Acute Lung Injury In Rats Research Articles

Proteomic analysis of therapeutic effects of Qingyi pellet on rodent severe acute pancreatitis-associated lung injury

Severe acute pancreatitis (SAP) is a common acute clinical abdomen syndrome which is characterized by pancreatic self-digestion. As one of the major complication of SAP, acute lung injury is the main reason of high mortality. The traditional Chinese medicine Qingyi pellet (QYT) has been widely used for SAP in clinic. In our study, we constructed the severe acute pancreatitis-associated lung injury (SAP-ALI) rat model and treated with QYT, then characterized the protein from the lung tissue by using a mass spectrometry-based proteomic strategy. Our results showed that, in the SAP group, 9 proteins exhibited obvious changes according to the proteomic analysis. Among the 9 proteins, 7 proteins (alpha-2-macroglobulin, Cathepsin S, ras-related protein RAP-1A, integrin beta, protein phosphatase 2A, Intercellular adhesion molecule 1 and p38) were up-regulated, and 2 proteins (adapter molecule Crk and stathmin) were down-regulated. Interestingly, the data of the QYT group showed that adapter molecule Crk and stathmin were up-regulated, but the other 7 proteins were down-regulated. The kyoto encyclopedia of genes shows that the proteins act on PI3K-AKT, chemokine signaling pathways, apoptosis, leukocyte transendothelial migration and focal adhesion. Therefore, the therapeutic effects of QYT on SAP-ALI are potentially through the additive and/or synergistic interactions of numerous components.

Novel insights into the role of LRRC8A in ameliorating alveolar fluid clearance in LPS induced acute lung injury

Leucine-rich repeat-containing 8A (LRRC8A) protein was recently identified as an essential component of volume-regulated anion channel which plays a central role in maintaining cell volume. The aim of this study was to elucidate the role of LRRC8A in alveolar fluid clearance (AFC) and the effect of inflammatory cytokines on LRRC8A and the underlying mechanism. Lipopolysaccharide (LPS) was used to generate a rat acute lung injury model. The results showed that the concentrations of IL-1β, TNF-α and IL-6 in bronchoalveolar lavage fluid increased significantly, but the expression of LRRC8A in the lung tissue decreased dramatically in the acute lung injury group followed by a decline in the AFC rate. Additionally, LRRC8A knockdown reduced AFC in normal rats. However, specific overexpression of LRRC8A in the lung could increase AFC. Furthermore, we observed the effects of LPS, IL-1β, TNF-α and IL-6 on the LRRC8A current in alveolar type II (ATII) cells, and IL-1β showed the greatest inhibition among them, which was involved in phospho-p38 activation. Overall, LRRC8A plays an essential role in the progression of AFC in LPS-induced acute lung injury, and chronic treatment with IL-1β or TNF-α could inhibit the function of LRRC8A in ATII cells by targeting phospho-p38. All of the findings suggested that LRRC8A could be a new partner in AFC and a potential target for the treatment of acute lung injury.

VEGF mediates fat embolism-induced acute lung injury via VEGF receptor 2 and the MAPK cascade

Fat embolism (FE) is a lethal medical emergency often caused by fracture of long bones and amputation of limbs. Vascular endothelial growth factor (VEGF) promotes angiogenesis and increases vascular permeability. We tested the hypothesis that VEGF plays a critical role in FE-induced acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). Fat tissues were collected from male Sprague-Dawley rats, and animal oil was extracted and mixed with water to form fatty micelles. The micelles were then injected into the tail vein to produce FE and ALI in rats. Lung weight gain was measured as the index of pulmonary edema. The expression of pulmonary VEGF was evaluated by real-time PCR and western blot analysis. Inducible nitric oxide synthase (iNOS) and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by western blot analyses. Interleukin-1β (IL-1β) was quantified by ELISAs. Hematoxylin and eosin staining was used to evaluate the pathological damage of ALI. In this study, we found that animal oil-induced FE significantly increased pulmonary VEGF expression and MAPK phosphorylation. We also evaluated the inflammatory response after FE and found that iNOS and IL-1β significantly increased after FE. Systemic administration of SU-1498, an antagonist of VEGF receptor 2 (VEGFR-2), significantly attenuated the FE-induced inflammatory response and histological damage. This study suggested that VEGF is involved in FE-induced ARDS via the VEGFR-2 and MAPK cascades, which induce IL-1β release and iNOS upregulation. Blockade of could be used to treat FE-induced pulmonary damage.

Dexmedetomidine attenuates lipopolysaccharide induced acute lung injury in rats by inhibition of caveolin-1 downstream signaling

ObjectiveToll-like receptor 4(TLR-4)/nuclear factor-kappa B(NF-κB) pathway plays an important role in inducing acute lung injury (ALI). Studies have proved Dexmedetomidine (Dex) inhibits inflammatory response to mitigate lipopolysaccharide (LPS)‐induced ALI and protect against multiorgan injury in various scenarios via restraining TLR-4/NF-κB signaling pathway. Many of the known downstream molecules have been orientated with a protein caveolin-1(Cav-1), which is supposed to take part in regulating TLR4-mediated inflammatory responses. However, its mechanisms have not been confirmed. The aim of this study is to evaluate the protective effects and potential mechanisms of Dex against LPS-induced ALI in male rats. MethodsMale rats received tail-vein injection of LPS to form ALI model. Rats were administrated with intraperitoneal injection Dex0.5 h before ALI. At 6 h after LPS injection, bronchoalveolar lavage fluid (BALF) and lung tissue were harvested. We stained the lung tissue sections with hematoxylin eosin (HE) staining to observe the histopathological damage and measure the ALI pathology score. We also measured the wet-to-dry(W/D) weight ratio of lung tissue. Lung myeloperoxidase (MPO) and inflammatory cytokines in the BALF were detected by Enzyme‐linked immunosorbent assay(ELISA). Protein levels of Cav-1, TLR-4 and NF-κB in lung tissue were tested by immunohistochemistry method. The mRNA expression of Cav-1, TLR4 and the NF-κB in lung tissue were measured to determine the related mechanisms by quantitative real-time polymerase chain reaction(RT-PCR). ResultsIt was indicated that Dex pretreatment markedly mitigated pathomorphologic changes and pathological lung injury scores. Besides, Dex pretreatment obviously decreased the W/D weight ratio of lung tissue, attenuated MPO activity significantly, along with LPS-stimulated augment of lung inflammatory cells infiltration in BALF. Moreover, compared with LPS model group, Dex pretreatment apparently increased the protein levels of Cav-1 downregulated by sepsis and decreased the protein levels of TLR-4 and NF-κB in lung tissue. Furthermore, Dex pretreatment apparently upregulated the expression of Cav-1 mRNA, restrained TLR4 and NF-κB mRNA. ConclusionDex pretreatment protects against LPS‐induced ALI via inhibiting the activation of the TLR-4/NF-kB signaling pathway by upregulating the expression of Cav-1 downregulated by sepsis.

Ulinastatin ameliorates LPS‑induced pulmonary inflammation and injury by blocking the MAPK/NF‑κB signaling pathways in rats.

Ulinastatin, a urinary trypsin inhibitor (UTI) is commonly used to treat patients with acute inflammatory disease. However, the underlying mechanisms of its anti‑inflammatory effect in acute lung injury(ALI) are not fully understood. The present study aimed to investigate the protective effect of UTI and explore its potential mechanisms by using a rat model of lipopolysaccharide(LPS)‑induced ALI. Rats were treated with 5mg/kg LPS by intratracheal instillation. The histological changes in LPS‑induced ALI was evaluated using hematoxylin and eosin staining and the myeloperoxidase(MPO) activity was determined using ELISA. The wet/dry ratio (W/Dratio) of the lungs was used to assess the severity of pulmonary edema and Evans blue dye was used to evaluate the severity of lung vascular leakage. The results demonstrated that LPS administration induced histological changes and significantly increased the lung W/Dratio, MPO activity and Evans blue dye extravasation compared with the control group. However, treatment with UTI attenuated LPS‑induced ALI in rats by modifying histological changes and reducing the lung W/Dratio, MPO activity and Evans blue dye extravasation. In addition, LPS induced the secretion of numerous pro‑inflammatory cytokines in bronchoalveolar lavage fluid (BALF), including tumor necrosis factor‑α, interleukin(IL)‑6, IL‑1β and interferon‑γ; however, these cytokines were strongly reduced following treatment with UTI. In addition, UTI was able to reduce cellular counts in BALF, including neutrophils and leukocytes. Western blotting demonstrated that UTI significantly blocked the LPS‑stimulated MAPK and NF‑κB signaling pathways. The results of the present study indicated that UTI could exert an anti‑inflammatory effect on LPS‑induced ALI by inhibiting the MAPK and NF‑κB signaling pathways, which suggested that UTI may be considered as an effective drug in the treatment of ALI.

P-Coumaric acid protects cardiac function against lipopolysaccharide-induced acute lung injury by attenuation of oxidative stress.

Objective(s):Acute lung injury (ALI) has a high mortality rate and is characterized by damage to pulmonary system giving rise to symptoms such as histological alteration, lung tissue edema and production of proinflammatory cytokine. p-Coumaric acid (p-CA), as a phenolic compound, that is found in many types of fruits and vegetables has been reported to exhibit a therapeutic effect in several inflammatory disorders. The aim of our study was evaluation of pretreatment with p-CA against heart dysfunction, oxidative stress and nuclear factor-erythroid 2 -related factor 2 (Nrf2) modifications following lipopolysaccharide (LPS)-induced acute lung inflammation. Materials and Methods:The rats were divided into four groups (n=8): Control, LPS (5 mg/kg, it), p-CA (100 mg/kg, IP), and LPS+pCA. Inflammatory response and oxidative stress were evaluated by measurement of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α) and malondialdehyde (MDA) levels in heart tissue. For evaluation of the effect of LPS on cardiac response, electrocardiography (ECG) and hemodynamic parameters were recorded.Results:A significant increase in lipid peroxidation (P<0.001, cytokine parameters (TNF-α and IL-6 (P<0.01), gene expression of Nrf2 (P<0.05), and antioxidant activity of superoxide dismutase and glutathione (P< 0.05) in addition to glutathione peroxidase (P<0.01) was demonstrated in heart tissue of ALI rats. LPS can impair cardiac function (in in vitro measurement of hemodynamic parameters by using Langendorff setup, and in in vivo measurement of ECG parameters), and pretreatment with p-CA recovered these parameters to control levels in heart. Pretreatment with p-CA causes modulation of cytokines and MDA level that protected cardiac injury caused by LPS in ALI model. Conclusion:Our results showed anti-inflammatory and antioxidative effect of p-CA on LPS-induced ALI.

The Protective Effects of Protease Inhibitor MG-132 on Sepsis-Induced Acute Lung Rats and Its Possible Mechanisms.

BackgroundThe aim of the present study was to investigate the protective effects of protease inhibitor MG-132 on sepsis-induced acute lung injury rats.Material/MethodsSprague Dawley rats were employed to induce sepsis by cecal ligation and puncture (CLP) method. Rats were divided into 4 groups: control, sham, model (CLP), and MG-132. Histopathology observation was detected by hematoxylin and eosin staining. The ratio of wet lung to dry lung (W/D) was calculated. In addition, the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay (ELISA). Also, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were evaluated. Western blotting was performed to measure the expression of hypoxia-inducible factor-1 α (HIF-1α). In order to assess the role of HIF-1α, YC-1, the inhibitor of HIF-1α, was used to treat the rats. The expression of phosphor-mTOR (p-mTOR), p-4EBP1, and p-EIF4E were evaluated by western blotting.ResultsObvious pathological injury and increasing ratio of W/D in the model group were observed. Both pathological injury and W/D were improved in the MG-132 group, and the greatest improvement could be seen in the YC-1+MG-132 group. Furthermore, the MDA levels in the MG-132 group was decreased, accompanied by an increase in SOD levels. The level of HIF-1α was increased in the model group while a decreased was detected in the MG-132 group. The levels of inflammatory factors were high in the model group, whereas the opposite result was found in the MG-132 group, and the lowest in were in the YC-1+MG-132 group. Furthermore, the expression levels of p-mTOR, p-4EBP1, and p-EIF4E proteins were downregulated in the MG-132 group compared to the model group, and the lowest was in the YC-1+MG-132 group.ConclusionsOur study suggested that MG-132 was able to protect against acute lung injury via inhibition of HIF-1α mediated mTOR/4EBP1/EIF4E pathway.

Acute lung injury in neonatal rats causes postsynaptic depression in nucleus tractus solitarii second-order neurons

Acute Lung Injury (ALI) alters pulmonary reflex responses, in part due to changes in modulation within the lung and airway neuronal control networks. We hypothesized that synaptic efficacy of nucleus tractus solitarii (nTS) neurons, receiving input from lung, airway, and other viscerosensory afferent fibers, would decrease following ALI. Sprague Dawley neonatal rats (postnatal days 9–11) were given intratracheal installations of saline or bleomycin (a well-characterized model that reproduces the pattern of ALI) and then, one week later, in vitro slices were prepared for whole-cell and perforated whole-cell patch-clamp experiments (postnatal days 16–21). In preparations from ALI rats, 2nd-order nTS neurons had significantly decreased amplitudes of both spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs), compared to saline controls. Rise and decay times of sEPSCs were slower in whole-cell recordings from ALI animals. Similarly, the amplitude of tractus solitarii evoked EPSCs (TS-eEPSCs) were significantly lower in 2nd-order nTS neurons from ALI rats. Overall these results suggest the presence of postsynaptic depression at TS-nTS synapses receiving lung, airway, and other viscerosensory afferent tractus solitarii input after bleomycin-induced ALI.

Keratinocyte Growth Factor-2 Is Protective in Oleic Acid-Induced Acute Lung Injury in Rats.

Objective The aim of this study was to examine the role of keratinocyte growth factor-2 (KGF-2) in oleic acid-induced acute lung injury (ALI) in rats. Methods Forty-five healthy adult male Sprague Dawley rats were divided into 3 groups. Rat ALI model was established by injection of 0.01 mL/kg oleic acid into the tail vein. Rats in the control group were injected with the same amount of normal saline (NS). In the ALI + KGF-2 group, 5 mg/kg of KGF-2 was instilled into the airway of rats 72 hours before the model preparation, and the control group and the ALI model group were instilled with the same amount of NS. The lung permeability index (LPI) and lung wet/dry weight (W / D) ratios were measured 8 hours after the model preparation. The permeability of pulmonary microvascular endothelium was evaluated by Evans blue leakage test. Histopathological changes were observed under light microscope and the ALI pathology score (LIS) was calculated. Ultrastructural changes of lung tissue were observed under electron microscope. The apoptosis was detected by TUNEL assay. The expression of Claudin-5, ZO-1, and VE Cadherin in lung tissue was qualitatively and quantitatively analyzed by immunohistochemistry, Western Blot, and qRT-PCR, respectively. Results The ALI model group had severe lung injury and obvious pathological changes, including alveolar septal thickening and inflammatory cell infiltration. TUNEL assay showed that the apoptosis of ALI group was significantly increased. The LIS score, lung W/D ratio, LPI, and Evans blue leakage were significantly higher than those in the control group; electron microscopy showed that the alveolar-capillary barrier was severely damaged in the ALI group. Compared with the control group, the expression of Claudin-5, ZO-1, and VE cadherin in the lung tissue of the ALI model group was significantly attenuated. After pretreatment with KGF-2, the degree of lung tissue damage was significantly reduced and the pathological changes were significantly improved. TUNEL assay showed that the apoptosis of ALI group was decreased. Lung W/D ratio, LPI, and Evans blue leakage decreased; electron microscopy showed that the alveolar-capillary barrier of ALI group recovered significantly. Compared with the ALI model group, the expression of Claudin-5, ZO-1, and VE cadherin in the lung tissue of the KGF-2 pretreatment group increased. Conclusion The results indicate that KGF-2 may attenuate oleic acid-induced ALI in rats by maintaining the pulmonary microvascular endothelial barrier, which is an effective ALI preventive measure.

Astragalin reduces lipopolysaccharide-induced acute lung injury in rats via induction of heme oxygenase-1.

Astragalin, a bioactive component of medicinal plants such as Rosa agrestis, has anti-inflammatory and antioxidant features. Induction of heme oxygenase (HO)-1 is an effective strategy to reduce excessive generated oxidants during the pathogenesis of acute lung injury (ALI). The aim of the present study is to investigate that whether the anti-inflammatory and antioxidant features of astragalin is HO-1 dependent in lipopolysaccharide (LPS)-induced ALI. Sprague-Dawley rats were used in animal study. Intratracheal LPS was performed to induce experimental ALI model. Astragalin was administrated 1h after LPS challenge. Human lung epithelial cells were used in cell study. Samples from rats were harvested at 24h post LPS challenge. Astragalin treatment inhibited LPS-induced inflammatory cells infiltration in the lung and pulmonary edema. Astragalin treatment markedly enhanced the activity of HO-1 compared with vehicle-treated group at 24h post LPS challenge. Levels of lipid hydroperoxide, a marker for oxidative stress, were decreased in astragalin-treated animals compared with vehicle-treated group. However, the protective effect of astragalin on LPS-induced ALI was abolished in an inhibitor of HO-1-treated animals. Moreover, the astragalin-induced the upregulation of HO-1 in human lung epithelial cells was inhibited when nuclear factor erythroid-2-related factor 2 (Nrf2) was silenced by small interfering RNA. Astragalin reduces LPS-induced ALI via activation of Nrf2/HO-1 pathway.

Role of endogenous heme oxygenase-1/carbon monoxide signaling pathway in endoplasmic reticulum stress during endotoxin-induced acute lung injury in rats

Objective To evaluate the role of endogenous heme oxygenase-1/carbon monoxide (HO-1/CO) signaling pathway in endoplasmic reticulum stress during endotoxin-induced acute lung injury(ALI) in rats. Methods Forty healthy clean-grade male Sprague-Dawley rats, aged 8 weeks, weighing 190-210 g, were divided into 4 groups (n=10 each) using a random number table method: control group (group C), ALI group, ALI plus ZnPP-IX group (group AZ), and ALI plus vehicle sodium bicarbonate group (group AV). ALI was induced by intravenously injecting lipopolysaccharide 5 mg/kg in anesthetized rats.At 30 min before establishing the model, ZnPP-IX 10 μmol/kg (diluted to 1 ml in 50 mmol/L sodium bicarbonate) was intraperitoneally injected in group AZ, and 50 mmol/L sodium bicarbonate 1 ml was intraperitoneally injected in group AV.After injecting lipopolysaccharide for 6 h, blood samples were collected from the common carotid artery for determination of plasma CO concentration, the rats were then sacrificed, and lungs were removed for microscopic examination of the pathological changes which were scored and for determination of CO level, wet to dry weight ratio (W/D ratio), cell apoptosis (by TUNEL), and expression of heme oxygenase-1 (HO-1), glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmie reticulum kinase (p-PERK), phosphorylated eukaryotic translation initiation factor 2 alpha (p-elF2), CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-12 in lung tissues (by Western blot). Apoptosis index (AI) was calculated. Results Compared with group C, the lung injury scores, W/D ratio, AI and CO levels in plasma and lung tissues were significantly increased, and the expression of HO-1, GRP78, p-PERK, p-elF2, CHOP and caspase-12 was up-regulated in the other three groups (P 0.05). Conclusion HO-1/CO signaling pathway produces endogenous protection possibly through inhibiting endoplasmic reticulum stress during endotoxin-induced ALI in rats. Key words: Heme oxygenase-1; Carbon monoxide; Endotoxins; Acute lung injury; Endoplasmic reticulum stress

Effects of dexmedetomidine on NLRP3 inflammasome during acute lung injury induced by blunt chest trauma and hemorrhagic shock-resuscitation in rats

Objective To evaluate the effects of dexmedetomidine on NLRP3 inflammasome during acute lung injury (ALI) induced by blunt chest trauma and hemorrhagic shock-resuscitation in rats. Methods Forty-five SPF healthy male Sprague-Dawley rats, weighing 200-220 g, were divided into 3 groups (n=15 each) using a random number table method: sham operation group (Sham group), ALI group and dexmedetomidine group (Dex group). A rat model of ALI was established by blunt chest trauma and hemorrhagic shock-resuscitation.Dexmedetomidine 5 μg·kg-1·h-1 was infused into the femoral vein after blunt chest trauma in Dex group.Blood samples were collected from the femoral artery at 6 h after blunt chest trauma for measurement of arterial oxygen partial pressure and concentrations of pro-inflammatory cytokines interleukin-1beta (IL-1β) and IL-18 in serum (by enzyme-linked immunosorbent assay). The oxygenation index was calculated.The rats were sacrificed and lungs were removed for examination of the pathological changes which were scored and for determination of the expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing CARD (ASC) in lung tissues (by Western blot) and activities of lactic dehydrogenase (LDH) and myeloperoxidase (MPO) in lung tissues (by colorimetry). Results Compared with Sham group, PaO2 and oxygenation index were significantly decreased, the expression of NLRP3, caspase-1 and ASC was up-regulated, the activities of LDH and MPO were increased, and the concentrations of IL-1β and IL-18 in serum were increased in ALI group (P<0.05). Compared with ALI group, PaO2 and oxygenation index were significantly decreased, the expression of NLRP3, caspase-1 and ASC was down-regulated, the activities of LDH and MPO were decreased, and the concentrations of IL-1β and IL-18 in serum were decreased in ALI group (P<0.05). Conclusion Dexmedetomidine can alleviate blunt chest trauma and hemorrhagic shock-resuscitation induced ALI in rats, and the mechanism is related to inhibition of NLRP3 inflammasome activation and reduction of inflammatory response. Key words: Dexmedetomidine; Acute lung injury; NLR family, pyrin domain-containing 3 protein

Overexpression of transcription factor EB regulates mitochondrial autophagy to protect lipopolysaccharide-induced acute lung injury.

Background:Acute lung injury (ALI) is characterized by an acute inflammatory process, and oxidative stress in the lung tissue leads to a lack of effective therapeutics. This study aimed to identify whether the overexpression of transcription factor EB (TFEB) regulates mitophagy to protect against lipopolysaccharide (LPS)-induced ALI.Methods:We detected the expression of inflammatory factors, cytochrome c (Cyt.c) and nicotinamide adenine dinucleotide phosphate (NADPH), and autophagy-related proteins and observed the changes in lung histopathology induced by ALI in rats and the changes in the cell ultrastructure of primary alveolar type II epithelial cells induced by changing the expression of TFEB in the context of ALI.Results:The overexpression of TFEB could reduce the expression of proinflammatory factors, such as IL-1 and IL-6, and increase the expression of anti-inflammatory factors, such as IL-10, both in vitro and in vivo. In addition, the overexpression of TFEB could reduce the Cyt.c and NADPH levels both in vivo and in vitro. The overexpression of TFEB could upregulate the expression of autophagy-related proteins, such as lysosomal-associated membrane protein 1 (LAMP1), microtubule-associated protein light chain 3B (LC3B), and Beclin both in vivo and in vitro, and promote mitochondrial autophagy. The overexpression of TFEB significantly improved the histopathologic changes induced by LPS-induced ALI in rats. However, low TFEB expression produced the opposite results.Conclusion:TFEB overexpression can decrease inflammation and mitochondrial damage in the lung tissue and alveolar epithelial cells through regulating mitochondrial autophagy to protect against LPS-induced ALI. Therefore, TFEB is likely a potential therapeutic target in LPS-induced ALI.

Effects and mechanism of fecal transplantation on acute lung injury induced by lipopolysaccharide in rats

Objective: To investigate the effect of fecal microbiota transplantation (FMT) on acute lung injury (ALI) induced by lipopolysaccharide (LPS) and its regulatory mechanism. Methods: Fifteen rats were divided into control group, LPS group and LPS+FMT group by random number table method. LPS group and LPS+FMT group were intraperitoneally injected with LPS to generate rat ALI model. After 24 h of modeling, feces (10 ml/kg) were given to the LPS+FMT group twice a day, and the control group and LPS group were given the same amount of normal saline. The intervention lasted for 2 days. After 24 h of the last fecal microbiota transplantation, arterial blood gas analysis was performed in each group. Then rats were sacrificed and enzyme-linked immunosorbent (ELISA) method was used to detect intercellular adhesion molecule 1 (ICAM-1) content in the serum and bronchoalveolar lavage fluid (BALF). The lung wet-dry weight ratio (W/D) was evaluated; HE staining and lung tissue pathology scoring, immunohistochemical detection of nuclear factor-kappa B (NF-κB) predominate nuclear expression and expression of ICAM-1 of alveolar epithelial cells were conducted; Western blot was used to detect the expression of proteins related to the intracellular phosphatidylinositol kinase (PI3K)/protein kinase (AKT) signaling pathway. Samples of rat feces were collected and DNA was extracted. Polymerase chain reaction (PCR) products of the V3 and V4 regions of the 16S ribosomal RNA gene (16SrDNA) were sequenced at high throughput, and bioinformatics analysis was conducted on the microbial community based on the operational classification unit. Results: The lung W/D and lung histopathological score of the LPS group were significantly higher than those of the control group, while the arterial partial oxygen pressure (PaO(2)) of the LPS group was significantly lower than that of the control group [(79.2±5.89 vs 95.2±2.77) mmHg, 1 mmHg=0.133 kPa](all P<0.05). The results of intestinal flora sequencing revealed that the diversity index of LPS group was significantly higher than that of the control group, while the lactobacillus of LPS group rats was significantly lower than that of the control group. The content of ICAM-1 in serum, BALF and its relative expression on the cell membrane in the LPS group was significantly higher than that in the control group [(8.64±0.87) vs (7.40±0.32) ng/L; (0.941±0.035) vs (0.739±0.079) ng/L; (0.250±0.010) vs (0.076±0.010)] (all P<0.05). Moreover, the relative expression levels of phosphorylated P65 (p-P65), p-PI3K and p-AKT nucleoprotein in the LPS group were significantly higher than those in the control group (4.89±0.27 vs 3.28±0.13, 0.265±0.030 vs 0.036±0.013 and 0.444±0.040 vs 0.109±0.016) (all P<0.05). The above injury effect was reduced after fecal fungus transplantation. The lung W/D and lung pathological score of LPS+FMT group were significantly lower than those of LPS group, and PaO(2) of LPS+FMT group was significantly higher than that of LPS group [(88.0±3.53) mmHg]. The results of intestinal flora sequencing revealed that the diversity index of LPS+FMT group was significantly lower than that of LPS group, and the lactobacillus genus of LPS+FMT group was significantly higher than that of LPS group. ICAM-1 in the blood serum ((7.44±0.46) ng/L), BALF (0.834±0.040) ng/L) and its relative expression on alveolar epithelial cell membrane (0.173±0.030), the relative expression of p-P65, p-PI3K and p-AKT protein of NF-κB in alveolar epithelial cells was down-regulated ((2.99±0.28, 0.090±0.013 and 0.206±0.018) in LPS+FMT group than those of LPS group, the differences were statistically significant (all P<0.05). Conclusion: Fecal transplantation can alleviate lipopolysaccharide-induced acute lung injury in rats, and its regulatory effect may be related to inhibiting the activation of PI3K/AKT/NF-κB signaling pathway and reducing the expression of inflammatory factor ICAM-1.

Protective Effect of Thymosin β4 against Abdominal Aortic Ischemia–Reperfusion-Induced Acute Lung Injury in Rats

Background and objectives: Ischemia–reperfusion (IR) caused by infrarenal abdominal aorta cross-clamping is an important factor in the development of ischemia–reperfusion injury in various distant organs. Materials and Methods: We investigated potential antioxidant/anti-inflammatory effects of thymosin beta 4 (Tβ4) in a rat model of abdominal aortic surgery-induced IR. Tβ4 (10 mg/kg, intravenous (i.v.)) was administered to rats with IR (90-min ischemia, 180-min reperfusion) at two different periods. One group received Tβ4 1 h before ischemia, and the other received 15 min before the reperfusion period. Results: Results were compared to control and non-Tβ4-treated rats with IR. Serum, bronchoalveolar lavage fluid and lung tissue levels of oxidant parameters were higher, while antioxidant levels were lower in the IR group compared to control. IR also increased inflammatory cytokine levels. Tβ4 reverted these parameters in both Tβ4-treated groups compared to the untreated IR group. Conclusions: Since there is no statistical difference between the prescribed results of both Tβ4-treated groups, our study demonstrates that Tβ4 reduced lung oxidative stress and inflammation following IR and prevented lung tissue injury regardless of timing of administration.

Effect of exercise training on heat shock protein 70 expression during endotoxin-induced acute lung injury in rats

Objective To evaluate the effect of exercise training on heat shock protein 70 (HSP70) expression during endotoxin-induced acute lung injury (ALI) in rats. Methods Thirty-two SPF healthy male Sprague-Dawley rats, aged 8 weeks, weighing 175-220 g, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), group ALI, low-intensity exercise training group (group ET1) and high-intensity exercise training group (group ET2). The rats in ET1 and ET2 groups received 2- and 4-week treadmill exercise training before establishing the ALI model, while the rats in C and ALI groups received no training.ALI was induced by intravenously injecting 5 mg/kg lipopolysaccharide via the tail vein in ALI, ET1 and ET2 groups, and the equal volume of normal saline was given instead in group C. The animals were sacrificed, and the lungs were harvested for microscopic examination of the pathological changes of lung tissues which were also scored and for determination of wet to dry weight ratio (W/D ratio), concentrations of total protein, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in bronchoalveolar lavage fluid (BALF), and expression of HSP70 and nuclear factor kappa B (NF-κB) in lung tissues by Western blot. Results Compared with group C, the W/D ratio and pathological changes of lung tissues were significantly increased, the concentrations of total protein, IL-1β and TNF-α in BALF were increased, the expression of NF-κB was up-regulated (P 0.05). Compared with group ALI, the W/D ratio and pathological changes of lung tissues were significantly decreased, the concentrations of total protein, IL-1β and TNF-α in BALF were decreased, the expression of HSP70 was up-regulated, and the expression of NF-κB was down-regulated in ET1 and ET2 groups (P<0.05). Conclusion Exercise training can attenuate the endotoxin-induced ALI through relieving the inflammatory responses, which may be related to up-regulating HSP70 expression in the lung of rats. Key words: Exercise; Endotoxemia; Acute lung injury; HSP70 heat-shock proteins

Tetramethylpyrazine alleviates LPS-induced inflammatory injury in HUVECs by inhibiting Rho/ROCK pathway

Endothelial dysfunction plays an important role in the pathogenesis of acute lung injury (ALI). Tetramethylpyrazine (TMP) has been reported to attenuate harmful changes in ALI rats. However, the effects of TMP on endothelial cell injury and its underlying mechanisms remain unknown. In this study, human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS) was used as an inflammatory injury model, also served as LPS group. HUVECs pretreated with TMP for 2 h before induced by LPS was served as LPS + TMP group. Untreated HUVECs was served as control group. After incubation with LPS for 12 h, cell viability and morphology, cell apoptosis rate, CD31-positive endothelial microparticles (EMPs) release, proinflammatory cytokines secretion, and ROCK II expression were evaluated. Compared with LPS group, TMP pretreatment improved cell viability and morphology. Besides, cell apoptosis rate, CD31-positive EMPs amount, TNF-α and IL-1β concentrates, and ROCK II mRNA and protein levels in LPS + TMP group were significantly decreased when compared with LPS group. To further confirm the mechanism, HUVECs in all the above groups were pretreated with Y27632 (ROCK II inhibitor) for 30 min before grouping, then treated as above. No significant differences in cell apoptosis rate, CD31-positive EMPs amount, and ROCK II expression between Y27632 + LPS group and Y27632 + LPS + TMP group were found. To sum up, our study found that TMP alleviated LPS-induced inflammatory injury in HUVECs by inhibiting Rho/ROCK pathway.

Role of miR-146a in bone marrow mesenchymal stem cells-induced reduction of acute lung injury in rats

Objective To evaluate the role of miR-146a in bone marrow mesenchymal stem cells (BMSCs)-induced reduction of acute lung injury (ALI) in rats. Methods A total of 105 clean-grade healthy male Wistar rats, weighing 170-190 g, were divided into 5 groups (n=21 each) using a random number table method: control group (group C), phosphate buffer solution group (group P), group ALI, BMSC group (group B) and BMSC plus miR-146a inhibitor group (group BM). ALI was induced by intraperitoneally injecting 5 mg/kg lipopolysaccharide (LPS) 0.5 ml in anesthetized rats.Phosphate buffer solution 0.5 ml was injected via the tail vein in group P. In group B, 1×104 cells/ml BMSC 0.5 ml was injected via the tail vein after establishing the model.In group BM, miR-146a inhibitor 50 mg/kg was injected via the tail vein after establishing the model, and 2 h later 1×104 cells/ml BMSC 0.5 ml was injected via the tail vein.Group C received no treatment.Blood samples were obtained from the abdominal aorta for blood gas analysis at 6, 24 and 48 h after injection of BMSC (T1-3), the chest was immediately opened, and the lung tissues were obtained for microscopic examination of pathologic changes and for determination of the wet/dry lung weight ratio (W/D ratio), expression of IRAK-1, nuclear factor kappa B (NF-κB) and interleukin-6 (IL-6) (by Western blot) and expression of miR-146a and IRAK-1 mRNA (by quantitative real-time polymerase chain reaction). Results Compared with group C, the pH value and PO2were significantly decreased, PCO2 and W/D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point (P<0.05), and the pathological changes of lung tissues were aggravated in ALI, B and BM groups.Compared with group P, the pH value and PO2 were significantly decreased, PCO2 and W/D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point (P<0.05), and the pathological changes of lung tissues were aggravated in ALI, B and BM groups.Compared with group ALI, the pH value and PO2 were significantly increased, PCO2 and W/D ratio were decreased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was down-regulated at each time point (P<0.05), and the pathological changes of lung tissues were attenuated in group B. Compared with group B, the pH value and PO2 were significantly decreased, PCO2 and W/D ratio were increased, and the expression of IRAK-1, NF-κB, IL-6 and miR-146a was up-regulated at each time point (P<0.05), and the pathological changes of lung tissues were aggravated in group BM. Conclusion miR-146a is involved in BMSCs-induced reduction of ALI in rats. Key words: MicroRNAs; Mesenchymal stem cell transplantation; Acute lung injury

Protective effects of the suppressed NF-κB/TLR4 signaling pathway on oxidative stress of lung tissue in rat with acute lung injury.

The pathogenesis of acute lung injury (ALI) is characterized by lung inflammation and lung oxidative stress. The study was conducted in order to investigate the effect toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) exhibited on oxidative stress in ALI. After the rats had been assigned into different groups, arterial blood, white blood cell (WBC), lung permeability index (LPI), wet/dry (W/D) ratio, TLR4 and NF-κB expression and superoxide dismutase (SOD), myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) were examined. Afterward, the correlation between the levels of TLR4 and NF-κB was determined. Decreased levels of PaO2 , SOD, MPO, and GSH accompanied by increased levels of PaCO2 , WBC number, LPI and W/D ratio, MDA and ROS, as well as TLR4 and NF-κB expressions in the ALI, ALI + NF-κB inhibitor, and ALI + phosphate buffer saline groups were found. Inhibition of NF-κB resulted in increased PaO2 and decreased PaCO2 levels, WBC number, and LPI and W/D ratio. Decreased expression of NF-κB increased SOD, GSH, and MPO, but decreased MDA and ROS. We also found that NF-κB inhibition resulted in the improvement of ALI in rats. TLR4 and NF-κB expressions were negatively correlated with levels of SOD, MPO, and GSH, and positively correlated with MDA and ROS levels. In summary, our findings provided evidence that inhibition of the TLR4/NF-κB signaling pathway decreases oxidative stress, thereby improving ALI. As a result, NF-κB signaling pathway has shown potential as a therapeutic target in ALI therapy.

Inhibition of MRP4 alleviates sepsis-induced acute lung injury in rats

This study was undertaken to examine the regulatory role of multidrug resistance-associated protein 4 (MRP4) in an experimental model of sepsis-induced acute lung injury in rats. Sepsis was induced by cecal ligation and puncture in anesthetized rats. Animals were then randomly assigned to receive intravenous injection of vehicle or MRP4 inhibitor (MK571, 20 mg/kg). The pathological changes were observed by hematoxylin and eosin staining. Lung water content, lung vascular permeability and inflammatory cell count in bronchoalveolar lavage fluid (BALF) were quantified. Serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were measured. In addition, lung tissue cyclic adenosine monophosphate (cAMP) levels were examined by enzyme-linked immunosorbent assay. Furthermore, the effects of MRP4 knockdown on lipopolysaccharide (LPS)-induced endothelial permeability and the cytoskeleton of rat pulmonary microvascular endothelial cells (PMVECs) were detected. The protein expression levels of MRP4, Rac1, VE-cadherin, β-catenin and ZO-1 were measured by Western blot analysis. MK571 significantly reduced lung tissue damage, lung water content and lung vascular permeability. Lung tissue cAMP levels were attenuated in MK571-treated animals compared with vehicle controls. MK571 also decreased sepsis-induced inflammatory cell accumulation in BALF. In addition, the MK571 group had significantly lower serum TNF-α and IL-6 levels compared with vehicle controls. Consistently, knockdown of MRP4 protected against LPS-induced increase in the endothelial permeability and the destruction of cytoskeleton in vitro. Furthermore, silencing MRP4 gene significantly reduced MRP4 protein expression and restored the protein expression of Rac1, VE-cadherin, β-catenin and ZO-1 in rat PMVECs in response to LPS stimulation. These data suggest that inhibition of MRP4 significantly alleviates sepsis-induced acute lung injury in rats.