Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: ER β inhibits oxalate-induced oxidative stress via modulating NOX2 expression. (a) The qRT-PCR analysis of NADPH oxidase subunits after knocking down ER β in HK-2 and HKC-8 cells. Cells were transduced with shER β or with shLuc as control after 72 hr incubation, and then treated with 0.75 mM oxalate for 6 hr. (b) Western blot analysis of NOX2 expression in renal cells with/without shER β after 6 hr exposure to 0.75 mM oxalate. (c) Rescue assay using HK-2 and HKC-8 cells with/without shER β . shNOX2 showed partially reversed knockdown of ER β - (shER β -) induced ROS production. All cells were exposed to 0.75 mM oxalate for 6 hr prior to collection for data analysis. Upper panels show representative images of DHE staining, and quantification is shown in the lower left panel. Western blot in the lower right panel shows NOX2 knockdown efficiency. (d) After treating with 0.75 mM oxalate for 6 hr, shNOX2 can partly reverse the silenced ER β -induced H 2 O 2 production in conditioned media. (e) Rescue assay using HK-2 and HKC-8 cells with/wthout shNOX2 showed partially reversed shER β -induced LDH release. All the cells in Figures – were exposed to 0.75 mM oxalate for 6 hr prior to collection for data analysis. For (a), (c), (d), and (e), data are presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Expressing, Quantitative RT-PCR, Transduction, Control, Incubation, Western Blot, Rescue Assay, Knockdown, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: Mechanism dissection on how ER β regulates NOX2 expression. (a) NOX2 mRNA levels were regulated by E2 or ER β antagonists in HK-2 and HKC-8 cells. Cells were treated for 6 hr with vehicle (control), 0.75 mM oxalate, 0.75 mM oxalate + 10 nM E2, 0.75 mM oxalate + 10 nM E2 + 10 μ M ICI, or 0.75 mM oxalate + 10 nM E2 + 10 μ M PHTPP for q-PCR of NOX2 mRNA levels. Bonferroni's test was applied for statistical analysis of results. (b) Predicted 3 potential EREs located in the 3 kb-NOX2 promoter region. (c) ChIP was performed in HK-2 cells, and only ERE I of the NOX2 promoter could be bound by ER β . (d) ERE I wild-type or mutant of NOX2 promoter reporter constructs were cotransfected with pRL-TK at a ratio of 1000 : 1 into HK-2-shLuc and HK-2-shER β cells. Luciferase reporter assay was performed after 48 hr incubation. For (a) and (d), data shown are mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01. n.s. = not significant.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Dissection, Expressing, Control, Mutagenesis, Construct, Luciferase, Reporter Assay, Incubation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: In vivo mouse model data confirm that ER β deficiency promotes renal CaOx crystal deposition via increasing the hepatic oxalate biosynthesis and renal oxidative stress. (a) Diagram describing the injection schedule for mock control, a selective ER β antagonist PHTPP, and glyoxylate (upper panel). Crystal staining (lower panels) showing the deposition of CaOx crystals in kidney tissues of the PHTPP-treated and mock-control mice. CaOx crystal formation was detected by Pizzolato staining. Crystallization in each kidney section was quantified by calculating the ratio (percent) of the area containing crystals to the entire kidney section using ImageJ software. (b) Staining (left panels) and quantification (right panels) results for CaOx crystal deposition as detected by Pizzolato staining in kidney tissues of the ER β KO and their WT female littermate mice. (c) Detection of 24 hr oxalate excretion in urine samples collected from the ER β KO and WT female mice prior to sacrifice. (d) Western blot of 60 μ g liver protein from ER β KO and WT mice probed with affinity-purified rabbit antibody raised against recombinant mouse AGT1 shows lower expression of AGT1 in the ER β KO mice. (e) Fresh-frozen renal tissues were stained for 30 min with DHE. Representative sections from the ER β KO and their WT female littermate mice are shown (left panels). Right bar graphs show quantification of superoxide generation. (f) H 2 O 2 concentration in urine sample from ER β KO and WT female mice. (g) Representative micrographs of NOX2 immunostaining in renal tissue from the ER β KO and their WT littermate mice. The NOX2 protein was mainly located in renal tubular cells. Quantification of NOX2 is shown in the right panel. Scores were classified as 0 to 3, based on the intensity of staining and the percentage of positive cells. (h) Inhibitory effect of apocynin treatment in mice. CaOx crystal formation in ER β KO mice was significantly decreased after in vivo apocynin treatment. (i) Apocynin treatment reduced H 2 O 2 concentration in urine samples from ER β KO mice. For (a)–(c) and (e)–(i), data are presented as mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: In Vivo, Injection, Control, Staining, Crystallization Assay, Software, Western Blot, Affinity Purification, Recombinant, Expressing, Concentration Assay, Immunostaining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: Suppressing ER β activity could increase renal CaOx crystal formation in HLP-induced rat model. (a) A diagram describing the injection schedule for PHTPP/DMSO treatment. (b) Crystal staining showing the deposition area of CaOx crystals in kidney tissues of the rats treated with PHTPP or vehicle control. CaOx crystal formation was detected by Pizzolato staining and imaged by polarized light optical microphotography. (c) The PHTPP-treated rats showed increased urine oxalate excretion compared to vehicle-treated control rats. Prior to sacrifice, we collected and detected 24 hr oxalate excretion in urine of rats treated with PHTPP or vehicle control. (d) IHC staining of AGT1 in liver tissues of the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed decreased AGT1 expression. (e) Detection of 24 hr urine H 2 O 2 levels in the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed higher H 2 O 2 levels. (f) IHC staining of 8-OHdG in kidney tissues of the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed increased 8-OHdG expression. (g) IHC staining of NOX2 in kidney tissues of the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed increased NOX2 expression as compared to control rats. For (b)–(g), data are presented as mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Activity Assay, Injection, Staining, Control, Immunohistochemistry, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: Scheme. The schematic illustration of the ER β signal regulation of CaOx crystal formation in the liver and kidney. For the liver part, ER β signaling promotes activity of AGT1, which decreases the biosynthesis of oxalate by converting glyoxylate into glycine decreasing CaOx crystal formation. For the kidney part, ER β has a protective effect in human renal tubular cells against oxalate-induced oxidative stress via suppression of NOX2 (a NADPH oxidase), which finally leads to decreasing CaOx crystal deposition on the damaged cell surface.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Gallic Acid Reduces Blood Pressure and Attenuates Oxidative Stress and Cardiac Hypertrophy in Spontaneously Hypertensive Rats

doi: 10.1038/s41598-017-15925-1

Figure Lengend Snippet: GA attenuates Nox2 expression in SHRs and angiotensin II-treated H9c2 cells. ( A ) The mRNA levels of Nox1, Nox2, and Nox4 were analyzed using qRT-PCR. The transcript levels have been normalized to 18 S and presented as a relative value. *** P < 0.001 versus WKY. # P < 0.05 and ### P < 0.001 versus SHR. ( B ) Representative immunoblots. Nox1, Nox2, and Nox4 protein expression in rat hearts from the WKY, SHR, and SHR+GA groups, was determined using western blot analysis. GAPDH was used as the loading control. ( C ) The protein expression of Nox1, Nox2, and Nox4 was quantified by densitometry. *** P < 0.001 versus WKY. # P < 0.05 versus SHR. ( D ) The mRNA levels of Nox1, Nox2, and Nox4 were determined using H9c2 cells treated with the vehicle (control), with vehicle + Ang II (1 µM), or with Ang II (1 µM) + GA (100 µM) for 9 h. The values are the mean ± SE of at least three independent experiments. ** P < 0.01 versus vehicle-treated cells. # P < 0.05 versus angiotensin II-treated cells. NS, not significant.

Article Snippet: Anti-Nox1 (GTX103888), -Nox2 (GTX63960), -Nox4 (GTX121929), and -ACE1 (GTX100923) antibodies were purchased from GeneTex Inc. (Irvine, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Current Issues in Molecular Biology

Article Title: Coptis chinensis Franch. Suppresses Invasive Pulmonary Aspergillosis by Augmenting NADPH-Dependent Neutrophil Extracellular Traps via Dual Modulation of Complement Activation and Gut Microbiota

doi: 10.3390/cimb48040424

Figure Lengend Snippet: Bayesian networks and KEGG suggest a CCF-biased reorganization toward CR3-SYK-NOX/NET pathways relative to IPA/AmB. The network analysis highlights recurrent CCF-associated hub genes, including Itgam, Syk, Rac2, and Cybb, together with pathway enrichment linked to oxidative and NET-related responses. ( A ) Hub gene networks identified in the IPA, CCF, and AmB groups by Bayesian network analysis. Genes were ranked by DEGREE value, with the top 10% defined as hub genes and marked in color; nodes with DEGREE >6 were placed at the core of the network. ( B ) KEGG pathway enrichment of hub genes in each group. Pathways were ranked by −log 10 ( p value). Bar colors represent different groups: IPA (dark blue), CCF (dark red), and AmB (light blue). The red box denotes the most significantly enriched KEGG pathway in the CCF group. ( C ) FPKM values of key CCF-regulated genes at +2, +4, and +6 dpi. Statistical analysis was conducted comparing all groups to the CCF group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 indicate levels of statistical significance. ( D ) qPCR validation of key CCF-regulated genes in each group. Statistical analysis was conducted using one-way ANOVA, comparing all groups to the CCF group. * p < 0.05, ** p < 0.01, **** p < 0.0001 indicate levels of statistical significance. Data are presented as mean ± SD from n = 3 mice per group.

Article Snippet: Primary antibodies used in Western Blot included Beta Actin Monoclonal antibody (66009-1-Ig, Proteintech, Wuhan, China), NCF4/p40phox Rabbit pAb (A2096, Abclonal, Wuhan, China), Anti-NOX2/gp91phox Recombinant Rabbit Monoclonal Antibody (ET1611-44, Huabio, Hangzhou, China), RAC2 Rabbit pAb (A1139, Abclonal, China) and Anti-NCF1/p47phox Rabbit pAb (GB11724, Servicebio, Wuhan, China).

Techniques: Biomarker Discovery

Journal: Current Issues in Molecular Biology

Article Title: Coptis chinensis Franch. Suppresses Invasive Pulmonary Aspergillosis by Augmenting NADPH-Dependent Neutrophil Extracellular Traps via Dual Modulation of Complement Activation and Gut Microbiota

doi: 10.3390/cimb48040424

Figure Lengend Snippet: CCF enhances NOX2 components and its benefit is partially attenuated by apocynin, consistent with oxidative pathway involvement. This figure combines protein, survival, and fungal-burden data to show partial pharmacological support for the role of oxidative responses in CCF-mediated protection. ( A ) Protein expression of NADPH oxidase subunits in lung tissue at +4 dpi as detected by Western blot. I, IPA group; C, CCF group; A, AmB group; M, Marker. Data are presented as mean ± SD from n = 5 mice per group. ( B ) Densitometric quantification of protein levels normalized to β-ACTIN. Statistical analysis was performed using one-way ANOVA with multiple comparisons. * p < 0.05. Data are presented as mean ± SD from n = 5 mice per group. ( C ) Survival curves of mice up to +8 dpi. CCF significantly improved survival compared to the IPA group. Co-treatment with the NADPH oxidase inhibitor apocynin partially reversed this effect. * p < 0.05, ** p < 0.01. Survival data were analyzed using the Kaplan–Meier method with n = 10 mice per group. ( D ) Fungal burden in lung tissues at +4 dpi measured by colony-forming unit counts. Statistical analysis was performed using one-way ANOVA with multiple comparisons. * p < 0.05. Data are presented as mean ± SD from n = 5 mice per group.

Article Snippet: Primary antibodies used in Western Blot included Beta Actin Monoclonal antibody (66009-1-Ig, Proteintech, Wuhan, China), NCF4/p40phox Rabbit pAb (A2096, Abclonal, Wuhan, China), Anti-NOX2/gp91phox Recombinant Rabbit Monoclonal Antibody (ET1611-44, Huabio, Hangzhou, China), RAC2 Rabbit pAb (A1139, Abclonal, China) and Anti-NCF1/p47phox Rabbit pAb (GB11724, Servicebio, Wuhan, China).

Techniques: Expressing, Western Blot, Marker

Journal: Current Issues in Molecular Biology

Article Title: Coptis chinensis Franch. Suppresses Invasive Pulmonary Aspergillosis by Augmenting NADPH-Dependent Neutrophil Extracellular Traps via Dual Modulation of Complement Activation and Gut Microbiota

doi: 10.3390/cimb48040424

Figure Lengend Snippet: CCF-treated mice show microbiota remodeling with enrichment of Clostridium sp., and observed Cybb/Syk associations are non-causal. The main features of this figure are the distinct clustering of gut microbial communities across groups, the selective enrichment of Clostridium sp. in the CCF group, and its positive correlation with the redox-related host genes Cybb and Syk . ( A ) Alpha diversity analysis using the Chao1 index and Shannon index reveals significant differences among CN, IPA, CCF, and AmB groups. ( B ) Principal coordinates analysis (PCoA) based on weighted UniFrac distances demonstrates distinct clustering of gut microbial communities across groups. ( C ) Taxonomic composition profiles at the family genus, and species levels. Bar plots display the relative abundance of dominant taxa within each group. ( D ) Histogram of LDA scores (log 10 ) for discriminative features, indicating significantly enriched taxa in each group. The colors represent different groups: CN (blue), IPA (red), CCF (green), and AmB (purple). ( E ) Cladogram representing the phylogenetic distribution of differentially abundant taxa among the groups. Colored nodes represent taxa significantly enriched in the corresponding groups: CN (blue), IPA (red), CCF (green), and AmB (purple). From inner to outer circles represent phylum, class, order, family, genus, and species levels. Taxa connected by the same color belong to the same species. ( F ) Comparison of the abundances of significantly different biomarkers: g__ Parasutterella and s__ Lactobacillus in the CN group, s__ Clostridium_sp in the CCF group, g__ Clostridia in the AmB group, and significantly deleted biomarkers g__ Muribaculaceae and g__ Lachnospiraceae in the CCF group. ( G ) Spearman correlation analysis between Clostridium sp. abundance and the expression levels of NADPH oxidase-related genes. Scatter plots show strong positive correlations with Cybb (R = 0.81, p = 0.0076) and Syk (R = 0.75, p = 0.021).

Article Snippet: Primary antibodies used in Western Blot included Beta Actin Monoclonal antibody (66009-1-Ig, Proteintech, Wuhan, China), NCF4/p40phox Rabbit pAb (A2096, Abclonal, Wuhan, China), Anti-NOX2/gp91phox Recombinant Rabbit Monoclonal Antibody (ET1611-44, Huabio, Hangzhou, China), RAC2 Rabbit pAb (A1139, Abclonal, China) and Anti-NCF1/p47phox Rabbit pAb (GB11724, Servicebio, Wuhan, China).

Techniques: Comparison, Expressing