Activation of TLR2 heterodimers-mediated NF-κB, MAPK, AKT signaling pathways is responsible for Vibrio alginolyticus triggered inflammatory response in vitro.

Vibrio alginolyticus is an important zoonotic marine pathogenic bacterium. Previous studies on the mechanism of innate immune against V. alginolyticus infection have been limited to aquatic animals, however, how V. alginolyticus activates mammalian immune cells has not been fully clarified. Here, ELISA combined RT-qPCR assays were used to detect the secretion and transcription level of pro-inflammatory cytokines and TLRs during V. alginolyticus infection of mice peritoneal macrophages (PMϕs). Western blotting was used to explore the phosphorylation levels of p38, JNK, ERK, AKT and NF-κB protein. Immunofluorescence assay was used to determine the location of NF-κB protein. Inhibition assay was used to study the role of up-regulated TLR in activated signaling pathways and the role of these pathways in the release of pro-inflammatory cytokines. Our data showed that V. alginolyticus can up-regulate the expression levels of IL-1ß, IL-6, IL-12 and TNF-α in PMϕs. In addition, V. alginolyticus stimulation activated the phosphorylation of p38, JNK and ERK were TLR2 heterodimers-dependent, whereas inhibitors of SB203580 (p38), SCH772984 (ERK) and SP600125 (JNK) significantly reduced IL-1ß, IL-6, IL-12 and TNF-α production. We further revealed that V. alginolyticus activated the signaling pathways of AKT via TLR2 heterodimers. The inhibitor of MK-2206 2HCl (AKT) negatively regulated the IL-1ß, IL-6 and TNF-α release levels. Moreover, V. alginolyticus infection of PMϕs resulted in TLR2 heterodimers-mediated activation of NF-κB and induced translocation of phosphorylated NF-κB protein from the cytoplasm into the nucleus via IκBα degradation. V. alginolyticus induced IL-1ß, IL-6, IL-12 and TNF-α release were blocked by the specific NF-κB inhibitor, BAY 11-7082. Taken together, our results suggested that activation of the TLR2 heterodimers-mediated downstream signaling pathways NF-κB, MAPK and AKT is responsible for inflammatory response during Vibrio alginolyticus infection in vitro.

Protective Effects of Scutellarin on Acute Alcohol Intestinal Injury.

The present study aims to investigate the roles of scutellarin (SCU) on acute alcohol intestinal injury. Mice were divided into six groups: alcohol, three administration, negative control and positive drug bifendate control. The administration group mice were intraperitoneally injected with SCU for 3 consecutive days followed by alcohol gavage at an interval of 1 h. After the mice were sacrificed, colon tissue damage was evaluated by histopathological examination; the activities of inducible nitric oxide synthase (iNOS) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected using biochemical kits; the levels of inflammatory cytokines mRNA were determined by real-time fluorescence quantitative PCR; the protein expression levels of hemeoxygenase-1 (HO-1) and phosphorylated nuclear factor-ĸB p65 were measured via western blotting. The results showed that alcohol induced severe colon morphological degradation, epithelia atrophy, and more inflammatory cells infiltration in the submucosa. SCU treatment prevented this process, especially in the middle and high dose groups. Alcohol treatment caused excessive lipid peroxidation product accumulation of MDA, restrained the activity of antioxidant enzyme CAT, induced HO-1 expression in the colon, whereas low dose SCU treatment significantly down-regulated the MDA level, enhanced the CAT level, and accelerated HO-1 signals. SCU prevented alcohol stimulation triggered inflammatory response in colon tissues through significantly downregulating the iNOS activity, transcript levels of Tnf-α, Il-1ß and Il-6, and phosphorylation levels of NF-κB p65. These findings suggest that SCU protects the colon via antioxidant and anti-inflammatory mechanisms, making it a promising drug against alcohol-induced colon damage.

NLRP3 inflammasome signal pathway involves in Vibrio harveyi-induced inflammatory response in murine peritoneal macrophages in vitro.

Vibrio harveyi, an important zoonotic pathogen, can infect wounds and cause inflammatory response. Understanding the inflammatory response pathways could facilitate the exploration of molecular mechanisms for treating V. harveyi infection. NLR family pyrin domain-containing 3 (NLRP3) inflammasome is involved in the interaction between hosts and pathogenic microorganisms and could be sensed by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Nonetheless, the function of NLRP3 inflammasome in V. harveyi infection remains unclear. In the present study, we established a V. harveyi infection model using murine peritoneal macrophages (PMs). Various techniques, including western blot analysis, enzyme-linked immunosorbent assay (ELISA), RT-qPCR, immunofluorescence, and inhibition assays, were used to explore the molecular mechanism of V. harveyi-induced inflammation. The results showed that many inflammatory cytokines participated in V. harveyi infection, with interleukin (IL)-1ß being the most abundant. Pan-caspase inhibitor pretreatment significantly decreased the secretion of IL-1ß in murine PMs. Moreover, the identification of V. harveyi involved a large number of NLR molecules, especially the NLRP3 receptor, and further studies revealed that NLPR3 inflammasome was activated by V. harveyi infection, as evidenced by puncta-like NLRP3 surrounding cell nuclear, ASC specks in the nucleus and cytoplasm, and ASC oligomerization. Inhibition of NLRP3 inflammasome impaired the release of mature IL-1ß in V. harveyi-infected murine PMs. Furthermore, blocking the secretion of mature IL-1ß could markedly decrease the release of other proinflammatory cytokines, including IL-6, IL-12, and tumor necrosis factor-α. Overall, these data indicated that NLRP3 inflammasome was activated in response to V. harveyi infection and enhanced inflammatory response by promoting IL-1ß secretion in murine PMs.

Abamectin causes cardiac dysfunction in carp via inhibiting redox equilibrium and resulting in immune inflammatory response and programmed cell death.

This study aims to investigate the effects of environmentally relevant concentrations of abamectin on the cardiac function of carp and the potential mechanisms. Here, male carp were exposed to abamectin, and cardiac function-related enzymatic markers were examined. Cardiac histopathology, redox equilibrium, inflammation, and cell death were evaluated. Abamectin exposure caused cardiac dysfunction by upregulating lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK), creatine Kinase MB isoenzyme (CK-MB) and white blood cells (WBCs), and decreasing red blood cells (RBCs) and hemoglobin (Hb). DHE staining and biochemical assays revealed that abamectin caused ROS release and oxidative stress by inhibiting Nrf2-ARE pathway. Histopathological and real-time fluorescence quantitative PCR (RT-qPCR) assays revealed that abamectin caused myocardial fiber swelling and inflammatory cell infiltration, enhanced pro-inflammatory cytokines tumor necrosis factor-α (Tnf-α), interleukin-1 beta (Il-1ß), and Il-6 levels and attenuated anti-inflammatory cytokines Il-10 and transforming growth factor beta 1 (Tgf-ß1) through activating NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome and nuclear factor kappa-B (NF-κB) pathway. Tunel staining showed that abamectin triggered cardiac apoptosis via activating p53-mediated mitochondrial apoptosis with elevated bcl2-associated X (Bax), reduced B-cell lymphoma-2 (Bcl-2), and activated Caspase-9 and Caspase-3. Immunoblot analysis revealed that abamectin activated autophagic flow by inhibiting mammalian target of rapamycin (mTOR), resulting in the conversion of LC3B from LC3-I to LC3-II, elevation of autophagy protein 5 (Atg5), and reduction of p62. Overall, abamectin caused cardiac dysfunction in carp via inhibiting redox equilibrium and resulting in immune inflammatory response and programmed cell death.

Scutellarin prevents acute alcohol-induced liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and inhibiting inflammation by regulating the AKT, p38 MAPK/NF-κB pathways. / ç¯ç›èŠ±ä¹™ç´ é€šè¿‡è°ƒèŠ‚Nrf2/HO-1通路抑制氧化应激以及通过调节AKT、p38 MAPK/NF-κBé€šè·¯æŠ‘åˆ¶ç‚Žç—‡ååº”é¢„é˜²æ€¥æ€§é ’ç²¾æ€§è‚æŸä¼¤.

Alcoholic liver disease (ALD) is the most frequent liver disease worldwide, resulting in severe harm to personal health and posing a serious burden to public health. Based on the reported antioxidant and anti-inflammatory capacities of scutellarin (SCU), this study investigated its protective role in male BALB/c mice with acute alcoholic liver injury after oral administration (10, 25, and 50 mg/kg). The results indicated that SCU could lessen serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and improve the histopathological changes in acute alcoholic liver; it reduced alcohol-induced malondialdehyde (MDA) content and increased glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) activity. Furthermore, SCU decreased tumor necrosis factor-|α (TNF-|α), interleukin-6 (IL-6), and IL-|1ß messenger RNA (mRNA) expression levels, weakened inducible nitric oxide synthase (iNOS) activity, and inhibited nucleotide-binding oligomerization domain (NOD)|-like receptor protein 3 (NLRP3) inflammasome activation. Mechanistically, SCU suppressed cytochrome P450 family 2 subfamily E member 1 (CYP2E1) upregulation triggered by alcohol, increased the expression of oxidative stress-related nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) pathways, and suppressed the inflammation-related degradation of inhibitor of nuclear factor-|κB (NF-|κB)|-|α (IκBα) as well as activation of NF|-|κB by mediating the protein kinase B (AKT) and p38 mitogen-activated protein kinase (MAPK) pathways. These findings demonstrate that SCU protects against acute alcoholic liver injury via inhibiting oxidative stress by regulating the Nrf2/HO-1 pathway and suppressing inflammation by regulating the AKT, p38 MAPK/NF-κB pathways.

Vibrio harveyi infections induce production of proinflammatory cytokines in murine peritoneal macrophages via activation of p38 MAPK and NF-κB pathways, but reversed by PI3K/AKT pathways.

Vibrio harveyi is a zoonotic pathogen that can infect humans through wounds and cause severe inflammatory responses. Previous studies have reported that the Toll like receptors (TLR) mediated MAPK, AKT and NF-κB signaling pathways are involved in innate immune system resistance to pathogen invasion. However, the molecular mechanism of these pathways, as well as their involvement in V. harveyi infection remains elusive. This study established a V. harveyi infection model using murine peritoneal macrophages (PMs). Various techniques, including western blotting, ELISA, RT-qPCR, immunofluorescence, inhibition assays, were used to explore the roles of TLRs, MAPK, AKT and NF-κB signaling pathways in V. harveyi-induced inflammatory responses. ELISA assays showed that V. harveyi infection triggered proinflammatory cytokines secretion in PMs. RT-qPCR and inhibition assays showed that TLR2 participated in V. harveyi infection and up-regulated the proinflammatory cytokines secretion in murine PMs. Western blotting data showed that the phosphorylation of p38, JNK, AKT, and NF-κB p65 were significantly increased partly mediated by TLR2. In addition, immunofluorescence assays revealed that the NF-κB p65 translocated into nucleus in response to V. harveyi infection. The secretion of IL-1ß, IL-6, IL-12, and TNF-α were considerably reduced when the p38 MAPK and NF-κB signaling pathways were blocked, whereas blocking of AKT significantly increased the expression of IL-1ß, IL-6, IL-12, and TNF-α. These findings indicate that V. harveyi infection induces inflammatory responses in murine PMs via activation of p38 MAPK and NF-κB pathways, which are partly mediated by TLR2, but are inhibited by PI3K/AKT pathways.

Immune Protective Evaluation Elicited by DNA Vaccination With Neospora caninum Dense Granules Proteins in Mice.

Neospora caninum, an obligate intracellular protozoan, is the major cause for neosporosis and brings serious economic losses to cattle breeding industries worldwide. After invasion, dense granules proteins are abundantly secreted and being important components of parasitophorous vacuole and intravacuolar network where N. caninum survives and replicates. The aim of the present study was to evaluate the protective immunity induced by DNA vaccines with genes encoding dense granules proteins 1 (GRA1), GRA4, GRA9, GRA14, GRA17, and GRA23 against N. caninum tachyzoites in BALB/C mice. Eukaryotic expressing plasmids of pcNcGRAs were constructed and the mice were intramuscularly immunized with pcNcGRAs followed by challenging infection with lethal doses of N. caninum. Immune responses were evaluated through monitoring the levels of serum antibodies, measurement of lymphocyte proliferation, and secretion of cytokines. Immune protection assays were carried out through monitoring survival time, body weight, and parasite burden in the brains. Results showed that all the pcNcGRA DNA vaccines could trigger remarkably specific humoral and cellular responses, with higher levels of IgG and IgG2a antibodies as well as obviously increased secretion of Th1-type IFN-γ cytokines. The immune protective efficacy revealed that pcNcGRA4, pcNcGRA14, and pcNcGRA17 DNA vaccines could individually increase the survival rate to 50, 37.5, and 25% in comparison with 0% in the control group; prolong the survival time more than 20.88 ± 11.12, 18.88 ± 10.83, and 16.63 ± 10.66 days compared with the control group of 4 ± 1.31 days; and decrease parasite burden in the brains to 297.63 ± 83.77, 471.5 ± 110.74, and 592.13 ± 102.2 parasites/100 ng comparing with 1221.36 ± 269.59 parasites/100 ng in the control group. These findings indicated that NcGRA4, NcGRA14, and NcGRA17 are potential vaccine candidates; NcGRA4 displayed better performance in immune protective efficacy and could be further combined with other advantageous antigens applied to the development of safe and effective DNA vaccines against N. caninum.

Vibrio alginolyticus Triggers Inflammatory Response in Mouse Peritoneal Macrophages via Activation of NLRP3 Inflammasome.

Vibrio alginolyticus is a food-borne marine Vibrio that causes gastroenteritis, otitis media, otitis externa, and septicemia in humans. The pathogenic mechanisms of V. alginolyticus have previously been studied in aquaculture animals; however, the underlying mechanisms in mammals remain unknown. In this study, an in vitro model of mouse peritoneal macrophages infected with V. alginolyticus was established. qPCR results revealed that V. alginolyticus induced the transcription levels of various cytokines, including IL-1ß, IL-12, IL-18, TNF-α, IL-17, IL-6, IFN-γ, and IL-10, and the secretion level of IL-1ß is the most significant. Inhibition assays with Ac-YVAD-CHO (a caspase-1 inhibitor) and Z-VAD-FMK (a pan-caspase inhibitor) were conducted to determine whether caspase-1 or caspase-11 is involved in V. alginolyticus-triggered IL-1ß secretion. Results showed that IL-1ß secretion was partly inhibited by Ac-YVAD-CHO and absolutely blocked by Z-VAD-FMK. To explore the sensed pattern recognition receptors, several NLR family members and the AIM2 receptor were detected and many receptors were upregulated especially NLRP3. Moreover, the NLRP3 protein displayed a puncta-like surrounding cell nucleus, which signified that the NLRP3 inflammasome was activated in response to V. alginolyticus infection. Inhibition assays with glyburide and CA-074 methyl ester (K+ outflow inhibitor and cathepsin B inhibitor) blocked IL-1ß secretion, which demonstrated the essential role of the NLRP3 inflammasome in inflammatory response. To better understand how V. alginolyticus affects IL-1ß release, the NLRP3 inflammasome was detected with doses ranging from 0.1 to 10 MOIs and time periods ranging from 3 to 12 h. Results showed that V. alginolyticus-mediated NLRP3 inflammasome activation was in a time- and dose-dependent manner and IL-1ß release peaked at MOI of 1 for 12 h. Most importantly, blocking the NLRP3 inflammasome with inhibitors and the use of NLRP3-/- and caspase-1/11-/- mice could attenuate pro-inflammatory cytokine secretion, such as IL-1ß, IL-6, IL-12, and TNF-α. Taken together, our study first found that the NLRP3 inflammasome plays vital roles in V. alginolyticus triggered inflammatory response in mouse peritoneal macrophages. This may provide reference information for the development of potential anti-inflammatory treatments against V. alginolyticus infection.

Higher energy optical transitions in semiconducting carbon nanotubes.

We have studied the high energy optical transitions of semiconducting single-walled carbon nanotubes using the nonorthogonal tight-binding model, which takes into account the exciton effect. It is found from our calculations that the exciton's binding energies for the high energy E(33) and E(44) transitions are large enough, indicating clearly they are also excitonic in nature. More importantly, the logarithmic Kane-Mele correction, successful for the low energy E(11) and E(22) transitions, is now found to fail for describing the many-body effects in the higher energy transitions, which is consistent with the experimental result of Araujo et al (2007 Phys. Rev. Lett. 98 067401). Finally, it is interesting to find the possibility of a crossover effect between the E(33) and E(44) energies for certain mod 1 chiralities and the family behavior of the many-body corrections in the E(33) and E(44) transitions, both of which are well supported by the recent experiment of Haroz et al (2008 Phys. Rev. B 77 125405).

Exciton effect in deformed carbon nanotubes.

The exciton states in deformed single-walled carbon nanotubes (SWNTs), under two kinds of strain, i.e., uniaxial and torsional, are theoretically studied in the Su-Schrieffer-Heeger (SSH) model, supplemented by long-range Coulomb interactions. It is found that for semiconducting zigzag tubes, the exciton binding energy E(b) and the (quasi-)continuum edge E(c) are very sensitive to the uniaxial strain, but not to the torsional one, showing two different kinds of variation behaviour of E(b) with increasing uniaxial strain, of which one decreases monotonically, and the other first increases and then decreases. Additionally, the excitons in torsionally distorted armchair tubes and uniaxially strained metallic zigzag tubes have also been studied, showing increased E(b) and E(c) with increasing strain.