Journal: Heliyon

Article Title: Effect of citronellol on oxidative stress, neuroinflammation and autophagy pathways in an in vivo model of Parkinson's disease

doi: 10.1016/j.heliyon.2022.e11434

Figure Lengend Snippet: Citronellol prevented production of pro-inflammatory factors in experimental animals. ELISA and western blotting were performed to analyze the effect of citronellol on pro-inflammatory markers in the midbrain of experimental animals. Administration of rotenone (2.5 mg/kg, i. p) for four weeks significantly enhanced secretion of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α and MMP-9. (a). Further, rotenone enhanced the expression of COX-2 and iNOS in midbrain of experimental animals (b). Quantitative evaluations of blots were done using ImageJ and their results were represented in histogram (c). However, administration of citronellol (25 mg/kg, oral) for four weeks significantly decreased the expression of pro-inflammatory factors. Data are represented as mean ± SD. ∗p < 0.05 compared to control, # p < 0.05 compared to rotenone alone treated group.

Article Snippet: Anti-inflammatory activity of citronellol was analyzed using commercially available ELISA kits from R&D systems, Minnesota, United States (IL-6: Cat. No. DY506, TNF-α: Cat. No. DY510, IL-1β: Cat. No. DY501).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Pathological neutrophil extracellular traps hinder postoperative anal fistula wound healing and are attenuated by Zuoqing granule via suppression of the Nox4 pathway

doi: 10.3389/fimmu.2025.1730184

Figure Lengend Snippet: ZQG attenuates PMA-induced NETosis and inflammatory responses in human neutrophils via targeting the Nox4/ROS pathway. (A) ZQG suppresses oxidative burst and NET formation. Representative immunofluorescence images showing intracellular ROS (stained by DHE, red) and NETosis (marked by CitH3, green). Nuclei are counterstained with DAPI (blue). Scale bars: 20 μm. (B) Quantitative analysis of intracellular ROS levels. (C) Quantification of NETosis, expressed as the percentage of Sytox Green + cells. (D) Western blot analysis demonstrating the effects of ZQG and si-Nox4 on the protein expression of Nox4, p-Akt, and PADI4. (E) Densitometric quantification of Western blot results. (F) qPCR analysis of Nox4, PI3K, Akt, and PADI4 mRNA expression. (G) ELISA analysis of pro-inflammatory cytokine levels (IL-2, IL-5, IL-6, IL-12, TNF-α) in cell culture supernatants. Data are presented as mean ± SEM (n=10 independent experiments). ***p < 0.001 vs. PMA group.

Article Snippet: The concentrations of interleukin-2 (IL-2), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF-α) in the supernatants were quantified using specific commercial rat ELISA kits (R&D Systems, Catalog # R6000B) according to the manufacturers’ instructions.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Interleukin 6 Signaling Regulates Promyelocytic Leukemia Protein Gene Expression in Human Normal and Cancer Cells

doi: 10.1074/jbc.m111.316869

Figure Lengend Snippet: FIGURE 2. Cell type-dependent differences in IL6-STAT3 activity and expression of PML. A, shown is immunoblot detection of total STAT1, STAT3, STAT5, tyrosine 701-phosphorylated STAT1, tyrosine 705-phosphorylated STAT3, and tyrosine 694-phosphorylated STAT5 in U2OS, HeLa, and BJ cells cultivated at standard conditions and BJ exposed to IFN (10 IU/ml) for 24 h (included as a positive control of STAT1 activation); GAPDH was used as a loading control. B, shown is immunoblot detection of total and p53 serine 15-phosphorylated p53 in U2OS, HeLa, and BJ cultivated at standard conditions; U2OS cell treated with a combination of 10 M BrdU and 10 M distamycin A (BrdU/DMA) are included as positive control; GAPDH was used as a loading control. C, PML mRNA levels were detected by real time qRT-PCR in U2OS, HeLa, and BJ cells cultivated at standard conditions. The values represent the average of two independent experiments performed in triplicate and are given as the -fold induction of PML mRNA levels relative to U2OS; -catenin (CTNNB1) was used as a reference gene. Asterisks (***) represent a p value 0.005. D, shown is immunofluorescence detection of endogenous level of H2AX, a marker of DNA damage response, in U2OS, HeLa, and BJ cells. BJ treated for 4 days with camptothecin (BJ CPT) were used as a positive control of H2AX formation. Bar, 15 m. E, shown is an estimation of IL6 and IL1 in medium of U2OS, HeLa, and BJ cells measured by FACS beads.

Article Snippet: Estimation of IL6 Biological Activity—To test the effectiveness of IL6 depletion mediated via IL6 antibody (2 g/ml; goat polyclonal antibody; R&D Systems, Inc., Minneapolis, MN), growth dependence of mouse hybridoma B9 cells on the presence of IL6 was utilized (46).

Techniques: Activity Assay, Expressing, Western Blot, Positive Control, Activation Assay, Control, Quantitative RT-PCR, Immunofluorescence, Marker

Journal: Journal of Biological Chemistry

Article Title: Interleukin 6 Signaling Regulates Promyelocytic Leukemia Protein Gene Expression in Human Normal and Cancer Cells

doi: 10.1074/jbc.m111.316869

Figure Lengend Snippet: FIGURE3.IL6-STAT3pathwaymodulatesbasalPMLgeneexpression.A,shownisefficiencyofSTAT3knockdownbyspecificsiRNAdetectedonmRNAgiven as the -fold induction of PML mRNA levels relative to BJ transfected with control nonspecific siRNA (-actin was used as a reference gene). B, shown is protein level by immunoblot detection of PML, total, and activated STAT3 in BJ cells 2 days after transfection. Ratios of major unmodified PML isoforms and GAPDH, estimated by densitometric analysis, are given on the right side of the PML immunoblot. Shown is down-regulation of PML after STAT3 knockdown, estimated at protein levels by immunofluorescence detection of PML NBs (C) and on mRNA levels by qRT-PCR in BJ cells (D) 2 days after transfection. The values represent the average of three independent experiments performed in triplicate and are given as -fold induction of PML mRNA levels relative to BJ transfected with control nonspecific siRNA. Bar, 15 m. E, shown is down-regulation of STAT3 activity measured as tyrosine 705-phosphorylated STAT3 2 days after depletion of IL6 from medium by IL6 antibody (IL6 AB, 2 g/ml ; antibody was at all times in the medium) in BJ cells; GAPDH was used as a loading control. F, down-regulation of PML mRNA levels was estimated by real time qRT-PCR in BJ cells 2 days after depletion of IL6 from medium by IL6 antibody (AB). The values represent the average of three independent experiments performed in triplicate and are given as -fold induction of PML mRNA levels relative to BJ without IL6 depletion; -actin was used as a reference gene. Asterisks (***) represent p values 0.005. G, time-course response of total and activated STAT3 and STAT5 levels was detected by immunoblotting as tyrosine 705-phosphorylated STAT3 and tyrosine 694-phosphorylated STAT5 in HeLa cells treated with IL6 (5000 pg/ml). H, time course response of PML mRNA levels in HeLa cells treated with IL6 (5000 pg/ml) estimated by RT-qPCR. The values represent the average of two independent experiments performed in triplicate and are given as -fold induction of PML mRNA levels relative to untreated HeLa cells. I, induction of PMLproteinlevelswasdetectedasPMLNBsbyindirectimmunofluorescenceinU2OScellsaftera2-and4-dayexposuretorecombinanthumanIL6(rhIL6;1000 pg/ml). Bar, 15 m.

Article Snippet: Estimation of IL6 Biological Activity—To test the effectiveness of IL6 depletion mediated via IL6 antibody (2 g/ml; goat polyclonal antibody; R&D Systems, Inc., Minneapolis, MN), growth dependence of mouse hybridoma B9 cells on the presence of IL6 was utilized (46).

Techniques: Transfection, Control, Western Blot, Knockdown, Immunofluorescence, Quantitative RT-PCR, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Interleukin 6 Signaling Regulates Promyelocytic Leukemia Protein Gene Expression in Human Normal and Cancer Cells

doi: 10.1074/jbc.m111.316869

Figure Lengend Snippet: FIGURE 4. ISRE element of PML promoter gene is involved in basal transcription of PML gene. Shown is luciferase reporter gene activity under the control of PML gene promoter (809 bp/633 bp relative to transcription start) in HeLa cells with an intact or deleted (595 bp/628 bp) ISRE DNA binding site (cells were harvested 24 h after transfection; the average values representing three independent experiments are given as arbitrary units (AU); error bars represent S.E.) (A), with or without IL6-depleting antibody (IL6 AB; 2 g/ml; average values representing two independent experiments are given as arbitrary units; error bars represent S.E.) (B), and with or without IL6 treatment (5000 pg/ml; average values representing two independent experiments are given as -fold induction; error bars represent S.E.) (C). Asterisks (*, **, and ***) represent p values 0.05, 0.01, and 0.005, respectively. D, shown is evaluation of STAT3 binding to PML ISRE element in untreated BJ, HeLa, and U2OS cells and in HeLa and U2OS cells treated with IL6 for 0.5, 1, and 6 h in HeLa and 48 h in U2OS cells, respectively, quantified with RT-qPCR after chromatin immunoprecipitation. The values quantified with RT-qPCR represent average of two independent experiments performed in triplicate and are expressed as -fold induction relative to HeLa untreated cells.

Article Snippet: Estimation of IL6 Biological Activity—To test the effectiveness of IL6 depletion mediated via IL6 antibody (2 g/ml; goat polyclonal antibody; R&D Systems, Inc., Minneapolis, MN), growth dependence of mouse hybridoma B9 cells on the presence of IL6 was utilized (46).

Techniques: Luciferase, Activity Assay, Control, Binding Assay, Transfection, Quantitative RT-PCR, Chromatin Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Interleukin 6 Signaling Regulates Promyelocytic Leukemia Protein Gene Expression in Human Normal and Cancer Cells

doi: 10.1074/jbc.m111.316869

Figure Lengend Snippet: FIGURE 5. NFB pathway controls PML expression through regulation of IL6 level. A, shown is efficiency of NEMO knockdown by specific siRNA detected on mRNA level 2 days after transfection in BJ. B, down-regulation of PML, NEMO, and the activated and total form of STAT3 in BJ detected on immunoblot 2 and 4 days after transfection of NEMO siRNA; GAPDH was used as loading control. Down-regulation of PML (D) and IL6 mRNA (D) levels quantified by qRT-PCR in BJ cells 2 days after NEMO knockdown is shown. The values represent the average of three independent experiments and are given as the -fold induction relative to BJ cells transfected with control nonspecific siRNA; error bars represent S.E. -Actin was used as a reference gene. E, shown is immunoblot detection of the STAT3-activated form after NEMO knockdown and NEMO knockdown with the addition of recombinant human IL6 protein (5000 pg/ml) into medium of BJ cells. Shown is down-regulation of PML (F), NEMO (G), and IRF1 (H) mRNA levels 2 days after NEMO knockdown and NEMO knockdown with the addition of recombinant human IL6 protein (5000 pg/ml) quantified by real time qRT-PCR in BJ cells. The values represent the average of two independent experiments and are shown as -fold induction relative to BJ cells transfected with control nonspecific siRNA; error bars represent S.E.; -actin was used as a reference gene. Shown is down-regulation of PML mRNA (I) and protein levels detected as PML NBs (J) 2 days after NEMO, STAT3, or combined NEMO and STAT3 knockdown by specific siRNAs in BJ cells. The mRNA values represent the average of two independent experiments and are shown as -fold induction relative to BJ cells transfected with control nonspecific siRNA; error bars represent S.E. -Actin was used as a reference gene. Bar, 15 m. Asterisks (*, **, and ***) represent p value 0.05, 0.01, and 0.005, respectively.

Article Snippet: Estimation of IL6 Biological Activity—To test the effectiveness of IL6 depletion mediated via IL6 antibody (2 g/ml; goat polyclonal antibody; R&D Systems, Inc., Minneapolis, MN), growth dependence of mouse hybridoma B9 cells on the presence of IL6 was utilized (46).

Techniques: Expressing, Knockdown, Transfection, Western Blot, Control, Quantitative RT-PCR, Recombinant

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA) analysis of IL-10 concentration in supernatant from Caco-2, MC-38 and Raw 264.7 cells treated with various amounts of PBS, IL-10 -mRNA or IL-10 -mRNA NPs for 24 h. ( B ) Western blot analysis of IL-10 expression in MC-38 cells treated with PBS, IL-10 -mRNA, blank NPs or IL-10 -mRNA NPs for 12 h. IL-10 -mRNA 750 ng/mL. ( C ) Confocal microscopy images of immunofluorescence staining of IL-10 expression in MC-38 cells treated with free IL-10 -mRNA or IL-10 -mRNA NPs. Hoechst (blue) was used to stain the cell nuclei. An Alexa Fluor 647-labeled antibody was used to stain IL-10. ( D ) Schematic illustration of how IL-10 -mRNA NPs directly transfect macrophages, inhibiting the polarization of macrophages to proinflammatory M1 phenotype in the presence of lipopolysaccharide (LPS), an inflammatory stimulator. ( E ) ELISA analysis of anti-inflammatory cytokine IL-10 and proinflammatory cytokine TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with PBS or IL-10 -mRNA NPs (w/wo LPS stimulation) following procedures illustrated in (D). ( F ) Schematic illustration of how IL-10 -mRNA NPs first transfect intestinal epithelial cells, then indirectly induce the repolarization of macrophages from proinflammatory M1 phenotype to anti-proinflammatory M2 phenotype. ( G ) ELISA analysis of IL-10 and TNF-α and IL-6 in supernatant from RAW 264.7 cells treated with supernatant from Caco-2 cells incubated with PBS or IL-10 -mRNA NPs following procedures illustrated in (F). ( H ) Ex vivo images of excised rat gastrointestinal (GI) tract at various time points from 15 min to 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100-55 coated). The left panel shows enlarged images of RNACaps at 1 h and 2 h (images 1–4). Cy5-mRNA: 50 μg per rat. Color scale, 0-255 gray value. ( I ) Confocal microscopy images of intestine sections from rats treated with Cy5-mRNA-RNACaps (purple) for 4 h. Hoechst (blue) was used to stain the cell nuclei. Scale bar, 100 μm. ( J ) Ex vivo images of excised intestines at 6 h post-administration. Rats were orally administered six Cy5-mRNA-RNACaps (L100 coated). Color scale, 0-255 gray value. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in ( E ) and ( G ). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data in (A, B, C, E, G, H, I, J) are representative of n = 3 independent experiments. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vitro, Ex Vivo, Imaging, In Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Western Blot, Expressing, Confocal Microscopy, Immunofluorescence, Staining, Labeling, Incubation

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to acute colitis rat models. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. Acute colitis was induced in rats by providing free access to drinking water supplemented with 6.0% (w/w) dextran sulfate sodium (DSS) for 8 days. Rats were treated with RNACaps on day 2, 5 and 8. ( B and C ) Relative body weight (B) and disease activity index (DAI) (C) of healthy (plain water-treated), DSS-treated or DSS plus IL-10 -mRNA-RNACap-treated rats were monitored daily. ( D ) Quantification of colon length on day 8. ( E to J) Colon tissue protein expression of IL-10 (E), tumor necrosis factor-alpha (TNF-α) (F), interleukin-1β (IL-1β) (G), IL-6 (H), IL-17A (I) and monocyte chemoattractant protein-1 (MCP-1) (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the colon tissue sections. Dashed box indicates inset. Scale bars, 500 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Activity Assay, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for the oral administration of IL-10 -mRNA-RNACaps in acute colitis rat models. Rats were given free access to drinking water supplemented with 8.0% (w/w) DSS for 10 days to induce colitis. Afterward, plain water was provided, and rats were treated with RNACaps ( IL-10 -mRNA: 25 μg in 3 RNACaps per rat.) on days 11, 14 and 17 or sulfasalazine (SSZ, standard therapy, 100 mg/kg/day) daily. ( B and C ) Relative body weight (B) and DAI (C) of healthy (plain water-treated), DSS-treated, DSS plus IL-10 -mRNA-RNACap-treated or DSS plus SSZ-treated rats were monitored daily. ( D ) Quantification of colon length on day 17. ( E to J ) Quantification tissue protein expression of IL-10 (E), TNF-α (F), IL-1β (G), IL-6 (H), IL-17A (I) and MCP-1 (J) by ELISA. ( K to P ) Quantification of protein expression in blood of IL-10 (K), TNF-α (L), IL-1β (M), IL-6 (N), IL-17A (O) and MCP-1 (P) by ELISA. ( Q ) H&E staining images of the corresponding colon tissue sections. Dashed box indicates inset. Scale bars, 100 μm and 400 μm. Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc analysis in (B to P). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. n = 5 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Science translational medicine

Article Title: Oral delivery of liquid mRNA therapeutics by engineered capsule for treatment of preclinical intestinal disease

doi: 10.1126/scitranslmed.adu1493

Figure Lengend Snippet: ( A ) Experimental timeline for oral administration of IL-10 -mRNA-RNACaps to rats for safety assessment. IL-10 -mRNA: 25 μg in 3 RNACaps per rat. ( B and C ) Analysis of blood chemistry, including aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) and complete blood count analysis, including white blood cell count (WBC), neutrophil (NE) (B), lymphocyte (LY), red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelets (PLT) (C). ( D ) The rats were euthanized at the end of the study, and the indicated organs were sectioned and stained with H&E. Scale bars, 100 μm and 400 μm. ( E and F ) Cytokine concentration in the serum 6 h following oral administration of Fluc -mRNA-RNACap ( Fluc -mRNA: 25 μg in 3 RNACaps per rat) to healthy rats were measured using ELISA and Luminex. Cytokines measured include IL-1ra, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, and IL-10 (E) or IL-12p70, IL-13, IL-17A, IL-18, IFN-γ, TNF-α, GM-CSF, and VEGF (F). Data are presented as mean ± S.D. Dots represent individual sample replicates. Statistical significance was evaluated by unpaired two-tailed Student’s t-test in (B, C, E, F). * P < 0.05, *** P < 0.001. n = 3 animals per group for all panels. All the schematic illustrations were created using Adobe Illustrator.

Article Snippet: Rat TNF-α ELISA (438204, Biolegend), Rat IL-1 beta/IL-1F2 ELISA (DY501-05, R&D Systems), Rat IL-6 ELISA (DY506-05, R&D Systems), Rat IL-17A ELISA (437904, Biolegend), Rat JE/MCP-1/CCL2 ELISA (DY3144-05, R&D Systems).

Techniques: In Vivo, Cell Characterization, Concentration Assay, Staining, Enzyme-linked Immunosorbent Assay, Luminex, Two Tailed Test