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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<sup>th</sup> 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.
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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.
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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.
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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.
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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.
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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 .