epithelial interactions, we used epithelial-specific MyD88 KO mice; MyD88 is a key adaptor protein downstream of microbe-TLR signalling. Notably, mice carrying truncated epithelial MyD88  to modulate LPS-induced cell shedding. Notably, mice receiving B. breve UCC2003-EPSdel showed no significant protection against cell shedding when compared with EPS-positive (i.e. B. breve UCC2003) colonized mice , suggesting an important role for this EPS in microbe–host cross-talk. Importantly, EPS structures can be recognized via TLR2 , and previous work with the polysaccharide A (PSA) capsule of Bacteroides fragilis highlights that PSA can modulate dendritic cell and T regulatory cell function via TLR2 signalling [B. breve (Yakult strain) can also induce IL-10 producing T regulatory cells via TLR2; however they did not determine if this was via an EPS-specific mechanisms [Bacillus subtilis have demonstrated that the EPS capsule of this bacterium is able to protect against intestinal inflammation in a murine model of colitis (in this instance via TLR4), providing further support for the likely role of bifidobacterial EPS in the effects observed in these studies [Lactobacillus also produces distinct EPSs, which are structurally similar to those observed in bifidobacteria [. Recently, within an in vitro system , the EPS from Lactobacillus acidophilus 5e2 was shown to increase IL-8 expression and also TLR2 expression , and additionally upregulation of TLR2 was found to potentially ‘sensitize’ epithelial cells to subsequent stimulation with peptidoglycan (a TLR2 agonist) [Lactobacillus or indeed B. breve UCC2003 potentially translocate across the epithelial barrier, it could by hypothesized they directly influence macrophage function. Previous studies have shown that L. casei Shirota can dampen down inflammatory macrophage responses, and L. rhamnosus EPS has also been shown to modulate macrophage function in vitro, but on this occasion induced proinflammatory responses [B. breve strains in TLR2 and/or TLR4 KO animals; unfortunately, previous work has shown that these mice do not respond to LPS and thus would not have a cell shedding response, making these further studies not possible. However, in studies using RNAscope, we found significant numbers of B. breve UCC2003 associated with the villi in colonized mice , suggesting that direct signalling interactions between the bacteria  and IECs may play an important role in modulating this response. These data, alongside our findings, suggest that B. breve EPS may regulate cell shedding by acting as TLR ligands via MyD88, leading to protective epithelial responses.Having determined the importance of host adaptor MyD88, we next sought to determine if there was a specific bifidobacterial molecule central to the observed protective response. Because we have previously shown that surface EPS of response , we invezed mice a,b, sugggnalling ,47. Addichanisms . Further studies . Notablybacteria . Recentlagonist) . Furtheragonist) . From a esponses ,53. Ideaeps gene cluster in B. breve UCC2003, which can express two genetically and importantly chemically distinct surfaces EPSs [B. breve UCC2003), but we also determined responses following EPS2 (i.e. B. breve UCC2003-EPSInv) colonization. Strikingly, and contrary to our expectations, we found that this isogenic strain was unable to confer protection against LPS-induced cell shedding . Importantly, EPSs are composed of repeating mono- or oligosaccharides linked by various glycosidic linkages, and the three-dimensional structures and other physiochemical features of EPSs can vary widely [B. breve EPSs  are important for function [in vitro studies on L. reuteri strains (DSM 17938 and L26 Biocenol) indicate both EPSs are high-molecular-weight d-glucan polysaccharides with differing spatial conformations, which may relate to induction of different cytokine responses. However, the direct chemical structures involved in this modulation have yet to be defined [To probe these EPS–epithelial interactions further, we took advantage of the bidirectional ces EPSs . All preshedding c,d. Impoy widely . The varposition ) could, function . Additio defined . Future  defined ,57.B. breve UCC2003 does not induce inflammatory host responses after colonization, which we hypothesize is to the advantage of the bacterium and host for maintaining efficient symbiosis and homeostasis [B. breve UCC2003) appeared to attenuate apoptosis-induced signalling activation, in stark contrast to mice colonized with the B. breve UCC2003-EPSdel strain, which had significantly elevated apoptotic gene expression (B. breve UCC2003-del compared with B. breve UCC2003-colonized mice. Bad is a pro-apoptotic (BH3-only) member of the bcl-2 family that antagonizes the anti-apoptosis proteins bcl-2, bcl-xl and bcl-2, allowing activation of bax/bak oligomers and release of cytochrome c from the mitochondria. Within the same pathway, Cycs encodes the haem protein cytochrome c, which forms a multiprotein complex called the apoptosome, which activates a cascade of caspases which cause apoptotic cell death [B. breve UCC2003, via EPS, may block intrinsic and extrinsic apoptosis signalling (via activation of MyD88) during inflammation to protect epithelial cells under highly apoptotic conditions.We have previously shown that EPS-positive eostasis . Interesll death . Traf5 ill death . We havell death . TNFRF9 ll death . These dB. breve UCC2003) are able to protect against pathologic cell shedding induced by IP injection of LPS, and that this protection appears to be independent of TNF-α production by resident tissue macrophages. Using wild-type and mutant B. breve, we have demonstrated that a specific EPS is able to confer this protection and, using knockout mice, have shown that this protection appears contingent on functional (MyD88) signalling downstream of the epithelial TLR family members and modulation of pro-apoptotic gene pathways. Understanding how health-promoting species of bacteria such as the Bifidobacterium genus interact with the intestinal epithelium and how these species confer their protective effects may drive progress towards understanding how pathologic cell shedding in IBD patients is linked to changes in the intestinal microbiota and how intervention strategies could positively impact disease progression. Future human studies could be considered to address issues of microbial dysbiosis, the relationship to the cell shedding response, to what extent microbial dysbiosis is linked to periods of remission and relapse in such patients, and how bifidobacterial supplementation could be used to reduce relapse in IBD patients.In summary, we have demonstrated that certain bifidobacteria (i.e.


Salvia miltiorrhiza is a traditional Chinese medicinal herb used for treating cardiovascular diseases. Depside salt from S. miltiorrhiza (DSSM) contains the following active components: magnesium lithospermate B, lithospermic acid, and rosmarinic acid. This study aimed to reveal the mechanisms of action of DSSM. After searching for DSSM-associated genes in GeneCards, Search Tool for Interacting Chemicals, SuperTarget, PubChem, and Comparative Toxicogenomics Database, they were subjected to enrichment analysis using Multifaceted Analysis Tool for Human Transcriptome. A protein-protein interaction (PPI) network was visualised; module analysis was conducted using the Cytoscape software. Finally, a transcriptional regulatory network was constructed using the TRRUST database and Cytoscape. Seventy-three DSSM-associated genes were identified. JUN, TNF, NFKB1, and FOS were hub nodes in the PPI network. Modules 1 and 2 were identified from the PPI network, with pathway enrichment analysis, showing that the presence of NFKB1 and BCL2 in module 1 was indicative of a particular association with the NF-κB signalling pathway. JUN, TNF, NFKB1, FOS, and BCL2 exhibited notable interactions among themselves in the PPI network. Several regulatory relationships  were also found in the regulatory network. Thus, DSSM exerts effects against cardiovascular diseases by targeting JUN, TNF, NFKB1, FOS, and BCL2. Salvia miltiorrhiza  is widely used as a traditional Chinese medicinal herb to prevent and treat vascular diseases  were associated with all MLB, RA, and LA; four genes  were associated with both MLB and RA; and four genes  were associated with both MLB and LA. Jun protooncogene (JUN), TNF, nuclear factor kappa B subunit 1 (NFKB1), and Fos protooncogene (FOS) were noted as particular hub nodes according to their BC, closeness centrality, and DC scores (κB) signalling pathway  and leishmaniasis (p = 4.72E−07) were particularly associated with the nodes in modules 1 and 2, respectively   and 108 relationship pairs (such asCL2/TNF) . JUN → TNF/FOS, FOS → NFKB1, and NFKB1 → BCL2/TNF) were found to be involved in the transcriptional regulatory network.In this study, a total of 73 DSSM-associated genes were identified from various databases. In the PPI network constructed on the basis of these genes and their interactions, JUN, TNF, NFKB1, and FOS were established as hub nodes according to their BC, closeness centrality, and DC scores. JUN, TNF, NFKB1, FOS, and BCL2 were also shown to interact among themselves in the PPI network. Moreover, two distinct modules (modules 1 and 2) of the PPI network were identified. In addition, several regulatory relationships  across endothelial cells, thus contributing to the reservoir of LDL in vascular walls [ TNF may be associated with the functions of DSSM in cardiovascular diseases.Prolonged anti-TNF-soriasis . Studiesrthritis , 29. TNF setting . TNF-α bar walls . These f (DN-c-Jun) gene transfer inhibits VSMC proliferation, and JUN is associated with the intimal hyperplasia induced by balloon injury [ FOS is a novel factor that determines the severity and development of atherosclerosis and is thus involved in tobacco toxicity in coronary artery disease (CAD) patients [ miR-146a-targeting FOS is a potential tool for treating enhanced inflammation-associated cardiac disorders [ JUN and FOS may also be targets of DSSM in cardiovascular diseases.Blocking the JNK pathway may provide a new strategy for treating the cardiomyocyte death induced by myocardial ischaemia/reperfusion , 33. Domn injury . Saliquepatients . Palomerisorders . Thus, J NFKB1 polymorphism (rs28362491) may be a genetic marker of CAD [κB, which is in turn correlated with the reduction of plasma inflammatory markers [ BCL2 expression, miR-21 promotes heart failure progression with preserved left ventricular ejection fraction and subsequently inhibits cardiac fibrosis [ miR-1 targets BCL2 functions in mediating cardiomyocyte apoptosis [ NFKB1 and BCL2 in module 1 indicated a particular association with the NF-κB signalling pathway, indicating that NFKB1 and BCL2 may be correlated with the effects of DSSM against cardiovascular diseases through this pathway.Studies involving Uygur and Han women in China have demonstrated that the DD genotype ofr of CAD , 38. The markers . By incrfibrosis . Moreovepoptosis . In this JUN, TNF, NFKB1, FOS, and BCL2 were also revealed as potential targets of DSSM for treating cardiovascular diseases. However, further experimental research should be performed to confirm these findings obtained by bioinformatic analysis.In conclusion, a total of 73 DSSM-associated genes were identified by conducting a search of public databases.


Despite new developments in cancer therapy, chemotherapy and radiotherapy remain the cornerstone of breast cancer treatment. Therefore, finding ways to reduce the toxicity and increase sensitivity is particularly important. Tumor necrosis factor alpha (TNF-α) exerts multiple functions in cell proliferation, differentiation and apoptosis. In the present study, we investigated whether TNF-α could enhance the effect of chemotherapy and radiotherapy against breast cancer cells.Cell growth was determined by MTT assay in vitro, and by using nude mouse tumor xenograft model in vivo. Cell cycle and apoptosis/necrosis were evaluated by flow cytometry. DNA damage was visualized by phospho-Histone H2A.X staining. mRNA expression was assessed by using real-time PCR. Protein expression was tested by Western blot assay.137-irradiation and strengthened G2/M and S phase cell cycle arrest induced by docetaxel and cisplatin respectively. Moreover, the up-regulation of RIP3 (a necroptosis marker) by 5-FU, and the activation of RIP3 by TNF-α, synergistically triggered necroptosis (programmed necrosis). Knockdown of RIP3 attenuated the synergetic effect of TNF-α and 5-FU.TNF-α strengthened the cytotoxicity of docetaxel, 5-FU and cisplatin against breast cancer cells both in vitro and in vivo. TNF-α activated NF-κB pathway and dependently up-regulated expressions of CyclinD1, CyclinD2, CyclinE, CDK2, CDK4 and CDK6, the key regulators participating in G1→S phase transition. As a result, TNF-α drove cells out of quiescent G0/G1 phase, entering vulnerable proliferating phases. Treatment of TNF-α brought more DNA damage after CsTNF-α presented radiotherapy- and chemotherapy-sensitizing effects against breast cancer cells. Breast cancer is by far the most frequent cancer among women worldwide and accounts for 30% of all new cancer cases in females with a greater incidence in women over the age of 60 years –5. AlthoTumor necrosis factor alpha (TNF-α) is a classical member of a ligand family of cytokines that include TNF, lymphotoxin-α (LTα), Fas ligand (FasL), CD40 ligand (CD40L), and TNF-related apoptosis-inducing ligand (TRAIL) . TNF-α iIn our present study, we investigate whether TNF-α could enhance the cytotoxicity of chemotherapeutics and radiotherapy against breast cancer cells. The mechanisms involved in the synergistic anticancer effect were also investigated.2. Cells were passaged every 2–3 days to obtain exponential growth.Human breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from American Type Culture Collection . Cells were maintained in RPMI-1640 medium , supplemented with 10% fetal calf serum (Gibco), 100 units/ml penicillin, and 100 mg/ml streptomycin. The cultures were incubated at 37 °C in a humidified atmosphere with 5% COTNF-α was purchased from Sigma . Docetaxel was purchased from Jiang Su Heng Rui Medicine CO., LTD. . 5-Flurouracil (5-FU) was purchased from Shanghai Xudong Haipu Pharmaceutical CO., LTD. . Cisplatin was purchased from Nanjing Pharmaceutical Factory CO., LTD. . Bay11-7082 was purchased from Sigma .4 cells per well. After treatment, MTT (Sigma) was added to each well to a final concentration of 0.5 mg/ml, followed by incubation at 37 °C for 4 h. The medium was then removed, and 800 μl of dimethyl sulfoxide (DMSO) was added per well. The absorbance in each well was measured at 490 nm using a microplate ELISA reader . The relative cell viability was calculated as follows: relative cell viability =  ×100%. The concentration that caused 50% growth inhibition (IC50) was calculated by the modified Kärbers method [50 = lg−1 [Xk − i (∑p − 0.5)], in which Xk represents the logarithm of the highest drug concentration; i is that of ratio of adjacent concentration; and ΣP is the sum of the percentage of growth inhibition at various concentrations.Cellular growth was evaluated by MTT (methyl thiazolyl tetrazolium) assay . Cells ws method  accordin7 cells). TNF-α   and NF-κB-dependent firefly luciferase gene  was purcApoptosis was evaluated using the Annexin V-FITC/PI Apoptosis Detection kit  according to the manufacturer’s instructions . The celTotal protein was extracted using a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, protease inhibitors , and phosphatase inhibitors . The protein extracts were separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and were transferred to polyvinylidene fluoride (PVDF) membranes . After 1-h blocking in 5% non-fat milk, the membranes were incubated overnight with primary antibodies, rabbit anti-RIP3  or mouse anti-β-actin , at 4 °C. Protein expression was determined using horseradish peroxidase—conjugated antibodies followed by enhanced chemiluminescence) (ECL) detection (Millipore). Protein band analysis was performed using Quantity One 4.6.2 software . β-actin was used as internal control.Target specific small interfering RNAs (siRNAs) were synthesized by Genepharma . The specific sequences were as follows: (1) Control-siRNA, 5′-UUCUCCGAACGUGUCACGUdTdT-3′; (2) RIP3-siRNA-1, 5′-UAACUUGACGCACGACAUCAGGCUGUU-3′; (3) RIP3-siRNA-2, 5′-GCAGUUGUAUAUGUUAACGAGCGGUCG-3′ . The trat test. A P value <0.05 was considered significant.Each experiment was performed a minimum of three times. Results were expressed as the mean value ± standard deviation (SD). Statistical analysis was performed by unpaired Student’s 50 values, 48 and 72 h post treatment with each chemotherapeutic, were presented in Table The growth inhibition effects of chemotherapeutics, docetaxel, 5-FU and cisplatin, against breast cancer cells were firstly evaluated by using MTT assays. As shown in Fig. 50 values of chemotherapeutics, chemotherapy sensitization effect of TNF-α was evaluated in vitro. Co-treatment with TNF-α strengthened the growth inhibition induced by docetaxel, 5-FU and cisplatin remarkably , a marker of DNA double-strand breaks (DSBs) –22, in bCancer cells in G0/G1 cell cycle have been proved to be quiescent and resistant to chemotherapy and radiotherapy. Since TNF-α sensitized chemotherapy and radiotherapy, we then performed cell cycle analysis to explore the effect of TNF-α on cell cycle distribution. As shown in Fig. Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E, CDK2, CDK4 and CDK6 are the key regulators participating in G1→S phase transition, and NF-κB is the main pathway at the downstream of TNF-α stimulation. Therefore, we then investigate whether TNF-α could regulate expressions of G1→S phase transition regulators through NF-κB pathway dependent manner. Activation of NF-κB pathway was firstly confirmed by luciferase report gene assays  . TherefoTo verify whether the chemotherapy sensitization effect of TNF-α on 5-FU was executed through a RIP3-dependent manner, we applied siRNAs targeting RIP3  . FurtherActually, TNF-α is a pleiotropic cytokine that plays a vital role in a wide variety of physiological processes . The ple5-FU can suppress thymidylate synthase and block DNA synthesis, causing a restraint of the transition from G1 phase to S phase . AlthougIn conclusion, we presently proved that, TNF-α present radiotherapy- and chemotherapy-sensitizing effect against breast cancer cells. TNF-α drove cells out of quiescent G0/G1 phase, entering vulnerable proliferating phases. As a result, TNF-α brought more DNA damage after irradiation and strengthened G2/M and S phase cell cycle arrest induced by docetaxel and cisplatin respectively. Moreover, the up-regulation of RIP3 by 5-FU, and the activation of RIP3 by TNF-α, synergistically triggered necroptosis. Taking together, our present investigation brought new strategies for breast cancer treatment through sensitized chemotherapy and radiotherapy by TNF-α.TNF-α presented radiotherapy- and chemotherapy-sensitizing effects against breast cancer cells.


Overexpression of cyclin D1 dependent kinases 4 and 6 (CDK4/6) is a common feature of many human cancers including leukemia. LEE011 is a novel inhibitor of both CDK4 and 6. To date, the molecular function of LEE011 in leukemia remains unclear.INK4a expression analysis. Gene expression profiles of LEE011 treated HL-60 cells were investigated using an Arraystar Human LncRNA array. Gene ontology and KEGG pathway analysis were then used to analyze the differentially expressed genes from the cluster analysis.Leukemia cell growth and apoptosis following LEE011 treatment was assessed through CCK-8 and annexin V/propidium iodide staining assays. Cell senescence was assessed by β-galactosidase staining and p161 arrest in seven of eight acute leukemia cells lines, the exception being THP-1 cells. β-Galactosidase staining analysis and p16INK4a expression analysis showed that LEE011 treatment can induce cell senescence of leukemia cells. LncRNA microarray analysis showed 2083 differentially expressed mRNAs and 3224 differentially expressed lncRNAs in LEE011-treated HL-60 cells compared with controls. Molecular function analysis showed that LEE011 induced senescence in leukemia cells partially through downregulation of the transcriptional expression of MYBL2.Our studies demonstrated that LEE011 inhibited proliferation of leukemia cells and could induce apoptosis. Hoechst 33,342 staining analysis showed DNA fragmentation and distortion of nuclear structures following LEE011 treatment. Cell cycle analysis showed LEE011 significantly induced cell cycle G1 arrest and cellular senescence in leukemia cells. LncRNA microarray analysis showed differentially expressed mRNAs and lncRNAs in LEE011-treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may open new lines of investigation regarding the molecular mechanism of LEE011 induced cellular senescence.We demonstrate for the first time that LEE011 treatment results in inhibition of cell proliferation and induction of GThe online version of this article (doi:10.1186/s12935-017-0405-y) contains supplementary material, which is available to authorized users. Acute leukemia is the most common pediatric malignancy constituting more than 30% of all childhood cancers . Approxi1 phase progression, with their activity strictly restricted to the G1-S phase [The proteins encoded by CDK4 and 6 are members of the Ser/Thr protein kinase family . Both CD-S phase –5. Mutat-S phase . It is n-S phase –10. CDK4-S phase , 12.1 arrest and growth inhibition in xenograft models of human tumor cell lines including breast, ovary, lung and multiple myeloma. Another CDK4/6 inhibitor, LY2835219, has been reported to inhibit CDK4 and 6 at very low concentrations, resulting in proliferation inhibition and G1 cell cycle arrest [Several pharmacological inhibitors of CDK4/6 have been developed and many are currently being tested in clinical trials. One CDK4/6 selective inhibitor, PD-0332991, causes Ge arrest . GCS-100e arrest . KBH-A42e arrest . Resultse arrest .LEE011 is a recently developed CDK4/6 inhibitor . LEE011 Leukemia cell lines HL-60, MV4-11, U937 and K562 were obtained from the American Type Culture Collection (ATCC). CCRF, 697 and SHI-1 cell lines (gifts from The Cyrus Tang Hematology center of Soochow University). NB4 and THP-1 cell lines (gifts from Hematology Institute of Soochow University). All cell lines were maintained at 37 °C in the RPMI 1640  supplemented with 10% fetal bovine serum . LEE011  was dissolved in DMSO .Bone marrow specimens were obtained at the time of diagnosis from 5 pediatric AML and 5 ALL patients between 2014 and 2015. Ethical approval was provided by the Children’s Hospital of Soochow University Ethics Committee (No. SUEC2013-022), and written informed consent was obtained from the parents or guardians. They will be given the opportunity to withdraw from the research at any time prior to the publication of the research findings. The matter of how data will be collected and stored, with reference to the data protection legislation will be clarified for participants, with information being stored in locked cabinets or on IT hardware protected with the highest security software. The main clinical and laboratory features of the patient cohort are summarized in Tables Cell proliferation analysis was almost same as introduced before . Leukemi4 cells) and were analyzed with a Beckman Gallios™ Flow Cytometer. Then these data was analyzed with cell cycle software (MultiCycle for Windows).Cell cycle analysis was also introduced before . LeukemiApoptosis assay was according to the manual operation of BD Annexin V Staining Kit . All the details have been introduced before .Cells were seeded into 6-well plates, and then treated with LEE011 (2 or 5 μM) and cultured at 37 °C for 24 h, stained with 0.1 µg/ml Hoechst 33,342  for 5 min, then observed with filters for blue fluorescence under fluorescence microscopy . Abnormal nuclear cells were counted between the RO3280 treatment group and DMSO control group .Leukemia cells were seeded into 6-well plates, and then treated with LEE011 (2 or 5 μM) and cultured at 37 °C for 24–72 h, senescence β-galactosidase staining analysis was according to the manufacture of senescence β-galactosidase staining kit . Staining cells were photographed with microscopy . Positive staining cells were counted between the LEE011 treatment group and DMSO control group.HL-60 cells were treated with 1 μM LEE011 and control group cells were treated with the same volume of DMSO 24 h later. Human LncRNA array analysis was performed by KangChen Bio-tech, Shanghai P. R. China. And experimental details were introduced by Yu et al. . RNA purhttp://www.geneontology.org). The lower the P value is, the more significant the GO term (a P ≤ 0.05 is recommended). Pathway analysis is a functional analysis that maps genes to Kyoto encyclopedia of genes and genomes (KEGG) pathways (http://www.genome.jp/kegg/) was introduced before [P value  denotes the significance of the pathway correlated to the conditions. The lower the P value is, the more significant the correlation .Gene ontology (GO) analysis introduced before  is a fund before . The P vINK4a  Company Ltd. Pudong, Shanghai, China), p21 Waf1/Cip1 ,GAPDH .For western blot analysis, protocol is introduced before . Blots wQuantitative real-time PCR was performed to determine the expression levels of dyes-regulated genes in 1 μM LEE011-treated HL-60 cells. Real-time PCR analysis was introduced before . cDNA syCR1L were F: 5′-GTCCTCCTTCTCCGATCAATGC-3′ and R: 5′-CTTAGCACTTGTCCAGACTGAG-3′; TCP11L2 were F: 5′-CTAAATGCTGACCCTCCTGAGT-3′ and R: 5′- GCCACCGGGAGTGAGAAAA-3′; CR1 were F: 5′-AGAGGGACGAGCTTCGACC-3′ and R: 5′-TCAGGACGGCATTCGTACTTT-3′; AMICA1 were F: 5′-GTTTCCCCGCCTGAGCTAAC-3′ and R: 5′-TTCTGGAAGCGCCCAATAGG-3′; MCM10 were F: 5′-AAGCCTTCTCTGGTCTGCG-3′ and R: 5′-CTGTGGCGTAACCTTCTTCAA-3′; CDK1 were F: 5′-AAACTACAGGTCAAGTGGTAGCC-3′ and R: 5′-TCCTGCATAAGCACATCCTGA-3′; DLGAP5 were F: 5′-AAGTGGGTCGTTATAGACCTGA-3′ and R: 5′-TGCTCGAACATCACTCTCGTTAT-3′; KIF20A were F: 5′-TGCTGTCCGATGACGATGTC-3′ and R: 5′-AGGTTCTTGCGTACCACAGAC-3′; S100A8 were F: 5′-CATGCCGTCTACAGGGATGA-3′ and R: 5′- GACGTCTGCACCCTTTTTCC-3′; IL8 were F: 5′-GAATGGGTTTGCTAGAATGTGATA-3′ and R: 5′-CAGACTAGGGTTGCCAGATTTAAC-3′; PLK1 were F: 5′- CTCAACACGCCTCATCCTC-3′ and R: 5′-GTGCTCGCTCATGTAATTGC-3′; MYBL2 were F: 5′-TGCCAGGGAGGACAGACAAT-3′ and R: 5′-CTGTACCGATGGGCTCCTGTT-3′; PADI4 were F: 5′-AGTGGCTTGCTTTCTTCTCCTGTG-3′ and R: 5′-AGCAGAACTGAGTGTGCAGTGCTA-3′. Expression of genes was normalized to endogenous GAPDH expression.6*Log2 (−ΔCt). Gene expression between the DMSO and the LEE011 samples were analyzed using Multi Experiment View (MEV) cluster software.Cluster analysis of the data was performed with gene cluster from the real-time PCR arrays. For gene expression quantification, we used the comparative Ct method. First, gene expression levels for each sample were normalized to the expression level of the housekeeping gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) within a given sample (−ΔCt). The relative expression of each gene was calculated using the equation: 10http://www.genechem.com.cn). RNAi products target-specific lentivirus designed to knockdown MYBL2 expression; sequences are 1# 5-CAGATCAGAAGTACTCCAT-3; for KIF20A, sequences are 1# 5- CAGAAGAATATAAGGCTGT-3; for PLK1, sequences are 1# 5-CAACCAAAGUCGAAUAUGA-3. The control sequence is 5-TTCTCCGAACGTGTCACGT-3. Lentivirus infection was according to the manufacture of Shanghai Genechem Co., Ltd. at a final concentration of 100–200 MOI (multiplicity of infection). Interference efficiency was measured by western blot at 3 days after transfection. The rest cells were harvested for further analysis.RNAi lentivirus was purchased from Shanghai Genechem Co., Ltd. .Western blot analysis showed that expression of CDK6 was very high in seven of nine of the leukemia cell lines investigated, the exceptions being THP-1 and U937 cells Fig. . Our resWe investigated apoptosis in leukemia cells following LEE011 treatment. Cells treated with LEE011 at 2 and 5 µM for 48 h showed more apoptotic features when compared to controls in seven leukemia cell lines, the exception being THP-1 Fig. c.Fig. 2AHoechst 33,342 staining analysis showed that DNA fragmentation and an increase in cells with nuclear abnormalities were observed after 24-h LEE011 treatment  related lncRNAs were upregulated, including BDNF-AS  and BDNF-AS1 (uc009yis.3). BDNF plays an important role in the aging process . BDNF he−13), immune system process (P = 1.71894E−13), defense response (P = 3.32706E−13) and response to stimulus (P = 2.09266E−12). For the downregulated transcripts , cell cycle (P = 1.62994E−88), cell cycle process (P = 7.37652E−84) and mitotic cell cycle process (P = 1.36566E−81). We further investigated the pathways in which these differentially expressed genes are involved through KEGG database analysis. The five most enriched pathways from KEGG analysis are shown in Fig. Gene ontology pathway enrichment analysis was performed for the differentially expressed genes identified through microarray analysis. Fisher’s exact test was used to determine whether the differential expression was greater than that expected by chance. For the upregulated transcripts Fig. a, the moss P = 1.1894E−13,ss P = 1.1894E−13,To identify the cellular senescence molecules implicated in the mechanism of LEE011, the expressions of certain dysregulated genes identified in the gene array were confirmed using real-time PCR and western blot analyses. Cluster analysis of the real-time PCR results showed dysregulated genes in HL-60 cells treated with 1 µM LEE011 for 24–72 h Fig. a. These The molecular function of MYBL2, PLK1 and KIF20A was also analyzed in HL-60 cells. RNA interference of MYBL2 significantly downregulated the expression of MYBL2. Cell proliferation was also inhibited when the expression of MYBL2 was downregulated by RNA interference Fig. b. Figure1 phase cells in the Si-MYBL2 group increased significantly , ribociclib (LEE011) and abemaciclib (LY2835219), have been clinically approved or are in late-stage clinical trials 91, riboc, 31. Mos1 phase cells increased significantly and nucleus became larger and irregular in the Si-MYBL2 group cells. These results imply that LEE011 induces senescence in AML cells partially through downregulation of the transcriptional expression of MYBL2. Therefore, our study may provide new clues into the mechanism of senescence induced by LEE011 in AML cells.MYBL2 is emerging as an important gene in cellular senescence. When cells are senescing, MYBL2 has been shown to consistently be the most downregulated gene . As repo1 arrest and cellular senescence in leukemia cells. The lncRNA microarray was used to identify mRNA and lncRNA expression profiles in LEE011 treated HL-60 cells and we demonstrated that LEE011 induces cellular senescence partially through downregulation of the expression of MYBL2. These results may provide new insights into the molecular mechanism of the anticancer effects of LEE011 and its potential as a candidate drug for leukemia; however, further research will be required to determine the underlying details.In this study, we have shown that LEE011 treatment resulted in inhibition of cell proliferation and induction of G


Thus, TNF-α has been defined as a therapeutic target for RA. Although some TNF-α antagonists including neutralizing monoclonal antibodies and soluble receptors have been approved to be successful in attenuating symptoms in patients suffering from RA, the long-term use of these passive immunization reagents could cause some problems like a variable degree of immunogenicity. In the present study, in order to wake up active immune responses of RA patients, we developed a recombinant TNF-α therapeutic vaccine (named mrTNF-PADRE) by coupling a 12-amino acid universal Pan HLA-DR Epitope (PADRE) to the protein. Codon optimization was performed to improve the secondary structure of mrTNF-PADRE mRNA to ensure its heterologous expression. As a result, a single codon synonymous mutation greatly elevated recombinant protein expression  in E. coli as compared with the undetectable expression of the unoptimized gene. Although expressed as insoluble inclusion bodies (IBs), the vaccine can be effectively prepared with a purity of over 95% by IBs washing and one-step gel-infiltration chromatography. By this strategy, a stable yield of 5.2 mg purified mrTNF-PADRE per gram of cell paste could be obtained.As a proinflammatory cytokine, tumor necrosis factor-alpha (TNF- However, aberrant TNF-α overexpression can induce chronic inflammation and subsequent tissue destruction and thus play important roles in various diseases including cachexia, Crohn's disease (CD), and rheumatoid arthritis (RA) [It is well known that TNF-eostasis , 2. By btis (RA) , 4.α, some anti-TNF-α monoclonal antibodies  and soluble TNF-α receptor have been successfully used to attenuate pain and symptoms in patients suffering from CD and RA [α greatly limited their practical applications [α drugs provide a predisposition of patients with RA and CD to lymphoma, although a causal relationship between them still cannot be established [By neutralizing the endogenous TNF-D and RA –7. Howevications . Many RAications , 10. In ablished –13.α spectrum.The strategy to develop protein autovaccines by coupling an exogenous T helper cell epitope has emerged recently and attracted much attention . This acα vaccine by fusion expressed TNF-α with the T helper cell epitope PADRE. The designated mrTNF-PADRE vaccine was expressed in Escherichia coli (E. coli) at high level after mRNA secondary structure optimization by a single codon synonymous mutation. The recombinant vaccine can be conveniently purified by IBs washing and one-step gel filtration chromatography. The final yields were about 5.2 mg purified mrTNF-PADRE per gram cell paste.Here, we constructed a recombinant TNF- E. coli strain BL21 (DE3) were purchased from Novagen . Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Sigma-Aldrich . Sephacryl S-300 was from GE Healthcare Life Sciences . Mouse anti-human TNF-α monoclonal antibody and HRP-conjugated rabbit anti-mouse IgG were from Abcam . Standard recombinant hTNF-α was purchased from National Institutes for Food and Drug Control . The fermentor was from B. Braun (Germany). All other reagents were of analytical grade.Restriction enzymes, T4 DNA ligase, and Phusion High-Fidelity DNA Polymerase were purchased from New England Biolabs . Oligonucleotides were synthesized by Beijing AuGCT Biological Technology Co., Ltd . The vector pET-22b andα autovaccine, the antigenic determinants of TNF-α were firstly predicted by Antigenic software based on the method of Kolaskar and Tongaonkar [ (recombinant TNF-PADRE) was constructed according to our previous strategy [α gene and replaced the TNF-α127–141 fragment. In order to splice the recombinant gene conveniently, two synonymous mutations were introduced into wild-type TNF-α gene and the sequence CAGCTG that encodes amino acid residues 125 and 126 (Gln-Leu) was mutated to CAATTG, the restriction enzyme Mun I recognition sequence. Then, the DNA coding for PADRE (AKFVAAWTLKAA) and the C-terminus fragment of TNF-α (TNF-α142–157) were obtained by oligonucleotide synthesis according to the sequences PTF and PTR listed in α1–126 was amplified from TNF-α gene with the primers P1 and P2  was confirmed by DNA sequencing.For codon optimization, the rTNF-PADRE gene was obtained from pET-22b-rTNF-PADRE plasmid with the primers P3 and P4 . Nde I a E. coli, pET-22b-rTNF-PADRE and pET-22b-mrTNF-PADRE vectors were transformed into BL21 (DE3) competent cells, respectively, and positive clones were determined by DNA sequencing. A single colony was then inoculated into 5 mL LB medium containing 100 μg/ml of ampicillin and cultured with 200 rpm shaking at 37°C overnight. The bacteria cultures (3 ml) were then transferred to 300 ml fresh LB medium and grown to an OD600 of 0.5. For protein expression, 1 mM IPTG  was added and samples (1 ml) were collected 4 h later and analyzed by SDS-PAGE. For protein purification, bacteria were cultured in large scale with a 5 L fermentor as previously described [To express the recombinant vaccine TNF-PADRE inescribed . The culμm; Invitrogen, USA) and Western blot analyses were carried out using an hTNF-α monoclonal antibody, followed by an HRP-labeled IgG. The target proteins were finally visualized with an enhanced chemiluminescence .Western blot was performed as previously described . In brieβ-mercaptoethanol, 50 mM Tris-HCl pH 8.5) at room temperature for 30 min. The final pellet of IBs was solubilized by homogenizing in 20 mM β-mercaptoethanol and 7 M guanidine HCl. The supernatant was then loaded onto a chromatography column (2.4 × 100 cm) packed with 500 ml of depyrogenated Sephacryl S-300 resin and equilibrated with 50 mM Tris-HCl, pH 8.5, 8 M urea, and 150 mM NaCl. To purify mrTNF-PADRE, the column was eluted with the equilibration buffer at a flow rate of 1.0 ml/min . The purified mrTNF-PADRE fraction was then refolded by dialyzing against the refolding buffers  β-mercaptoethanol, 1/105 (v/v) 0.5 M CuSO4) at 20°C for 8 h and then against PBS at 20°C for 8 h twice. After removing the aggregated particles by centrifugation, the final products were lyophilized and stored at −70°C.mrTNF-PADRE was purified as previously described but optimized . In brieμg) in PBS was injected onto a 7.5 mm × 300 mm G2000SW column (TOSOH) at a flow rate of 0.5 ml/min. Peaks were detected by monitoring at a wavelength of 275 nm. The purity of mrTNF-PADRE was calculated as a percentage of the total peak area. N-terminus amino acid sequencing was finished by Shanghai Shenggong Biological Engineering Co., Ltd. .Size exclusion chromatography- (SEC-) HPLC analyses were performed on a Waters HPLC apparatus. The sample  subcutaneously (s.c.) without adjuvant, and saline was used as the negative control. Sera were collected 10 days after the last immunization and antibody titers were analyzed by enzyme-linked immunosorbent assay (ELISA).The recombinant vaccine was used to immunize mice with the protocol as previously described . In brie in vitro neutralization assay, we detected the ability of antisera to inhibit TNF-α-stimulated fibroblast-like synoviocytes (FLS) proliferation. FLS cells were separated from the synovial tissues of RA patients and cultured as previously described [α (100 ng/mL), either alone or in the presence of the antisera, and then cultured at 37°C with 5% CO2 in humid air. Normal mouse serum was used as a negative control. Cell proliferation was measured with a BrdU assay kit  according to the manufacturer's protocol. The absorbance was measured at 450 nm using a multiwell microplate reader.For theescribed . FLS celα vaccine, we firstly predicted the antigenic determinants of TNF-α by antigenic software based on the method of Kolaskar and Tongaonkar [α. Thus, the PADRE T helper epitope was designed to be cloned between residues 126 and 147 of TNF-α without destroying its antigenic determinants. α was replaced by PADRE. However, this recombinant vaccine failed to be expressed in E. coli  was introduced into the reverse primer P4 as shown in After analyzing the rTNF-PADRE gene with software, we found that the secondary structure of its mRNA is folded in a very complicated manner which may suppress translation complex entry and movement through the mRNA chain . To attaα monoclonal antibody  strain. The recombinant colonies BL21 (DE3)/pET-22b-mrTNF-PADRE were cultured in 5 ml LB medium and mrTNF-PADRE expression was induced by IPTG. As shown in antibody . This inantibody . Resultsβ-mercaptoethanol at 4°C overnight. The solubilized mrTNF-PADRE was purified by gel filtration chromatography with Sephacryl S-300 resin , which are in accordance with the standard of SFDA.We also detected the amount of residualα with the titer reaching a level of 1 : 100,000  in mice. In this study, a human TNF-α autovaccine was constructed according to this strategy by introducing PADRE to human TNF-α. Unfortunately, the recombinant gene rTNF-PADRE failed to be expressed in E. coli.Here, we want to develop an autovaccine to neutralize the endogenous TNF-se TNF-α , 20. Fin E. coli via different mechanisms such as preventing the depletion of rare tRNAs and improving the secondary structure and stability of mRNA. We analyzed the gene of rTNF-PADRE and the predicted secondary structure of its mRNA is too complicated to be expressed. Thus, a single codon synonymous mutation (TCT→AGT) was introduced into the gene. As a result, the optimized mrTNF-PADRE vaccine was successfully expressed in E. coli at a high level (about 30% of the whole cell lysate).Codon optimization often improves expression of exogenous recombinant genes inα recombinant vaccine by introducing the epitope PADRE to this protein. The recombinant mrTNF-PADRE was efficiently and stably expressed in E. coli at high level and was conveniently purified by a one-step gel filtration method after thorough IBs washing. In addition, the vaccine can induce high titer of neutralizing antibodies which can inhibit TNF-α-stimulated FLS proliferation. Considering the fact that TNF-α belongs to a huge superfamily and the elicited antibodies may cross-react with other family members, rational designed pharmacodynamics experiments in animal models are still needed.Taken together, we expressed a human TNF-