Crotalaria ferruginea extract attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting MAPK/NF-κB signaling pathways

Objective:To evaluate the anti-inflammatory activity of Crotalaria ferruginea extract(CFE)and its mechanism.Methods:An intratracheal lipopolysaccharide(LPS)instillationinduced acute lung injury(ALI)model was used to study the antiinflammatory activity of CFE in vivo.The LPS-induced shock model was used to analyze the effect of CFE on survival.LPS-stimulated RAW264.7 cell model was used to investigate the anti-inflammatory activity of CFE in vitro and the effects on mitogen-activated protein kinase(MAPK)or nuclear factor-κB(NF-κB)signaling pathways.Results:CFE administration decreased the number of inflammatory cells,reduced the levels of tumor necrosis factor-α(TNF-α),monocyte chemotactic protein-1(MCP-1),interleukin-6(IL-6),and interferon-γ,and diminished protein content in the bronchoalveolar lavage fluid of mice.CFE also reduced lung wet-to-dry weight ratio,myeloperoxidase,and lung tissue pathological injury.CFE preadministration improved the survival rate of mice challenged with a lethal dose of LPS.CFE reduced LPS-activated RAW264.7 cells to produce nitric oxide,TNF-α,MCP-1,and IL-6.Furthermore,CFE inhibited nuclear translocation and phosphorylation of NF-κB P65,extracellular signal-regulated kinase,c-Jun N-terminal kinases,and P38 MAPKs.Conclusions:CFE exhibits potent anti-inflammatory activity in LPS-induced ALI mice,LPS-shock mice,and RAW264.7 cells,and its mechanism may be associated with the inhibition of NF-κB and MAPK signaling pathways.Crotalaria ferruginea may be a useful therapeutic drug for the treatment of ALI and other respiratory inflammations.

Protective Effect of Genistein on Lipopolysaccharide-induced Acute Lung Injury in Rats

Summary： To investigate the protective effect of genistein on endotoxin-induced acute lung injury in rats, and explore the underlying mechanisms, 32 male Sprague-Dawley rats were randomly divided into 4 experimental groups： saline control, genistein alone, lipopolysaccaride alone, and genistein pretreatment. Each treatment group consisted of eight animals. Animals were observed for 6 h after LPS challenge, and the wet/dry （W/D） weight ratio of the lung and bronchoalveolar lavage fluid （BALF） protein content were used as a measure of lung injury. Neutrophil recruitment and activation were evaluated by BALF cellularity and myeloperoxidase （MP（）） activity. RT-PCR analysis was performed in lung tissue to assess gene expression of ICAM-1. The histopathological changes were also observed using the HE staining of lung tissue. Our results showed that lung injury parameters, including the wet/dry weight ratio and protein content in BALF, were significantly higher in the LPS alone group than in the saline control group （P〈0.01）. In the LPS alone group, a larger number of neutrophils and greater MPO activity in cell-free BAL and lung homogenates were observed when compared with the saline control group （P〈0.01）. There was a significant increase in lung ICAM-1 mRNA in response to LPS challenge （P〈0. 01, group L versus group S）. Genistein pretreatment significantly attenuated LPS-induced changes in these indices. LPS caused extensive lung damage, which was also lessened after genistein pretreatment. All above-mentioned parameters in the genistein alone group were not significantly different from those of the saline control group. It is concluded that genistein pretreatment attenuated LPS-induced lung injury in rats. This beneficial effect of genistein may involves, in part, an inhibition of neutrophilic recruitment and activity, possibly through an inhibition of lung ICAM-1 expression.

Lipopolysaccharide Challenge Induces Long Pentraxin 3 Expression in Mice Independently from Acute Lung Injury

Objective To determine whether the onset of acute lung injury(ALI) induces the up-regulation of pentraxin 3(PTX3) expression in mice and whether PTX3 concentration in the biofluid can help recognizing sepsis-induced ALI. Methods Wild-type C57BL/6 mice(12-14 weeks old) were randomly divided into 3 groups. Mice in the group 1(n=12) and group 2(n=12) were instilled with lipopolysaccharide via intratracheal or intraperitoneal routes, respectively. Mice in the group 3(n=8) were taken as blank controls. Pulmonary morphological and functional alterations were measured to determine the presence of experimental ALI. PTX3 expression in the lung was quantified at both protein and m RNA levels. PTX3 protein concentration in blood and bronchoalveolar lavage fluid was measured to evaluate its ability to diagnose sepsis-induced ALI by computing area under receiver operator characteristic curve(AUROCC). Results ALI was commonly confirmed in the group 1 but never in the other groups. PTX3 expression was up-regulated indiscriminately among lipopolysaccharide-challenged mice. PTX3 protein concentration in the biofluid was unable to diagnose sepsis-induced ALI evidenced by its small AUROCC. PTX3 concentration in bronchoalveolar lavage fluid did not correlate with that in serum. Conclusions Lipopolysaccharide challenges induced PTX3 expression in mice regardless of the presence of ALI. PTX3 may act as an indicator of inflammatory response instead of organ injury per se.

Establishment of Acute Lung Injury Model Induced by Intraperitoneal Injection of Lipopolysaccharide in Rats

The model of acute lung injury(ALI)was established by intraperitoneal administration,but there was no time-point observation and comparison.ALI model was established by intraperitoneal injection of lipopolysaccharide(LPS)at the concentration of 10 mg·kg^-1 (10 mg LPS dissolved in 1 mL normal saline to prepare 1 mL·kg^-1solution)in rats.The control group(CG)was intraperitoneally injected with saline of the same dose.In the LPS group,lung tissues were collected at 4,6,8,12 and 24 h after administration.Then,the morphology changes,the ratio of wet-to-dry weight(W/D),the expression of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)proteins,the levels of malondialdehyde(MDA),the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH)were measured.To verify the success of the model,the degrees of lung injury via Western blot,RT-PCR,ELISA and other techniques were detected at different time points,and the severe time of the ALI model established was deterimined by intraperitoneal administration,which provided a stable model basis for the study of the pathogenesis of ALI in the future.The results showed that the lung injury occurred in LPS group.W/D and lung pathological changes at 12 and 24 h of LPS group were significantly different from those in the CG.Compared with the CG,the expression of IL-1βand TNF-αproteins and the content of MDA in lung tissues of LPS group increased and most significant difference was found at 12 and 24 h(p<0.01).Compared with the CG,the activities of SOD and GSH in LPS 12 h group decreased significantly(p<0.01).In conclusion,inflammation and oxidative damage were the main causes of the ALI in rats.Lung injury was most obvious 12 h after intraperitoneal injection of 10 mg·kg^-1 LPS.

Moderate hypothermia attenuates lung in flammation inlipopolysaccharide- induced acute lung injury in rats

Objective To investigate the role of moderate h.vpothennia in the lung inflammation of rat acute lung injury induced by lipopolysaccharide(LPS). Methods A rat model of acute lung injury (ALl) was established by in-tin-tracheal instillation of lipopolysaccharide ( 1.5 mg/kg, 0.5 ml) at 16 h after LPS ( 1.0 mg/kg) intraperitoneal adrninis-tmtion. Thirty-four male Sprague Dawley rats were randomly divided into four groups: control group, receiving saline only;LPS group, receiving LPS; hypothennia group, treated with hypothennia without LPS; LPS + hypothennia group, treated with LPS and cooled to 32.5℃-33.0℃ as PaO2/FiO2. was below 300 mmHg. Hemodynamics and blood gases were record-ed every hour throughout the study. Rats were killed 4 h after ALl, and lung lavage was performed to measure the tumor ne-crosis factor α(TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) concentrations in bronchoalveolar lavage fluid (BALF) by using enzyme-linked immunosorbent assay (ELISA). Results PaO2/FiO2 was significantly decreased and PaCO2 was increased in the LPS group as compared to their baseline values( P＜0.01). Treatment with hypothermia inhib-ited the increase in PaCO2( P＜0.05) but had no effect on PaO2/FiO2 in the presence of LPS. The administration of LPS significantly increased the concentrations of TNF-α, IL-6 and IL-10 in BALF as compared to the control experiment( P＜0.05, P＜0.01 ). Moderate hypothermia reduced the expressions of TNF-α and IL-6 ( P＜0.01 ) but had no effect on the production of IL-10 ( P＞0.05). Conclusion Moderate hypothermia significantly inhibits proinflammatory cytokine ex-pressions in lipopolysaccharide-induced acute lung injury.

Multiomics reveal human umbilical cord mesenchymal stem cells improving acute lung injury via the lung-gut axis

BACKGROUND Acute lung injury(ALI)and its final severe stage,acute respiratory distress syndrome,are associated with high morbidity and mortality rates in patients due to the lack of effective specific treatments.Gut microbiota homeostasis,including that in ALI,is important for human health.Evidence suggests that the gut microbiota improves lung injury through the lung-gut axis.Human umbilical cord mesenchymal cells(HUC-MSCs)have attractive prospects for ALI treatment.This study hypothesized that HUC-MSCs improve ALI via the lung-gut microflora.AIM To explore the effects of HUC-MSCs on lipopolysaccharide(LPS)-induced ALI in mice and the involvement of the lung-gut axis in this process.METHODS C57BL/6 mice were randomly divided into four groups(18 rats per group):Sham,sham+HUC-MSCs,LPS,and LPS+HUC-MSCs.ALI was induced in mice by intraperitoneal injections of LPS(10 mg/kg).After 6 h,mice were intervened with 0.5 mL phosphate buffered saline(PBS)containing 1×10^(6) HUC-MSCs by intraperitoneal injections.For the negative control,100 mL 0.9%NaCl and 0.5 mL PBS were used.Bronchoalveolar lavage fluid(BALF)was obtained from anesthetized mice,and their blood,lungs,ileum,and feces were obtained by an aseptic technique following CO_(2) euthanasia.Wright’s staining,enzyme-linked immunosorbent assay,hematoxylin-eosin staining,Evans blue dye leakage assay,immunohistochemistry,fluorescence in situ hybridization,western blot,16S rDNA sequencing,and non-targeted metabolomics were used to observe the effect of HUC-MSCs on ALI mice,and the involvement of the lung-gut axis in this process was explored.One-way analysis of variance with post-hoc Tukey’s test,independent-sample Student’s t-test,Wilcoxon rank-sum test,and Pearson correlation analysis were used for statistical analyses.RESULTS HUC-MSCs were observed to improve pulmonary edema and lung and ileal injury,and decrease mononuclear cell and neutrophil counts,protein concentrations in BALF and inflammatory cytokine levels in the serum,lung,and ileum of ALI mice.Especially,HUC-MSCs decreased Evans blue concentration and Toll-like receptor 4,myeloid differentiation factor 88,p-nuclear factor kappa-B(NF-κB)/NF-κB,and p-inhibitorαof NF-κB(p-IκBα)/IκBαexpression levels in the lung,and raised the pulmonary vascular endothelial-cadherin,zonula occludens-1(ZO-1),and occludin levels and ileal ZO-1,claudin-1,and occludin expression levels.HUC-MSCs improved gut and BALF microbial homeostases.The number of pathogenic bacteria decreased in the BALF of ALI mice treated with HUCMSCs.Concurrently,the abundances of Oscillospira and Coprococcus in the feces of HUS-MSC-treated ALI mice were significantly increased.In addition,Lactobacillus,Bacteroides,and unidentified_Rikenellaceae genera appeared in both feces and BALF.Moreover,this study performed metabolomic analysis on the lung tissue and identified five upregulated metabolites and 11 downregulated metabolites in the LPS+MSC group compared to the LPS group,which were related to the purine metabolism and the taste transduction signaling pathways.Therefore,an intrinsic link between lung metabolite levels and BALF flora homeostasis was established.CONCLUSION This study suggests that HUM-MSCs attenuate ALI by redefining the gut and lung microbiota.

Anti-inflammatory and Analgesic Effects of Bi-yuan-ling Granules

Bi-yuan-ling granule（BLG） is a traditional Chinese medicine compound composed mainly of baicalin and chlorogenic acid. It has been demonstrated to be clinically effective for various inflammatory diseases such as acute rhinitis, chronic rhinitis, atrophic rhinitis and allergic rhinitis. However, the underlying mechanisms of BLG against these diseases are not fully understood. This study aimed to explore the anti-inflammatory and analgesic activities of BLG, and examine its protective effects on mouse acute lung injury（ALI）. The hot plate test and acetic acid-induced writhing assay in Kunming mice were adopted to evaluate the pain-relieving effects of BLG. The anti-inflammatory activities of BLG were determined by examining the effects of BLG on xylene-caused ear swelling in Kunming mice, the cotton pellet-induced granuloma in rats, carrageenan-induced hind paw edema and lipopolysaccharide（LPS）-induced acute lung injury（ALI） in mice. The results showed that BLG at 15.5 mg/g could significantly relieve the pain by 82.5%（P〈0.01） at 1 h after thermal stimulation and 91.2%（P〈0.01） at 2 h after thermal stimulation. BLG at doses of 7.75 and 15.5 mg/g reduced the writhing count up to 33.3%（P〈0.05） and 53.4%（P〈0.01）, respectively. Additionally, the xylene-induced edema in mice was markedly restrained by BLG at 7.75 mg/g（P〈0.05） and 15.5 mg/g（P〈0.01）. BLG at 5.35 and 10.7 mg/g significantly reduced paw edema by 34.8%（P〈0.05） and 37.9%（P〈0.05） at 5 h after carrageenan injection. The granulomatous formation of the cotton pellet was profoundly suppressed by BLG at 2.68, 5.35 and 10.7 mg/g by 15.4%, 38.2%（P〈0.01） and 58.9%（P〈0.001）, respectively. BLG also inhibited lung W/D ratio and the release of prostaglandin E2（PGE2） in ALI mice. In addition, the median lethal dose（LD_（50））, median effective dose（ED_（50）） and half maximal inhibitory concentration（IC50） of BLG were found to be 42.7, 3.2 and 12.33 mg/g, respectively. All the findings suggest that BLG has significantly anti-inflammatory and analgesic effects and it may help reduce the damage of ALI.

Lipopolysaccharide “Two-hit” Induced Refractory Hypoxemia Acute Respiratory Distress Model in Rats

To establish a stable and reliable model of refractory hypoxemia acute respiratory distress syndrome （ARDS） and examine its pathological mechanisms, a total of 144 healthy male Wistar rats were randomized into 4 groups： group Ⅰ （saline control group）, group Ⅱ （LPS intravenous ＂single-hit＂ group）, group Ⅲ （LPS intratracheal ＂single-hit＂ group） and Group IV （LPS ＂two-hit＂ group）. Rats were intravenously injected or intratracheally instilled with a large dose of LPS （10 mg/kg in 0.5 mL） to simulate a single attack of ARDS, or intraperitoneally injected with a small dose of LPS （1 mg/kg） followed by tracheal instillation with median dose of LPS （5 mg/kg） to establish a ＂two-hit＂ model. Rats in each group were monitored by arterial blood gas analysis and visual inspection for three consecutive days. Arterial blood gas values, lung wet/dry weight ratio and pathological pulmonary changes were analyzed to determine the effects of each ALI/ARDS model. Concentrations of TNF-α, IL-1 and IL-10 in the bronchoalveolar lavage fluid （BALF） and blood plasma were meastired by using enzyme-linked immunosorbent assays （ELISA）. Our resulsts showed that single LPS-stimulation, whether through intravenous injection or tracheal instillation, could only induce ALl and temporary hypoxemia in rats. A two-hit LPS stimulation induces prolonged hypoxemia and specific pulmonary injury in rats, and is therefore a more ideal approximation of ARDS in the animal model. The pathogenesis of LPS two-hit-induced ARDS is associated with an uncontrolled systemic inflammatory response and inflammatory injury. It is concluded that the rat ARDS model produced by our LPS two-hit method is more stable and reliable than previous models, and closer to the diagnostic criteria of ARDS, and better mimics the pathological process of ARDS.

Protective Effect of Curcumin on Endotoxin-induced Acute Lung Injury in Rats

To investigate the protective effect of curcumin on endotoxin-induced acute lung injury in rats, and explore the underlying mechanisms, 24 male Wistar rats were randomly divided into 4 experimental groups： sham-vehicle （S）, sham-curcumin （C）, lipopolysaccharide （LPS）-vehicle （L）, and curcumin-lipopolysaccharide （C-L） groups. The wet/dry （W/D） weight ratio of the lung and bronchoalveolar lavage （BAL） fluid protein content were used as measures of lung injury. Neutrophil recruitment and activation were evaluated by BAL fluid cellularity and myeloperoxidase （MPO） activity in cell-free BAL and lung tissue. The levels of cytokine-induced neutrophil chemoattractant-1 （CINC-1） in lung tissues were measured by ELISA. were observed by using the HE staining. Our results the wet/dry weight ratio and protein content in BALE The histopathological changes of lung tissues showed that lung injury parameters, including were significantly higher in the L group than in the S group （P〈0.01）. In the L group, higher numbers of neutrophils and greater MPO activity in cell-free BAL and lung homogenates were observed when compared with the S group （P〈0.01）. There was a marked increase in CINC-1 levels in lung tissues in response to LPS challenge （P〈0.01, L group vs S group）. Curcumin pretreatment significantly attenuated LPS-induced changes in these indices. LPS caused extensive morphological lung damage, which was also lessened after curcumin pretreatment. All the above-mentioned parameters in the C group were not significantly different from those of the S group. It is concluded that curcumin pretreatment attenuates LPS-induced lung injury in rats. This beneficial effect of curcumin may involves, in part, inhibition of neutrophilic recruitment and activity, possibly through inhibition of lung CINC-1 expression.

Effects of methyl palmitate and lutein on LPS-induced acute lung injury in rats

AIM: To investigate the effects of methyl palmitate and lutein on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in rats and explore the possible mechanisms. METHODS: Male Sprague-Dawley rats were divided into 4 groups:(1) control;(2) LPS;(3) Methyl palmitate; and(4) Lutein groups. Methyl palmitate(300 mg/kg, ip) was administered 3 times per week on alternating days while lutein(100 mg/kg, oral) was given once daily. After 1 wk of vehicle/methyl palmitate/lutein treatment, ALI was induced by a single dose of LPS(7.5 mg/kg, iv). After 24 h of LPS injection, animals were sacrificed then biochemical parameters and histopathology were assessed. RESULTS: Treatment with methyl palmitate attenuated ALI, as it significantly decreased the lung wet/dry weight(W/D) ratio, the accumulation of the inflammatory cells in the bronchoalveolar lavage fluid(BALF) andhistopathological damage. However, methyl palmitate failed to decrease lactate dehydrogenase(LDH) activity in BALF. On the other hand, lutein treatment produced significant anti-inflammatory effects as revealed by significant decrease in accumulation of inflammatory cells in lung, LDH level in BALF and histopathological damage. Methyl palmitate and lutein significantly increased superoxide dismutase(SOD) and reduced glutathione(GSH) activities with significant decrease in the lung malondialdehyde(MDA) content. Importantly, methyl palmitate and lutein decreased the level of the inflammatory cytokine tumor necrosis factor-α(TNF-α) in the lung. Lutein also reduced LPS-mediated overproduction of pulmonary nitrite/nitrate(NO-2/NO-3), which was not affected by methyl palmitate pretreatment. CONCLUSION: These results demonstrate the potent protective effects of both methyl palmitate and lutein against LPS-induced ALI in rats. These effects can be attributed to potent antioxidant activities of these agents, which suppress inflammatory cell infiltration and regulated cytokine effects.

Anti-Inflammatory Effects of Redlining Injection (热毒宁注射液)on Lipopolysaccharide-lnduced Acute Lung Injury of Rats

Objective： To evaluate the protective effects of Reduning Injection （热毒宁注射液, RDN）, a patent Chinese medicine, on lipopolysaccharide （LPS）-induced acute lung injury （ALl） in rats and its underlying mechanisms of action. Methods： Sixty male Sprague-Dawley rats were randomly divided into 6 groups, including normal control, model, dexamethasone （DEX, 5 mg/kg）, RDN-H （720 mg/kg）, RDN-M （360 mg/kg） and RDN-L （180 mg/kg） groups, with 10 rats in each group. Rats were challenged with intravenous injection of LPS 1 h after intraperitoneal treatment with RDN or DEX. At 6 h after LPS challenge, lung tissues and bronchoalveolar lavage fluid （BALF） were collected, and the number of inflammatory cells was determined. The right lungs were collected for histopathologic examination, measurement of gene and protein expressions, superoxide dismutase （SOD） and myeloperoxidase （MPO） activities. Results： In vivo pretreatment of RDN （360, 720 mg/kg） significantly reduced the weight of wet to dry （W/D） ratio of lung, protein content in BALF, and led to remarkable attenuation of LPS-induced histopathological changes in the lungs. Meanwhile, RDN enormously decreased BALF total inflammatory cells, especially neutrophil and macrophage cell numbers. Moreover, RDN increased SOD activity, inhibited MPO activity, alleviated LPS-induced tumor neurosis factor-o~ （TNF-o~） and inducible nitric oxide synthase （iNOS） expression in lung tissues. Furthermore, RDN （720 mg/kg） efficiently weakened nuclear factor- kappa B （NF- K B） gene and protein expression. Conclusion： Anti-inflammatory effects of RDN was demonstrated to be preventing pulmonary neutrophil infiltration, lowering MPO activity, TNF-oL and iNOS gene expression by inhibiting NF- K B activity in LPS-induced ALl.

Inactivation of mammalian target of rapamycin （mTOR） by rapamycin in a murine model of lipopolysaccharide-induced acute lung injury

Background The mammalian target of rapamycin （mTOR） pathway, a key cellular signaling pathway associated with various cellular functions, has distinct roles in the inflammatory process. In this study, the mTOR inhibitor rapamycin （Rapa） was used to test whether inhibition of mTOR activation attenuates lipopolysaccharide （LPS）-induced acute lung injury （ALl） in a murine model.Methods Mice pretreated with Rapa or vehicle were given LPS intratracheally. Local cell numbers and inflammatory cytokines present in the bronchoalveolar lavage fluid （BAL）, wet-to-dry weight ratio, histopathology of the lungs, and survival were evaluated.Results The phosphorylation of S6, a major downstream target of mTOR, had a 3-fold increase in lung tissue after LPS stimulation, but the increase was blocked by Rapa. Rapa reduced the levels of TNF-α （LPS vs. LPS ＋ Rapa,（1672.74±193.73） vs. （539.17±140.48） pg/ml, respectively; P 〈0.01） and IL-6 （LPS vs. LPS ＋ Rapa： （7790.88±1170.54）vs. （1968.57±474.62） pg/ml, respectively; P 〈0.01） in the BAL fluid. However, Rapa had limited effects on the overall severity of ALI, as determined by the wet-to-dry weight ratio of the lungs, number of neutrophils in the BAL fluid, and changes in histopathology. In addition, Rapa failed to reduce mortality in the LPS-induced ALI model.Conclusions We confirmed that mTOR was activated during LPS-induced ALI and strongly inhibited by Rapa.Although Rapa reduced the levels of the mediators of inflammation, the overall severity and survival of the ALI murine model were unchanged.

Changes in intestinal microflora in rats with acute respiratory distress syndrome

AIM:To implement high-throughput 16S rDNA sequencing to study microbial diversity in the fecal matter of rats with acute lung injury/acute respiratory distress syndrome(ALI/ARDS).METHODS:Intratracheal instillation of lipopolysaccharide was used to induce ALI,and the pathological changes in the lungs and intestines were observed.D-lactate levels and diamine oxidase(DAO)activities were determined by enzymatic spectrophotometry.The fragments encompassing V4 16S rDNA hypervariable regions were PCR amplified from fecal samples,and the PCR products of V4 were sequenced by Illumina MiSeq.RESULTS:Increased D-lactate levels and DAO activities were observed in the model group(P<0.01).Sequencing results revealed the presence of 3780 and4142 species in the control and model groups,respectively.The percentage of shared species was 18.8419%.Compared with the control group,the model group had a higher diversity index and a lower number of species of Fusobacteria(at the phylum level),Helicobacter and Roseburia(at the genus level)(P<0.01).Differences in species diversity,structure,distribution and composition were found between the control group and early ARDS group.CONCLUSION:The detection of specific bacteria allows early detection and diagnosis of ALI/ARDS.

The microRNAs expression changes rapidly in mice lung tissue during lipopolysaccharide-induced acute lung injury

MicroRNAs （miRNAs） are a novel class of-22-nt, .non-coding, short single-stranded RNAs producedfrom endogenous genes by the sequential action of the RNase III enzymes Drosha and Dicer. Increasing evidence shows that miRNAs play a critical role in regulating inflammatory and innate immunity.1,2 However, whether miRNAs are involved in the altered function of innate immune cells and ultimately contribute to the development of acute lung injury （ALl） remains largely unknown. It is worth studying the expression profiles of miRNAs in mouse lungs stimulated by lipopolysaccharide （LPS）, which may be very important in the regulation of inflammatory mediators during ALI. In the present study, we investigated the expression profiles of miRNAs in LPS-induced ALI in mice.

Protectin D1 promotes resolution of inflammation in a murine model of lipopolysaccharide-induced acute lung injury via enhancing neutrophil apoptosis

Background Protectin D1 （PD1）,derived from docosahexaenoic acid,has been shown to control and resolve inflammation in some experimental models of inflammatory disorders.We investigated the protective roles of protectin D1 in pulmonary inflammation and lung injury induced by lipopolysaccharide （LPS）.Methods Mice were randomly assigned to six groups （n=6 per group）：sham-vehicle group,sham-PD1 group,shamzVAD-fmk group,LPS-vehicle group,LPS-PD1 group,and LPS-PD1-zVAD-fmk group.Mice were injected intratracheally with 3 mg/kg LPS or saline,followed 24 hours later by intravenous injection of 200 μg/mouse PD1 or vehicle.At the same time,some mice were also injected intraperitoneally with the pan-caspase inhibitor zVAD-fmk.Seventy-two hours after LPS challenge,samples of pulmonary tissue and bronchoalveolar lavage fluid were collected.Optical microscopy was used to examine pathological changes in lungs.Cellularity and protein concentration in bronchoalveolar lavage fluid were analyzed.Lung wet/dry ratios and myeloperoxidase activity were measured.Apoptosis of neutrophils in bronchoalveolar lavage fluid （BALF） was also evaluated by flow cytometry.Results Intratracheal instillation of LPS increased neutrophil counts,protein concentration in bronchoalveolar lavage fluid and myeloperoxidase activity,it induced lung histological injury and edema,and also suppressed apoptosis of neutrophils in BALF.Posttreatment with PD1 inhibited LPS-evoked changes in BALF neutrophil counts and protein concentration and lung myeloperoxidase activity,with the outcome of decreased pulmonary edema and histological injury.In addition,PD1 promoted apoptosis of neutrophils in BALF.The beneficial effects of PD1 were blocked by zVAD-fmk.Conclusion Posttreatment with PD1 enhances resolution of lung inflammation during LPS-induced acute lung injury by enhancing apoptosis in emigrated neutrophils,which is,at least in part,caspase-dependent.

Protectin DX Exhibits Protective Effects in Mouse Model of Lipopolysaccharide-Induced Acute Lung Injury

Background：Acute lung injury （ALI） is a severe disease with high mortality and poor prognosis.Protectin DX （PDX）,a pro-resolving lipid mediator,exhibits protective effects in ALI.Our experiment aimed to explore the effects and related mechanisms of PDX in mice with ALI induced by lipopolysaccharide （LPS）.Methods：BALB/c mice were randomly divided into five groups：sham,LPS,LPS plus 1 ng ofPDX （LPS ＋ PDX-1 ng）,LPS plus 10 ng ofPDX （LPS ＋ PDX-10 ng）,and LPS plus 100 ng ofPDX （LPS ＋ PDX-100 ng）.Bronchoalveolar lavage fluids （BALFs） were collected after 24 h,and total cells,polymorphonuclear leukocytes,monocyte-macrophages,and lymphocytes in BALF were enumerated.The concentration of interleukin （IL）-1 β3,IL-6,IL-10,tumor necrosis factor-alpha （TNF-α）,macrophage inflammatory protein （MIP）-1 cα,and MIP-2 in BALF was determined,and histopathological changes of the lung were observed.The concentration of protein in BALF and lung wet/dry weight ratios were detected to evaluate pulmonary edema.After determining the optimal dose of PDX,neutrophil-platelet interactions in whole blood were evaluated by flow cytometry.Results：The highest dose of PDX （100 ng/mouse） failed to provide pulmonary protective effects,whereas lower doses of PDX （1 ng/mouse and 10 ng/mouse）,especially 1 ng PDX,alleviated pulmonary histopathological changes,mitigated LPS-induced ALI and pulmonary edema,inhibited neutrophil infiltration,and reduced pro-inflammatory mediator （IL-1β,IL-6,TNF-α,and MIP-lα） levels.Meanwhile,1 ng PDX exhibited pro-resolving functions in ALI including upregulation of monocyte-macrophage numbers and anti-inflammatory mediator IL-l 0 levels.The flow cytometry results showed that PDX could inhibit neutrophil-platelet interactions in ALI.Conclusion：PDX exerts protective effects in LPS-inducedALI by mitigating pulmonary inflammation and abrogating neutrophil-platelet interactions.

Baicalin inhibits inflammation of lipopolysaccharide-induced acute lung injury via toll like receptor-4/myeloid differentiation primary response 88/nuclear factor-kappa B signaling pathway

OBJECTIVE:To explore the effect and mechanism of baicalin in the treatment of acute lung injury(ALI)by in vivo and in vitro experiments.METHODS:ALI was induced by instilling 10 mg/m L lipopolysaccharide(LPS)into the airway of rats.Different doses of baicalin(50 and 100 mg·kg^(-1)·d^(-1 ))were administered by gavage one day before modeling.RESULTS:Baicalin significantly reduced the permeability of the alveolocapillary membrane,alleviated tissue injury and inflammatory infiltration,and inhibited the secretion of inflammatory factors and the infiltration of neutrophils.The decline in these inflammations was related to the inhibition of the toll like receptor-4(TLR4)/myeloid differentiation factor 88(My D88)/nuclear factor-kappa B(NF-κB)/nod-like receptor pyrin containing 3(NLRP3)signaling pathway and the mitogen-activated protein kinase(MAPK)signaling pathway.CONCLUSIONS:Baicalin inhibits the secretion of inflammatory factors by inhibiting the TLR4-My D88-NF-κB/NLRP3 pathway and the MAPK signaling pathway.Thus,it reduces lung bronchial epithelial layer,alveolar damage,and pulmonary edema as detected in the in vivo and in vitro experiments.Therefore,baicalin may be a potential preventive and therapeutic drug for ALI.

Protective effects of Rabdosia japonica var. glaucocalyx extract on lipopolysaccharide-induced acute lung injury in mice

The present study was designed to evaluate the protective effects of ethanol extracts of Rabdosia japonica var. glaucocalyx(Maxim.) Hara(RJ) on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice and the possible underlying mechanisms of action. The mice were orally administrated with RJ extract(16, 32 or 64 mg?kg–1) daily for consecutive7 days before LPS challenge. The ung specimens and the bronchoalveolar lavage fluid(BALF) were collected for histopathological examinations and biochemical analyses. Pretreatment with RJ significantly enhanced superoxide dismutase(SOD) activity and reduced the wet-to-dry weight(W/D) ratio, the levels of nitric oxide(NO) and protein leakage, and myeloperoxidase(MPO) activity in mice with ALI, in a dose-dependent manner. RJ reduced complement deposition and significantly attenuated LPS-induced ALI by reducing productions of inflammatory mediators, such as tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β). The results demonstrated that RJ may attenuate LPS-induced ALI via reducing the production of pro-inflammatory mediators, and reducing complement deposition and radicals.

Effect of Palrnatine on lipopolysaccharide-induced acute lung injury by inhibiting activation of the Akt/NF-κB pathway

Inflammation plays an important role in the development of acute lung injury(ALI).Severe pulmonary inflammation can cause acute respiratory distress syndrome(ARDS)or even death.Expression of proinflammatory interleukin-1β(IL-1β)and inducible nitric oxide synthase(iNOS)in the process of pulmonary inflammation will further exacerbate the severity of ALI.The purpose of this study was to explore the effect of Palrnatine(Pa)on lipopolysaccharide(LPS)-induced mouse ALI and its underlying mechanism.Pa,a natural product,has a wide range of pharmacological activities with the potential to protect against lung injury.Western blotting and quantitative real-time polymerase chain reaction(qRT-PCR)assays were performed to detect the expression and translation of inflammatory genes and proteins in vitro and in vivo.Immunoprecipitation was used to detect the degree of P65 translocation into the nucleus.We also used molecular modeling to further clarify the mechanism of action.The results showed that Pa pretreatment could significantly inhibit the expression and secretion of the inflammatory cytokine IL-1β,and significantly reduce the protein level of the proinflammatory protease iNOS,in both in vivo and in vitro models induced by LPS.Further mechanism studies showed that Pa could significantly inhibit the activation of the protein kinase B(Akt)/nuclear factor-κB(NF-κB)signaling pathway in the LPS-induced ALI mode and in LPS-induced RAW264.7 cells.Through molecular dynamics simulation,we observed that Pa was bound to the catalytic pocket of Akt and effectively inhibited the biological activity of Akt.These results indicated that Pa significantly relieves LPS-induced ALI by activating the Akt/NF-κB signaling pathway.

Jinyinqingre Oral Liquid alleviates LPS-induced acute lung injury by inhibiting the NF-κB/NLRP3/GSDMD pathway

Acute lung injury(ALI)is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers,resulting in high incidence and mortality rates.Currently,there is a lack of safe and effective drugs for the treatment of ALI.In a previous clinical study,we observed that Jinyinqingre oral liquid(JYQR),a Traditional Chinese Medicine formulation prepared by the Taihe Hospital,Affiliated Hospital of Hubei University of Medicine,exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings.However,the potential role of JYQR in ALI/acute respiratory distress syndrome(ARDS)and its anti-inflammatory mechanism remains unexplored.Thus,the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide(LPS)-induced ALI and an in vitro RAW264.7 cell model.JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues.Additionally,JYQR administration led to a noteworthy reduction in total protein levels within the BALF,a decrease in MPAP,and attenuation of pleural thickness.These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI.Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins,namely NLRP3 and GSDMD,as well as proinflammatory cytokine levels in mice and RAW2647 cells.Consequently,JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway.JYQR exerts a protective effect against LPS-induced ALI in mice,and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway.