Umbilical Vein Endothelial Cell Injury Research Articles

Heracleifolinosides A-F, New Triterpene Glycosides from Cimicifuga heracleifolia, and Their Inhibitory Activities against Hypoxia and Reoxygenation

Six new 9,19-cycloartane triterpene glycosides, heracleifolinosides A-F (1-6), and one new chromone, norkhelloside (7), were isolated from the rhizome of Cimicifuga heracleifolia, together with 15 known compounds (8-22). The structures of the new compounds were elucidated by means of spectroscopic methods including 2D NMR and mass spectrometry. The extracts of C. heracleifolia and all the isolated compounds were tested for activities against hypoxia and reoxygenation injury in human umbilical vein endothelial cells. Heracleifolinoside B (2) is effectively resistant to hypoxia and reoxygenation-induced human umbilical vein endothelial cell injury, with cell viabilities of 61.95 ± 2.04 %, 77.04 ± 4.44 %, and 83.65 ± 3.29 % at concentrations of 1, 10, and 100 µM, respectively.

Chemical constituents and activities of total flavonoids from Yushen Tang

To study the chemical constituents and activity of total flavonoids contained in Yushen Tang,in order to lay a foundation for defining active consituents of the prescription and their mechanism. The activity of total flavonoids contained in Yushen Tang were evaluated by in vitro H2O2-induced human umbilical vein endothelial cell injury experiment. The chemical constituents were separated and purified by such methods as silica column chromatography, macroporous resin chromatography, sephadex and HPLC preparation,and their structures were identified on the basis of their spectral data and physicochemical properties. Total flavonoids contained in Yushen Tang showed the effects in inhibiting hydrogen peroxide-induced human umbilical vein endothelial cell (HUVEC) injury and down-regulating PAI-1 expression (P<0.05). Nine flavonoids were separated from total flavonoids contained in Yushen Tang and identified as calycosin (1), linarin (2), 3',4',7 -trihydroxyflavone-7-O-beta-D-glucopyranoside (3), 7,3'-dihydroxy-4'-methoxyflavone-7-glucoside (4), quercetin (5), kaempferol (6), rutin (7), pratensein-7-O-beta-D-glucoside (8) and 7-O-glucosyl liquiritigenin (9). Total flavonoids contained in Yushen Tang showed the effect in inhibiting HUVEC injury. All of the nine flavonoids were separated from Yushen Tang for the first time, and compounds 1,3,4,8,9 may be derived from astragalus mongholicus, while compounds 4,5-7 may be derived from herba cepbalanoplosis segeti and hairyvein agrimony. Among them, compounds 3,4 and 9 were seperated from single ingredient of the prescription for the first time.

Protective effects of penehyclidine hydrochloride on lipopolysaccharide-induced human umbilical vein endothelial cells injury and its mechanism

Objective To investigate the protective effects of penehyclidine hvdrochloride (PHC)on lipopolysaccharide ( LPS)-induced human umbilical vein endothelial cells (HUVECs) injury and the underlying mechanism.Methods Cultured HUVECs were divided into five groups:control group,LPS group,LPS/PHC ( 10 μg/L) group,LPS/PHC (25 μg/L) group,LPS/PHC (50 μg/L) group.Cell viability was measured by methyl thiazol tetrazolium (MTT) colorimetric method.The concentrations of lactate dehydrogenase (LDH) and nitric oxide (NO) were measured respectively by using chemical colorimetric method and nitrate reduction method.The expression of inducible nitric oxide synthase (iNOS) mRNA and protein was detected hy using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively.The activity of nuclear factor-KB (NF-KB) and signal transducer and activators of transcription 3 (STAT3) were measured by enzyme linked immunosorbent assay (ELISA).Results As compared with control group,cell viability [(60.31 ± 8.76 )％ vs 100.00％] was decreased and the concentrations of LDH [(326 ±52) μmol/L vs ( 125 ±25) μmoL/L] and NO [(55.49 ± 10.16) μmol/L vs ( 12.13 ±11.02) μmol/L] were increased in LPS group ( P ＜0.01 ) ; PHC could revere these effects on HUVECs (P ＜0.05).At the same time,PHC reduced the expression of iNOS mRNA and protein,and the activity of NF-KB and STAT3 ( P ＜ 0.05 ).Conclusion PHC can protect the endothelial function by reducing the concentration of NO.Inhibiting the NF-KB and STAT3 signal pathway may be the underlying mechanism. Key words: Penehyclidine hydrochloride; Lipopolysaccharide; Human umbilical vein endothelial cells; Inducible nitric oxide synthase; Nitric oxide

Protective effect of α -lipoic acid on oxidized low density lipoprotein-induced human umbilical vein endothelial cell injury

AbstractThe present study investigated the effect and possible mechanisms of α -lipoic acid (LA) in preventing endothelial cell injury induced by oxidized low-density lipoprotein (oxLDL). A model of human umbilical vein endothelial cell (HUVEC) injury was established by incubating the HUVECs with 200µg/ml oxLDL. HUVECs were pre-treated with 0.1, 0.2 or 0.5mmol/l of LA in the presence of oxLDL for 24h. Apoptosis and cellular surface ceramide content were investigated separately by flow cytometry and by LC-MS/MS. LOX-1, Bcl-2 and CRP protein expression levels were evaluated by western blotting. LOX-1 mRNA expression was evaluated by RT-PCR assay. The results showed that oxLDL induced cytotoxicity in both concentration-dependent and time-dependent manners. LA boosted the cell survival rate and significantly reduced the content of MDA and lactate dehydrogenase (LDH) leakage. Apoptotic rates were significantly reduced by the addition of LA compared to oxLDL group. LA might also have inhibited ceramide generation induced by oxLDL in a dose-dependent manner. Furthermore, LA down-regulated LOX-1 protein and mRNA expression and up-regulated Bcl-2 protein expression levels in a dose-dependent manner. Expression of CRP protein was weak and undetectable. These results suggested that LA exhibited cytoprotective effects against oxLDL by decreasing apoptotic rates and decreasing cellular surface ceramide content, two effects that are related to decreased LOX-1 expression, and also by stimulating the expression of Bcl-2 protein. The cytoprotective effects are not thought to be due to inhibited C-reactive protein (CRP) protein expression in HUVECs.

Curculigoside attenuates human umbilical vein endothelial cell injury induced by H2O2

Vessel endothelium injury caused by reactive oxygen species (ROS) including H(2)O(2) plays a critical role in the pathogenesis of cardiovascular disorders. Therefore, agents or antioxidants that can inhibit production of ROS has highly clinical values in cardiovascular therapy. Curculigoside is the major bioactive compounds present in Curculigo orchioides, and possess potent antioxidant properties against oxidative stress insults through undefined mechanism(s). The present study was designed to test the hypothesis that curculigoside can inhibit H(2)O(2)-induced injury in human umbilical vein endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with curculigoside in the presence/absence of hydrogen peroxide (H(2)O(2)). The protective effects of curculigoside OP-D against H(2)O(2) were evaluated. HUVECs incubated with 400 μM H(2)O(2) had significantly decreased the viability of endothelial cells, which was accompanied with apparent cells apoptosis, the activation of caspase-3 and the upregulation of p53 mRNA expression. In addition, H(2)O(2) treatment induced a marked increase of MDA, LDH content and in intracellular ROS, decreased the content of nitric oxide (NO) and GSH-Px activities in endothelial cells. However, pretreatment with 0.5.5,10 μM curculigoside resulted in a significant recovery from H(2)O(2)-induced cell apoptosis. Also, it decreased other H(2)O(2)-induced damages in a concentration-dependent manner. Furthermore, pretreatment with curculigoside decreased the activity of caspase-3 and p53 mRNA expression, which was known to play a key role in H(2)O(2)-induced cell apoptosis. The present study shows that curculigoside can protect endothelial cells against oxidative injury induced by H(2)O(2), suggesting that this compound may constitute a promising intervention against cardiovascular disorders.

Anti-ischemia Steroidal Saponins from the Seeds of Allium fistulosum

Six new furostanol saponins (1-6), named fistulosaponins A-F, three known furostanol saponins (7-9), and seven known aromatic compounds were isolated from seeds of Welsh onion (Allium fistulosum). The structures of these compounds were characterized by spectroscopic analyses including 2D NMR spectroscopy, mass spectrometry, and acid hydrolysis. The protective effect of the saponins on hypoxia-induced human umbilical vein endothelial cell injury was evaluated.

Protective effects of prostaglandin E1 on human umbilical vein endothelial cell injury induced by hydrogen peroxide

To investigate the protective effects of prostaglandin E(1) (PGE(1)) against H(2)O(2)-induced oxidative damage on human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with PGE(1) (0.25, 0.50, and 1.00 micromol/L) for 24 h and exposed to H(2)O(2) (200 micromol/L) for 12 h, and cell viability was measured by the MTT assay. LDH, NO, SOD, GSH-Px, MDA, ROS, and apoptotic percentage were determined. eNOS expression was measured by Western blotting and real-time PCR. PGE(1) (0.25-1.00 micromol/L) was able to markedly restore the viability of HUVECs under oxidative stress, and scavenged intracellular reactive oxygen species induced by H(2)O(2). PGE(1) also suppressed the production of lipid peroxides, such as MDA, restored the activities of endogenous antioxidants including SOD and GSH-Px, and inhibited cell apoptosis. In addition, PGE(1) significantly increased NO content, eNOS protein, and mRNA expression. PGE(1) effectively protected endothelial cells against oxidative stress induced by H(2)O(2), an activity that might depend on the up-regulation of NO expression.

Protein kinase Cβ_2 mediated high glucose-induced human umbilical vein endothelial cells injury via regulation of peroxisome proliferator-activated receptor α

Objective To investigate the roles of protein kinase C(PKC)β_2 and PPARα in the mRNA expression of vascular endothelial growth factor(VEGF)and vascular cell adhesion molecule-1(VCAM-1)in human umbilical vein endothelial cells(HUVECs)exposed to high glucose,and to explore their relationship.Methods The HUVECs were divided into eight groups:normal glucose(NG,5 mmoL/L D-glucose)group,high glucose(HG,25 mmol/L D-ucose)group,osmotic control(L,NG+20 mmol/L L-glucose)group,normal glucose transfected with empty vector(NN,NG+AdS-null)group,high glucose transfected with PKCβ_2(HB,HG+Ad5-PKCβ_2)group,high glucose plus fenofibrate(HF,HG+40 μmol/L fenofibrate)group,and HB plus fenofibrate (HBF,HB+40μmol/L fenofibrate)group.HUVECs were incubated with fenofibrate for 20 minutes as HF20 group.All cells in various groups were cultured for 6 days.The expressions of VEGF and VCAM-1 mRNA were detected by RT-PCR.PPARα protein expression was tested by Western blot.The expression and traaslocation of PKCβ_2 protein were observed by confocal laser scanning microscopo.Results (1)HG increased VEGF and VCAM-1 mRNA expressions,with 1.91 and 1.56 folds of NG group,respectively(both P<0.05).VEGF and VCAM-1 mRNA expressions in HB group further increased,with 2.59 and 2.07 folds of NG group,respectively(both P<0.05).Fenofibrate significantly decreased VEGF and VCAM-1 mRNA expressions,with 68%and 74%of HG group,respectively(both P<0.05).There were no significant difierences in the expressiong of VEGF,VCAM-1 mRNA between HF20 and HG groups.(2)The protein expression of PPARα decreased by 20%in HG group compared with NG group,and further decreased in HB group,being 78%of HG group.Compared with HG group,PPARαexpression increased by 13%in HF group(P<0.05).(3)HG induced PKCβ_2 translocation from cytosol to nucleus and quantitative analysis showed the ratio of plasma/nuclear fluorescence intensity in HG group decreased by 37% compared with NG group(P<0.05).The PKCβ_2 translocation was more obvious in HB group than in HG group.Conclusion Hiigll glucose stimulates VEGF and VCAM-1 mRNA expressions in HUVECs via PKCβ_2 activationdependent PPARα pathway. Key words: Protein kinase Cβ_2; PPARα; Human umbilical vein endothelial ceHs; Glucose; Retinopathies; diabctic

Stachydrine, a Major Constituent of the Chinese Herb Leonurus Heterophyllus Sweet, Ameliorates Human Umbilical Vein Endothelial Cells Injury Induced by Anoxia-Reoxygenation

Stachydrine is a major constituent of Chinese herb leonurus heterophyllus sweet, which is used in clinics to promote blood circulation and dispel blood stasis. Our study aimed to investigate the role of stachydrine in human umbilical vein endothelial cells (HUVECs) injury induced by anoxia-reoxygenation. Cultured HUVECs were divided randomly into control group, anoxia-reoxygenation (A/R) group and 4 A/R+stachydrine groups. HUVECs in the control group were exposed to normoxia for 5 hours, while in all A/R groups, HUVECs underwent 3 hours anoxia followed by 2 hours reoxygenation, and HUVECs in the 4 A/R+stachydrine groups were treated with 10(-8) M, 10(-7) M, 10(-6) M and 10(-5) M (final concentration) of stachydrine respectively. After anoxia-reoxygenation, tissue factor (TF) was over-expressed, cell viability and the concentrations of SOD, GSH-PX and NO were declined, while LDH, MDA and ET-1 were over-produced (p < 0.05 to 0.001 vs. the control group). However, in stachydrine treated groups, TF expression was inhibited at both mRNA and protein levels, while the declined cell viability and SOD, GSH-PX, NO as well as the enhanced LDH, MDA and ET-1 levels occurred during anoxia-reoxygenation were ameliorated and reversed effectively (p < 0.05 to 0.01 versus A/R group). Consequently, our findings indicate that TF plays an important role in the development of anoxia-reoxygenation injury of HUVECs, stachydrine ameliorates HUVECs injury induced by anoxia-reoxygenation and its putative mechanisms are related to inhibition of TF expression.

Antisense Oligodeoxynucleotide Against Tissue Factor Inhibits Human Umbilical Vein Endothelial Cells Injury Induced by Anoxia-Reoxygenation

Aims: The role of antisense oligodeoxynucleotide against tissue factor (aODN/TF) in cultured human umbilical vein endothelial cells (HUVECs) subjected to anoxia-reoxygenation (A/R) was investigated. Methods: HUVECs were divided randomly into control group, A/R group, aODN/TF+A/R group, sense oligodeoxynucleotide (sODN/TF) + A/R group and mismatched oligodeoxynucleotide (mODN/TF) + A/R group, in the latter 3 groups, HUVECs were transfected with aODN/TF, sODN/TF and mODN/TF respectively. HUVECs in all A/R groups underwent 3 hrs of anoxia and followed by 2 hrs of reoxygenation. In order to investigate the potential mechanisms of how increased TF may contribute to A/R injury in HUVECs, another set of HUVECs were incubated with human recombinant active site blocked factor VII (FVIIai) during A/R. Results: After A/R, TF expression at both mRNA and protein level was increased, furthermore, cell viability and the concentrations of SOD, GSH-PX and NO were declined, while LDH, MDA and ET-1 were overproduced (P<0.05 to 0.001 versus control group). In HUVECs of aODN/TF+A/R group, however, TF expression was inhibited, while the declined cell viability and the concentrations of SOD, GSH-PX, NO as well as the enhanced LDH, MDA and ET-1 levels occurred during A/R were ameliorated and reversed effectively (P<0.05 to 0.01 versus those in other A/R groups). The results also showed that ROS was increased and PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase were all activated after A/R (P<0.001 versus HUVECs under normoxia), while FVIIai inhibited the increment of ROS, PAR-1, PAR-2, p38 MAP kinase and p42/44 MAP kinase, and improved the changes of TF:C, MDA, SOD, GSH-PX, cell viability and LDH occurred during A/R (P<0.05 to 0.001 versus HUVECs without FVIIai treatment). Conclusion: Tissue factor plays an important role in the development of HUVECs injury induced by anoxia-reoxygenation, inhibition of TF with antisense oligodeoxynucleotide is an effective approach to ameliorate the damage.

High glucose induced human umbilical vein endothelial cell injury: involvement of protein tyrosine nitration

The dysfunction and further damage of endothelium play an important role in the development and progression of diabetic vascular complications. Protein tyrosine nitration is involved in endothelial cell injury induced by high glucose. Little is known about protein nitration in human umbilical vein endothelial cells (ECV304) induced by high glucose. In the present article, exposure of ECV304 to 30 mM high glucose (HG30) and 40 mM high glucose (HG40) or hemin-nitrite-H2O2 system for 72 h, the cell injury in ECV304 induced by high glucose and exogenous nitrating agent was studied. After 72 h treatment, it was found that high glucose stimulated ECV304 injury in a dose-dependent manner, including reducing cell viability, increasing malondialdehyde (MDA) content, decreasing glutathione (GSH) content, increasing intracellular reactive oxygen species (ROS), increasing the production of nitric oxygen (NO) (increased nitrite content in cell and nitrate content in medium) and generating protein tyrosine nitration. It was also found that protein tyrosine nitration could induce cell injury further. By comparison the protein tyrosine nitration induced by high glucose condition and extrinsic factors (hemin-nitrite-H2O2 system), it may be speculated that protein is nitrated selectively to generate nitrotyrosine in diabetic vascular complications.

Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-κB activation

Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C. Artesunate (AS), a water-soluble derivative of dihydroartemisinin, has recently been demonstrated to protect against LPS-induced human umbilical vein endothelial cell (HUVEC) activation and injury by inhibiting tumor necrosis factor-alpha (TNF-α) mRNA expression. In the present study, heat-killed Escherichia coli was used to induce sepsis in the animal models. We observed that AS could protect mice against a lethal challenge with heat-killed E. coli in a dose-dependent manner. This protection was associated with reductions in serum TNF-α and measurable endotoxin levels. In addition, the treatment of murine peritoneal macrophage cells with AS strongly inhibited the release of TNF-α and IL-6 induced by CpG oligodeoxynucleotide (CpG ODN), LPS, or heat-killed E. coli in a dose-dependent manner. Experiments using affinity sensor technology revealed that AS could not directly bind to CpG ODN or LPS. Moreover, AS could not neutralize LPS in vitro. Further, flow cytometry revealed that AS could not alter the binding of CpG ODN to cell surfaces but could promote CpG ODN accumulation within RAW264.7 cells. Furthermore, AS reduced the expressions of TLR4 and TLR9 mRNA that were stimulated by LPS, CpG ODN, or heat-killed E. coli and inhibited heat killed E. coli-induced NF-κB activation. In conclusion, our results demonstrated that AS-mediated protection against a lethal heat-killed E. coli challenge was associated with a reduction in proinflammatory cytokine release and endotoxin levels via a mechanism involving a decrease in TLR4, TLR9 mRNA expression and NF-κB activation.

Protective effect of Huaxia shallot preparation on human umbilical vein endothelial cell injury induced by oxidized low density lipoprotein and its mechanism

To observe the protective effect of Huaxia shallot preparation on human umbilical vein endothelial cell (HUVEC) injury induced by oxidized low density lipoprotein (Ox-LDL) in vitro. Ox-LDL was prepared and identified, and HUVECs were cultured. After 2-hour intervention of different drugs and 24-hour following intervention of Ox-LDL, the number of HUVECs was observed by phase contrast optical microscope and the activity of the HUVECs was observed by methyl thiazolyl tetrazolium (MTT) technique. Superoxide dismutase (SOD) activity and nitric oxide (NO) content were assayed by respective kit. The protein expressions and mRNA levels of peroxisome proliferators activated receptor gamma(PPAR-gamma) and endothelial nitric oxide synthase (eNOS) were measured by western blot technique and reverse transcription polymerase chain reaction (RT-PCR). Ox-LDL could increase the apoptosis rate of the HUVECs and decrease the NO release as compared with the blank control group (P<0.05). These effects induced by Ox-LDL were all significantly inhibited by Huaxia shallot preparation. It could up-regulate the protein expressions and mRNA levels of PPAR-gamma and eNOS significantly (P<0.05). Huaxia shallot preparation could decrease the apoptosis rate of the HUVECs. Ox-LDL may be involved in the initiation and progression of atherosclerosis by injuring the endothelial cells directly and may cause the endothelial dysfunction. Huaxia shallot preparation can protect against Ox-LDL induced endothelial cell injury by up-regulating the protein expressions and mRNA levels of PPAR-gamma and eNOS. It suggests that Huaxia shallot preparation may play a role in the prevention and treatment of cardiovascular disease.

Protective effects of silybin on human umbilical vein endothelial cell injury induced by H 2O 2 in vitro

Oxidative injury induces cellular and nuclear damages that lead to cell injury. Agents or antioxidants that can inhibit production of reactive oxygen species can prevent injury. We tested the hypothesis that silybin can inhibit H 2O 2-induced injury in human umbilical vein endothelial cells. Eighteen hours of treatment with 750 μmol l − 1 H 2O 2 significantly stimulated expression of caspase-3 and cell apoptosis. In addition, it is observed that H 2O 2 increased the amounts of malondialdenhyde (MDA), the dehydrogenase (LDH) leakage, and decreased the activity of GSH-Px and NO contents in ECV-304 cells. In the H 2O 2 apoptosis model, the addition of 6.25–25 mg/L of silybin, which has in vitro radical scavenging activity, partially restored cell viability with a reduction in H 2O 2-induced apoptotic DNA damage, and decreased the expression of caspase-3. Moreover, it decreased other H 2O 2-induced damage in a concentration-dependent manner. The endothelial cell apoptosis was detected by AO/EB dual staining as well as flow cytometry, and the activity of pro-apoptotic factor caspase-3 was detected by immunocytochemical method. Our results suggest that the antioxidant, silybin, protects ECV-304 cells against H 2O 2-induced injury probably through its antioxidant activity, increasing the NO content, the activity GSH-Px and inhibiting signaling pathways mediated by caspase-3.

Protective effects of icariin on human umbilical vein endothelial cell injury induced by H 2O 2 in vitro

Icariin is a flavonoid isolated from Epimedium and is considered to be the major pharmacological active component of Epimedii Herba. In the present investigation, we studied and confirmed the protective activity of icariin on H 2O 2-induced injury in human umbilical vein endothelial cell line: ECV-304. Eighteen-hour treatment with 750 μmol l −1 H 2O 2 significantly decreased the viability of ECV-304 cells, which was accompanied with apparent apoptotic features, including distinct cell morphological alteration and the increase of caspase-3 expression. In addition, it is observed that H 2O 2 increased the amounts of malondialdenhyde (MDA) and the dehydrogenase (LDH), and decreased the content of nitric oxide (NO) in ECV-304 cells. However, pretreatment with 0.1–50 μmol l −1 icariin resulted in a significant recovery from H 2O 2-induced cell apoptosis. Also, it decreased other H 2O 2-induced damage in a concentration-dependent manner. Furthermore, pretreatment with icariin decreased the expression of caspase-3, which was known to be involved as a key role executor in H 2O 2-induced cell apoptosis. The endothelial cells apoptosis were detected by acridine orange/ethidium bromide (AO/EB) dual staining as well as flow cytometry, and the expression of pro-apoptotic factor caspase-3 were detected by immunocytochemical method. Taken together, these data suggest that protective effects of icariin against oxidative injuries of ECV-304 cells may be achieved via decreasing of caspase expression.

Effect of the NADPH oxidase inhibitor apocynin on septic lung injury in guinea pigs.

Reactive oxygen species (ROS) produced by NADPH oxidase activation in neutrophils play a major role in mediating sepsis-induced acute lung injury. To provide insight into whether the NADPH oxidase inhibitor apocynin might attenuate oxidant-induced lung injury, we examined the effect of apocynin on (1) sepsis-induced lung injury in guinea pigs, (2) ROS generation by LPS-stimulated neutrophils measured by chemiluminescence (CL), and (3) LPS-stimulated neutrophil-mediated human umbilical vein endothelial cell (HUVEC) injury assessed by 51Cr release. Sepsis-induced lung injury in guinea pigs was assessed by comparing 125I-labeled albumin concentrations in lung tissue and bronchoalveolar lavage (BAL) fluid relative to plasma (L/P and BAL/P), lung wet-to-dry weight ratios, and the number of neutrophils in BAL fluid. The lung wet-to-dry weight ratio, L/P, and the number of neutrophils in BAL fluid decreased after pretreatment and post-treatment with apocynin. BAL/P decreased upon pretreatment but not upon post-treatment with apocynin. Apocynin at concentrations from 10 to 100 micrograms/ml significantly reduced LPS-stimulated neutrophil CL and neutrophil-mediated HUVEC 51Cr release. We conclude that the NADPH oxidase inhibitor apocynin attenuates (1) sepsis-induced lung injury in guinea pigs, (2) neutrophil ROS generation measured by CL, and (3) neutrophil-mediated HUVEC injury assessed by 51Cr release.