PRMT2 silencing regulates macrophage polarization through activation of STAT1 or inhibition of STAT6.

BACKGROUND: Macrophages play significant roles in innate immune responses and are heterogeneous cells that can be polarized into M1 or M2 phenotypes. PRMT2 is one of the type I protein arginine methyltransferases involved in inflammation. However, the role of PRMT2 in M1/M2 macrophage polarization remains unclear. Our study revealed the effect and mechanism of PRMT2 in macrophage polarization. METHODS: Bone marrow-derived macrophages (BMDMs) were polarized to M1 or M2 state by LPS plus murine recombinant interferon-γ (IFN-γ) or interleukin-4 (IL-4). Quantitative polymerase chain reaction (qPCR), western blot and flow cytometry (FCM) assay were performed and analyzed markers and signaling pathways of macrophage polarization. RESULTS: We found that PRMT2 was obviously upregulated in LPS/IFN-γ-induced M1 macrophages, but it was little changed in IL-4-induced M2 macrophages. Furthermore, PRMT2 konckdown increased the expression of M1 macrophages markers through activation of STAT1 and decreased the expression of M2 macrophages markers through inhibition of STAT6. CONCLUSIONS: PRMT2 silencing modulates macrophage polarization by activating STAT1 to promote M1 and inhibiting STAT6 to attenuate the M2 state.

ATAXIA TELANGIECTASIA MUTATED PROTECTS AGAINST LIPOPOLYSACCARIDE-INDUCED BLOOD-BRAIN BARRIER DISRUPTION BY REGULATING ATK/DRP1-MEDIATED MITOCHONDRIAL HOMEOSTASIS.

ABSTRACT: Background: Protein kinase ataxia telangiectasia mutated (ATM) regulates the function of endothelial cells and responds quickly to endotoxin. However, the function of ATM in lipopolysaccharide (LPS)-induced blood-brain barrier (BBB) disruption remains unknown. This study aimed to investigate the role and underlying mechanism of ATM in the regulation of the BBB function in sepsis. Methods: We used LPS to induce BBB disruption in vivo and to establish an in vitro model of cerebrovascular endothelial cells. Blood-brain barrier disruption was assessed by measuring Evans blue leakage and expression of vascular permeability regulators. To investigate the role of ATM, its inhibitor AZD1390 and clinically approved doxorubicin, an anthracycline that can activate ATM, were administered as scheduled. To explore the underlying mechanism, protein kinase B (AKT) inhibitor MK-2206 was administered to block the AKT/dynamin-related protein 1 (DRP1) pathway. Results: Lipopolysaccharide challenge induced significant BBB disruption, ATM activation, and mitochondrial translocation. Inhibiting ATM with AZD1390 aggravated BBB permeability as well as the following neuroinflammation and neuronal injury, while activation of ATM by doxorubicin abrogated these defects. Further results obtained in brain microvascular endothelial cells showed that ATM inhibition reduced the phosphorylation of DRP1 at serine (S) 637, promoted excessive mitochondrial fission, and resulted in mitochondrial malfunction. By activating ATM, doxorubicin increased the protein binding between ATM and AKT and promoted the phosphorylated activation of AKT at S473, which could directly phosphorylate DRP1 at S637 to repress excessive mitochondrial fission. Consistently, the protective role of ATM was abolished by the AKT inhibitor MK-2206. Conclusions: Ataxia telangiectasia mutated protects against LPS-induced BBB disruption by regulating mitochondrial homeostasis, at least in part, through the AKT/DRP1 pathway.

Siglec-G Deficiency Ameliorates Hyper-Inflammation and Immune Collapse in Sepsis via Regulating Src Activation.

Hyper-inflammation during acute phase and sequential hypo-inflammation during immunosuppressive phase in macrophages/monocytes lead to multiorgan failure syndrome and immune collapse of sepsis, in which toll-like receptor (TLR)-triggered inflammatory responses play a major role. Here, we reported that Siglecg deficiency attenuated TLR4-triggered pro-inflammatory cytokine production and increased anti-inflammatory cytokine [interleukin-10 [IL-10]] production in vivo and in vitro at both acute and immunosuppressive phases. Siglecg deficiency also protected mice from lipopolysaccharide (LPS)-induced sepsis with less inflammation in the lung and less tissue destruction in the spleen. Siglec-G inhibited proto-oncogene tyrosine-protein kinase Src (Src) activation via recruiting and activating tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP1) through immunoreceptor tyrosine-based inhibitory motif (ITIM) domain. Src could inhibit TLR4-induced inflammatory cytokines and promote anti-inflammatory cytokine IL-10. Mechanical investigation showed that Src could interact with and phosphorylate STAT3. Src could also promote HIF1α degradation through activating GSK3ß. Our study reveals that Siglec-G orchestrates TLR-induced inflammation, which outlines that blocking Siglec-G or activating Src may be a promising strategy for both acute and chronic inflammatory diseases.

Isolation and Antibiotic Resistant Research of Tetragenococcus halophilus from Xuanwei Ham, A China High-Salt-Fermented Meat Products.

We assessed the prevalence of antibiotic resistant and antibiotic resistance genes for 49 Tetragenococcus halophilus (T. halophilus) strains isolated from Xuawei ham in China. The antibiotic resistance phenotype was detected by the Bauer-Kirby (K-B) method and the results showed that 49 isolates can be considered completely susceptible to penicillin, ampicillin, amoxicillin, cefradine, cefotaxime, tetracyclines, minocycline, doxycycline, and vancomycin, but resistant to gentamicin, streptomycin, neomycin, polymyxinB, cotrimoxazole. This resistance was sufficiently high to consider the potential for acquisition of transmissible determinants. A total of 32 isolates were resistant to ofloxacin, 4 isolates were resistant to ciprofloxacin and chloramphenicol, and 2 isolates were resistant to ceftazidime and ticarcillin. The antibiotic resistance genes were detected by routine polymerase chain reaction (PCR). Among the 26 antibiotic resistance genes, 5 varieties of antibiotic resistance genes, including acrB, blaTEM, AAda1, SulII, and GyrB were detected and the detection rates were 89.79%, 47.7%, 16.33%, 77.55%, and 75.51%, respectively. The potential acquisition of transmissible determinants for antibiotic resistance and antibiotic resistance genes identified in this study necessitate the need for a thorough antibiotic resistance safety assessment of T. halophilus before it can be considered for use in food fermentation processes.

Identification of the Pol Gene as a Species-Specific Diagnostic Marker for Qualitative and Quantitative PCR Detection of Tricholoma matsutake.

Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake.

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice.

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C13H8N4OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1 h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.

Ginsenoside Rg1 protects against sepsis-associated encephalopathy through beclin 1-independent autophagy in mice.

BACKGROUND: Sepsis-associated encephalopathy (SAE), a commonly complicated syndrome, is associated with increased mortality in patients with sepsis. Currently, no specific diagnostic test or effective intervention exists to improve long-term consequences on cerebral function. Ginsenoside Rg1 (Rg1), a major component in ginseng, was reported to have pleiotropic properties including anti-inflammation and neuroprotection. The aim of our study was to investigate the protective effect of Rg1 on SAE and the potential mechanism. MATERIALS AND METHODS: SAE model was prepared by inducing cecal ligation and puncture (CLP) in mice. Rg1 was injected 1 h before the CLP operation. Survival rate within 7 d after operation was analyzed. Surviving mice were subjected to Morris water maze tests and the brains were collected for histopathologic evaluation and immunohistochemistry. The hippocampus was obtained for Western blot, real time polymerase chain reaction, and enzyme-linked immunosorbent assay analysis. RESULTS: Rg1 improved the postoperative survival rate and protected against sepsis-associated learning and memory impairments (Morris water maze). Besides, Rg1 was able to attenuate brain histopathologic changes (hematoxylin and eosin staining), suppress Iba1 activation, decrease the expressions of inflammatory cytokines (tumor necrosis factor α, interleukin 1ß, and interleukin 6), and reduce neuronal apoptosis (cleaved caspase 3 activation) in hippocampus. Furthermore, the mechanism study showed that Rg1 suppressed the expressions of light chain 3-II and p62 in hippocampus but not beclin 1. CONCLUSIONS: These findings suggested that Rg1 improved the survival rate and ameliorated cognitive impairments partially through regulating cerebral inflammation and apoptosis. In addition, the action mechanism might be noncanonical beclin 1-independent autophagy pathway. Rg1 may be a promising treatment strategy for SAE.

The Specific Protein Kinase R (PKR) Inhibitor C16 Protects Neonatal Hypoxia-Ischemia Brain Damages by Inhibiting Neuroinflammation in a Neonatal Rat Model.

BACKGROUND Brain injuries induced by hypoxia-ischemia in neonates contribute to increased mortality and lifelong neurological dysfunction. The specific PKR inhibitor C16 has been previously demonstrated to exert a neuroprotective role in adult brain injuries. However, there is no recent study available concerning its protective role in hypoxia-ischemia-induced immature brain damage. Therefore, we investigated whether C16 protects against neonatal hypoxia-ischemia injuries in a neonatal rat model. MATERIAL AND METHODS Postnatal day 7 (P7) rats were used to establish classical hypoxia-ischemia animal models, and C16 postconditioning with 100 ug/kg was performed immediately after hypoxia. Western blot analysis was performed to quantify the phosphorylation of the PKR at 0 h, 3 h, 6 h, 12 h, 24 h, and phosphorylation of NF-κB 24h after hypoxia exposure. The TTC stain for infarction area and TUNEL stain for apoptotic cells were assayed 24 h after the brain hypoxia. Gene expression of IL-1ß, IL-6, and TNF-α was performed at 3 h, 6 h, 12 h, and 24 h. RESULTS The level of PKR autophosphorylation was increased dramatically, especially at 3 h (C16 group vs. HI group, P<0.01). Intraperitoneal C16 administration reduced the infarct volume and apoptosis ratio after this insult (C16 group vs. HI group<0.01), and C16 reduced proinflammatory cytokines mRNA expression, partly through inhibiting NF-κB activation (C16 group vs. HI group<0.05). CONCLUSIONS C16 can protect immature rats against hypoxia-ischemia-induced brain damage by modulating neuroinflammation.

Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltration of M2 macrophages.

Ginsenoside Rg1 (Rg1), the major effective component of ginseng, has been reported to have potent anti-inflammatory properties. However, the effect of ginsenoside Rg1 on lipopolysaccharide (LPS) -induced acute lung injury (ALI) in mice was unknown. The present study was designed to investigate the protective role of Rg1 on LPS-induced ALI and explore the potential mechanisms. The mice were divided randomly into four groups: the sham group, the LPS group and the LPS+Rg1 (40 mg/kg or 200mg/kg) pretreatment groups. All mice received Rg1 or an equivalent volume of phosphate buffer saline (PBS) intraperitoneally 1h before LPS administration. Edema quantification, histology, and apoptosis were detected 6h after LPS administration. The number of inflammatory cells, the percentage of alternative activated (M2) macrophages and the exudate quantification in bronchoalveolar lavage fluid (BALF) were evaluated. The caspase 3 expression, and the levels of phosphorylated IκB-α and p65 were tested. The results showed that the Rg1 pretreatment group markedly improved lung damage, modulated the infiltration of neutrophils and M2 macrophages, prevented the production of protein and proinflammatory cytokines in BALF, and inhibited apoptosis in lung. We also found that Rg1 suppressed NF-κB and caspase 3 activation. These data suggest that Rg1 plays a protective role against LPS-induced ALI by ameliorating inflammatory responses, regulating the infiltration of M2 macrophages, and inhibiting pulmonary cell apoptosis.