Acute Lung Injury Model Group Research Articles

Exploring the treatment of sepsis-associated acute lung injury with Liangge Powder via ERK1/2 and PI3K/AKT pathways: based on network pharmacology and whole animal experimentation

Objective: To investigate the therapeutic effect and mechanism of Liangge Powder against sepsis-induced acute lung injury (ALI) . Methods: From April to December 2021, the key components of Liangge Powder and its targets against sepsis-induced ALI were analyzed by network pharmacology, and to enrich for relevant signaling pathways. A total of 90 male Sprague-Dawley rats were randomly assigned to sham-operated group, sepsis-induced ALI model group (model group), Liangge Powder low, medium and high dose group, ten rats in the sham-operated group and 20 rats in each of the remaining four groups. Sepsis-induced ALI model was established by cecal ligation and puncture. Sham-operated group: gavage with 2 ml saline and no surgical treatment. Model group: surgery was performed and 2 ml saline was gavaged. Liangge Powder low, medium and high dose groups: surgery and gavage of Liangge Powder 3.9, 7.8 and 15.6 g/kg, respectively. To measure the wet/dry mass ratio of rats lung tissue and evaluate the permeability of alveolar capillary barrier. Lung tissue were stained with hematoxylin and eosin for histomorphological analysis. The levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL) -6 and IL-1β in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The relative protein expression levels of p-phosphatidylinositol 3-kinase (PI3K), p-protein kinase B (AKT), and p-ertracellular regulated protein kinases (ERK) were detected via Western blot analysis. Results: Network pharmacology analysis indicated that 177 active compounds of Liangge Powder were selected. A total of 88 potential targets of Liangge Powder on sepsis-induced ALI were identified. 354 GO terms of Liangge Powder on sepsis-induced ALI and 108 pathways were identified using GO and KEGG analysis. PI3K/AKT signaling pathway was recognized to play an important role for Liangge Powder against sepsis-induced ALI. Compared with the sham-operated group, the lung tissue wet/dry weight ratio of rats in the model group (6.35±0.95) was increased (P<0.001). HE staining showed the destruction of normal structure of lung tissue. The levels of IL-6 [ (392.36±66.83) pg/ml], IL-1β [ (137.11±26.83) pg/ml] and TNF-α [ (238.34±59.36) pg/ml] were increased in the BALF (P<0.001, =0.001, <0.001), and the expression levels of p-PI3K, p-AKT and p-ERK1/2 proteins (1.04±0.15, 0.51±0.04, 2.31±0.41) were increased in lung tissue (P=0.002, 0.003, 0.005). The lung histopathological changes were reduced in each dose group of Liangge Powder compared with the model group. Compared with the model group, the wet/dry weight ratio of lung tissue (4.29±1.26) was reduced in the Liangge Powder medium dose group (P=0.019). TNF-α level [ (147.85±39.05) pg/ml] was reduced (P=0.022), and the relative protein expression levels of p-PI3K (0.37±0.18) and p-ERK1/2 (1.36±0.07) were reduced (P=0.008, 0.017). The wet/dry weight ratio of lung tissue (4.16±0.66) was reduced in the high-dose group (P=0.003). Levels of IL-6, IL-1β and TNF-α[ (187.98±53.28) pg/ml, (92.45±25.39) pg/ml, (129.77±55.94) pg/ml] were reduced (P=0.001, 0.027, 0.018), and relative protein expression levels of p-PI3K, p-AKT and p-ERK1/2 (0.65±0.05, 0.31±0.08, 1.30±0.12) were reduced (P=0.013, 0.018, 0.015) . Conclusion: Liangge Powder has therapeutic effects in rats with sepsis-induced ALI, and the mechanism may be related to the inhibition of ERK1/2 and PI3K/AKT pathway activation in lung tissue.

Taurine Alleviates LPS-Induced Acute Lung Injury by Suppressing TLR-4/NF-κB Pathway.

Taurine has the function of immune regulation, relieving acute and chronic inflammation caused by various agents, and maintaining cell homeostasis. This investigation focused on the protective functions of taurine targeting acute lung injury (ALI) induced by LPS. Sixty male SD rats aged 6-7weeks were segregated at random: blank control group (C group), taurine control group (T group), ALI model group (LPS group), and taurine prevention groups (LPST1, LPST, LPST3 Groups). C group and LPS group were given normal drinking water, while T group and LPST group were given 2% taurine in drinking water. LPST1 group was given 1% taurine in drinking water while. LPST3 group was given 3% taurine in drinking water. On the 14th and 28th day, LPS group and LPST1, LPST, and LPST3 groups were subjected to injection of LPS (25 mg/kg) into the trachea of rats. Serum, peripheral blood, lung tissue, and bronchoalveolar lavage fluid (BALF) were collected at 6h post-LPS injection. The wet/dry ratio (W/D) of lung was measured by hot drying method. The population of white blood cells and the abundance of inflammatory-related cells within peripheral blood were counted by an automatic blood cell analyzer. The population of white blood cells within BALF was counted by a white blood cell counting plate combined with Swiss Giemsa staining, while the proportion of related white blood cells was calculated. BCA reagent was used to determine the protein concentration in BALF. The levels of pro-inflammatory factors (IL-1 β, IL-6, IL-18, TNF - α), anti-inflammation factors (IL-10, IL-4), and taurine within serum and lung tissue were detected by ELISA. Lung structural tissue alterations were observed through HE staining techniques. Myeloperoxidase (MPO) activities within lung tissue were detected through colorimetry. Protein expression levels of TLR4, MyD88, NF-κ Bp65, NF-κ Bp-p65, MCP-1, together with CD68 within lung tissue, were analyzed by Western blot (WB) and immunohistochemistry (IHC). The taurine pretreatment group contained significantly reduced W/D, MPO activity, and the number of inflammatory cells in BALF induced by LPS. In addition, compared with ALI model group, the taurine pretreatment group contained significantly reduced levels of pro-inflammatory factors in lung tissue, increased levels of anti-inflammatory factors, and decreased expression levels of key proteins in TLR-4/NF-κ B pathway. Taurine can protect rats from ALI by inhibiting the activation of neutrophils, macrophages, and TLR-4/NF-κ B signaling pathway.

2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-d-glucoside alleviates lipopolysaccharide-induced acute lung injury in rats

To observe the protective effect of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-d-glucoside (TSG) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and explore the underlying mechanism. Thirty-six SD rats were randomized equally into 4 groups: the normal control group, ALI model group, and low- and high-dose TSG groups (treated with 50 and 100 mg/kg TSG via intragastric administration, respectively). In all but the normal control group, the rats were subjected to tail vein injection of LPS to induced ALI. The rats were euthanized at 6 h after the injection for pathological examination of the lungs. The wet/dry weight ratio (W/D) of the lungs were calculated, and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the lung tissues and serum levels of TNF-α and IL-6 were determined. Western blotting was performed to detect the levels of NF-κB p65 in the lungs. Compared with those in LPS group, the TSGtreated rats showed significantly milder lung pathologies (P < 0.001) and had lower serum TNF-α and IL-6 levels (P < 0.001) and W/D of the lung tissues (P < 0.001), higher SOD activity (P < 0.001) and lower MDA content in the lungs (P < 0.001), and significantly lower expression of NF-κB p65 in the lungs (P < 0.001). None of these indices showed significant differences between the lowand high-dose TSG treatment groups (P>0.05). TSG can ameliorate LPS-induced ALI in rats possibly by suppressing the NF-κB pathway to improve the antioxidant capacity and decrease the release of inflammatory factors.

The Preventive Effects of Lactobacillus casei on Acute Lung Injury Induced by Lipopolysaccharide.

Lactobacillus has been reported to inhibit acute lung injury (ALI). However, the molecular mechanism of Lactobacillus casei (L. casei) in preventing ALI has not been identified, so we investigated whether L. casei pretreatment could inhibit the activation of TLR4/MyD88/NF-κB signaling pathway following ALI. ALI model was established by intraperitoneal injection of 2 mg/kg lipopolysaccharide (LPS) to female BALB/c mice. In L. casei LC2W group, mice were intragastrically administrated L. casei LC2W for a week, before the ALI modeling. The serum of normal BALB/c mice after intragastric administration of L. casei LC2W was used for in vitro cell assays. The serum was pre-incubated with mouse macrophage cell line (RAW264.7) and human lung cell line (HLF-A), then LPS was added to co-incubate. Compared with ALI model group, L. casei LC2W pretreatment significantly reduced lung pathological damage, the number of neutrophils and total cells in bronchoalveolar lavage fluid. Besides, L. casei LC2W pretreatment could significantly reverse the abnormal expression of ICAM-1, IL-6, TNF-α and IL-10 in lung tissue and serum, plus, L. casei LC2W significantly reduced the phosphorylation levels of IRAK-1 and NF-κB p65. In vitro, the serum decreased the up-regulation of IL-6 and TNF-α in cell lines induced by LPS. In conclusion, L. casei LC2W intragastric administration pretreatment could significantly improve LPS-induced ALI in mice, probably through circulation to reach the lungs so as to inhibit the inflammatory response induced by activation of TLR4/MyD88/NF-κB signaling pathway.

Effects of Dexmedetomidine on Inflammatory Factors and Mitogen Activated Protein Kinase-Related Signaling Pathways of Alveolar Lavage Fluid in Mice with Lipopolysaccharide-Induced Acute Lung Injury

To investigate the effects and mechanisms of Dexmedetomidine on inflammatory factors and mitogenactivated protein kinase related signaling pathways in alveolar lavage fluid of mice with acute lung injury induced by lipopolysaccharide. 36 clean-grade male Kunming mice were randomly divided into blank control group (normal control group), lipopolysaccharide-induced acute lung injury model group, and lipopolysaccharide+dexmedetomidine group, with 12 mice in each group. The lipopolysaccharide group mice were intraperitoneally injected with 0.1 ml 5 mg kg-1 Lipopolysaccharide, lipopolysaccharide+Dexmedetomidine mice were injected with 25 μg kg-1. Dexmedetomidine intraperitoneally with 0.1 ml and 1 h later with 5 mg kg-1. Lipopolysaccharide injected with 0.1 ml intraperitoneally and the blank control group mice were intraperitoneally injected with normal saline of the same volume as the lipopolysaccharide group. All mice in each group were sacrificed after intraperitoneal injection for 6 h, and their lung tissues were separated by thoracotomy and their bronchoalveolar lavage fluid was recovered for subsequent detection. Analysis groups of mice lung tissue pathology, compare groups of mice lung tissue pathology score, lung wet weight/dry weight (W)/(D), mitogen-activated protein kinase pathways (p38 lightning, extracellular signal regulating kinase, c-jun amino terminal kinase) and bronchoalveolar lavage fluid protein expression level of tumor necrosis factor alpha beta, interleukin-1β, myeloperoxidase, protein concentration. In the blank control group, lung tissue structure was basically normal and alveolar structure was intact. lipopolysaccharide group mice had obvious lung tissue damage, and inflammatory cells appeared in the alveoli, with significantly increased exudation of red blood cells and proteins. Lipopolysaccharide+Dexmedetomidine group mice lung tissue damage significantly improved, inflammatory cell infiltration decreased, and red blood cell, protein exudation significantly decreased. Lung histopathological scores and bronchoalveolar lavage fluid levels of tumor necrosis factor alpha, interleukin- 1β and myeloperoxidase in lipopolysaccharide group were significantly higher than those in blank control group (p<0.05). The lung histopathological scores and the levels of tumor necrosis factor alpha, interleukin- 1β and myeloperoxidase in bronchoalveolar lavage fluid of lipopolysaccharide+Dexmedetomidine group were significantly lower than those of lipopolysaccharide group (p<0.05). The protein concentration in W/D and bronchoalveolar lavage fluid in lipopolysaccharide group was significantly higher than that in blank control group (p<0.05). Protein concentration in W/D and bronchoalveolar lavage fluid of lipopolysaccharide+Dexmedetomidine group was significantly lower than that of lipopolysaccharide group (p<0.05). The protein expression levels of extracellular signal regulating kinase, c-jun amino terminal kinase and P-P38 in THE lipopolysaccharide group were significantly higher than those in the blank control group (p<0.05). The protein expression levels of extracellular signal regulating kinase, c-jun amino terminal kinase and P-P38 in the lipopolysaccharide+Dexmedetomidine group were significantly lower than those in the lipopolysaccharide group (p<0.05). There was no significant difference in extracellular signal regulating kinase, c-jun amino terminal kinase and p38 protein expression levels in each group (p>0.05). Dexmedetomidine can significantly alleviate acute lung injury induced by lipopolysaccharide in mice, and its mechanism may be realized by blocking the activation of mitogen-activated protein kinaserelated signaling pathway and thereby reducing inflammatory response, providing a new target for the treatment of sepsis induced acute lung injury in the futurey.

Protective effect of FOXP3-mediated miR-146b-5p/Robo1/NF-κB system on lipopolysaccharide-induced acute lung injury in mice.

BackgroundAs a key transcription factor, forkhead box protein 3 (FOXP3) plays an important role in the development and function of natural cluster of differentiation 4 [CD4 (+)] regulatory T cells (Treg cells). However, the function of FOXP3 in Lipopolysaccharide (LPS)-induced acute lung injury (ALI) through regulating miR-146b-5p is unclear. This research aimed to disclose the regulatory effect of the FOXP3-mediated miR-146b-5p/Roundabout 1 (Robo1)/NF-κB system on LPS-induced ALI in mice.MethodsThe mice were subjected to 5 mg/kg of LPS via intratracheal instillation to induce ALI and generate the ALI model. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, the ALI + miR antagomir group and ALI + miR antagomir+ FOXP3 group. Lung tissue injury were detected by hematoxylin and eosin (HE) staining. Lung wet/dry weight ratio, total cells in bronchoalveolar lavage fluid (BALF), total protein in BALF and the polymorphonuclear leukocyte (PMN) in BALF were detected. The levels of tumor necrosis factor-α (TNF-α), Interleukin 6 (IL-6) and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA) kit. The dual-luciferase reporter assay were used to detect the target relationship between FOXP3 and Robo1. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, ALI + Robo1 group and ALI + FOXP3+ Robo1 group. The protein levels of FOXP3, Robo1 and p-p65 were detected by western bolt. The mRNA levels of miR-146b-5p and Robo1 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsAlthough protein expression levels of FOXP3 were significantly down-regulated in the ALI model, the increased FOXP3 levels promoted an increase in miR-146b-5p. Compared with the control group, the ALI model group exhibited severe histopathologic injury, such as thickening of the alveolar wall, pulmonary congestion, and decreased alveolar numbers. By mediating the overexpression of miR-146b-5p, FOXP3 also increased alveolar clearance and inhibited inflammatory responses in the ALI model. Importantly, Robo1 is a potential target of miR-146b-5p.ConclusionsFOXP3 could inhibit NF-κB activation, reduce lung pathological damage, and inhibit inflammatory responses by mediating the miR-146b-5p/Robo1/NF-κB system in the ALI model. These results may provide a new potential target for the treatment of ALI disease.

Phagocytosis of alveolar macrophages is suppressed in a mouse model of lipopolysaccharide-induced acute lung injury

To investigate the changes in phagocytic function of alveolar macrophages (AMs) in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explore the possible mechanism. Kunming mice were randomly divided into normal control group and ALI (induced by LPS instillation in the airway) model group. AMs were obtained from bronchoalveolar lavage fluid in both groups, and phagocytosis of the AMs was observed using flow cytometry and fluorescence microscopy. Western blotting and ELISA were used to detect the expression and secretion of IL-33 in the lung tissue of the mice. We also detected the secretion of IL-33 by an alveolar epithelial cell line MLE-12 in response to stimulation with different concentrations of LPS. The AMs from the normal control mice were treated with different concentrations of LPS and IL-33, and the changes in the phagocytic activity of the cells were observed. Compared with those in normal control group, the percentage of AMs phagocytosing fluorescent microspheres was significantly decreased, and the expression of IL-33 in lung tissue and IL-33 level in the bronchoalveolar lavage fluid were significantly increased in ALI mice (P < 0.01). LPS (100-1000 ng/mL) obviously promoted the secretion of IL-33 in cultured MLE-12 cells (P < 0.01). Both LPS (10-500 ng/mL) and IL-33 (100 ng/mL) significantly inhibited the phagocytic activity of the AMs from normal control mice (P < 0.01). The phagocytic activity of AMs is weakened in ALI mice possibly due to direct LPS stimulation and the inhibitory effect of the alarmin IL-33 produced by LPS-stimulated alveolar epithelial cells.

Effect of human placental mesenchymal stem cells transplantation on pulmonary vascular endothelial permeability and lung injury repair in mice with acute lung injury

To investigate the effects of human placental mesenchymal stem cells (hPMSCs) transplantation on pulmonary vascular endothelial permeability and lung injury repair in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). The hPMSCs were isolated from the human placental tissue by enzyme digestion and passaged. The cell phenotype of the 3rd generation hPMSCs was detected by flow cytometry. Twenty-four 6-week-old healthy male C57BL/6 mice were randomly divided into 3 groups ( n=8). The mice were instilled with LPS in the airway to prepare an ALI model in the ALI model group and the hPMSCs treatment group, and with saline in the control group. At 12 hours after LPS infusion, the mice were injected with 3rd generation hPMSCs via the tail vein in hPMSCs treatment group and with saline in the ALI model group and the control group. At 24 hours after injection, the lung tissues of all mice were taken. The pathological changes were observed by HE staining. The wet/dry mass ratio (W/D) of lung tissue was measured. The Evans blue leak test was used to detect the pulmonary vascular endothelial permea bility in mice. The expression of lung tissue permeability-related protein (VE-cadherin) was detected by Western blot. Flow cytometry examination showed that the isolated cells had typical MSCs phenotypic characteristics. Mice in each group survived. The alveolar structure of the ALI model group significantly collapsed, a large number of inflammatory cells infiltrated, and local alveolar hemorrhage occurred; while the alveolar structure collapse of the hPMSCs treatment group significantly improved, inflammatory cells infiltration significantly reduced, and a few red blood cells were in the interstitial lung. W/D and exudation volume of Evans blue stain were significantly higher in the ALI model group than in the control group and the hPMSCs treatment group ( P<0.05), in the hPMSCs treatment group than in the control group ( P<0.05). The relative protein expression of VE-cadherin was significantly lower in the ALI model group than in the control group and the hPMSCs treatment group ( P<0.05), and in the hPMSCs treatment group than in the control group ( P<0.05). Intravenous injection of hPMSCs can effectively reduce the increased pulmonary vascular endothelial permeability mediated by LPS, relieve the degree of lung tissue damage, and play a therapeutic role in ALI mice.

Influence regulation of inflammatory immune response by interleukin-17 lipopolysaccharide-induced acute lung injury in mice

To explore the immunomodulatory effects of interleukin-17 (IL-17) on acute lung injury (ALI) induced by lipopolysaccharide (LPS). Thirty-six SPF-class C57BL/6 mice were divided into normal saline control group (NS group) and LPS-induced ALI model group (LPS group, LPS 5 mg/kg intratracheal drip) according to random number table method, with 18 mice in each group. Six mice were sacrificed at 2, 6 and 24 hours after model reproduction, and peripheral blood, lung and spleen tissues were harvested. After staining with hematoxylin-eosin (HE), the pathological changes of lung tissue were observed under microscope and the infiltration level of lymphocytes, neutrophils and macrophages in the alveolar wall and tracheal wall were detected. Immunohistochemistry was used to detect the protein expression of IL-17 in alveolar wall and tracheal wall, and the correlation between IL-17 expression and lymphocytes, neutrophils and macrophages infiltration in alveolar wall and tracheal wall were analyzed. The level of IL-17 in lung tissue homogenate was determined by enzyme linked immunosorbent assay (ELISA). Flow cytometry was used to detect the proportion of CD4+IL-17+ helper T cells (Th17 cells) in CD4+ T cells in peripheral blood, lung tissue and spleen tissue. (1) Microscopy showed that the lung tissue structure of NS group was basically normal at each time after model reproduction, and there was no obvious inflammatory cell infiltration, while the lung tissue edema and inflammatory reaction were gradually aggravated in the LPS group, and the lung injury score was significantly higher than that in NS group at each time (2 hours: 4.47±1.42 vs. 1.10±0.55, 6 hours: 7.93±2.14 vs. 1.23±0.50, 24 hours: 12.67±2.67 vs. 1.20±0.61, all P < 0.01). (2) Immunohistochemistry showed that the protein expression of IL-17 in alveolar wall and tracheal wall of LPS group increased gradually with time, while that in NS group was negative or weak positive. Quantitative analysis showed that the immunohistochemical staining score of IL-17 protein in alveolar wall and tracheal wall of LPS group were higher than those of NS group (alveolar wall: 2.70±1.40 vs. 0.90±0.37 at 2 hours, 5.10±1.76 vs. 1.17±0.59 at 6 hours, 9.67±1.32 vs. 1.10±0.45 at 24 hours; tracheal wall: 2.87±0.89 vs. 0.90±0.39 at 2 hours, 4.97±1.48 vs. 1.10±0.41 at 6 hours, 8.67±1.54 vs. 1.03±0.29 at 24 hours; all P < 0.05). (3) Correlation analysis showed that the protein expression of IL-17 in alveolar wall and tracheal wall were positively correlated with the degree of lymphocyte, neutrophil and macrophage infiltration (alveolar wall: r value was 0.632, 0.550, 0.466; tracheal wall: r value was 0.695, 0.662, 0.575, respectively; all P < 0.01). (4) IL-17 content (μg/L) in lung tissue homogenate was significantly higher than that in NS group at each time after model reproduction (2 hours: 1.37±0.14 vs. 1.01±0.18, 6 hours: 1.65±0.19 vs. 1.11±0.18, 24 hours: 1.92±0.36 vs. 1.17±0.24, all P < 0.01). (5) The proportion of Th17 cells in the peripheral blood, lung tissue and spleen tissue of the LPS group were higher than those of the NS group at each time after model reproduction [peripheral blood: (2.62±0.62)% vs. (1.42±0.40)% at 2 hours, (3.74±0.43)% vs. (1.27±0.32)% at 6 hours, (4.44±0.65)% vs. (1.59±0.45)% at 24 hours; lung tissue: (2.32±0.44)% vs. (1.50±0.25)% at 2 hours, (3.66±0.36)% vs. (1.33±0.24)% at 6 hours, (4.60±0.54)% vs. (1.60±0.27)% at 24 hours; spleen tissue: (1.49±0.36)% vs. (0.69±0.21)% at 2 hours, (2.58±0.55)% vs. (0.59±0.18)% at 6 hours, (3.76±0.57)% vs. (0.65±0.26)% at 24 hours; all P < 0.01]. IL-17 is involved in the inflammatory immune regulation of ALI mice.

Effects of keratinocyte growth factor-2 on expressions of chemokine FKN and tight junction protein claudin-5 in lung of rats with acute lung injury

To observe the effects of keratinocyte growth factor-2 (KGF-2) on the expressions of chemokine FKN and tight junction protein claudin-5 in lung tissue of rats with acute lung injury (ALI). Healthy male Sprague-Dawley (SD) rats were randomly divided into normal saline (NS) control group, ALI model group and KGF-2 pretreatment group, with 10 rats in each group. The rat ALI model was reproduced by injection of 0.01 mL/kg oleic acid into the tail vein, and the rats in NS control group were injected with the same amount of NS. The rats in KGF-2 pretreatment group were instilled with 5 mg/kg KGF-2 in the airway at 72 hours before the model reproduction, and the rats in the NS control group and the ALI model group were instilled with the same amount of NS. The abdominal aortic blood of rats was collected at 8 hours after model reproduction, and then the rats were sacrificed, bronchoalveolar in left lung was lavage, and the bronchoalveolar lavage fluid (BALF) was collected for determination of protein levels in plasma and BALF, and the lung permeability index (LPI) was calculated. The lung tissues were harvested, after hematoxylin-eosin (HE) staining, the histopathological changes were observed under light microscope, and the ALI pathology score (LIS) was calculated. The lung wet/dry weight (W/D) ratio was determined. Immunohistochemistry and Western Blot were used to qualitatively and quantitatively analyze the expressions of FKN and claudin-5 in the lung tissue. The correlation between two variables was analyzed by linear or curve fitting correlation analysis. In the ALI model group, the lung tissue was severely damaged, and obvious pathological changes were observed, including thickened alveolar space and inflammatory cell infiltration, and LIS score, lung W/D ratio and LPI were significantly higher than those of the NS control group (LIS: 3.56±0.28 vs. 0.62±0.36, lung W/D ratio: 6.37±0.29 vs. 4.39±0.33, LPI: 3.46±0.69 vs. 0.98±0.17, all P < 0.01). Compared with the NS control group, the positive expression of FKN in the lung tissue of the ALI model group was significantly increased, and the expression level was significantly increased (FKN/GAPDH: 0.97±0.18 vs. 0.62±0.04, P < 0.01); the positive expression of claudin-5 was significantly decreased, and the expression level was significantly decreased (claudin-5/GAPDH: 0.56±0.11 vs. 1.06±0.13, P < 0.01). There was a significant negative correlation between FKN and claudin-5 protein expression (r = -0.817, P = 0.025). After pretreatment with KGF-2, the degree of lung tissue damage was significantly reduced, and the pathological changes were significantly improved, and the LIS score, lung W/D ratio and LPI were significantly lower than those of the ALI model group (LIS: 2.41±0.17 vs. 3.56±0.28, lung W/D ratio: 5.45±0.55 vs. 6.37±0.29, LPI: 2.42±0.19 vs. 3.46±0.69, all P < 0.01). Compared with the ALI model group, the positive expression of FKN in the lung tissue of KGF-2 pretreatment group was significantly decreased, and the expression level was significantly decreased (FKN/GAPDH: 0.79±0.03 vs. 0.97±0.18, P < 0.01); the positive expression of claudin-5 was significantly increased, and the expression level was significantly increased (claudin-5/GAPDH: 0.80±0.05 vs. 0.56±0.11, P < 0.01). There was still a significant negative correlation between FKN and claudin-5 protein expression (r = -0.847, P = 0.012). KGF-2 may restore the expression of tight junction protein claudin-5 by down-regulating the expression of chemokine FKN, thereby reducing the damage of blood barrier in ALI.

VEGF165 transfected endothelial progenitor cells mediated by lentivirus alleviated ALI in rats

To investigate the protective effects of vascular endothelial growth factor-165 (VEGF165) transfected the endothelial progenitor cells (EPCs) mediated by lentivirus on acute lung injury (ALI) in rats. The mononuclear cells from the male Sprague-Dawley (SD) rats were isolated and cultured to get the EPCs for study. The lentivirus vector carrying the human VEGF165 gene was constructed. According to the random number table method, 90 male SD rats were divided into ALI model group, phosphate buffer solution (PBS) group, EPCs treatment group, none transfected EPCs treatment group and VEGF165 transfected EPCs treatment group, and the rats in each group were subdivided into 4, 12 and 48 hours subgroups, with 6 rats in each subgroup. The rat model of ALI was reproduced by intravenous injection of oleic acid (0.15 μL/g). Then each treatment group was given PBS, EPCs, none transfected EPCs and VEGF165 transfected EPCs respectively with the same volume of 0.2 mL. For the groups with cells, about 1×106 cells were contained. Abdominal aortic blood and lung tissue were harvested at 4, 12 and 48 hours. Arterial blood gas analysis was performed. The lung wet/dry weight ratio (W/D) was calculated. The expressions of induced nitric oxide synthase (iNOS), endothelin-1 (ET-1) and VEGF165 were determined by enzyme-linked immunosorbent assay (ELISA). After dyed with hematoxylin-eosin (HE), the lung tissue pathology was observed and the lung injury score was performed. Compared with the ALI model group, the arterial partial pressure of oxygen (PaO2) in EPCs, none transfected EPCs and VEGF165 transfected EPCs treatment groups was significantly increased from 4 hours, and lung W/D, expressions of iNOS and ET-1 were significantly decreased, and VEGF165 expression was significantly increased. Compared with the EPCs treatment group, the increase in PaO2, the decrease in lung W/D and expressions of iNOS and ET-1, and the increase in VEGF165 expression in VEGF165 transfected EPCs treatment group were more significant [4 hours: PaO2 (mmHg, 1 mmHg = 0.133 kPa) was 82.84±10.69 vs. 72.34±9.36, lung W/D ratio was 4.83±0.23 vs. 5.55±0.37, iNOS (ng/mg) was 8.77±1.10 vs. 14.84±1.34, ET-1 (ng/mg) was 103.41±5.66 vs. 153.08±5.12, VEGF165 (ng/mg) was 130.56±12.16 vs. 83.03±5.95; 12 hours: PaO2 (mmHg) was 91.67±6.81 vs. 78.5±8.81, lung W/D ratio was 4.44±0.35 vs. 5.32±0.25, iNOS (ng/mg) was 7.23±0.24 vs. 14.04±1.18, ET-1 (ng/mg) was 91.98±3.52 vs. 125.99±7.55, VEGF165 (ng/mg) was 164.49±5.71 vs. 96.61±6.12]; individual parameters reached valley value or peak value at 48 hours [lung W/D ratio was 4.26±0.30 vs. 4.89±0.15, iNOS (ng/mg) was 5.79±0.85 vs. 12.72±1.10, ET-1 (ng/mg) was 74.53±7.10 vs. 108.33±5.84, VEGF165 (ng/mg) was 237.43±10.79 vs. 134.24±11.99, all P < 0.05]. Over time, lung tissue injury in each group was gradually increased, and the lung injury score was gradually increased. The lung injury score at 48 hours in the EPCs, none transfected EPCs and VEGF165 transfected EPCs treatment groups were lower than that in the ALI model group. Compared with the EPCs treatment group, the VEGF165 transfected EPCs treatment group had a lower score at 48 hours (8.50±1.05 vs. 10.50±1.05, P < 0.05). The transplantation of EPCs which were transfected with VEGF165 mediated by lentivirus could obviously improve the oxygen pressure, reduce the lung water seepage, decrease the iNOS and ET-1 expressions in lung tissue, and had obvious protective effects on ALI.

Effects of calcitonin gene-related peptide on aquaporin 1 and aquaporin 5 expressions in young rats with acute lung injury

Objective To explore the effect of calcitonin gene-related peptide (CGRP) on expressions of aquaporin (AQP)1 and AQP 5 in young rats with acute lung injury(ALI) caused by lipopolysaccharide(LPS). Methods Eighty-four young rats were randomly divided into control group, ALI model group and CGRP group.The rats in ALI model group were given intraperitoneal injection of LPS (5 mg/kg)for 2, 6, 12, 24 hours; while the rats in CGRP group were given intraperitoneal CGRP (1 mg/kg) after 1 h injection of LPS.At 2 h, 6 h, 12 h and 24 h, all rats were sacrificed and lung tissues were obtained.The histopathological changes in lung tissues were evaluated by adopting hematoxylin-eosin staining, and wet/dry(W/D) was measured.The mRNA and protein levels of AQP1 and AQP5 in lung tissues were detected by adopting fluorogenic quantitative PCR(qPCR) and Western blot. Results Pathological stain showed that rats in control group had a normal lung tissue structure, and LPS made lung tissue edema, narrowing the alveolar cavity and inflammatory cell infiltration.CGRP attenuated the effect of LPS on rat′s lung.The W/D ratio of lung tissue was significantly higher than that in the control group, and CGRP reduced the W/D ratio of lung tissue.qPCR showed that the mRNA levels of AQP1 and AQP5 from rats in ALI group (0.009±0.001 and 0.055±0.006) decreased compared with those in the control group (0.035±0.002 and 0.167±0.006) and CGRP group (0.024±0.002 and 0.134±0.012) (all P<0.001). Western blot results showed after 24 h injection of LPS, both AQP1 and AQP5 levels from ALI group (0.397±0.041 and 0.215±0.029) were significantly lower than those in the control group (0.850±0.020 and 0.741±0.032) (all P<0.001), and their levels in CGRP group (0.593±0.065 and 0.461±0.039) were also lower than those in the control group, but higher than those in ALI group (all P<0.001). Conclusion CGRP can enhance AQP1 and AQP5 levels and reduce pulmonary edema, and it has a protective effect on rats with acute lung injury. Key words: Acute lung injury; Calcitonin gene-related peptide; Aquaporin

Polysaccharides from Arnebia euchroma Ameliorated Endotoxic Fever and Acute Lung Injury in Rats Through Inhibiting Complement System.

Arnebiaeuchroma (Royle) Johnst (Ruanzicao) is a traditional Chinese herbal medicine (TCM). It is extensively used in China and other countries for treatment of inflammatory diseases. It is known that hyper-activated complement system involves in the fever and acute lung injury (ALI) in rats. In our preliminary studies, anti-complementary activity of crude Arnebiaeuchroma polysaccharides (CAEP) had been demonstrated in vitro. This study aimed to investigate the role and mechanism of crude Arnebiaeuchroma polysaccharides (CAEP) using two animal models, which relate with inappropriate activation of complement system. In lipopolysaccharide (LPS)-induced fever model, the body temperature and leukocytes of peripheral blood in rats were significantly increased, while the complement levels of serum were remarkably decreased. CAEP administration alleviated the LPS-induced fever, reduced the number of leukocytes, and improved the levels of complement. Histological assay showed that there were severe damages and complement depositions in lung of the ALI rats. Further detection displayed that the oxidant stress was enhanced, and total hemolytic activity and C3/C4 levels in serum were decreased significantly in the ALI model group. Remarkably, CAEP not only attenuated the morphological injury, edema, and permeability in the lung but also significantly weakened the oxidant stress in bronchoalveolar lavage fluid (BALF) in the ALI rats. The levels of complement and complement depositions were improved by the CAEP treatment. In conclusion, the CAEP treatment ameliorated febrile response induced by LPS and acute lung injury induced by LPS plus ischemia-reperfusion. CAEP exerted beneficial effects on inflammatory disease potentially via inhibiting the inappropriate activation of complement system.

Impact of TREM-2 gene silencing on inflammatory response of endotoxin-induced acute lung injury in mice.

Acute lung injury (ALI) is one of the critical clinical respiratory diseases, of which infection is the main cause and the first risk factor. This study investigated the impact of triggering receptor of myeloid cells expression (TREM)-2 gene silencing on inflammatory response of endotoxin-induced ALI in mice. Lentivirus-mediated TREM-2-shRNA was transfected into healthy male C57BL/6 mice, and the lipopolysaccharide-induced ALI model was established. The immunohistochemistry, immunofluorescence, fluorescence quantitative PCR, western blot, and ELISA were applied to detect the pathological changes of lung tissue and expressions of TREM-2, tumor necrosis factor-α (TNF-α), and interleukin 10 (IL-10) in bronchoalveolar lavage fluid. The lentivirus group, saline control group, ALI model group, blank control group, and negative control group were set up at the same time. Results found that, in lentivirus group, the pathological change of lung tissue was significantly lighter than ALI model group (P < 0.05), and the expression of TREM-2 was significantly reduced compared with all control groups (P < 0.05). The levels of TNF-α and IL-10 were significantly increased than all control groups (P < 0.05), while above indexes in negative control group and blank control group showed no significant difference with ALI group (P > 0.05). This study indicates that TREM-2 has a protective effect on inflammatory response of endotoxin-induced ALI in mice, which has provided new potential targets for prevention and treatment of ALI.

Influence of resolvin E1 on inflammatory response in mice with acute lung injury

Objective To evaluate the intluence ot resolvin E1 ( RvE1 ) on intlammatory response in mice with acute lung injury (ALI).Methods The animal model of ALI was established. After 12 h,histological changes,wet-to-dry (W/D) weight ratio of the lungs and the contents of tumor necrosis factorα (TNF-α) in bronchoalveolar lavage fluid (BALF) and nuclear factor-κB (NF-κB) in lung homogenate were observed.Results Histological study showed the lung injury was significantly alleviated in RvE1-treated group.W/D ratio (4.637 ±0.125) g/g and contents of TNF-α (217.2 ±30.1 ) ng/L in BALF of RvE1-treated group were significantly decreased as compared with ALI model group [for W/D ratio:(4.906 ± 0.176) g/g,and for contents of TNF-α:(372.2 ± 20.6) ng/L] ( P ＜ 0.05 ).The level of NF-κB in RvE1-treated group was significantly lower than in ALI model control group.Conclusion RvE1 can inhibit inflammatory response and alleviate lung injury in mice with ALI. Key words: Resolvin E1; Acute lung injury; Inflammatory response; Nuclear factor-κB

Effects of penehyclidine hydrochloride on acute lung injury in rats

盐酸戊已奎醚(penehyclidine hydrochloride,PHCD)是一类新型选择性抗胆碱药,与其他抗胆碱药所不同的是,该药效持续作用时间长,心血管副作用小等特点.该药已成功应用于临床救治有机磷农药中毒[1],并推荐用于麻醉前给药[2],但对内毒素性急性肺损伤(ALI)的影响尚未见文献报道.本实验通过大鼠内毒素性ALI模型,观察盐酸戊二奎醚对肺NF-κB、一氧化氮合酶(NOS)和肺和血清中一氧化氮(NO)及肺病理形态学的影响,探讨盐酸戊二奎醚对ALI的防治效应及作用机制,并为临床应用提供理论依据。