A New Galactoglucomannan from the Mycelium of the Medicinal Parasitic Fungus Cordyceps cicadae and Its Immunomodulatory Activity In Vitro and In Vivo.

A new galactoglucomannan (C-0-1) was purified from the medicinal parasitic fungus of Cordyceps cicadae using an anion-exchange column and gel permeation column. The results of high-performance liquid chromatography and high-performance gel permeation chromatography indicated that C-0-1 consists of galactose, glucose, and mannose in a ratio of 5:1:4 and has a molecular weight of 23.3 kDa. The combined structural elucidation analysis methods including partial acid hydrolysis, methylation analysis, and NMR experiments revealed that C-0-1 was a comb-like polysaccharide with a core structure including (1→2)-α-D-Manp residues in the backbone and branches at O-6 of the main chain. (1→4)-α-D-Glcp, (1→2)-ß-D-Galf, (1→2,6)-ß-D-Galf, and terminal ß-Galf were located at the side chains. An in vitro experiment using RAW 264.7 cells indicated that C-0-1 exhibits good immunomodulatory activity by enhancing inducible nitric oxide synthase secretion and the production of some major inflammatory cytokines. On inhibiting the cytokine production using anti-pattern recognition receptors antibodies, it was revealed that the activation of macrophages is mainly carried out by C-0-1 through the mannose receptor. Toll-like receptor 4 and Toll-like receptor 2 were also involved in this identification process. An in vivo experiment on immunosuppressive mice treated with cyclophosphamide indicated that C-0-1 improves the secretion of serum-related cytokines (IFN-γ, TNF-α, IL-2, IL-4, and IL-10) and affects the balance of T helper cells Th1/Th2. Given the structural and bioactivity similarity between Cordyceps cicadae and Cordyceps sinensis, we can conclude that Cordyceps cicadae could be used as an important medicinal fungus like Cordyceps sinensis.

Astaxanthin attenuated hyperuricemia and kidney inflammation by inhibiting uric acid synthesis and the NF-κ B/NLRP3 signaling pathways in potassium oxonate and hypoxanthine-induced hyperuricemia mice.

Inflammation is an important pathological feature of hyperuricemia, which in turn aggravates hyperuricemia. Astaxanthin is a carotenoid with strong antioxidant capacity and possesses many biological activities. This study was aimed to evaluate the effect of astaxanthin (ASX) on hyperuricemia and kidney inflammation in potassium oxonate (PO) and hypoxanthine (HX)-induced hyperuricemic mice. Male ICR mice were administered intragastrically with PO and HX (250 mg/kg, respectively) for 14 days. ASX was given by gavage one hour after PO and HX administration. ASX treatment significantly reversed PO and HX-induced hyperuricemia and kidney inflammation in mice as evidenced by decreased serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), and inflammatory factors (IL-1ß, IL-6, and TNF-α) and increased activities of antioxidant enzymes (CAT, SOD and GSH-Px). Furthermore, ASX administration effectively inhibited the activities of key enzymes related to UA synthesis (xanthine oxidase (XOD) and adenosine deaminase (ADA)) and modulated the protein expressions of NF-κ B p65, p-NF-κ B p65, Iκ Bα, p-Iκ Bα, NLRP3, ASC, Caspase-1, and cleavedCaspase-1 involved in inflammation pathways. Our results suggested that ASX improved hyperuricemia and kidney inflammation induced by PO and HX, probably by reducing UA synthesis and suppressing the NF-κ B and NLRP3 pathways simultaneously.

MicroRNA-155 Participates in Smoke-Inhalation-Induced Acute Lung Injury through Inhibition of SOCS-1.

Smoke inhalation causes acute lung injury (ALI), a severe clinical disease with high mortality. Accumulating evidence indicates that microRNA-155 (miR-155) and suppressor of cytokine signaling 1 (SOCS-1), as mediators of inflammatory response, are involved in the pathogenesis of ALI. In this paper, we explored the proinflammatory mechanism of miR-155 in smoke-inhalation-induced ALI. Our data revealed that smoke inhalation induces miR-155 expression, and miR-155 knockout (KO) significantly ameliorates smoke-inhalation-induced lung injury in mice. Neutrophil infiltration and myeloperoxidase (MPO), macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) expressions were decreased in miR-155-/- mice after smoke inhalation as well. Real-time RT-PCR and immunoblotting results showed that SOCS-1 level was remarkably increased in miR-155-/- mice after smoke exposure. Furthermore, the experiments performed in isolated miR-155 KO pulmonary neutrophils demonstrated that the lack of SOCS-1 enhanced inflammatory cytokines (MIP-2 and KC) secretion in response to smoke stimulation. In conclusion, smoke induces increased expression of miR-155, and miR-155 is involved in inflammatory response to smoke-inhalation-induced lung injury by inhibiting the expression of SOCS-1.

High Molecular Weight Hyaluronan Suppresses Macrophage M1 Polarization and Enhances IL-10 Production in PM2.5-Induced Lung Inflammation.

PM2.5 is particulate matter with a diameter of 2.5 µm or less. Airway macrophages are the key players regulating PM2.5-induced inflammation. High molecular weight hyaluronan (HMW-HA) has previously been shown to exert protective effects on PM2.5-induced acute lung injury and inflammation. However, little is known about the detailed mechanism. In this study, we aimed to determine whether HMW-HA alleviates PM2.5-induced pulmonary inflammation by modulating macrophage polarization. The levels of M1 biomarkers TNF-α, IL-1ß, IL-6, CXCL1, CXCL2, NOS2 and CD86, as well as M2 biomarkers IL-10, MRC1, and Arg-1 produced by macrophages were measured by ELISA, qPCR, and flow cytometry. In addition, the amount of M1 macrophages in lung tissues was examined by immunofluorescence of CD68 and NOS2. We observed a decline in PM2.5-induced M1 polarization both in macrophages and lung tissues when HMW-HA was administered simultaneously. Meanwhile, western blot analysis revealed that PM2.5-induced JNK and p38 phosphorylation was suppressed by HMW-HA. Furthermore, in vitro and in vivo studies showed that co-stimulation with HMW-HA and PM2.5 promoted the expression and release of IL-10, but exhibited limited effects on the transcription of MRC1 and ARG1. In conclusion, our results demonstrated that HMW-HA ameliorates PM2.5-induced lung inflammation by repressing M1 polarization through JNK and p38 pathways and promoting the production of pro-resolving cytokine IL-10.

Low-molecular-mass hyaluronan induces pulmonary inflammation by up-regulation of Mcl-1 to inhibit neutrophil apoptosis via PI3K/Akt1 pathway.

Although low-molecular-mass hyaluronan (LMMHA) has been implicated in pulmonary inflammatory diseases, the signalling pathway of LMMHA (200 000 molecular weight) that initiates the inflammatory response in lung is still unknown. In this study, we evaluate the role of phosphoinositide 3-kinase (PI3K) and its downstream signalling pathway in LMMHA-induced lung inflammatory responses. Our results indicate that pharmacological inhibition of PI3K or genetic deletion of Akt1 enhances neutrophil apoptosis, attenuates neutrophil influx into the lungs of mice and diminishes the expression of pro-inflammatory factors such as interleukin-6, keratinocyte cell-derived chemokine and pro-matrix metalloproteinase-9 in bronchoalveolar lavage fluid after intratracheal administration of LMMHA. More importantly, we found that PI3K/Akt1 participates in LMMHA-induced inflammatory responses, which are mainly mediated by the myeloid leukaemia cell differentiation protein (Mcl-1). Our study suggests that LMMHA induced significantly increased levels of inflammatory factors in bronchoalveolar lavage fluid and activation of the PI3K/Akt1 pathway, which up-regulates the expression of the anti-apoptotic protein Mcl-1 and inhibits the activation of caspase-3, thereby suppressing neutrophil apoptosis to trigger lung inflammation. These findings reveal a novel molecular mechanism underlying sterile inflammation and provides a new potential target for the treatment of pulmonary disease.

SOCS-1 ameliorates smoke inhalation-induced acute lung injury through inhibition of ASK-1 activity and DISC formation.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of apoptosis and pro-inflammatory cytokine signaling, two major contributors to the pathogenesis of ALI. We have found that SOCS-1 protects lung epithelial cells from smoke-induced apoptosis through two mechanisms. One is that SOCS-1 enhances degradation of ASK-1 and diminishes cleavage of pro-caspase-3 to repress smoke-triggered apoptosis in lung epithelial cells. The other is that SOCS-1 represses smoke-triggered DISC formation through altering TRADD-caspase-8 interaction rather than TNFR-1-TRADD interaction or TNFR-1-TRAF-2 interaction. In conclusion, SOCS-1 relieves smoke inhalation-induced lung injury by repressing ASK-1 and DISC-mediated epithelium apoptosis.

The Therapeutic Effect and the Possible Mechanism of C-Phycocyanin in Lipopolysaccharide and Seawater-Induced Acute Lung Injury.

Background: Seawater drowning-induced acute lung injury (ALI) is a severe clinical condition characterized by increased alveolar-capillary permeability, excessive inflammatory response, and refractory hypoxemia. C-phycocyanin (C-PC), a biliprotein found in blue-green algae such as spirulina platensis, is widely used in the food and dietary nutritional supplement fields due to its beneficial pharmacological effects. Previous studies have revealed that C-PC has anti-inflammatory, antioxidant, and anti-apoptotic activities. Purpose: Therefore, this study investigated the protective effect and underlying mechanisms of C-PC on lipopolysaccharide (LPS) and seawater (SW) induced ALI (SW and LPS-induced ALI). Methods: An SW and LPS mouse model of ALI mice was established through intratracheal administration of 5mg/kg LPS and 25% SW. Different doses of C-PC (100, 200 and 400 mg/kg) were administered by intraperitoneal injection for seven days. In addition, gap junction communication in RAW264.7 and MLE-12 cells was determined following stimulation with 25% SW and 10 µg/ml LPS after treatment with C-PC (120 µg/ml). Moreover, the arterial partial pressure of oxygen, lung wet/dry weight ratios, total protein content and MPO levels in the bronchoalveolar lavage fluid (BALF), and the histopathologic and ultrastructure staining of the lung tissues were determined. The oxidative stress index, levels of the pro-inflammatory mediators, epithelial cell viability and apoptosis, and the regulatory effect of C-PC on the NF-κB/NLRP3 axis were investigated. Results: The results showed that C-PC significantly alleviated pathological damages, suppressed oxidative stress, inflammation and apoptosis, and enhanced the viability of epithelial cells in the lung tissues. Furthermore, C-PC was shown to inhibit activation of the NF-κB/NLRP3 pathway and the formation of the NLRP3 inflammasome complex. Conclusions: In conclusion, C-PC shows promising therapeutic value in SW and LPS-induced ALI/ARDS, providing new insight into ALI/ARDS treatment.

Ameliorative Effects of Peptide Phe-Leu-Ala-Pro on Acute Liver and Kidney Injury Caused by CCl4 via Attenuation of Oxidative Stress and Inflammation.

Acute liver injury (ALI) and acute kidney injury (AKI) are significantly affected by the antioxidant status. In the present study, the protective effect and mechanism of the collagen peptide Phe-Leu-Ala-Pro (FLAP) in mice with ALI and AKI induced by carbon tetrachloride (CCl4) were examined. The results showed that FLAP effectively improved the liver mass index, the renal mass index, and the histopathological morphology. FLAP treatment significantly decreased the levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (BUN), and creatinine (CRE) but increased the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). The protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), p-protein kinase B (p-AKT), and p-phosphatidylinositol-3-kinase (p-PI3K) in the liver and kidneys were significantly up-regulated after FLAP treatment. FLAP down-regulated the levels of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), and nuclear factor-κ B (NF-κB) in liver and kidney tissues. Thus, FLAP may play a protective role in ALI and AKI by attenuating oxidative stress and inflammation mediated by the Nrf2/anti-response element (ARE) and PI3K/AKT/NF-κB pathways.

Astragalus polysaccharide strengthens the inflammatory and immune responses of Brucella suis S2-infected mice and macrophages.

Brucella infection is one of the most serious zoonoses worldwide, affecting humans and domestic and wild animals. Astragalus polysaccharide (APS) is extracted from astragalus, which exhibits bioactive properties, including immunomodulation and anti-tumour and antiviral activity. The present study revealed that APS treatment promoted macrophage activation, the production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-12 and interferon-γ, and Brucella clearance in murine macrophages and spleens. APS treatment was also demonstrated to protect the integrity of macrophages during infection with live attenuated Brucella suis strain 2 (B. suis S2). The results from in vitro experiments were consistent with the findings from the in vivo study, showing the elevated secretion of TNF-α and nitric oxide in APS-treated murine peritoneal macrophages following B. suis S2 infection. The current study demonstrated the potential of APS in the control and treatment of Brucella infection, and the enhancement of host inflammatory and immune responses.

Protective effect of polysaccharides of sea cucumber Acaudina leucoprocta on hydrogen peroxide-induced oxidative injury in RAW264.7 cells.

The aim of this experiment was to investigate the protective effects of polysaccharides of sea cucumber Acaudina leucoprocta (ALP) against hydrogen peroxide (H2O2) induced oxidative injury in RAW264.7 cells. Analysis of monosaccharide composition and structure of one fraction from ALP (ALPN) were analyzed by High Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared Spectoscopy (FT-IR). The results showed that ALPN contain sulfate groups, which is sulfated polysaccharides. The results from MTT assay indicated that ALPN could markedly increase viability of cells compared with RAW264.7 cells exposed to H2O2. Moreover, ALPN significantly increased the levels of catalase (CAT), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD), decreased the production of malondialdehyde (MDA) and lactate dehydrogenase (LDH) in RAW264.7 cells. The data from RT-PCR showed that ALPN (300 µg/mL) could increase the gene expression levels of SOD1 and GPX1. ALPN could also observably increase the protein expression level of Nrf2 and decrease the protein expression level of Keap1 with western blot. Collectively, this study suggested that polysaccharides of sea cucumber Acaudina leucoprocta (ALP) could effectively protect RAW264.7 cells against H2O2-induced oxidative injury. This protection mechanism may be related to activation of the Nrf2/Keap1 signaling pathway.

High molecular weight hyaluronan attenuates fine particulate matter-induced acute lung injury through inhibition of ROS-ASK1-p38/JNK-mediated epithelial apoptosis.

Inhalation of fine particulate matter (PM2.5) is asscoiated with lung injury. High molecular weight hyaluronan (HMW-HA) is an essential constituent of extracellular matrix (ECM), exhibiting anti-oxidative and anti-inflammatory properties when administered by injection, inhalation, nebulization or gene delivery of HA synthases. The aim of the present study is to determine whether HMW-HA alleviates PM2.5-induced acute lung injury (ALI) and investigate the underlying mechanisms. We observed that HMW-HA suppressed pathological injury, inflammation, oxidative stress, edema and epithelial damage caused by PM2.5 in the lungs of the rats. The protective mechanism of HMW-HA was further explored in vitro. The results elucidated that reactive oxygen species (ROS) was involved in PM2.5-induced cell apoptosis, and HMW-HA mitigated the oxidative potential of PM2.5, subsequently inhibiting phosphorylation of ASK1 at Thr845, downstream phosphorylation of p38 and JNK, and eventual apoptosis. Our study indicates that HMW-HA is a promising strategy in the prevention of PM2.5-induced pulmonary damage.

SOCS-1 Suppresses Inflammation Through Inhibition of NALP3 Inflammasome Formation in Smoke Inhalation-Induced Acute Lung Injury.

Smoke inhalation leads to acute lung injury (ALI), a devastating clinical problem associated with high mortality rates. Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of proinflammatory cytokine signaling. We have found that adenoviral gene transfer of SOCS-1 ameliorates smoke inhalation-induced lung injury in C57BL/6 mice. We also found that the release of adenosine triphosphate (ATP) was increased post smoke exposure, while oxidized ATP, an inhibitor of purinergic P2X7 receptor, suppressed smoke-induced NALP3 inflammasome assembly, caspase-1 activation, and K+ efflux. Similar to oxidized ATP, high protein level of SOCS-1 dampened the formation of NALP3 inflammasome and the activation of caspase-1 and IL-1ß induced by smoke exposure in mouse alveolar macrophages. In conclusion, SOCS-1 relieves smoke inhalation-induced pulmonary inflammation and injury by inhibiting NALP3 inflammasome formation.

Extraction, characterization, and biological activity of polysaccharides from Sophora flavescens Ait.

Four water-soluble polysaccharides, designated as SF1, SF2, SF3 and SF4, were efficiently extracted from the roots of Sophora flavescens by mechanochemistry under the conditions of rotational speed of 400rpm, grinding time of 10min, powder to ball weight ratio of 1:20, and Na2CO3 loading of 7wt%. The results obtained indicated that all of these four acid heteropolysaccharides are composed of rhamnose, arabinose, xylose, mannose, glucose and galactose, with the average molecular weights of 400.9, 98.6, 99.3, 42.7kDa, respectively. In vitro, SF4 showed the most significant scavenging activity on superoxide radical, ABTS, and DPPH radical, while SF3 had the most significant scavenging activity on hydroxyl radical. Immunological tests demonstrated that SF1, SF2, SF3 and SF4 significantly stimulated nitric oxide production without cytotoxicity in macrophages and promoted splenocyte proliferation. These data suggest that the four polysaccharides fractions have the potential as novel natural sources of antioxidative and immunopotentiating agents.

[Pattern recognition and activation effect of mast cells in vitro infected by Brucella suis S2].

OBJECTIVE: To study the pattern recognition and activation effect of mast cells infected by Brucella (B.) suis S2. METHODS: Mast cells derived from bone marrow in vitro were infected by B.suis S2. The change in the cell morphology was observed with Wrights-Giemsa's staining, and cell degranulation was tested with toluidine blue staining. The extracellular levels of histamine, IFN-γ and IL-12 of mast cells at 1 and 12 h after infection were detected by indirect ELISA. The uptake pattern of mast cells to B.suis S2 was determined by laser-scanning confocal microscopy. The expressions of TLR4 and TLR8 mRNA were detected by RT-PCR at 12 and 24 h after infection by B.suis S2, and the TLR4 and TLR8 protein expressions were detected by flow cytometry at 24 h. RESULTS: The form of mast cells infected by B.suis S2 was obviously changed. Significant degranulation was observed at 1 h, and at 1, 12 h post-infection by B.suis S2, the content of histamine secreted by mast cells was significantly higher than that of normal control group (P<0.05), and IFN-γ and IL-12 were not found. At 30 min and 1 h, B.suis S2 bound to the mast cell surface and were not uptaken into the mast cells. Compared with the control group, the expression of TLR4 mRNA increased after 12 h infection by B.suis S2, and was reduced at 24 h. The expression of TLR4 protein rose at 24 h, but the expression of TLR8 mRNA and protein did not alter at 12 and 24 h after infection by B.suis S2. CONCLUSION: B.suis S2 can bind to the cell surface and activate mast cells, cause their degranulation, induce the release of histamine, but IFN-γ and IL-12 were not found during the observing time. The mechanism may be that B.suis S2 can be recognized by mast cells through TLR4 but can not be phagocytosed by mast cells.

[Comparison of macrophages and dendritic cells infected by Brucella S(2)].

Objective To make a comparison of the characteristics between macrophages and dendritic cells (DC) infected by Brucella suis (B. suis) S(2);. Methods Wrights-Giemsa's stainning was used to observe the cell morphology and calculate the phagocytic rate. ELISA was employed to detect the expressions of IL-12 and TNF-α in cell culture supernatants as well as the contents of IFN-γ and IL-4 in the co-culture with T cells. With annexin-V-FITC/PI double staining, the cell apoptosis rate was determined by flow cytometry. Results 1 h after infected by B. suis S(2);, the phagocytic rate of macrophages was (43.6±4.8)%, which was significantly higher than that of the DC (16.3±2.7)% (P<0.05). The apoptosis rates of normal macrophages and macrophages 6, 12 and 24 h after infected by B. suis S(2); were (3.09±1.21)%, (19.89±1.36)%, (22.73±2.21)% and (42.44±3.12)%, respectively, which were dramatically higher than those of the DC at the corresponding time points, being (1.82±0.01)%, (3.76±0.13)%, (7.87±0.56)% and (9.08±0.23)%, respectively (P<0.05). The levels of IL-12 secreted by macrophages 24 and 48 h after infected by B. suis S(2); were significantly lower than those by the DC (P<0.01). At 24, 48 and 72 h, the levels of TNF-α secreted by macrophages were dramatically lower than those by the DC (P<0.01), and the levels of IFN-γ in the co-culture supernatants of macrophages and T cells were significantly lower than those in DC and T cell co-culture (P<0.01). Conclusion Macrophages have a better ability in phagocytosing B. suis S(2); than DC and the apoptosis rate of macrophages is higher than that of DC after infected by B. suis S(2);, but in activating and inducing the cellular immune response and presenting antigen, DC are stronger than macrophages.