Salvianolic acid B protects against acute lung injury by decreasing TRPM6 and TRPM7 expressions in a rat model of sepsis

This study aimed to investigate the protective effects of salvianolic acid B (SA‐B) on acute lung injury (ALI) through decreasing the expressions of channel kinase's TRPM6 and TRPM7. Wistar Septic rat models were established by lipopolysaccharide (LPS), which were separated into the control, lipopolysaccharide (LPS), SA‐B, SA‐B + si‐TRPM6, SA‐B + si‐TRPM7, si‐TRPM6, and si‐TRPM7 groups. Arterial blood gas, protein content, total white blood cell (WBC) count and the percentage of polymorphonuclear neutrophils (PMN%) were measured. Levels of TNF‐α and IL‐6 levels in bronchoalveolar lavage fluid (BALF) were monitored. Lung coefficient, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity were conducted by MPO and SOD kit. The mRNA expressions of TRPM6 and TRPM7 were detected by qRT‐PCR. Compared with the control group, the PaO₂ and PaO₂/FiO₂ values exhibited decreases in other group, while the PaCO₂ value_, protein content, total WBC, PMN%, TNF‐α, IL‐6 levels and lung coefficient values all increased. MPO activity in lung tissue increased, while SOD activity decreased. TRPM6 and TRPM7 expressions increased significantly. Compared with the LPS group, the SA‐B, SA‐B + si‐TRPM6, SA‐B + si‐TRPM7, si‐TRPM6, and si‐TRPM7 groups had increased PaO₂ and the PaO₂/FiO₂, while decreased PaCO_2, protein content, total WBC, PMN%, TNF‐α, IL‐6 levels, and lung coefficient. MPO activity in lung tissue decreased while SOD activity increased. Decreased mRNA expressions of TRPM6 and TRPM7 in the SA‐B, SA‐B + si‐TRPM6, and SA‐B + si‐TRPM6 groups were observed. Through decreasing the expressions of the channel kinase TRPM6 and TRPM7, SA‐B protects against ALI in septic rats.     

Enhanced protection against lipopolysaccharide‐induced acute lung injury by autologous transplantation of adipose‐derived stromal cells combined with low tidal volume ventilation in rats

Adipose‐derived stromal cells (ADSCs) showed excellent capacity in regeneration and tissue protection. Low tidal volume ventilation (LVT) strategy demonstrates a therapeutic benefit on the treatment of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). This study, therefore, aimed to undertaken determine whether the combined LVT and ADSCs treatment exerts additional protection against lipopolysaccharide (LPS)‐induced ALI in rats. The animals were randomized into seven groups: Group I (control), Group II (instillation of LPS at 10 mg/kg intratracheally), Group III (LPS+LVT 6 ml/kg), Group IV (LPS+intravenous autologous 5 × 10⁶ ADSCs which were pretreated with a scrambled small interfering RNA [siRNA] of keratinocyte growth factor [KGF] negative control), Group V (LPS+ADSCs which were pretreated with a scrambled siRNA of KGF, Group VI (LPS+LVT and ADSCs as in the Group IV), and Group VII (LPS+LVT and ADSCs as in the Group V). We found that levels of tumor necrosis factor‐α, transforming growth factor‐β1, and interleukin (IL)‐1β and IL‐6, the proinflammatory cytokines, were remarkably increased in LPS rats. Moreover, the expressions of ENaC, activity of Na, K‐ATPase, and alveolar fluid clearance (AFC) were obviously reduced by LPS‐induced ALI. The rats treated by ADSCs showed improved effects in all these changes of ALI and further enhanced by ADSCs combined with LVT treatment. Importantly, the treatment of ADSCs with siRNA‐mediated knockdown of KGF partially eliminated the therapeutic effects. In conclusion, combined treatment with ADSCs and LVT not only is superior to either ADSCs or LVT therapy alone in the prevention of ALI. Evidence of the beneficial effect may be partly due to improving AFC by paracrine or systemic production of KGF and anti‐inflammatory properties.     

Effect of an acute moderate‐exercise session on metabolic and inflammatory profile of PPAR‐α knockout mice

Peroxisome proliferator‐activated receptors (PPARs) play a major role in metabolism and inflammatory control. Exercise can modulate PPAR expression in skeletal muscle, adipose tissue, and macrophages. Little is known about the effects of PPAR‐α in metabolic profile and cytokine secretion after acute exercise in macrophages. In this context, the aim of this study was to understand the influence of PPAR‐α on exercise‐mediated immune metabolic parameters in peritoneal macrophages. Mice C57BL/6 (WT) and PPAR‐α knockout (KO) were examined in non‐exercising control (n = 4) or 24 hours after acute moderate exercise (n = 8). Metabolic parameters (glucose, non‐esterified fatty acids, total cholesterol [TC], and triacylglycerol [TG]) were assessed in serum. Cytokine concentrations (IL‐1β, IL‐6, IL‐10, TNF‐α, and MCP‐1) were measured from peritoneal macrophages cultured or not with LPS (2.5 μg/mL) and Rosiglitazone (1 μM). Exercised KO mice exhibited low glucose concentration and higher TC and TG in serum. At baseline, no difference in cytokine production between the genotypes was observed. However, IL‐1β was significantly higher in KO mice after LPS stimulus. IL‐6 and IL‐1β had increased concentrations in KO compared with WT, even after exercise. MCP‐1 was not restored in exercised KO LPS group. Rosiglitazone was not able to reduce proinflammatory cytokine production in KO mice at baseline level or associated with exercise. Acute exercise did not alter mRNA expression in WT mice. Conclusion: PPAR‐α seems to be needed for metabolic glucose homeostasis and anti‐inflammatory effect of acute exercise. Its absence may induce over‐expression of pro‐inflammatory cytokines in LPS stimulus. Moreover, moderate exercise or PPAR‐γ agonist did not reverse this response.     

Salidroside suppressing LPS‐induced myocardial injury by inhibiting ROS‐mediated PI3K/Akt/mTOR pathway in vitro and in vivo

The purpose of the present study was to investigate the effect of salidroside (Sal) on myocardial injury in lipopolysaccharide (LPS)‐induced endotoxemic in vitro and in vivo. SD rats were randomly divided into five groups: control group, LPS group (15 mg/kg), LPS plus dexamethasone (2 mg/kg), LPS plus Sal groups with different Sal doses (20, 40 mg/kg). Hemodynamic measurement and haematoxylin and eosin staining were performed. Serum levels of creatine kinase (CK), lactate dehydrogenase, the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH‐px), glutathione, tumour necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) were measured after the rats were killed. iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway proteins were detected by Western blot. In vitro, we evaluated the protective effect of Sal on rat embryonic heart‐derived myogenic cell line H9c2 induced by LPS. Reactive oxygen species (ROS) in H9c2 cells was measured by flow cytometry, and the activities of the antioxidant enzymes CAT, SOD, GSH‐px, glutathione‐S‐transferase, TNF‐α, IL‐6 and IL‐1β in cellular supernatant were measured. PI3K/Akt/mTOR signalling was examined by Western blot. As a result, Sal significantly attenuated the above indices. In addition, Sal exerts pronounced cardioprotective effect in rats subjected to LPS possibly through inhibiting the iNOS, COX‐2, NF‐κB and PI3K/Akt/mTOR pathway in vivo. Furthermore, the pharmacological effect of Sal associated with the ROS‐mediated PI3K/Akt/mTOR pathway was proved by the use of ROS scavenger, N‐acetyl‐l ‐cysteine, in LPS‐stimulated H9C2 cells. Our results indicated that Sal could be a potential therapeutic agent for the treatment of cardiovascular disease.     

Resveratrol decreases CD45+CD206− subtype macrophages in LPS‐induced murine acute lung injury by SOCS3 signalling pathway

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are life‐threatening condition in critically ill patients. Resveratrol (Res), a natural polyphenol, has therapeutic effect in animal model with ALI; however, whether Res attenuates ALI through modulation of macrophage phenotypes in the animal model remains unknown. We in this study treated LPS‐induced murine ALI with 30 mg/kg Res and observed significantly reduced severity of ALI in the Res‐treated mice 48 hours after Res treatment. Neutrophil infiltrates were significantly reduced, accompanied with lower infiltration of CD45⁺Siglec F^? phenotype macrophages, but higher population of CD45⁺Siglec F⁺ and CD45⁺CD206⁺ alternatively activated macrophages (M2 cells) in the Res‐treated mice with ALI. In addition, the expression of IL‐1beta and CXCL15 cytokines was suppressed in the treated mice. However, Res treatment in mice with myeloid cell‐restricted SOCS3 deficiency did not significantly attenuate ALI severity and failed to increase population of both CD45⁺Siglec F⁺ and CD45⁺CD206⁺ M2 subtype macrophages in the murine ALI. Further studies in wild‐type macrophages revealed that Res treatment effectively reduced the expression of IL‐6 and CXCL15, and increased the expression of arginase‐1, SIRT1 and SOCS3. However, macrophages’ lack of SOCS3 expression were resistant to the Res‐induced suppression of IL‐6 and CXCL15 in vitro. Thus, we conclude that Res suppressed CD45⁺Siglec F^? and CD45⁺CD206^? M1 subtype macrophages through SOCS3 signalling in the LPS‐induced murine ALI.     

S‐allyl cysteine,an active ingredient of garlic,attenuates acute liver dysfunction induced by lipopolysaccharide/ d‐galactosamine in mouse: Underlying mechanisms

In the present study, beneficial effect of S‐allyl cysteine (SAC) was evaluated in the lipopolysaccharide/d ‐galactosamine (LPS/d ‐Gal) model of acute liver injury (ALI). To mimic ALI, LPS and d ‐Gal (50 μg/kg and 400 mg/kg, respectively) were intraperitoneally administered and animals received SAC per os (25 or 100 mg/kg/d) for 3 days till 1 hour before LPS/d ‐Gal injection. Pretreatment of LPS/d ‐Gal group with SAC‐lowered activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase and partially reversed inappropriate alterations of hepatic oxidative stress‐ and inflammation‐related biomarkers including liver reactive oxygen species, malondialdehyde, and hepatic activity of the defensive enzyme superoxide dismutase, ferric reducing antioxidant power (FRAP), toll‐like receptor‐4 (TLR4), cyclooxygenase 2, NLR family pyrin domain containing 3 (NLRP3), caspase 1, nuclear factor κB (NF‐κB), interleukin 1β (IL‐1β), IL‐6, tumor necrosis factor‐α, and myeloperoxidase activity. Additionally, SAC was capable to ameliorate apoptotic biomarkers including caspase 3 and DNA fragmentation. In summary, SAC can protect liver against LPS/d ‐Gal by attenuation of neutrophil infiltration, oxidative stress, inflammation, apoptosis, and pyroptosis which is partly linked to its suppression of TLR4/NF‐κB/NLRP3 signaling.     

Site‐specific and endothelial‐mediated dysfunction of the alveolar‐capillary barrier in response to lipopolysaccharides

Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar‐capillary barrier (ACB) in the lung. In this study, we analyse the site‐specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co‐cultures of H441 with endothelial cells cultured on Transwells^® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro‐inflammatory cytokines and immune‐modulatory molecules were evaluated by ELISA and semiquantitative real‐time PCR. Liquid chromatography–mass spectrometry‐based proteomics (LS‐MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co‐cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro‐inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro‐inflammatory cytokines such as IL‐6 in OEC and in turn induced the reduction of TEER and an increase in SP‐A expression in H441 monoculture, and H441/OEC co‐cultures after LPS treatment from basolateral compartment. LS‐MS‐based proteomics revealed factors associated with LPS‐mediated lung injury such as ICAM‐1, VCAM‐1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial–endothelial crosstalk in the ACB in ALI/ARDS.     

Targeting myeloid differentiation protein 2 by the new chalcone L2H21 protects LPS‐induced acute lung injury

Acute inflammatory diseases are the leading causes of mortality in intensive care units. Myeloid differentiation 2 (MD‐2) is required for recognizing lipopolysaccharide (LPS) by toll‐like receptor 4 (TLR4), and represents an attractive therapeutic target for LPS‐induced inflammatory diseases. In this study, we report a chalcone derivative, L2H21, as a new MD2 inhibitor, which could inhibit LPS‐induced inflammation both in vitro and in vivo. We identify that L2H21 as a direct inhibitor of MD‐2 by binding to Arg⁹⁰ and Tyr¹⁰² residues in MD‐2 hydrophobic pocket using a series of biochemical experiments, including surface plasmon response, molecular docking and amino acid mutation. L2H21 dose dependently inhibited LPS‐induced inflammatory cytokine expression in primary macrophages. In mice with LPS intratracheal instillation, L2H21 significantly decreased LPS‐induced pulmonary oedema, pathological changes in lung tissue, protein concentration increase in bronchoalveolar lavage fluid, inflammatory cells infiltration and inflammatory gene expression, accompanied with the decrease in pulmonary TLR4/MD‐2 complex. Meanwhile, administration with L2H21 protects mice from LPS‐induced mortality at a degree of 100%. Taken together, this study identifies a new MD2 inhibitor L2H21 as a promising candidate for the treatment of acute lung injury (ALI) and sepsis, and validates that inhibition of MD‐2 is a potential therapeutic strategy for ALI.     

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte‐derived dendritic cells

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro‐inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte‐derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF‐α, ‐β and ‐γ were significantly upregulated in the H5N1 group. Pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐18) were highly upregulated in early mid (IL‐1), and late (IL‐6) phases of H5N1 virus infection. IL‐8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus‐induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV‐infected cells.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

硫化氢对大鼠内毒素性急性肺损伤肺泡表面活性物质的影响

目的：观察脂多糖（LPS）所致内毒素性急性肺损伤（ALI）大鼠肺泡表面活性物质（PS）的变化及硫化氢（H2S）对PS的影响,探讨H2S对肺脏的作用机制。方法：雄性SD大鼠共48只,随机分为6组（n=8）：空白对照组、LPS组、LPS＋NaHS低、中、高剂量组、LPS＋PPG组。空白对照组给予生理盐水,LPS组给予LPS,LPS＋NaHS低、中、高剂量组和LPS＋PPG组分别在给予LPS3h时腹腔注射低、中、高剂量氢硫化钠（NariS）或炔丙基甘氨酸（PPG）。各组均于给予生理盐水或LPS6h时电镜下观察肺泡Ⅱ型上皮细胞（AEC-Ⅱ）的形态改变,检测血浆中H2S含量、肺组织中胱硫醚-γ-裂解酶（CSE）活性、肺泡灌洗液（BALF）中总蛋白（1P）和总磷脂（TPL）含量、及肺组织中肺泡表面活性蛋白A、B、C（SP-A、B、C）mRNA表达的变化。结果：①与空白对照组比较,LPS组AEC-Ⅱ超微结构明显受损,血浆中H2S含量、肺组织中CSE活性、BALF中TPL的含量、及肺组织中SP-A、B、CmRNA表达均明显降低（P〈0．05,P〈0．01）,BALF中TP的含量明显增加（P〈0．01）;②与LPS组比较,LPS＋NaHS低、中、高剂量组,AEC-Ⅱ超微结构均有所恢复,血浆中H2S含量、肺组织中CSE海性、SP-AmRNA表达均明显升高（P〈0．05,P〈0．01）;LPS＋NaHS中、高剂量组BALF中吼含量明显增高,SP-BmRNA表达升高（P〈0．05）;LPS＋NaHS高剂量组BALF中,IP含量明显降低（P〈0．05）;LPS＋NaHS各剂量组SP-CznRNA表达无明显变化;③与LPS组比较,LPS＋PPG组AEC-Ⅱ超微结构仍损伤严重,血浆中H2S含量、肺组织中CSE活性、BALF中TPL的含量、及肺组织中SP-A、B、CmRNA表达均明显降低（P〈0．05）,BALF中TP的含量明显升高（P〈0．05）。结论：PS降低是内毒素性ALI的重要病理生理过程,H2S对LPS诱导的ALI有保护性作用,其机制可能与H2S对PS的调节有关。     

8‐Methoxypsoralen protects bovine mammary epithelial cells against lipopolysaccharide‐induced inflammatory injury via suppressing JAK/STAT and NF‐κB pathway

Bovine mastitis is the most common disease in dairy cattle. Bacterial infections are the main cause of mastitis. Lipopolysaccharide (LPS), a major structural component of the cell wall of Escherichia coli, is a good inducer used to replicate inflammation models. 8‐Methoxypsoralen (8‐MOP), a formerly considered photosensitizing agent, has been used in immunotherapy. This study investigated the protective effects of 8‐MOP on LPS‐induced inflammatory injury in bovine mammary epithelial cells (BMECs). LPS treatment (50 μg/mL for 12 hr) caused a decrease in cell viability, morphological damage, and cell apoptosis. Pretreatment with 8‐MOP at concentrations of 25 and 50 μg/ml significantly attenuated LPS‐induced inflammation in BMECs. qRT‐PCR analysis revealed that the messenger RNA expression of inflammatory cytokines and chemokine (interleukin‐1β [IL‐1β], IL‐6, tumor necrosis factor‐α, and IL‐8) was suppressed by 8‐MOP in LPS‐stimulated BMECs. Western blot analysis showed that 8‐MOP could also reduce the protein levels of cyclooxygenase‐2 and promote the translocation of high‐mobility group box 1 from the nucleus to cytoplasm. Furthermore, the anti‐inflammatory property of 8‐MOP was mediated by inhibiting nuclear factor kappa‐light‐chain‐enhancer of activated B cells activation and STAT1 phosphorylation. Taken together, 8‐MOP could protect cells from inflammatory injury induced by LPS, and may be a potential agent against bovine mastitis.     

FCRL3 promotes IL‐10 expression in B cells through the SHP‐1 and p38 MAPK signaling pathways

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that is caused by the interaction of genetic and environmental factors. Current studies have shown that Fc‐receptor like‐3 (FCRL3) is closely related to MS, but the specific role of FCRL3 in MS has not yet been clarified. This study further found that FCRL3 and interleukin 10 (IL‐10) expression was downregulated in MS patients, but the expression of these proteins was higher in the remission phase than that in the acute phase. The C allele of rs7528684 was associated with MS, and the CC genotype could lead to the upregulation of FCRL3 expression and the increase in IL‐10 secretion. Further in vitro experiments with B cells found that lipopolysaccharide (LPS) promoted FCRL3 expression in a dose‐ and time‐dependent manner, thereby promoting IL‐10 secretion. LPS regulated Src homology region 2 domain‐containing phosphatase‐1 (SHP‐1) expression and p38 mitogen‐activated protein kinase (MAPK) pathway activation through FCRL3, and FCRL3 upregulated the SHP‐1 expression and p38 phosphorylation levels. When SHP‐1 small interfering RNA or a p38 pathway inhibitor was added, the effect of FCRL3 on IL‐10 secretion was significantly inhibited. In addition, FCRL3 inhibited the secretion of inflammatory factors (tumor necrosis factor‐α, IL‐1β, IL‐6, and IL‐8); after inhibiting the expression of IL‐10, the abovementioned effects of FCRL3 were blocked. These results suggest that FCRL3 can activate the SHP‐1 and p38 MAPK pathways and then promote the secretion of IL‐10 in B cells, thus inhibiting the secretion of inflammatory factors. Therefore, FCRL3 may play an immunoprotective role in MS, and it will be an effective target for the diagnosis and treatment of MS.     

Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

Microglia rapidly respond to CNS injury and disease and can assume a spectrum of activation states. While changes in gene expression and production of inflammatory mediators have been extensively described after classical (LPS-induced) and alternative (IL4-induced) microglial activation, less is known about acquired de-activation in response to IL10. It is important to understand how microglial activation states affect their migration and invasion; crucial functions after injury and in the developing CNS. We reported that LPS-treated rat microglia migrate very poorly, while IL4-treated cells migrate and invade much better. Having discovered that the lamellum of migrating microglia contains a large ring of podosomes – microscopic structures that are thought to mediate adhesion, migration and invasion – we hypothesized that IL4 and IL10 would differentially affect podosome expression, gene induction, migration and invasion. Further, based on the enrichment of the KCa2.3/SK3 Ca²⁺-activated potassium channel in microglial podosomes, we predicted that it regulates migration and invasion. We found both similarities and differences in gene induction by IL4 and IL10 and, while both cytokines increased migration and invasion, only IL10 affected podosome expression. KCa2.3 currents were recorded in microglia under all three activation conditions and KCNN3 (KCa2.3) expression was similar. Surprisingly then, of three KCa2.3 inhibitors (apamin, tamapin, NS8593), only NS8593 abrogated the increased migration and invasion of IL4 and IL10-treated microglia (and invasion of unstimulated microglia). This discrepancy was explained by the observed block of TRPM7 currents in microglia by NS8593, which occurred under all three activation conditions. A similar inhibition of both migration and invasion was seen with a TRPM7 inhibitor (AA-861) that does not block KCa2.3 channels. Thus, we conclude that TRPM7 (not KCa2.3) contributes to the enhanced ability of microglia to migrate and invade when in anti-inflammatory states. This will be an important consideration in developing TRPM7 inhibitors for treating CNS injury.     

Maresin Conjugates in Tissue Regeneration 1 improves alveolar fluid clearance by up‐regulating alveolar ENaC,Na, K‐ATPase in lipopolysaccharide‐induced acute lung injury

Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a newly identified macrophage‐derived sulfido‐conjugated mediator that stimulates the resolution of inflammation. This study assessed the role of MCTR1 in alveolar fluid clearance (AFC) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Rats were intravenously injected with MCTR1 at a dose of 200 ng/rat, 8 hours after administration of 14 mg/kg LPS. The level of AFC was then determined in live rats. Primary rat ATII (Alveolar Type II) epithelial cells were also treated with MCTR1 (100 nmol/L) in a culture medium containing LPS for 8 hours. MCTR1 treatment improved AFC (18.85 ± 2.07 vs 10.11 ± 1.08, P < .0001) and ameliorated ALI in rats. MCTR1 also significantly promoted AFC by up‐regulating epithelial sodium channel (ENaC) and Na⁺‐K⁺‐adenosine triphosphatase (Na, K‐ATPase) expressions in vivo. MCTR1 also activated Na, K‐ATPase and elevated phosphorylated‐Akt (P‐Akt) by up‐regulating the expression of phosphorylated Nedd4‐2 (P‐Nedd4‐2) in vivo and in vitro. However, BOC‐2 (ALX inhibitor), KH7 (cAMP inhibitor) and LY294002 (PI3K inhibitor) abrogated the improved AFC induced by MCTR1. Based on the findings of this study, MCTR1 may be a novel therapeutic approach to improve reabsorption of pulmonary oedema during ALI/acute respiratory distress syndrome (ARDS).     

Ganglioside GM3 suppresses lipopolysaccharide‐induced inflammatory responses in rAW 264.7 macrophage cells through NF‐κB,AP‐1, and MAPKs signaling

Gangliosides are known to specifically inhibit vascular leukocyte recruitment and consequent interaction with the injured endothelium, the basic inflammatory process. In this study, we have found that the production of nitric oxide (NO), a main regulator of inflammation, is suppressed by GM3 on murine macrophage RAW 264.7 cells, when induced by LPS. In addition, GM3 attenuated the increase in cyclooxyenase‐2 (COX‐2) protein and mRNA levels in lipopolysaccharide (LPS)‐activated RAW 264.7 cells in a dose‐dependent manner. Moreover, GM3 inhibited the expression and release of pro‐inflammatory cytokines of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) in RAW 264.7 macrophages. At the intracellular level, GM3 inhibited LPS‐induced nuclear translocation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and activator protein (AP)‐1 in RAW 264.7 macrophages. We, therefore, investigated whether GM3 affects mitogen‐activated protein kinase (MAPK) phosphorylation, a process known as the upstream signaling regulator. GM3 dramatically reduced the expression levels of the phosphorylated forms of ERK, JNK, and p38 in LPS‐activated RAW 264.7 cells. These results indicate that GM3 is a promising suppressor of the vascular inflammatory responses and ganglioside GM3 suppresses the LPS‐induced inflammatory response in RAW 264.7 macrophages by suppression of NF‐κB, AP‐1, and MAPKs signaling. Accordingly, GM3 is suggested as a beneficial agent for the treatment of diseases that are associated with inflammation.