OBJECTIVETo investigate the effect of combined application of Xuebijing Injection ( , XBJ) and resolvin D1 (RvD1) on survival rate and the underlying mechanisms in mice with sepsisinduced lung injury.

METHODSThe cecal ligation and puncture (CLP) method was used to develop a mouse sepsis model. Specific pathogen free male C57BL/6 mice were randomly divided into 5 groups (n=20 each): sham, CLP, CLP+XBJ, CLP+RvD1 and CLP+XBJ+RvD1. After surgery, mice in the CLP+XBJ, CLP+RvD1 and CLP+XBJ+RvD1 groups were given XBJ (25 μL/g body weight), RvD1 (10 ng/g body weight), and their combination (the same dose of XBJ and RvD1), respectively. In each group, 12 mice were used to observe 1-week survival rate, while the rest were executed at 12 h. Whole blood was collected for flow cytometric analysis of leukocyte adhesion molecules CD18, lung tissues were harvested for observing pathological changes, and testing the activity of myeloperoxidase (MPO) and the expression of intercellular cell adhesion molecule 1 (ICAM-1).

RESULTSCompared with the CLP group, the histopathological damage of the lung tissues was mitigated, MPO activity was decreased in the CLP+XBJ and CLP+RvD1 groups (P<0.05). In addition, the 1-week survival rate was improved, proportion of CD18-expressing cells in whole blood and ICAM-1 protein expression in lung tissue were decreased in the CLP+XBJ+RvD1 group (P<0.05 or P<0.01).

CONCLUSIONSXBJ together with RvD1 could effectively inhibit leukocyte adhesion, reduce lung injury, and improve the survival rate of mice with sepsis.

Animals ; CD18 Antigens ; metabolism ; Cell Adhesion ; drug effects ; Docosahexaenoic Acids ; administration & dosage ; pharmacology ; therapeutic use ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Injections ; Intercellular Adhesion Molecule-1 ; metabolism ; Leukocytes ; drug effects ; metabolism ; pathology ; Lung ; drug effects ; enzymology ; pathology ; Lung Injury ; blood ; complications ; drug therapy ; Male ; Mice, Inbred C57BL ; Peroxidase ; metabolism ; Sepsis ; blood ; complications ; drug therapy ; Survival Analysis

BackgroundAcute 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).

MethodsBALB/c mice were randomly divided into five groups: sham, LPS, LPS plus 1 ng of PDX (LPS + PDX-1 ng), LPS plus 10 ng of PDX (LPS + PDX-10 ng), and LPS plus 100 ng of PDX (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)-1β, IL-6, IL-10, tumor necrosis factor-alpha (TNF-α), macrophage inflammatory protein (MIP)-1α, 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.

ResultsThe 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-1α) levels. Meanwhile, 1 ng PDX exhibited pro-resolving functions in ALI including upregulation of monocyte-macrophage numbers and anti-inflammatory mediator IL-10 levels. The flow cytometry results showed that PDX could inhibit neutrophil-platelet interactions in ALI.

ConclusionPDX exerts protective effects in LPS-induced ALI by mitigating pulmonary inflammation and abrogating neutrophil-platelet interactions.

Acute Lung Injury ; chemically induced ; drug therapy ; Animals ; Chemokine CXCL2 ; metabolism ; Docosahexaenoic Acids ; therapeutic use ; Flow Cytometry ; Interleukin-10 ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; toxicity ; Lung ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effect of docosahexaenoic acid (DHA) on apoptosis, migration and invasion of cervical cancer cell lines.

METHODScervical cancer cell lines Hela and Siha in logarithmic phase were treated different concentrations of DHA. The morphological changes of the cells were observed microscopically and cell apoptosis was observed using Hoechst 33258 fluorescent staining. MTT assay was used to evaluate the effect of DHA in suppressing cell growth, and flow cytometry was employed to analyze the changes of cell apoptotic rate following DHA stimulations. Wound healing assay and Transwell migration assay were used to evaluate the migration of the cell lines. The expression levels of Bax, Bcl-2 cleaved caspase3, MMP-9 and VEGF proteins were detected by Western blotting.

RESULTSDHA exposure of the cells caused obvious morphological changes and dose-dependently increased the number of apoptotic bodies in the cells. MTT assay showed that DHA inhibited the growth of the cancer cells in a time- and concentration-dependent manner. DHA also effectively suppressed migration and invasion of the cancer cells. The cells exposed to DHA showed significantly down-regulation of Bcl-2, MMP-9 and VEGF proteins and up-regulation of cleaved-caspase 3 and Bax.

CONCLUSIONDHA can promote cervical carcinoma cell apoptosis by down-regulating the anti-apoptotic proteins Bax, Bcl-2 and cleaved-caspase3 and suppress cell invasion by decreasing MMP-9 and VEGF expressions.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; drug effects ; Cell Movement ; Cell Proliferation ; Docosahexaenoic Acids ; pharmacology ; Down-Regulation ; Female ; HeLa Cells ; drug effects ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; Vascular Endothelial Growth Factor A ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the effects of docosahexaenoic acid (DHA) on the expressions of TNF-α, IL-6, and leukotriene B4 (LTB4) in serum and expression of NF-κB in pulmonary tissue of rats with severe scald injury.

METHODSOne hundred and sixty SD rats were divided into sham injury (A), sham injury+DHA (B), scald (C), and scald+DHA (D) groups according to the random number table, with 40 rats in each group. Rats in groups A and B were sham injured, while rats in groups C and D were inflicted with 30% TBSA full-thickness scald on the back. Rats in groups B and D were injected with 0.5 mg/mL DHA solution with the dosage of 1 mL/kg via tail vein 5 minutes post injury, while rats in groups A and C with normal saline solution 1 mL/kg. At post injury hour (PIH) 3, 6, 12, 24, and 48, pulmonary tissue and abdominal aorta blood were collected from 8 rats in each group. The serum levels of TNF-α, IL-6, and LTB4 were determined with ELISA, and the protein expression of NF-κB p65 in pulmonary tissue was determined with Western blotting. Data were processed with analysis of variance of factorial design and LSD-t test.

RESULTS(1) The serum levels of TNF-α and IL-6 of rats in group A were similar to those of group B at each time point (with tTNF-α values from 0.223 to 0.947, tIL-6 values from 0.767 to 2.084, P values above 0.05). Compared with those of group A, the serum levels of TNF-α and IL-6 of rats in groups C and D were significantly higher at each time point (with tTNF-α values from 11.800 to 40.357, tIL-6 values from 10.334 to 39.321, P values below 0.01). The serum levels of TNF-α and IL-6 of rats in group D were significantly lower than those of group C at each time point (with tTNF-α values from -17.643 to -8.331, tIL-6 values from -21.596 to -6.332, P values below 0.01). The serum levels of TNF-α and IL-6 in groups C and D both showed a trend of increase earlier and decrease later, and they peaked at PIH 12, respectively (360.4 ± 13.2), (306.8 ± 7.2) pg/mL and (265.4 ± 12.3), (230.5 ± 2.2) pg/mL. (2) The serum level of LTB4 in group A was similar to that of group B at each time point (with t values from 0.787 to 1.096, P values above 0.05). The serum level of LTB4 was significantly higher in groups C and D than in group A at each time point (with t values from 7.501 to 38.962, P values below 0.01). The serum level of LTB4 in group D was obviously lower than that of group C at each time point (with t values from -19.244 to -2.532, P values below 0.01). The serum level of LTB4 in groups C and D both showed a trend of increase earlier and decrease later, and it peaked at PIH 12, (4.59 ± 0.29) and (2.85 ± 0.32) ng/mL respectively. (3) The protein expression of NF-κB p65 in pulmonary tissue in group A was similar to that of group B at each time point (with t values from 0.847 to 1.256, P values above 0.05). The protein expression of NF-κB p65 was significantly higher in groups C and D than in group A at each time point (with t values from 15.167 to 98.074, P values below 0.01). The protein expression of NF-κB p65 in group D was obviously lower than that of group C at each time point (with t values from -37.190 to -14.415, P values below 0.01). The protein expression of NF-κB p65 in groups C and D both showed a trend of increase earlier and decrease later, and it peaked at PIH 12, respectively 4.46 ± 0.12 and 2.94 ± 0.21.

CONCLUSIONSParenteral supply of DHA to rats with severe scald injury can reduce the levels of TNF-α, IL-6, and LTB4 in serum and decrease the expression of NF-κB in pulmonary tissue, thus alleviating the inflammation response.

Animals ; Blotting, Western ; Burns ; Cytokines ; Docosahexaenoic Acids ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; Interleukin-6 ; blood ; Leukotriene B4 ; blood ; Lung ; metabolism ; pathology ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; Soft Tissue Injuries ; Tumor Necrosis Factor-alpha ; blood ; genetics ; Up-Regulation ; physiology

BACKGROUNDProtectin 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).

METHODSMice were randomly assigned to six groups (n = 6 per group): sham-vehicle group, sham-PD1 group, sham-zVAD-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.

RESULTSIntratracheal 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.

CONCLUSIONPosttreatment 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.

Acute Lung Injury ; chemically induced ; drug therapy ; immunology ; Animals ; Apoptosis ; drug effects ; Docosahexaenoic Acids ; therapeutic use ; Inflammation ; drug therapy ; Lipopolysaccharides ; toxicity ; Male ; Mice ; Mice, Inbred BALB C ; Neutrophils ; cytology ; drug effects ; Peroxidase ; metabolism

BACKGROUNDA variety of inflammatory mediators and effector cells participate together in acute lung injury, and lead to secondary injury that is due to an inflammatory cascade and secondary diffuse lung parenchyma injury. Inflammation is associated with an oxidative stress reaction, which is produced in the development of airway inflammation, and which has positive feedback on inflammation itself. Resolvin D1 can reduce the infiltration of neutrophils, regulate cytokine levels and reduce the inflammation reaction, and thereby promote the resolution of inflammation. The purpose of this study is to investigate the effects of resolvin D1 on an inflammatory response and oxidative stress during lipopolysaccharide (LPS)-induced acute lung injury.

METHODSLPS (3 mg/kg) was used to induce the acute lung injury model. Pretreatment resolvin D1 (100 ng/mouse) was given to mice 30 minutes before inducing acute lung injury. Mice were observed at 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days and 7 days after LPS was administrated, then they were humanely sacrificed. We collected bronchoalveolar lavage fluid (BALF) and the lung tissues for further analysis. Paraffin section and HE staining of the lung tissues were made for histopathology observations. Parts of the lung tissues were evaluated for wet-to-dry (W/D) weight ratio. tumor necrosis factor (TNF)-α, inter leukin (IL)-1β, IL-10 and myeloperoxidase (MPO) were detected by enzyme-linked immunosorbent assay (ELISA). A lipid peroxidation malondialdehyde (MDA) assay kit was used to detect MDA. A total superoxide dismutase assay kit with WST-1 was used to analyze superoxide dismutase (SOD). We determined the apoptosis of neutrophils by Flow Cytometry. A real-time quantitative PCR Detecting System detected the expression of mRNA for heme oxygenase (HO)-1.

RESULTSPretreatment with resolvin D1 reduced the pathological damage in the lung, decreased the recruitment of neutrophils and stimulated their apoptosis. It markedly decreased the expressions of TNF-α, IL-1β and increased the expressions of IL-10, and decreased the production of MDA and increased the expressions of SOD. The mRNA expression of HO-1 was also significantly increased.

CONCLUSIONSResolvin D1 displays potent anti-inflammatory actions by regulating cytokines, inhibiting aberrant neutrophil recruitment and stimulating apoptosis of neutrophils. Resolvin D1 can also relieve the injury due to oxidative stress. The mechanisms might be related to increase HO-1 expression.

Acute Lung Injury ; chemically induced ; drug therapy ; immunology ; Animals ; Bronchoalveolar Lavage Fluid ; immunology ; Docosahexaenoic Acids ; therapeutic use ; Interleukin-10 ; metabolism ; Interleukin-1beta ; metabolism ; Lipopolysaccharides ; toxicity ; Male ; Mice ; Mice, Inbred BALB C ; Oxidative Stress ; drug effects ; Peroxidase ; metabolism ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

BACKGROUNDCigarette smoke induced airway inflammation plays a role in pathogenesis of airway inflammation. Resolvin-D1 derived from omega-3 polyunsaturated fatty acids is an endogenous anti-inflammatory and proresolving lipid mediator. Resolvin-D1 ameliorated inflammatory responses in lung injury, asthma, peritonitis and atherosclerosis. We investigated whether resolvin-D1 suppressed the productions of chemokines and oxidative stress induced by cigarette smoke extract (CSE) in vitro and its possible mechanism.

METHODSWe examined the proinflammatory chemokine interleukin-8 and hydrogen peroxide (H2O2) productions induced by CSE in 16 human bronchial epithelial (16HBE) cells after resolvin-D1 treatment and their mechanisms. 16HBE cells were treated with resolvin-D1 at up to 10 nmol/L, for 30 minutes before CSE up to 16% (v/v) exposure. Release of interlukin-8 proteins was assessed by enzyme linked immunosort assay (ELISA) and its mRNA level by RT-PCR. We evaluated extracellular H2O2 expression in the supernatant. Phosphorylation of NF-κB/p65 and degradation of I-κB in 16HBE cells were determined by Western blotting analysis and NF-κB DNA binding activity by electrophoretic mobility shift assay (EMSA).

RESULTS16HBE cells treated with 8% CSE showed significantly higher interlukin-8 production. Resolvin-D1 pretreatment inhibited CSE induced interlukin-8 production (mRNA and protein) in a dose and time dependent manner. Extracellular H2O2 level decreased after resolvin-D1 treatment. Resolvin-D1 attenuated CSE triggered I-κB degradation and NF-κB/p65 activation dose dependently and inhibited NF-κB DNA binding activity.

CONCLUSIONResolvin-D1 inhibits CSE induced interlukin-8 and H2O2 production in 16HBE cells by modulating NF-κB activation and has therapeutic potential for pulmonary inflammation.

Blotting, Western ; Cell Line ; Cell Survival ; drug effects ; Docosahexaenoic Acids ; pharmacology ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hydrogen Peroxide ; metabolism ; Interleukin-8 ; metabolism ; NF-kappa B ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smoking ; adverse effects

Three long-chain polyunsaturated fatty acids, docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3) and arachidonic acid (ARA, 20:4n-6), are the most biologically active polyunsaturated fatty acids in the body. They are important in developing and maintaining the brain function, and in preventing and treating many diseases such as cardiovascular disease, inflammation and cancer. Although mammals can biosynthesize these long-chain polyunsaturated fatty acids, the efficiency is very low and dietary intake is needed to meet the requirement. In this study, a multiple-genes expression vector carrying mammalian A6/A5 fatty acid desaturases and multiple-genes expression vector carrying mammalian Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases coding genes was used to transfect HEK293T cells, then the overexpression of the target genes was detected. GC-MS analysis shows that the biosynthesis efficiency and level of DHA, EPA and ARA were significantly increased in cells transfected with the multiple-genes expression vector. Particularly, DHA level in these cells was 2.5 times higher than in the control cells. This study indicates mammal possess a certain mechanism for suppression of high level of biosynthesis of long chain polyunsaturated fatty acids, and the overexpression of Δ6/Δ5 fatty acid desaturases and Δ6/Δ5 fatty acid elongases broke this suppression mechanism so that the level of DHA, EPA and ARA was significantly increased. This study also provides a basis for potential applications of this gene construct in transgenic animal to produce high level of these long-chain polyunsaturated fatty acid.
Acetyltransferases ; genetics ; metabolism ; Arachidonic Acid ; biosynthesis ; Docosahexaenoic Acids ; biosynthesis ; Eicosapentaenoic Acid ; biosynthesis ; Fatty Acid Desaturases ; genetics ; metabolism ; Fatty Acid Synthases ; genetics ; metabolism ; Fatty Acids, Unsaturated ; biosynthesis ; Genetic Vectors ; HEK293 Cells ; Humans ; Transfection

OBJECTIVETo investigate the protective effects of docosahexaenoic acid (DHA) and nervonic acid (NA) on the learning and memory abilities in rats exposed to 1-bromopropane (1-BP) and their action mechanisms.

METHODSForty male Wistar rats (specific pathogen-free) were randomly divided into 4 groups (n = 10 for each), i.e., solvent control group, 1-BP (800 mg/kg) group, NA (150 mg/kg) + 1-BP (800 mg/kg) group, and DHA (500 mg/kg) + 1-BP (800 mg/kg) group. The rats were given respective test substances by gavage for 7 d. The Morris water maze (MWM) test was performed from days 8 to 12 to evaluate the rats' learning and memory abilities. After MWM test, rats were sacrificed in the next day, and cerebral cortex was quickly dissected and homogenized in an ice bath. The supernatant of the obtained homogenate was collected to measure the content of glutathione (GSH) and malondialdehyde (MDA) and the activities of glutathione reductase (GR) and γ-glutamate cysteine ligase (γ-GCL).

RESULTSThe MWM spatial navigation test showed that the 1-BP group had significantly longer escape latency and significantly longer total swimming distance compared with the control group (P<0.05), while the DHA+1-BP group had significant decreases in escape latency and total swimming distance compared with the 1-BP group (P<0.05). The spatial probe test showed that the number of platform crossings was significantly greater in the DHA+1-BP group and NA+1-BP group than in the 1-BP group (P<0.05); compared with the control group, the 1-BP group had a significantly lower ratio of time spent in the zone around the platform to total time (P < 0.05), and the ratio was significantly higher in the DHA+1-BP group than in the 1-BP group (P < 0.05). Compared with the control group, the 1-BP group had a 18.1% decrease in GSH content, and DHA could significantly reverse 1-BP-induced decrease in GSH content (P < 0.05). Compared with the 1-BP group, the DHA+1-BP group and NA+1-BP group had significantly decreased MDA content (P < 0.05), the DHA+1-BP group had significantly increased GR activity (P < 0.05), and the NA+1-BP group had significantly increased γ-GCL activity (P < 0.05).

CONCLUSIONThe rats exposed to 1-BP have oxidative stress in the brain and impaired cognitive function. DHA and NA can reduce 1-BP-induced cognitive function impairment in rats, possibly by increasing the activities of GR and γ-GCL and the content of GSH in the brain.

Animals ; Behavior, Animal ; Brain ; drug effects ; Docosahexaenoic Acids ; pharmacology ; Fatty Acids, Monounsaturated ; pharmacology ; Glutamate-Cysteine Ligase ; metabolism ; Glutathione ; metabolism ; Glutathione Reductase ; metabolism ; Hydrocarbons, Brominated ; toxicity ; Male ; Malondialdehyde ; metabolism ; Maze Learning ; drug effects ; Memory ; drug effects ; Oxidative Stress ; Rats ; Rats, Wistar

OBJECTIVETo investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA).

METHODSCoronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16, 17-epoxydocosapentaenoic acid (16, 17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration.

RESULTSBK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2 ± 2.7)% of total potassium currents (n = 20). DHA could activate BK channels, and its 50% effective concentration (EC(50)) was (0.23 ± 0.03) µmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16, 17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC(50) was (19.7 ± 2.8) nmol/L.

CONCLUSIONDHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.

Animals ; Coronary Vessels ; cytology ; drug effects ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Docosahexaenoic Acids ; pharmacology ; Fatty Acids, Unsaturated ; pharmacology ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Proadifen ; pharmacology ; Rats ; Rats, Sprague-Dawley