Effect of Modified Shengjiangsan on Oxidative Stress and Apoptosis in MN Rats via Regulating HIF-1α/NOX4 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention effect of modified Shengjiangsan on hypoxia-inducible factor-1<italic>α </italic>（HIF-1<italic>α</italic>）/nicotinamide adenine dinucleotide phosphate oxidase 4 （NOX4） signaling pathway in membranous nephropathy （MN） rats and to explore its mechanism to reduce oxidative stress and apoptosis in renal tissues. Method:Cationized bovine serum albumin （C-BSA） was injected into the tail vein of rats to replicate the MN model. Rats were randomly divided into a model group， a modified Shengjiangsan group， and a benazepril group after modeling， and administered by gavage once a day accordingly. At the end of the 4^th week， the 24-h urine total protein （UTP）， urea nitrogen （BUN）， and serum creatinine （SCr） levels of each group were detected. Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， and reactive oxygen species （ROS） in renal tissues of rats. In situ end labeling（TUNEL） staining was used to detect the cell apoptosis rate. The mRNA and protein expression levels of HIF-1<italic>α</italic> and NOX4 were detected by real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）and Western blot， respectively. The immunohistochemistry method was used to detect the protein expression levels of B-cell lymphomas -2 （Bcl-2）， B-cell lymphomas xl （Bcl-xl）， Bcl-2 associated X protein （Bax）， Bcl-2 cell death regulator antibody （Bim）. Result:Compared with the normal group， the model group showed increased UTP （<italic>P</italic><0.05）， decreased SOD， elevated MDA and ROS （<italic>P</italic><0.05）， up-regulated mRNA and protein expression of HIF-1<italic>α</italic> and NOX4 （<italic>P</italic><0.05）， enhanced protein expression of Bax and Bim， declining protein expression of Bcl-xl and Bcl-2 （<italic>P</italic><0.05）， and increased cell apoptosis in renal tissues. Compared with the model group， the modified Shengjiangsan group and the benazepril group displayed declining UTP （<italic>P</italic><0.05）， up-regulated SOD， decreased MDA and ROS （<italic>P</italic><0.05）， down-regulated mRNA and protein expression of HIF-1<italic>α</italic> and NOX4 （<italic>P</italic><0.05）， diminished protein expression of Bax and Bim， elevated protein expression of Bcl-xl and Bcl-2 （<italic>P</italic><0.05）， and reduced cell apoptosis in renal tissues （<italic>P</italic><0.05）. Conclusion:The protective effect of modified Shengjiangsan on the kidney is presumedly achieved by reducing the oxidative stress and apoptosis in renal tissues of MN rats via inhibiting the HIF-1<italic>α</italic>/NOX4 signaling pathway.

Effect of Albiziae Flos and Polygalae Radix Alone and Their Combination on Depression-like Behavior and CREB and NOX2 Expression in Hippocampus of Chronic Unpredictable Stress-induced Rats / 中国实验方剂学杂志

Objective:To observe the effects of Albiziae Flos （AF） and Polygalae Radix （PR） alone and their combination on the improvement of depression-like behavior in rats with chronic unpredictable stress （CUS） as well as on hippocampal ultrastructure and the expression of cyclic adenosine monophosphate response element binding protein （CREB） and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2）， to explore their action mechanisms. Method:Seventy-two Wistar rats were randomly divided into the normal group， model group， AF group， PR group， AF-PR group， and fluoxetine group. Rats in all groups except for the normal group were exposed to CUS and separated feeding to induce depression. Since the first day of modeling， rats in the AF group， PR group， AF-PR group were provided with the corresponding decoction containing 1.05 g·kg^-1 total crude drug by gavage， the ones in the fluoxetine group with 2.1 mg·kg^-1 fluoxetine hydrochloride aqueous solution， and those in the normal group and model group with the distilled water， for 28 successive days. The open field test and forced swimming test were performed 1 d before modeling and 7， 14， 21， 28 d after modeling， respectively. The morphological changes in hippocampus were observed under an electron microscope on the 28^th day. The superoxide dismutase （SOD） and malondialdehyde （MDA） levels in hippocampus were detected by ultraviolet spectrophotometry， and the expression levels of CREB and NOX2 were detected by real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:The behavioral experiment results showed that the number of horizontal activities and sugar water consumption in the model group declined as compared with those in the normal group， while the immobility time in the forced swimming test was prolonged （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group exhibited elevated number of horizontal activities， increased sugar water consumption but shortened immobility time in the forced swimming test （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the AF group or PR group， the AF-PR group showed significantly different behavioral indexes （<italic>P</italic><0.05）. Morphological results showed that the mitochondria of the model group were obviously swollen and the ultrastructure of the hippocampus was destroyed. By contrast， the hippocampal ultrastructure in each administration group was close to normal. The comparison with the normal group revealed that the activity of SOD in the hippocampus of the model group was significantly reduced， whereas the content of MDA was elevated （<italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group displayed increased activity of SOD and decreased content of MDA in the hippocampal tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with AF or PR alone， the herbal pair AF-PR resulted in significant differences in the above-mentioned indexes （<italic>P</italic><0.05， <italic>P</italic><0.01）. The results of Real-time PCR and Western blot demonstrated that NOX2 expression in the hippocampus of the model group was up-regulated in comparison with that in the normal group， while the CREB expression was down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group all showed diminished NOX2 expression but elevated CREB expression in the hippocampal tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. The protein expression levels of NOX2 and CREB in the AF group or PR group were significantly different from those in the AF-PR group （<italic>P</italic><0.01）. Conclusion:AF and PR alone and their combination improve the depression-like behavior of rats exposed to CUS， which may be related to the reduction of oxidative stress， the up-regulation of CREB expression， and the down-regulation of NOX2 expression in hippocampus.

Effects of NF-κB/NOX1 signaling pathway in TNF-α-induced apoptosis of A549 cells / 中国职业医学

OBJECTIVE: To investigate the effects of nuclear transcription factor-κB(NF-κB)/amide adenine dinucleotide phosphate oxidase 1(NOX1) signaling pathway in tumor necrosis factor-α(TNF-α) induced apoptosis of A549 cells. METHODS: i) A549 cells were stimulated with TNF-α at the concentrations of 0.00, 0.25, 0.50, and 1.00 nmol/L. CCK-8 assay was used to detect the cell viability to screen the optimal stimulating concentration of TNF-α. ii) A549 cells at logarithmic growth stage were randomly divided into four groups, the control group, the TNF-α group, the BAY11-7082(NF-κB inhibitor) group and the TNF-α+BAY11-7082 group. The cells in the control group were not treated. The TNF-α and BAY11-7082 groups were stimulated with 0.50 nmol/L TNF-α and 5 μmol/L BAY11-7082, respectively. The TNF-α+BAY11-7082 group was stimulated by both TNF-α and BAY11-7082. After 24 hours of culture, the cell survival rate was detected by CCK-8 assay. Flow cytometry was used to detect cell apoptotic rate, and Western blotting was used to detect the relative expression of NF-κB(p65) and NOX1 proteins. RESULTS: i) When A549 cells were stimulated with TNF-α at the concentration of 0.50 nmol/L, the cell proliferative activity was reduced and the cell apoptosis was promoted. This concentration was selected as the stimulation dose of TNF-α in subsequent experiments. ii) The survival rate of A549 cells in the TNF-α group decreased(P<0.05), the apoptotic rate and the protein expressions of NF-κB(p65) and NOX1 increased in TNF-α group(all P<0.05) compared with the control group. In BAY11-7082 group, the survival rate and the relative expression of NF-κB(p65) and NOX1 of A549 cells were decreased(all P<0.05), and the apoptotic rate of A549 cells was increased(P<0.05) compared with the control group. A549 cells in TNF-α+BAY11-7082 group changed from a long spindle shape to an irregular one. The cell survival rate increased(P<0.05), the apoptotic rate and the relative expression of NF-κB(p65) and NOX1 decreased(all P<0.05) compared with the TNF-α group. CONCLUSION: NF-κB/NOX1 signaling pathway is involved in A549 cells apoptosis induced by TNF-α.

Inhibitory effects of NADPH oxidase 4 inhibitor on VEGF expression in hypoxia-induced human RPE cells / 中华实验眼科杂志

Objective@#To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.@*Methods@#The cultured APRE-19 cells were divided into Avastin group and VAS2870 group, and then the Avastin group was subdivided into the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining, and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.@*Results@#The relative expression of NOX4 was 0.657±0.153, 1.000±0.200, 1.206±0.300, 1.260±0.200 and 1.413±0.273, and the relative expression of VEGF-A was 0.821±0.110, 1.210±0.100, 0.672±0.100, 0.340±0.120 and 0.300±0.130 in the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, respectively, with statistically significant differences among the groups (F=17.631, P<0.001; F=4.777, P<0.05). The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P<0.001). The relative expression of VEGF-A protein in the cells of the 0.25, 0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P<0.05). The expression of NOX4 protein in the cells was 0.970±0.120, 1.060±0.130, 0.880±0.130, 0.567±0.135 and 0.450±0.120, and the relative expression of VEGF-A protein was 0.387±0.135, 0.627±0.125, 0.370±0.140, 0.363±0.140 and 0.160±0.100 in the normoxia control group, hypoxia control group, 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group, respectively, with statistically significant differences among them (F=12.933, P<0.001; F=4.948, P<0.05). The relative expression of VEGF-A protein in the 1, 3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P<0.05).@*Conclusions@#NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.

Inhibitory effects of NADPH oxidase 4 inhibitor on VEGF expression in hypoxia-induced human RPE cells / 中华实验眼科杂志

Objective To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.Methods The cultured APRE-19 cells were divided into Avastin group and VAS2870 group,and then the Avastin group was subdivided into the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group,3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining,and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.Results The relative expression of NOX4 was 0.657± 0.153,1.000±0.200,1.206 ± 0.300,1.260± 0.200 and 1.413 ± 0.273,and the relative expression of VEGF-A was 0.821±0.110,1.210±0.100,0.672±0.100,0.340±0.120 and 0.300±0.130 in the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,respectively,with statistically significant differences among the groups (F =17.631,P＜ 0.001;F=4.777,P＜0.05).The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P＜0.001).The relative expression of VEGF-A protein in the cells of the 0.25,0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P＜0.05).The expression of NOX4 protein in the cells was 0.970±0.120,1.060±0.130,0.880±0.130,0.567±0.135 and 0.450±0.120,and the relative expression of VEGF-A protein was 0.387±0.135,0.627±0.125,0.370±0.140,0.363±0.140 and 0.160±0.100 in the normoxia control group,hypoxia control group,1 μmol/ml VAS2870 intervention group,3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group,respectively,with statistically significant differences among them (F =12.933,P＜ 0.001;F =4.948,P＜ 0.05).The relative expression of VEGF-A protein in the 1,3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P＜0.05).Conclusions NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.

Effect of benazepril on nuclear factor E2 related factor 2, nicotinamide adenine dinucleotide phosphate oxidase and reactive oxygen species in rats with hepatic fibrosis / 中华肝脏病杂志

Objective@#To study the effect of benazepril on the expression of nuclear factor E2 related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and reactive oxygen species (ROS) concentration in rats with hepatic fibrosis and to explore the possible antifibrotic mechanism of benazepril.@*Methods@#Twenty-two healthy male Sprague-Dawley rats were randomly divided into 3 groups: control group (6 rats), model group (8 rats) and benazepril treatment group (8 rats). Two rats died during modeling and treatment in the model group and the benazepril treatment group, and a model of hepatic fibrosis induced by carbon tetrachloride (CCL4) was established. The rats in benazepril group were given benazepril for 8 weeks by gastric gavage. The assessment of liver tissue damage in each group was measured using conventional hematoxylin-eosin and Masson staining. The mRNA level of Nrf2, NOX4 in liver tissue was detected by RT-PCR, and serum ROS concentration was determined by colorimetry. All data were expressed in mean ± standard deviations, and were analyzed using SPSS21.0 statistical software. The data were compared using one-way analysis of variance, and the LSD-t method was used for pairwise comparison between the two groups. The correlation analysis was performed by Spearman’s correlation analysis.@*Results@#In the liver of the model group, with the aggravation of liver fibrosis the expression of Nrf2mRNA, NOX4 mRNA and ROS concentration were higher than control group [(4.01 ± 3.40), (31.78 ± 3.96), (1.82 ± 0.46) μg/ ml vs. (0.12 ± 0.11), (2.03 ± 0.31), (1.56±0.84) μg/ml, P < 0.05]. After benazepril treatment, NOX4 mRNA expression and ROS concentration were decreased than the model group [(15.93 ± 5.01), (0.78 ± 0.44) μg/ml vs. (31.78 ± 3.96), (1.82 ± 0.46) μg /ml, P < 0.05], while Nrf2 mRNA expression was higher than the model group [(6.69 ± 4.86) vs. (4.01 ± 3.40), P < 0.05]. There was a positive correlation between Nrf2 and NOX4, Nrf2 and ROS, and NOX4 and ROS (r = 0.616, 0.411, 0.802, P < 0.05).@*Conclusion@#Benazepril may exert an anti-hepatic fibrosis effect by activating Nrf2 expression, or may inhibit the ROS-mediated oxidative stress in response to NOX4.

The role of nicotinamide-adenine dinucleotide phosphate oxidase NOX family in acute lung injury / 中华危重病急救医学

Acute?lung?injury?(ALI)?and?its?severe?form,?acute?respiratory?distress?syndrome?(ARDS),?are?common?critical?syndromes.?The?causes?of?the?syndrome?are?complex?and?diverse.?The?main?pathological?features?are?the?diffuse?inflammatory?and?protein-rich?pulmonary?edema?caused?by?destruction?of?the?blood-air?barrier.?Reactive?oxygen?species?(ROS)?mediate?oxidative?damage?by?oxidizing?bio-macromolecules,?including?lipids,?proteins?and?nucleic?acid.?Among?many?systems?producing?ROS,?nicotinamide-adenine?dinucleotide?phosphate?(NADPH)?oxidase-mediated?ROS?is?the?main?source,?and?its?functional?subunit?is?the?transmembrane?subunit?NOX?family.?The?distribution?of?NOX?family?proteins?in?lung?tissue?is?cell?type?dependent.?NOX-derived?ROS?is?involved?in?the?defense?function?of?lung?tissue?and?related?to?the?occurrence?and?development?of?ALI/ARDS.?This?review?mainly?describes?the?cell?distribution,?activation?factors,?and?its?relationship?with?the?occurrence?and?development?of?ALI?of?the?NOX?family.

Adrenomedullin and bronchopulmonary dysplasia / 国际儿科学杂志

Bronchopulmonary dysplasia (BPD) is a common respiratory disease in premature infants characterized by alveolar and pulmonary vascular dysplasia.There are currently no effective preventive and therapeutic measures.Adrenomedullin (ADM) is a vasodilator peptide produced by endothelial and smooth muscle cells of the systemic and pulmonary circulation,which promotes alveolar growth and pulmonary angiogenesis.ADM protects the respiratory system through three signaling pathways:(1) it promotes pulmonary vascular endothelial cell proliferation through extracellular regulated protein kinases (ERK);(2) attenuates lung cell apoptosis and improves cell proliferation through protein kinase B (PKB) pathway;(3) it down-regulates NADPH oxidase 4 (NOX4) expression in lung tissue to inhibit oxidative stress and improves BPD.So ADM signaling axis may be a potential therapeutic target for human infants with BPD.It provides theoretical basis for clinical prevention and treatment of BPD in premature infants.

Role of NADPH oxidase-mediated activation of microglia in the comorbidity of neuropathic pain and depression / 临床麻醉学杂志

Objective To observe the variation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and microglia in the comorbidity of neuropathic pain and depression and discuss the related mechanism.Methods The spared nerve injury model was used.Forty-four male adult Sprague Dawley rats were randomly divided into the following four groups (n=11 each): sham+vehicle group (group SV), sham+APO group (group SA), SNI+vehicle group (group SNV), SNI+APO group (group SNA).In groups SA and SNA, rats were intraperitoneally injected with apocynin (APO) 15 mg/kg 24 hours and 1 hour before SNI and continued once daily until the 14th day.The rats in the other two groups received the equal volume of vehicle.The mechanical withdrawal threshold (MWT) was tested 1 day before SNI and 7 days and 14 days after SNI, and the open field test, the forced swimming test and the sucrose preference test were performed 14 days after SNI.The prefrontal cortex were collected 2 hour after the behavior tests.The expression of gp91phox was detected by Western blot and the expression of Iba1 and gp91phox were detected by double-immunofluorescance staining.Results The reduced MWT, the increased immobility time, the decreased sucrose consumption and the increased content of gp91phox were observed in group SNV compared with groups SV, SA and SNA (P<0.05).The expression of Iba1 and gp91phox were increased in group SNV.The total travel distance in the open field test and the total liquid consumption in the sucrose preference test had no significant difference among the four groups.Conclusion Neuropathic pain may induce depressive behaviors and activate NADPH oxidase in the prefrontal cortex.Moreover, the inhibition of NADPH oxidase by APO can alleviate neuropathic pain and depression, which is potentially related to the activation of microglia.

Effect of plumbagin on levels of Nox4/ROS andα-SMA in human hepatic stellate cells / 中国病理生理杂志

AIM:To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide ade -nine dinucleotidephosphate oxidase 4 ( Nox4 ) , reactive oxygen species ( ROS ) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro.METH-ODS:HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium-and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups .After incubated with each drug for 72 h, the mRNA expression of Nox4 was detec-ted by RT-PCR.ROS levels were tested by in situ loading probe method.The protein contents of Nox4 and α-SMA were measured by Western blot .RESULTS:Compared with model group , after treated with plumbagin for 72 h, the mRNA ex-pression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01).CONCLUSION:Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels .

Effect of high glucose on NADPH oxidase expression and reactive oxygen species generation in human umbilical vein endothelial cells / 中南大学学报(医学版)

Objective: To explore the effect of high glucose on NADPH oxidase (NOX) expression and intracellular reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were divided into a control group, a mannite group, a glucose group and aglucose plus diphenylene iodonium (DPI) group. Intracellular ROS was detected by lfow cytometry. RNA and protein expression of NOX in HUVECs was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot, respectively. Results: 1) Compared with the control group, the intracellular ROS were signiifcantly increased in the glucose group (P0.05,n=3); 2) Compared with the control group, the mRNA and protein expression of NOX4 in the glucose group were signiifcantly increased (P0.05); 3) there were no signiifcant difference in the mRNA and protein expression of NOX2, NOX4, p22phox, p67phox and rac between the glucose plus DPI group and the control group (allP>0.05). Conclusion: High glucose may increases intracellular ROS generation by increasing the expression of NOX4 in HUVECs, which might mediate the oxidative stress.

Inhibitor of NADPH oxidase slow photoreceptor cell death in the retinal degeneration of rd mice / 中华实验眼科杂志

Background Our previous study demonstrated that microglial activation is closely associated with photoreceptor apoptosis in rd mice.Recent studies on central nervous system (CNS) showed that activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the microglia activation and neural cell death.However,the mechanism of NADPH oxidase during the retinal degeneration and the effect of NADPH oxidase inhibitor on photoreceptor apoptosis are concerned.Objective The aim of this study was to further explore the production of reactive oxygen species (ROS) by NADPH oxidase in the retinal degenerative process of rd mice and protection of NADPH oxidase inhibitor on photoreceptors.Methods Sixty rd mice at postnatal day 9 (P9) were randomized into the experimental group and the control group by throwing coins method.Apocynin,a NADPH oxidase inhibitor,was intraperitoneally injected in the dose of 10 mg/kg (0.01 ml/kg) once daily for 5 days (P13) in the experimental group,and the equal amount of PBS was used in the same way in the control group,and 10 C57BL/6N mice without injection of any drugs served as the wild type mice group.All the mice were sacrificed in P14 for the preparation of retinal sections.The expression of ROS in the retina was detected by dihydroethidium (DHE) fluorescence staining.Expression level of rhodopsin mRNA in the photoreceptor of the mice was determined by real-time PCR,and the thickness of retinal outer nuclear layer (ONL) in the mice of the experimental group and the control group was measured using hematoxylin & eosin staining.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology.Results DHE staining showed that the ROS presented with the red fluorescence in the mouse retinas.In the rd mice of the experimental group,the ROS fluorescence intensity was dramatically enhanced in comparison with C57BL/6N mice,but weakened in comparison with the rd mice of the control group.Real-time PCR revealed that the relative expressing level of rhodopsin mRNA in the photoreceptor was (4.21±0.33) in the experimental group and (0.93±0.24) in the control group,showing a significant difference between them (t =2.360,P =0.000).The thickness value of retinal ONL was (35.95±1.63)μm in the mice of the experimental group,which was significantly higher than that in the mice of the control group ([23.17±1.38] μm) (t=3.850,P=0.016).Conclusions In the retinal degeneration of rd mice,activation of NADPH oxidase increases the ROS production.Apocynin can slow the apoptosis procedure of photoreceptor cells of rd mice.

Changes of NADPH oxidase expression in retina of rd mice / 中华实验眼科杂志

Background Studies showed that activation of microglia-derived nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in the neurodegenerative diseases and neural cell death in central nervous system.The effect of NADPH on cone degeneration have been determined in rd rats,its role in rod degeneration is relatively less studied.Objective This study was to study the expression of NADPH oxidase in the retinal degenerative process in rd mice and further explore its role in the photoreceptor degeneration.Methods rd Mice at postnatal day 8 (P8),P10,P12,P14,P16 and P18 were collected.The mice were sacrificed,and retinal sections,RNAs and proteins were prepared in above-mentioned time points.The expressions of the gp91 phox,a major subunit of NADPH oxidase,in transcript level and protein level in the retinas were semi-quantitatively detected by real-time PCR and Western blot respectively.Expression of gp91phox was localized in the rd retinas as ageing by immunohistochemstry,and the co-expression of gp91phox with CD11b,a specific marker of microglial cells,was assayed by immunofluorescent double labeling.The C57BL/6N mice were served as controls.The use and care of the animals complied with the Guideline of ARVO.Results Real-time PCR showed that gp91phox mRNA was not expressed in the retinas of C57BL/6N mice.Gp91phox mRNA was found to have less expressed in retinas of P8 rd mice.With aging,the expression level of gp91phox mRNA (gp91phox mRNA/β-actin) in rd mouse retinas was gradually increased with the highest level in P14 mice(1.136±0.370).A significant difference was seen in the gp91 phox mRNA expression among various groups of mice (F=17.81,P =0.00),and gp91phox mRNA expression was significantly elevated in P10,P12,P14,P16 and P18 rd mice compared with P8 rd mice(all at P＜0.05).The expression level of gp91phox protein (A value) in the retinas presented with a similar trend in the rd mice,with a significant difference among the various ages of rd mice and C57BL/6N mice (F =354.00,P＜0.01).The expression level of gp91 phox protein was increased in the rd mice in comparison with the C57BL/6N mice (all at P＜0.05).Immunochemistry revealed that the positive response cells for gp91phox increased in the inner layers of retinas in P10 rd mice and peaked in P14 mice.Immunofluorescent double labeling exhibited that gp91phox were seen to present a co-expression with CD11b,showing an orange fluorescence.Conclusions Expression of NADPH oxidase in the rctinas in the rd mice up-regulates and is parallels to the microglial activation and photoreceptor degeneration,suggesting that NADPH oxidase plays a role in the retinal dystrophy associated with microglial activation.

TNF-alpha stimulated IL-8 and IL-10 expression in monocytes from patients with chronic granulomatous disease / 소아과

PURPOSE: Patients with chronic granulomatous disease (CGD) have genetic mutations in a component of the NADPH oxidase enzyme that is necessary for the generation of the superoxide anion. The profound defect in innate immunity is reflected by the patients susceptibility to catalase-positive bacteria and fungi. In addition, CGD patients display signs of persistent inflammation, which is not associated only with deficient superoxide anion production. The aim of this study was to elucidate the cytokine responses in CGD patients after TNF-alpha stimulation. METHODS: Heparinized blood samples were collected from 8 CGD patients and 10 healthy volunteers. Monocytes (1x10(6) cell/well) isolated by the magnet cell isolation system were incubated with a constant amount of TNF-alpha (10 ng/mL) at 37degrees C for 6 h. Incubated cells were harvested at 60-min intervals for IL-8 and IL-10 mRNA analysis, and the supernatant was collected at the same intervals to determine IL-8 and IL-10 expression. Monocytes from healthy volunteers were also incubated with antioxidants followed by TNF-alpha stimulation for IL-8 and IL-10 expression. RESULTS: In CGD patients, a high expression of IL-8 together with a significantly higher IL-10 expression than in the healthy controls was seen after TNF-alpha stimulation. Moreover, normal monocytes treated with antioxidants exhibited increased IL-8 responses. CONCLUSION: The absence of phagocyte-derived reactive oxidants in CGD might be associated with a dysregulated production of pro- and antiinflammatory cytokines. Additional research related to reactive oxidants is needed to clarify the role of cytokines in CGD patients.