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: 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

Journal: Inflammation

Article Title: Zmiz1-Mediated SUMOylation of NLRP3 Inflammasome Regulates Satellite Glial Cell Activation and Neuronal Autophagy in Trigeminal Neuralgia

doi: 10.1007/s10753-025-02330-4

Figure Lengend Snippet: Zmiz1 regulation of NLRP3 further exacerbates TN symptoms. Note: ( A ) Measurement of mechanical allodynia threshold in TN model rats; ( B ) ELISA to detect pro-inflammatory cytokine levels in TN model rats; ( C ) RT-qPCR to measure pro-inflammatory cytokine expression levels in TN model rats; ( D ) H&E staining to assess inflammation and demyelination in the trigeminal nerve tissue of TN model rats (scale bar: 100 μm); ( E ) WB analysis to detect the expression levels of inflammasome activation proteins in TN model rats; ( F ) TUNEL assay to evaluate apoptosis in demyelinated trigeminal nerve tissue of TN model rats (scale bar: 100 μm). Data are presented as mean ± SD (* p < 0.05 compared to the Sham group, $ p < 0.05 compared to the shNC group, % p < 0.05 compared to the OE-NC + PBS group, # p < 0.05 compared to the OE-Zmiz1 + PBS group, using ANOVA and Tukey’s multiple comparison test), n = 6.

Article Snippet: Cultured supernatants were centrifuged at 1500 × g for 15 min, and IL-1β, TNF-α, and Interleukin-6 (IL-6) (rat, E-UNEL-R0030, Elabscience) ELISA antibodies were utilized according to the instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Staining, Activation Assay, TUNEL Assay, Comparison

Journal: Inflammation

Article Title: Zmiz1-Mediated SUMOylation of NLRP3 Inflammasome Regulates Satellite Glial Cell Activation and Neuronal Autophagy in Trigeminal Neuralgia

doi: 10.1007/s10753-025-02330-4

Figure Lengend Snippet: Molecular mechanism figure of the Zmiz1/NLRP3 signaling axis in TN. Note: Schematic illustration summarizing the proposed mechanism: Zmiz1 promotes SUMOylation of NLRP3, enhancing SGC activation and inflammatory cytokine release, while suppressing neuronal autophagy—contributing to TN progression.

Article Snippet: Cultured supernatants were centrifuged at 1500 × g for 15 min, and IL-1β, TNF-α, and Interleukin-6 (IL-6) (rat, E-UNEL-R0030, Elabscience) ELISA antibodies were utilized according to the instructions.

Techniques: Activation Assay

Journal: Bioengineered

Article Title: Silencing of specificity protein 1 protects H9c2 cells against lipopolysaccharide-induced injury via binding to the promoter of chemokine CXC receptor 4 and suppressing NF-κB signaling.

doi: 10.1080/21655979.2022.2026548

Figure Lengend Snippet: Figure 2. Silencing of SP1 or CXCR4 ameliorates LPS-induced inflammatory response and apoptosis in H9c2 cells. (a) qRT-PCR and (b) Western blot analysis of CXCR4 expression in H9c2 cells transfected with si-CXCR4, pcDNA-CXCR4, or their matched controls. H9c2 cells were transfected with si-SP1, si-CXCR4, or their corresponding controls, and then treated with 10 μg/mL of LPS for 24 h. (c) Cell viability was examined using CCK-8 assay. (d) The release of LDH was examined using a commercial kit. (e) qRT-PCR and (f) ELISA assays were performed to determine the levels of TNF-α, IL-8, and IL-6 in H9c2 cells. (g) TUNEL staining was carried out to evaluate the apoptosis of H9c2 cells. (h) The activity of caspase-3 was measured to assess cell apoptosis. ***P < 0.001.

Article Snippet: The levels of TNF-α, IL-8, and IL-6 were determined with the rat TNF-α ELISA kit (Solarbio), rat IL-8 ELISA kit (Solarbio), and rat IL-6 ELISA kit (Solarbio), respectively, according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Activity Assay

Journal: Bioengineered

Article Title: Silencing of specificity protein 1 protects H9c2 cells against lipopolysaccharide-induced injury via binding to the promoter of chemokine CXC receptor 4 and suppressing NF-κB signaling.

doi: 10.1080/21655979.2022.2026548

Figure Lengend Snippet: Figure 3. Overexpression of CXCR4 abolishes the protective effects of SP1 silencing on LPS-induced injury in H9c2 cells. H9c2 cells were transfected with si-SP1 alone or together with pcDNA-CXCR4, and then treated with 10 μg/mL of LPS for 24 h. (a) Cell viability was examined using CCK-8 assay. (b) The release of LDH was examined using a commercial kit. (c) qRT-PCR and (d) ELISA assays were performed to determine the levels of TNF-α, IL-8, and IL-6 in H9c2 cells. (e) TUNEL staining was used to evaluate the apoptosis of H9c2 cells. (f) The activity of caspase-3 was measured to assess cell apoptosis. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The levels of TNF-α, IL-8, and IL-6 were determined with the rat TNF-α ELISA kit (Solarbio), rat IL-8 ELISA kit (Solarbio), and rat IL-6 ELISA kit (Solarbio), respectively, according to the manufacturer’s instructions.

Techniques: Over Expression, Transfection, CCK-8 Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Activity Assay

Journal: Bioengineered

Article Title: Silencing of specificity protein 1 protects H9c2 cells against lipopolysaccharide-induced injury via binding to the promoter of chemokine CXC receptor 4 and suppressing NF-κB signaling.

doi: 10.1080/21655979.2022.2026548

Figure Lengend Snippet: Figure 6. The NF-κB signaling pathway is involved in mediating the effects of SP1/CXCR4 axis on LPS-induced H9c2 cell injury. (a) H9c2 cells were transfected with si-SP1, pcDNA-CXCR4, or si-CXCR4 alone or in combination, and then treated with (10 μg/mL) LPS for 24 h. Western blotting was carried out to evaluate the expression of p-p65 and p65 in H9c2 cells. (b) H9c2 cells were treated with (10 μg/mL) LPS alone or together with (10 μg/mL) DHMEQ, and then subjected to CCK-8 assay. (c) The release of LDH was examined in H9c2 cells stimulated with LPS alone or together with DHMEQ. (d) qRT-PCR and (e) ELISA assays were performed to determine the levels of TNF-α, IL-8, and IL-6 in H9c2 cells stimulated with LPS alone or together with DHMEQ. (f) TUNEL staining was carried out to evaluate the apoptosis of H9c2 cells stimulated with LPS alone or together with DHMEQ. (g) The activity of caspase-3 was measured to assess the apoptosis of H9c2 cells stimulated with LPS alone or together with DHMEQ. ***P < 0.001.

Article Snippet: The levels of TNF-α, IL-8, and IL-6 were determined with the rat TNF-α ELISA kit (Solarbio), rat IL-8 ELISA kit (Solarbio), and rat IL-6 ELISA kit (Solarbio), respectively, according to the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Expressing, CCK-8 Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining, Activity Assay