PURPOSE: Hyperglycemia increases reactive oxygen species (ROS) and the resulting oxidative stress plays a key role in the pathogenesis of diabetic complications. Nicotinamide dinucleotide phosphate (NADPH) oxidase is one of the major sources of ROS production in diabetes. We, therefore, examined the possibility that NADPH oxidase activation is increased in various tissues, and that the antioxidant N-acetylcysteine (NAC) may have tissue specific effects on NADPH oxidase and tissue antioxidant status in diabetes. MATERIALS AND METHODS: Control (C) and streptozotocin-induced diabetic (D) rats were treated either with NAC (1.5 g/kg/day) orally or placebo for 4 weeks. The plasma, heart, lung, liver, kidney were harvested immediately and stored for biochemical or immunoblot analysis. RESULTS: levels of free 15-F2t-isoprostane were increased in plasma, heart, lung, liver and kidney tissues in diabetic rats, accompanied with significantly increased membrane translocation of the NADPH oxidase subunit p67phox in all tissues and increased expression of the membrane-bound subunit p22phox in heart, lung and kidney. The tissue antioxidant activity in lung, liver and kidney was decreased in diabetic rats, while it was increased in heart tissue. NAC reduced the expression of p22phox and p67phox, suppressed p67phox membrane translocation, and reduced free 15-F(2t)-isoprostane levels in all tissues. NAC increased antioxidant activity in liver and lung, but did not significantly affect antioxidant activity in heart and kidney. CONCLUSION: The current study shows that NAC inhibits NADPH oxidase activation in diabetes and attenuates tissue oxidative damage in all organs, even though its effects on antioxidant activity are tissue specific.
Acetylcysteine/*therapeutic use ; Animals ; Antioxidants/*metabolism ; Diabetes Mellitus, Experimental/*drug therapy/*metabolism ; Heart/drug effects ; Kidney/drug effects/metabolism ; Liver/drug effects/metabolism ; Lung/drug effects/metabolism ; Male ; NADPH Oxidase/*metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the 4-hydroxynonenal (4-HNE) expression changes and the impact of ulinastatin (UTI) METHODS: Seventy-two healthy Sprague-Dawley rats were randomly divided into three groups: the control group, poisoning group and treatment group, with 24 rats in each group. The model of lung injury was established by intragastric PQ (80 mg/kg) administration in poisoning group and treatment group, and 1 mL saline was administered intragastrically in the control group. The rats in treatment group were injected intraperitoneally with UTI (100 000 U/kg) 30 minutes after PQ administration, and the rats in the control group and poisoning group were intraperitoneally injected with the same volume of saline. After different treatments, the pathological changes and the expression of 4-HNE in lung tissue was detected in 12, 24, and 72 h in three groups.

RESULTSIn the poisoning group and treatment group, the expression of 4-HNE in lung tissue of rats were increased in 12 h after poisoning and reached the peak in 48 h; in 72 h after poisoning, the expression of 4-HNE in lung tissue were decreased, but they were still high. Compared with the control group, the expression of 4-HNE in lung tissue of rats were significantly increased in the poisoning group and treatment group (P < 0.05). And compared with the poisoning group, the expression of 4-HNE in lung tissue of rats were significantly decreased in the treatment group (P < 0.01). The pathological changes were observed, including alveolar capillary expansion, diffuse alveolar hemorrhage and alveolar inflammation cell infiltration, were found in lungs of rats in poisoning group and treatment group. There is no significant change in the control group. Compared with the control group, the expression of 4-HNE in lung tissue significantly increased in poisoning group and treatment group (P < 0.01), but the expression in treatment group was lower than in poisoning group (P < 0.01).

CONCLUSIONThe expression of 4-HNE increased in PQ intoxicated rats. UTI may reduce the expression of 4-HNE and reduce lung injury in PQ intoxicated rats.

Aldehydes ; metabolism ; Animals ; Glycoproteins ; pharmacology ; Lung ; drug effects ; metabolism ; pathology ; Lung Injury ; metabolism ; pathology ; Paraquat ; poisoning ; Rats ; Rats, Sprague-Dawley

To study the potential effect of Dioscorea nipponica(DN) in intervening peripheral system of rats based on metabolomic analysis. The identification of the potential intervention targets of DN in peripheral system may facilitate its safe application and therapeutic potential exploitation. Totally 20 male SD rats were randomly divided into the blank group and the DN-treated groups, with 10 rates in each group. The DN-treated group was orally administrated with DN extracts once a day for 5 days, with the dose of 80 mg x kg(-1) (equivalent to 15 g crude drug in human), and the blank group was given equal volume of saline once a day for 5 days. Heart, liver, spleen, lung, and kidney tissues and serum samples were collected from each rat 24 h later after the last administration. The ultra-performance liquid chromatography/quadrupole time-of-flight-mass spectrometry based metabolomics was used to investigate the effect of DN in intervening peripheral system of rats. After the treatment with DN, 5 modulated metabolites in heart tissue, 6 in liver tissue, 5 in spleen tissue, 3 in lung tissue, 5 in kidney tissue and 6 in serum sample were identified and considered as the potential intervention targets of DN. Effect of DN in regulating some endogenous metabolites was beneficial for protecting peripheral system, while that in other endogenous metabolites produced potential toxicity to peripheral system. The metabolomic analysis revealed the coexistence of protective and toxic effects of DN on peripheral system, which may be a practical guidance for its safe application and beneficial to the expansion of its application scope.
Animals ; Dioscorea ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Heart ; drug effects ; Kidney ; chemistry ; drug effects ; metabolism ; Liver ; chemistry ; drug effects ; metabolism ; Lung ; chemistry ; drug effects ; metabolism ; Male ; Metabolomics ; Rats ; Rats, Sprague-Dawley ; Spleen ; drug effects ; metabolism

OBJECTIVETo investigate whether the cellular gap junctional communication(GJIC) down-regulation in alveolar epithelial cells (CCL-64 cells) induced by silica-stimulated pulmonary alveolar macrophages (PAM) supernatant is related with the phosphorylation states of connexin 43(Cx43) protein.

METHODWestern-blot analysis was used to identify phosphorylated Cx43 species.

RESULTSWestern-blot analyses of SiO2- and phorbol 12-myristate 13-acetate(TPA)-treated CCL-64 cells showed the same phosphorylation states of Cx43 as the control group. There were no Cx43 protein in nucleus of CCL-64 cells.

CONCLUSIONThe inhibition on GJIC induced by SiO2 and TPA in CCL-64 cells may not be brought about by altering the phosphorylation states of Cx43.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; metabolism ; Gap Junctions ; drug effects ; Lung ; drug effects ; metabolism ; Mink ; Phosphorylation ; Silicon Dioxide ; toxicity ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo investigate whether cellular gap-junctional communication(GJIC) down-regulation and the internalization of connexin 43(Cx43) in Chinese hamster lung fibroblasts (CHL) induced by silica-stimulated pulmonary alveolar macrophages (PAM) supermatant is related with the phosphorylation states of Cx43 protein.

METHODSWestern-blot analysis was used to identify phosphorylated Cx43 species.

RESULTSSamples from membrane protein, total protein and nucleoprotein in CHL cells with 50-500 micrograms/ml doses of silica-stimulated PAM supernatants showed NP, P1, P2, P3 four immunoreactive bands of Cx43 protein by contrast with the control group and 0 microgram/ml SiO2 group. And with the dose of SiO2 increased, the increment of the levels of P2 and P3 was observed. Moreover, the groups treated with SiO2 and protein kinase C inhibitor, Palmitoyl-DL-Camitine chloride (PMC), simutaneously showed reduced level of P2 and P3, as compared with the groups treated with SiO2 only.

CONCLUSIONThe inhibition of GJIC and the internalization of Cx43 by SiO2 in CHL cells may relate to the changes of phosphorylation states of Cx43, and its mechanism may be similar to that of phorbol 12-myristate 13-acetate (TPA), i.e. via PKC activation pathway.

Animals ; Cell Communication ; drug effects ; Cell Line ; Connexin 43 ; metabolism ; Cricetinae ; Gap Junctions ; drug effects ; Lung ; drug effects ; metabolism ; Phosphorylation ; Silicon Dioxide ; toxicity

Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.
Carcinoma, Non-Small-Cell Lung/pathology* ; Cell Line, Tumor ; Cell Movement/drug effects* ; Cell Proliferation/drug effects* ; Humans ; Isoflavones/pharmacology* ; Lung Neoplasms ; MicroRNAs/metabolism* ; Ubiquitin-Protein Ligases/metabolism*

BACKGROUND/AIMS: Peroxiredoxin (Prx) belongs to a ubiquitous family of antioxidant enzymes that regulates many cellular processes through intracellular oxidative signal transduction pathways. Silica-induced lung damage involves reactive oxygen species (ROS) that trigger subsequent toxic effects and inflammatory responses in alveolar epithelial cells resulting in fibrosis. Therefore, we investigated the role of Prx in the development of lung oxidant injury caused by silicosis, and determined the implication of ROS in that process. METHODS: Lung epithelial cell lines A549 and WI26 were treated with 1% silica for 0, 24, or 48 hours, following pretreatment of the A549 cells with N-acetyl-L-cysteine and diphenylene iodonium and no pretreatment of the WI26 cells. We transfected an HA-ubiquitin construct into the A549 cell line and then analyzed the cells via Western blotting and co-immunoprecipitation. RESULTS: Silica treatment induced cell death in the A549 lung epithelial cell line and selectively degraded Prx I without impairing protein synthesis in the A549 cells, even when the ROS effect was blocked chemically by N-acetyl-L-cysteine. A co-immunoprecipitation study revealed that Prx I did not undergo ubiquitination. CONCLUSIONS: Silica treatment induces a decrease of Prx I expression in lung epithelial cell lines regardless of the presence of ROS. The silica-induced degradation of Prx does not involve the ubiquitin-proteasomal pathway.
Cell Line ; Epithelial Cells/drug effects/metabolism ; Humans ; Lung/chemistry/*drug effects/metabolism ; Peroxiredoxins/analysis/*physiology ; Protein Isoforms ; Reactive Oxygen Species/metabolism ; Silicon Dioxide/*toxicity ; Ubiquitin/metabolism

OBJECTIVETo observe the effects of complement fragment C3f on expression and secretion of collagen I, III and transforming growth factor( TGF)-beta1 in human embryonic lung fibroblast (MRC-5) cells.

METHODSMRC-5 cells were cultured with C3f (the synthetic 17 peptides fragments of complement C3). The extracellular and intracellular expression levels of type I, III collagens and TGF-beta1 in MRC-5 cultures were detected by ELISA and immunohistochemistry, respectively.

RESULTSThe expression levels of type I, III collagen and TGF-beta1 in the supernatant of MRC-5 cultures decreased significantly with the concentrations of C3f as compared with controls (P < 0.05). Also the expression level of TGF-beta1 in MRC-5 cytoplasm reduced significantly as compared with controls (P < 0.05).

CONCLUSIONThe results of present in vitro study showed that the complement fragment C3f could reduce the formation of TGF-beta1 and type I, III collagens in MRC-5 cells, and inhibit the lung tissue fibrosis.

Cell Line ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Complement C3b ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; cytology ; drug effects ; embryology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo confirm the antioxidant protective effect of peroxiredoxin 6 (Prdx6) in acute lung injury in mice.

METHODSLung injury or lung alveolar type II epithelial cell (AEC II) injury models were induced in mice by 100% O2 exposure or H2O2 treatments. Mice and AEC II cell survival rate or BALF analysis were applied for evaluating the degree of acute lung injury. Western Blot assay was used to determine Prdx6 or Gpx1 protein expression in lung. Annexin V staining method was applied to detect cell apoptosis on cultured AEC II cell, and thiobarbituric acid reactive substance (TBARS) measurement and diphenyl-1-pyrenyl phospholine (DPPP) assays were separately used to measure the level of lipid peroxidation in mice lung and AEC II cell membrane.

RESULTSUnder 100% O2 exposure, Prdx6-/- mice presented 24 h shorter survival time compared to wild type (WT) mice, on the contrary, Prdx6 gene over-expressed (Tg Prdx6) mice showed enhanced mice survival; meanwhile, the degree of AEC II cell injury had H2O2-dose dependent pattern with interactive relationship of Prdx6 protection. Under 100% O2 exposure for 72 h, it caused 7-fold decreased Gpx1 expression in Prdx6-/- mouse lung with no remarkable decrease of Prdx6 expression in Gpx1-/- mice. The percentage of apoptotic cells was significantly increased in AEC II cells from Prdx6-/- mice, and the percentage of AEC II apoptotic cells from Tg Prdx6 kept consistently around 10% under H2O treatments; also, the lipid peroxidation level of AEC II cell membrane was the highest in the group of Prdx6-/- mice, which was about 2 or 4-fold increased compared to the groups of WT or Tg Prdx6, separately; meanwhile, the lipid peroxidation level in Prdx6-/- mice, was also the highest compared to the other groups.

CONCLUSIONSPrdx6 plays a critical role in defending acute oxidative lung injury and its function of defending cell apoptosis and cell membrane lipid peroxidation suggests its unique cell-based protective effect.

Acute Lung Injury ; metabolism ; prevention & control ; Animals ; Antioxidants ; metabolism ; Apoptosis ; Hydrogen Peroxide ; metabolism ; Lipid Peroxidation ; Lung ; drug effects ; metabolism ; Male ; Mice ; Mice, Knockout ; Peroxiredoxin VI ; metabolism

OBJECTIVE: To explore the effect of ulinastain on the expression of hemeoxy genase-1 (HO-1) in oil acid-induced acute lung injury in rats. METHODS: The animal model of acute lung injury was established by oil acid. Thirty SD rats were randomly divided into 3 groups: the blank control group (A), the acute lung injury group (B) and the acute lung injury group (C) followed by injecting 100 mL/kg ulinastatin. Each group consisted of 10 rats. Group A were given 0.2 mL/kg natural solution through the trial vein; Group B and C were given 0.2 mL/kg oil-acid through trial vein, while group C were injected 100mL/kg ulinastatin by the peritoneal cavity after injecting oil acid. After 4 hours, the rates of respiration were counted and blood samples were cramped out through the heart puncture for blood gas analysis. The expressions of hemeoxygenase-1 and the pathologic construction changes were determined by HE staining in the lower right lung of rats in the 3 groups. RESULTS: The respiration dysfunction caused by oil acid could be prominently improved by ulinastain. There was only a little expression of hemeoxygenase-1 in the lung of Group A, but the expression increased in Group B and significatively increased in Group C. CONCLUSION Ulinastatin may protect the rats from acute lung injury through increasing the expression of HO-1.
Acute Lung Injury ; chemically induced ; metabolism ; Animals ; Glycoproteins ; pharmacology ; Heme Oxygenase (Decyclizing) ; metabolism ; Lung ; drug effects ; metabolism ; Male ; Oleic Acid ; adverse effects ; Rats ; Rats, Sprague-Dawley