Resveratrol inhibits the progression of premature senescence partially by regulating v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) and sirtuin 1 (SIRT1).

OBJECTIVE: To explore the effect of resveratrol in premature senescence and reveal its anti-premature senescence mechanisms through network pharmacology. METHODS: In this study, the H2O2-induced bone marrow mesenchymal stem cells (BMMSCs) premature senescence model is applied. Cell counting kit-8 assay, ß-galactosidase staining and flow cytometry are conducted to detect the proliferation, senescence and apoptosis of BMMSCs. Bioinformatics analyses are used to screen and validate molecular targets of resveratrol acting on premature senescence. Dual-luciferase reporter assay is conducted to verify the interaction between v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) and sirtuin 1 (SIRT1). RT-qPCR and western blot are adopted to detect mRNA and protein levels of RELA, SIRT1, senescence-related genes and apoptosis-related genes. RESULTS: First, we proved that resveratrol alleviated the H2O2-induced senescence of BMMSCs. Then, bioinformatics analysis revealed that RELA was the downstream target of resveratrol and SIRT1 was the downstream target of RELA, respectively, involved in premature aging. RELA/SIRT1 may be the potential target of resveratrol for premature senescence. Notably, rescue experiments indicated that resveratrol inhibited premature senescence partially through targeting regulation RELA/SIRT1. CONCLUSION: In our study, we confirm the functional role of the resveratrol-RELA- SIRT1 axis in the progression of premature senescence, which provides a latent target for premature senescence treatment.

Effects of ribosomal associated protein hnRNP D in gemcitabine-resistant pancreatic cancer.

OBJECTIVE: To investigate the role of ribosomal associated protein hnRNP D in resistance to gemcitabine (GEM) in pancreatic cancer cells. METHODS: The expressions of hnRNP D in clinical pancreatic cancer tissues were detected by immunohistochemistry. The proliferation of pancreatic cancer cell lines (PANC-1, BXPC-3, SW1990 and ASPC-1) were measured by CCK8 assay. IC50 of each cell line was calculated and compared. The expressions of hnRNP D protein in pancreatic cancer cell lines were detected by Western Blot assay. The change of hnRNP D expression was confirmed by qPCR and Western Blot after the expressions of hnRNP D in PANC-1 cells being down-regulated by miRNA. And than the apoptosis rate and cell cycle of PANC-1 cells were detected by flow cytometry, while the expressions of apoptosis-related proteins cleaved caspase3, P-Akt, AKT and P65 were detected by Western Blot. RESULTS: HnRNP D protein expressed in clinical pancreatic tissues widely. The IC50 of GEM in PANC-1 was the highest while in BXPC-3 was the lowest. And the expression of hnRNP D protein in PANC-1 was the highest while in BXPC-3 was the lowest. After miRNA interfering, the expressions of hnRNP D protein and gene were significantly decreased in PANC-1 cells. The decrease of hnRNP D expression promoted cell apoptosis and inhibited the cell transformation to the S phase in cell cycle. Under the intervention of GEM, cleaved caspase3 expression was significantly increased, while p-Akt, AKT and P65 expression was significantly decreased. CONCLUSION: HnRNP D was associated with resistance to GEM in pancreatic cancer cells. Decreasing of hnRNP D expression promoted cell apoptosis induced by GEM.

The effect of N-acetyl cysteine and doxycycline on TNF-α-Rel-a inflammatory pathway and downstream angiogenesis factors in the cornea of rats injured by 2-chloroethyl-ethyl sulfide.

BACKGROUND: Cornea injury of sulfur mustard (SM) is considered as the most devastating injuries to the eye. This study aimed to evaluate the single and combined effects of N-acetyl cysteine (NAC) and doxycycline on the inflammatory pathway and cornea neovascularization (CNV) in the rat model of SM-injured cornea. MATERIALS AND METHODS: The right cornea of male Sprague-Dawley rats was subjected to 2-chloroethyl-ethyl sulfide (CEES). Rats were topically treated with a single and combined of 0.5% NAC and 12.5 µg/ml doxycycline and examined at 3rd, 15th, and 21st days. The activity of three antioxidant enzymes was analyzed in the cornea of different groups. Real-time PCR was performed to measure gene expression of inflammatory factors (tnf-α, rel-a & cxcl-1) and angiogenesis factors (vegf-a, mmp2,9) in the cornea lysates. The histological and opacity assessments were also carried out. RESULTS: The activity of antioxidant enzymes significantly declined 3 days after the CEES damage. NAC eye drop recovered the enzyme activity on the 21st day of treatment (p-value < .05). The expression of tnf-α and rel-a genes significantly increased after CEES cornea exposure, while NAC declined their expression on the 7th and 21st days. The CNV score and angiogenesis factor expression were decreased in the long term by single and combined treatments (p-value < .05), but the infiltration of inflammatory cells was not completely amended. CONCLUSION: NAC and doxycycline eye drop could improve the CNV complication. Also, NAC was an effective treatment against the inflammatory pathway involved in CEES-injured cornea.

The protective effect of piperine against isoproterenol-induced inflammation in experimental models of myocardial toxicity.

Myocardial infarction (MI) eventually exacerbates inflammatory response due to the release of inflammatory and pro-inflammatory factors. The aim of this study is to explore the protective efficacy of piperine supplementation against the inflammatory response in isoproterenol (ISO)-induced MI. Masson Trichome staining was executed to determine myocardial tissue architecture. Immunohistochemistry was performed for IL-6, TNF-α. RT-PCR studies were performed to ascertain the gene expression of IL-6, TNF-α, iNOS, eNOS, MMP-2, MMP-9, and collagen-III. Western blotting was performed to determine expression of HIF-1α, VEGF, Nrf-2, NF-ƙB, Cox-2, p-38, phospho-p38, ERK-1/2, phospho-ERK-1/2, and collagen-I. HIF-1α, VEGF, and iNOS expression were significantly upregulated with concomitant decline in eNOS expression in the heart myocardial tissue of rats received ISO alone whereas piperine pretreatment prevented these changes in ISO administered rats. Current results revealed ROS-mediated activation of MAPKs, namely, p-p38, p-ERK1/2 in the heart tissue of ISO administered group. Piperine pretreatment significantly prevented these changes in ISO treated group. NF-κB is involved in the modulation of gene expressions responsible for tissue repair. ISO-induced NF-κB-p65 expression was significantly reduced in the group pretreated with piperine and mitigated extent of myocardial inflammation. A significant increase in cardiac fibrosis upon ISO treatment was reported due to the increased hydroxyproline content, MMP-2 & 9 and upregulation of collagen-I protein compared to control group. All these cardiac hypertrophy markers were decreased in 'piperine pretreated ISO administered group' compared to group received ISO injection. Current findings concluded that piperine as a nutritional intervention could prevent inflammation of myocardium in ISO-induced MI.

Montelukast ameliorates Concanavalin A-induced autoimmune hepatitis in mice via inhibiting TNF-α/JNK signaling pathway.

BACKGROUND: Concanavalin A (ConA) is a well-established model to induce autoimmune hepatitis (AIH) in mice which mimics pathological alterations that occur in human. The pathogenesis of ConA-induced AIH involves many signaling pathways. Montelukast is a leukotriene receptor antagonist that is mainly used in the management of asthma. The antioxidant, anti-inflammatory and anti-apoptotic effects of montelukast have been reported in previous studies. Lately, montelukast has been documented to confer protection against various inflammatory diseases. Up to date, no study has explored the effect of montelukast on AIH induced by ConA. AIM AND METHOD: This study aims to detect the protective effects of montelukast (10 mg/kg) on ConA (20 mg/kg)- induced AIH in mice and to demonstrate its hepatoprotective mechanisms. Hepatic function, histological changes, oxidative stress, inflammation, autophagy, and apoptotic markers were investigated. RESULTS: Hepatic function and histological data revealed that treatment with montelukast significantly attenuated ConA-induced hepatic damage. Montelukast significantly reduced JNK level and NFκB p65 expression, and inhibited proinflammatory cytokines (TNF-α and IL-6) as well as oxidative stress (MDA, NO, and GSH). Moreover, inflammatory cells (CD4+ infiltration and the levels of apoptotic markers (Bax and caspase-3) besides autophagy biomarkers (Beclin1 and LC3) were reduced. CONCLUSION: Our results suggest that montelukast could be a potential therapeutic drug against the ConA-induced AIH through its anti-oxidant, anti-inflammatory, anti- autophagy as well as anti-apoptotic properties.

MiR-143 Inhibits Osteoclastogenesis by Targeting RANK and NF-κB and MAPK Signaling Pathways.

OBJECTIVE: To explore the effect of miRNA-143 on osteoclast formation and provide new ideas for the treatment of osteoporosis. METHODS: Mice macrophage lines RAW264.7 cells after transfection were divided into four groups: control group, RANKL group, RANKL combined with miR-143 mimics group and RANKL combined with miR-NC group. TARCP staining was used to observe the effect of miR-143 on osteoclast formation. The expression of RANK, TRAF6 and NFATc-1 in the upstream of RANKL pathway was detected by real-time quantitative PCR (RT qPCR) and Western blotting (WB). The binding of miR-143 to TNFRSF11A was detected by double Luciferase Reporter Analysis. The effect of miR-143 on the expression of NF-κB (p65, I-κB-α) signal pathway in osteoclasts was detected. The effects of I-BET151 on the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src were detected. RESULTS: The positive level of osteoclasts in RANKL group and RANKL combined with miR-NC group was significantly higher than that of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of RANK, TRAF6, NFATc-1, TRACP, MMP-9, CtsK and c-Src in RANKL group and RANKL combined with miR-NC group were significantly higher than those of RANKL combined with miR-143 mimics group and control group (P < 0.05). The expression levels of I-κB-α were significantly lower than that of RANKL combined with miR-143 mimics group and control group (P<0.05). CONCLUSION: MiR-143 can inhibit the expression of RANK, TRAF6 and downstream NFATc-1 in the RANKL pathway, thereby inhibiting the RANK/RANKL pathway. MiR-143 can inhibit the signal pathway of NF-κB (p65, I-κB-α). MiR-143 inhibits the expression of osteoclast-specific genes TRACP, MMP 9, CtsK and c-Src. That is to say, miR-143 inhibits osteoclast formation by targeting RANK, NF- κB and MAPK signaling pathways.

Cysteinyl Leukotriene Receptor 2 is Involved in Inflammation and Neuronal Damage by Mediating Microglia M1/M2 Polarization through NF-κB Pathway.

Microglia activation plays a key role in regulating inflammatory and immune reaction during cerebral ischemia and it exerts pro-inflammatory or anti-inflammatory effect depending on M1/M2 polarization phenotype. Cysteinyl leukotriene 2 receptor (CysLT2R) is a potent inflammatory mediator receptor, and involved in cerebral ischemic injury, but the mechanism of CysLT2R regulating inflammation and neuron damage remains unclear. Here, we found that LPS and CysLT2R agonist NMLTC4 significantly increased microglia proliferation and phagocytosis, up-regulated the mRNA expression of M1 polarization markers (IL-1ß, TNF-α, IFN-γ, CD86 and iNOS), down-regulated the expression of M2 polarization markers (Arg-1, CD206, TGF-ß, IL-10, Ym-1) and increased the release of IL-1ß and TNF-α. CysLT2R selective antagonist HAMI3379 could antagonize these effects. IL-4 significantly up-regulated the mRNA expression of M2 polarization markers, and HAMI3379 further increased IL-4-induced up-regulation of M2 polarization markers expression. Additionally, LPS and NMLTC4 stimulated NF-κB p50 and p65 proteins expression, and promoted p50 transfer to the nucleus. Pre-treatment with HAMI3379 and NF-κB signaling inhibitor Bay 11-7082 could reverse the up-regulation of p50 and p65 proteins expression, and inhibited p50 transfer to the nucleus. The conditional medium of BV-2 cells contained HAMI3379 could inhibit SH-SY5Y cells apoptosis induced by LPS and NMLTC4. These results were further confirmed in primary microglia. The findings indicate that CysLT2R was involved in inflammation and neuronal damage by inducing the activation of microglia M1 polarization and NF-κB pathway, inhibiting microglia M1 polarization and promoting microglia polarization toward M2 phenotype which may exerts neuroprotective effects, and targeting CysLT2R may be a new therapeutic strategy against cerebral ischemia stroke.

Effect of lncRNA H19 on the apoptosis of vascular endothelial cells in arteriosclerosis obliterans via the NF-κB pathway.

OBJECTIVE: To explore the effect of long non-coding ribonucleic acid (lncRNA) H19 on the apoptosis of vascular endothelial cells in arteriosclerosis obliterans (ASO) via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. PATIENTS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured, and lncRNA H19 was inhibited by Si-H9 and overexpressed by H19-OE. Then, the apoptosis rate was detected by flow cytometry, the target of lncRNA H19 was detected by dual luciferase reporter gene assay, and changes in the protein level were determined via Western blotting (WB). RESULTS: LncRNA H19 exhibited high expression in serum of patients with ASO, and compared with that in congeneric normal mice, the expression of lncRNA H19 in ASO mice rose. Besides, the proliferation ability of cells transfected with H19-OE was markedly strengthened, and H19-OE treatment could down-regulate the expression level of the apoptin, active cysteinyl aspartate-specific proteinase-3 (Caspase-3). In addition, lncRNA H19 bound to micro ribonucleic acid (miR)-19a in a targeted way. After lncRNA H19 was overexpressed, the expression of the NF-κB pathway key factors, p38 and p65, were notably increased, and the nuclear translocation of p65 was significantly enhanced after transfection with miR-19a. CONCLUSIONS: LncRNA H19 promotes the proliferation of vascular endothelial cells in ASO and inhibits the apoptosis of them via the NF-κB pathway.

Activation of EMT in colorectal cancer by MTDH/NF-κB p65 pathway.

Epithelial-mesenchymal transition (EMT) leads to tumor dissemination and metastasis. Metadherin (MTDH) is an oncogene that plays an important role in metastasis regulation. This study tries to investigate the effect of MTDH gene up-regulation on the activation of EMT in colorectal cancer (CRC) cells and identify the role of NF-κB p65. The CaCO2 cells were divided into three groups: one control group of cultured CaCO2 cells (C1), and two groups of CaCO2 cells co-transfected using human MTDH expression plasmid with either siRNA targeting human NF-κB p65 or its negative control (C2 and C3 respectively). The gene modification was confirmed by qPCR and the effect of gene modification on CRC aggravation was studied. MTDH up-regulation significantly promoted CRC cell proliferation, activated anaerobic respiration (glucose consumption and lactate production), and increased gene expression of multidrug resistance gene (MDR1), Snail transcription factor and NF-κB p65, but decreased the gene expression of E-cadherin. Moreover, MTDH up-regulation led to a significant increase in the acquisition of surface markers of CRC stem cells. Interference with NF-κB p65 gene expression reversed the action of MTDH gene up-regulation on MDR1 and E-cadherin gene expression and anaerobic respiration. Moreover, NF-κB p65 interference significantly decreased MTDH-induced cell proliferation and acquisition of surface markers of CRC stem cells but didn't affect the Snail transcription factor. MTDH-dependent EMT in CRC is activated via NF-κB p65 and is mediated by up-regulation of Snail. These results identify a pathway by which MTDH regulates NF-κB p65 induced EMT during CRC cell metastasis.

Glucocorticoids promote the development of azoxymethane and dextran sulfate sodium-induced colorectal carcinoma in mice.

BACKGROUND: Stress has been suggested as a promoter of tumor growth and development. Glucocorticoids (GCs) are the main stress hormones and widely prescribed as drugs. However, the effect of GCs on the development and progression of colorectal carcinoma (CRC) is unclear. METHODS: We evaluated the effect of corticosterone (CORT) on azoxymethane and dextran sulfate sodium (AOM/DSS)-induced carcinogenesis in the colorectum of C57BL/6 strain mice. Plasma level of CORT was detected by radioimmunoassay. The expression of proliferation markers (Ki-67 and PCNA), nuclear factor (NF)-κB p65 and phosphoto-p65 (P-p65), as well as cyclooxygenase (COX)-2 were determined by immunohistochemistry. Inflammation in colorectum was evaluated by histopathology. RESULTS: CORT feeding in drinking water of mice not only significantly elevated plasma CORT concentration, but also significantly increased the incidence and neoplasms burden (number and size of neoplasms) in colorectum. CORT also significant enhanced the expression of cell proliferation marker (Ki-67 and PCNA), NF-κB p65 and P-p65 as well as COX-2 in colorectal neoplasm of AOM/DSS-treated mice. CONCLUSION: In this study, we have found for the first time that CORT at stress level potentially promotes the growth and development of AOM/DSS-induced colorectal adenoma and carcinoma in mice. Up-regulation of NF-κB and COX-2 may be involved in the promoting effect of CORT.

Phase I study of ruxolitinib in previously treated patients with low or intermediate-1 risk myelodysplastic syndrome with evidence of NF-kB activation.

Therapeutic options for patients with lower-risk myelodysplastic syndrome (MDS) who have failed prior therapies are limited particularly after hypomethylating agent. Several studies have indicated that deregulation of innate immunity signaling is critical in the pathogenesis of MDS. This process involves Toll-like receptor stimulation, cytokine overexpression, and nuclear factor-kB (NF-kB) activation. Since ruxolitinib, a JAK1/JAK2 inhibitor, suppresses NF-kB expression, we conducted a phase 1 dose-escalation study to determine the safety and efficacy of ruxolitinib in previously treated lower-risk MDS patients with evidence of NF-kB activation. Nineteen patients, 8 with chronic myelomonocytic leukemia and 11 with MDS, were enrolled. No dose limiting toxicity was observed and the maximum tolerated dose was 20 mg twice daily. Responses were restricted to MDS patients with an overall response rate of 22% [hematological improvement in platelets (HI-P) = 2, hematological improvement in erythrocytes (HI-E) = 1, partial cytogenetic response (PCyR) = 1]. Of these patients, 2 relapsed (HI-P and PCyR) and 2 continue to be in HI-P and HI-E, respectively, with ongoing therapy. Meaningful improvement in bone marrow dysplasia was only seen in a patient who achieved HI-E. Phosphorylated p65 (pp65) decreased in 6 of 15 patients (40%) including the 2 patients with continued response to treatment and increased in a patient who relapsed after a short-lived HI-P. This suggests potential correlation between reduction in pp65 expression and response duration. In conclusion, ruxolitinib was well-tolerated in previously treated lower-risk MDS patients with evidence of NF-kB activation and resulted in low but significant frequency of responses. (NCT01895842).

Effect of two recombinant Trichinella spiralis serine protease inhibitors on TNBS-induced experimental colitis of mice.

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn's disease (CD), is a chronic autoimmune disease. Parasitic infections and their products have been shown to have protective effects on autoimmune diseases, including IBD. In this experiment, 96 male BALB/c mice aged 6-8 weeks were divided randomly into two large groups: prevention and therapy. The changes in the various indicators of colitis were detected to demonstrate that Trichinella spiralis serine protease inhibitors can relieve the inflammatory severity of 2,4,6-trinitrobenzenesulphonic acid solution (TNBS)-induced colitis and to explore possible immunological mechanisms. Results showed that the disease activity index (DAI) score, myeloperoxidase (MPO) activity, macroscopic and microscopic damage degrees of colon all decreased significantly, interferon (IFN)-γ expression decreased, interleukin (IL)-4 expression increased, nuclear factor kappa B (NF)-κB expression decreased and the percentage of CD4+ CD25+ forkhead box protein 3 (FoxP3+ ) regulatory T cells (Treg ) cells in the spleen. MLN increased significantly compared to the phosphate-buffered saline (PBS)/2,4,6-trinitrobenzenesulphonic acid solution (TNB) group. We found the same results with the T. spiralis Kazal-type serine protease inhibitors (TsKaSPI)+TNBS and TsAdSPI+TNBS groups in the large prevention group and the large therapy group, compared to the TNBS+PBS group with the TNBS+TsKaSPI and TNBS+TsAdSPI groups. Immunization with TsKaSPI and TsAdSPI on the CD models showed an intervention effect, possibly because TsKaSPI and TsAdSPI induced a T helper type 2 (Th2)-type immune response and balanced the TNBS-induced Th1-type immune response.

Ginkgolide B ameliorates NLRP3 inflammasome activation after hypoxic-ischemic brain injury in the neonatal male rat.

INTRODUCTION: Perinatal hypoxic-ischemic (HI) insult is an important cause of brain injury in neonates. The development of novel treatment strategies for neonates with HI brain injury is urgently needed. Ginkgolide B (GB) is a main component of Ginkgo biloba extracts with a long history of use in traditional Chinese medicine. However, it is unknown whether GB could play a protective role in hypoxic stress in immature animals. METHODS: Using neonatal hypoxic-ischemic (HI) brain injury model of rat pups, neurological score, infarct size, and brain edema were evaluated after HI injury. The activation of microglia and the production of IL-1ß and IL-18 were detected by immunohistochemistry and ELISA, respectively. A priming signal (NF-κB P65) and an activation signal (Caspase-1) of NLRP3 inflammasome activation were detected by western blot analyses. RESULTS: GB administrated 30 min prior to ischemia induction can improve neurological disorder, reduce infarct volume and alleviate cerebral edema. Compared with the HI groups, GB inhibited the activation of microglia and decreased the production of IL-1ß and IL-18 in neocortex. Furthermore, GB reduced NLRP3 expression mainly in microglia, and significantly inhibited the expression of Caspase-1 and the nuclear translocation of NF-κB P65, preventing NLRP3 inflammasome activation. CONCLUSIONS: GB ameliorates hypoxic-ischemic brain injury in the neonatal male rat via inhibiting NLRP3 inflammasome activation.

Fibrin patch influences the expression of hypoxia-inducible factor-1α and nuclear factor-κBp65 factors on ischemic intestinal anastomosis.

The intestinal anastomotic failure is one of the most severe complications in gastrointestinal surgery. Despite the great surgical improvements during the last decade, anastomotic leak rates remain practically the same, with a dramatically high grade of morbidity for patients. Leakages are usually the final consequence of ischemia in the anastomosis, leading to tissue hypoxia. In response to hypoxia, the cell orchestrates a variety of coordinated responses in order to restore oxygen homeostasis. The molecular mechanism of hypoxia sensitivity involves oxygen sensing hydroxylases, prolyl-hydroxylases, orchestrating two main transcription factors related to induction of inflammation and angiogenesis, namely nuclear factor-κB and hypoxia-inducible factors. The immunohistochemical expression of two transcription factors hypoxia-inducible factors-1α and nuclear factor-κB p65 has already been described in several disorders, including wound healing, asthma and chronic obstructive lung disease, rheumatoid arthritis, cancer, inflammatory bowel disease, and acute colitis. In the surgical field, fibrin sealants have been widely used to prevent leaks in lung surgery and they might also be useful as a reinforcement of sutures in intestinal anastomosis. The commercial fibrin sealant patches are hemostatic and adhesive surgical agents mainly derived from human plasma. We herein report the results of a prospective randomized experimental study on pigs. We performed a high-risk leakage model of bowel anastomosis, causing a significant devascularization of 10-15 cm of the bowel wall before performing a conventional colo-ileal anastomosis. We randomized the animals to receive a covering of the anastomosis with a fibrin patch (case group) or not (control group). We report the changes in the immunohistochemical expression of the proteins involved in tissue response to hypoxia in the experimental model. Our results indicate that the fibrin patch delays the healing response, promoting a longer lasting inflammation in the surgical bed. Nevertheless, the fibrin patches effectiveness to reduce dehiscence shown in clinical practice suggests that this delay does not negatively affect patients' outcome. Impact statement The consequences of the anastomotic failure are dramatic for patients. Understanding how the ever-increasing use of fibrin sealant, that seems to have a beneficial effect on the anastomoses, interacts with the tissue and the healing process can help to justify its use and encourage research on how to improve this effect even more. We feel that the present work shows that the patch can improve healing by complex mechanisms other than the mere contention and physical support of the intestine. Furthermore, research is needed to confirm our preliminary findings.

Transcriptomic analysis reveals Toxoplasma gondii strain-specific differences in host cell response to dense granule protein GRA15.

Growth and replication of the protozoan parasite Toxoplasma gondii within host cell entail the production of several effector proteins, which the parasite exploits for counteracting the host's immune response. Despite considerable research to define the host signaling pathways manipulated by T. gondii and their effectors, there has been limited progress into understanding how individual members of the dense granule proteins (GRAs) modulate gene expression within host cells. The aim of this study was to evaluate whether T. gondii GRA15 protein plays any role in regulating host gene expression. Baby hamster kidney cells (BHK-21) were transfected with plasmids encoding GRA15 genes of either type I GT1 strain (GRA15I) or type II PRU strain (GRA15II). Gene expression patterns of transfected and nontransfected BHK-21 cells were investigated using RNA-sequencing analysis. GRA15I and GRA15II induced both known and novel transcriptional changes in the transfected BHK-21 cells compared with nontransfected cells. Pathway analysis revealed that GRA15II was mainly involved in the regulation of tumor necrosis factor (TNF), NF-κB, HTLV-I infection, and NOD-like receptor signaling pathways. GRA15I preferentially influenced the synthesis of unsaturated fatty acids in host cells. Our findings support the hypothesis that certain functions of GRA15 protein are strain dependent and that GRA15 modulates the expression of signaling pathways and genes with important roles in T. gondii pathophysiology. A greater understanding of host signaling pathways influenced by T. gondii effectors would allow the development of more efficient anti-T. gondii therapeutic schemes, capitalizing on disrupting parasite virulence factors to advance the treatment of toxoplasmosis.

Expression analysis of Akirin-2, NFκB-p65 and ß-catenin proteins in imatinib resistance of chronic myeloid leukemia.

OBJECTIVE: Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and ß-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and ß-catenin in imatinib resistance of CML and to test if any direct physical protein-protein interaction exists between NFkB and both ß-catenin and Akirin-2. METHODS: RT-PCR and western blot were performed to determine Akirin-2, NFκB-p65 and ß-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and ß-catenin proteins to MDR1 promoter region, respectively. RESULTS: ß-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein-protein interaction could not be found between NFκB-p65 and Akirin-2 or ß-catenin proteins. Nuclear ß-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells. DISCUSSION: We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and ß-catenin genes may provide an opportunity to overcome imatinib resistance in CML.

Celastrol attenuates incision-induced inflammation and pain associated with inhibition of the NF-κB signalling pathway via SARM.

AIM: This study aimed to investigate whether celastrol (CEL) could alleviate incision-induced pain and decipher its possible mechanism. MATERIALS AND METHODS: Sprague-Dawley rats were randomly divided into five groups: naïve, vehicle, CEL (5 µg/paw, 10 µg/paw and 20 µg/paw). CEL or vehicle was administered intraplantarly before plantar surgical incision. Histological examinations of skin tissues were performed after HE staining. Additionally, immunohistochemical staining, RT-PCR and western blot were performed to analyse macrophages, proinflammatory cytokines, SARM and NF-κB expression, respectively. Moreover, the previous mentioned factors were re-evaluated after suppressing SARM expression by shRNA. KEY FINDINGS: The plantar incision rats displayed pain-related behaviours and inflammatory infiltration in the skin. The mRNA levels of proinflammatory cytokines, such as IL-1ß, IL-6, and TNFα were significantly upregulated in the skin of surgical rats. The expression of sterile α- and armadillo-motif-containing protein (SARM) was downregulated and nuclear factor kappa-B (NF-κB) was activated. Interestingly, CEL could partially restore the pain-related behavioural changes. Furthermore, molecular mechanism of CEL was explored, that included significantly reduction of proinflammatory cytokines mRNA expressions, a significant decrease of p-p65 and p65 levels and a markedly increase of SARM and IkBα expressions in skin tissues. However, supression SARM by shRNA partially eliminated those protective effect of CEL. SIGNIFICANCE: Our data suggest that intraplantarly administration of CEL attenuates inflammatory and acute pain. This finding could be attributed to regulation of the NF-κB signalling pathway via SARM. These results provide pre-clinical evidence supporting the use of CEL in the treatment of surgical pain.

The peptide lycosin-I attenuates TNF-α-induced inflammation in human umbilical vein endothelial cells via IκB/NF-κB signaling pathway.

OBJECTIVE: The peptide lycosin-I has anti-bacterial and anti-cancer capacities. However, the anti-inflammatory activity of lycosin-I remains unknown. We investigated whether lycosin-I could attenuate inflammation. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with lycosin-I before exposure to tumor necrosis factor-α (TNF-α). The expression of intercellular cell adhesion molecule-1 (ICAM-1), nuclear transcription factor-kappa B (NF-κB) p65 and inhibitory subunit of NF-κB alpha (IκBα) was evaluated by western blot. The expression of interleukin-6 (IL-6) and interleukin-8 (IL-8) was detected by quantitative RT-PCR or ELISA. Immunofluorescence analysis was used to determine the impact of lycosin-I on NF-κB pathway. C57BL/6 mice were pretreated with lycosin-I before exposure with lipopolysaccharide (LPS). RESULTS: Lycosin-I significantly reduced the TNF-α-enhanced expression of IL-6, IL-8 and ICAM-1. Lycosin-I also inhibited the human monocyte cells adhesion to HUVECs. We further demonstrated that lycosin-I could effectively suppress the reaction of endothelial cells to TNF-α by inhibiting IκBα degradation. Subsequently, the phosphorylation and translocation of NF-κB p65 could also be attenuated. Furthermore, lycosin-I exhibited a significant protection of C57BL/6 mice against LPS-induced death. CONCLUSIONS: Our results suggested that the anti-inflammatory activity of lycosin-I was associated with NF-κB activation and lycosin-I had potential to be a novel therapeutic candidate for inflammatory diseases.

miR-25 inhibits sepsis-induced cardiomyocyte apoptosis by targetting PTEN.

To investigate the regulatory mechanism of miR-25 in sepsis-induced cardiomyocyte apoptosis. Rats models of sepsis were established by cecal ligation and puncture (CLP). Lipopolysaccharide (LPS)-induced cardiomyocyte was used as an in vitro model of sepsis. The expressions of miR-25, tensin homolog deleted on chromosome 10 (PTEN), Toll-like receptors 4 (TLR4), and p-p65 were analyzed by quantitative real-time PCR (qRT-PCR) and Western blot, respectively. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) assay. The relationship between miR-25 and PTEN was measured by luciferase reporter assays. MiR-25 expression in serum of CLP rats and LPS-induced cardiomyocyte was decreased, while the contents of TNF-α and IL-6 were increased. Moreover, the expressions of PTEN, TLR4, and p-p65 in LPS-induced cardiomyocyte were significantly increased. Overexpression of miR-25 increased the survival rate of rats, inhibited LPS-increased cardiomyocyte apoptosis, reversed the increased expression of PTEN, TLR4, p-p65, TNF-α, and IL-6 induced by LPS. The luciferase assay demonstrated that PTEN was a target of miR-25. Additionally, pcDNA-PTEN reversed the inhibitory effect of miR-25 mimic on cardiomyocyte apoptosis, while TAK-242 (TLR-4 inhibitor) countered this effect. miR-25 reduced LPS-induced cardiomyocyte apoptosis by down-regulating PTEN/TLR4/NF-κB axis.

Anticancer Efficacy of Targeted Shikonin Liposomes Modified with RGD in Breast Cancer Cells.

Shikonin (SHK) has been proven to have a good anti-tumor effect. However, poor water solubility and low bioavailability limit its wide application in clinical practice. In this study, to overcome these drawbacks, RGD-modified shikonin-loaded liposomes (RGD-SSLs-SHK) were successfully prepared. It exhibited excellent physicochemical characteristics including particle size, zeta potential, encapsulation efficiency, and delayed release time. Meanwhile, the targeting activity of the RGD-modified liposomes was demonstrated by flow cytometry and confocal microscopy in the αvß3-positive MDA-MB-231 cells. Besides exhibiting greater cytotoxicity in vitro, compared with non-targeted shikonin-loaded liposomes (SSLs-SHK), RGD-SSLs-SHK could also evidently induce apoptosis by decreasing the expression of Bcl-2 and increasing the expression of Bax. It could also inhibit cell proliferation, migration, invasion, and adhesion by reducing the expression of MMP-9 and the level of NF-κB p65, but did not affect the expression of MMP-2 in the MDA-MB-231 cells. Therefore, these findings indicated that the strategy to use RGD-modified liposomes as carriers for targeted delivery of shikonin is a very promising approach to achieve breast cancer targeted therapy.