Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: Effects of RosA on H1N1 virus-mediated lung injury in vivo. A Chemical structure of RosA. B At day 7 p.i., gross examination of the lungs. White arrows indicate the lungs with edema and hemorrhage. C Lung index (lung/body weight ratio) was used to assess the severity of exudation and edema. D At day 7 p.i., lung histological changes elicited by the H1N1 viruses were examined by H&E staining. Black arrows show: (i) bronchi with epithelial sloughing; (ii) peribronchitis; (iii) perivasculitis; (iv) alveolar collapse and leukocyte infiltration. E Histological scoring of lung injury. F Representative immunofluorescence images of Bax (pink) and Bcl2 (red) expression in lung SpC + (green) alveolar epithelial cells. G Quantitative analysis of fluorescence intensities for Bax and Bcl2 in SpC + alveolar epithelial cells. H Representative immunofluorescence images of active caspase 3 (red) and TUNEL assay (green) in lung SpC + (pink) alveolar epithelial cells. I Quantitative analysis of fluorescence intensities for active caspase 3 and TUNEL in SpC + alveolar epithelial cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: One-step TUNEL in Situ apoptosis kit (Green, FITC) was obtained from Elabscience Biotechnology Co., Ltd (E-CK-A320; Wuhan, China).

Techniques: Virus, In Vivo, Staining, Immunofluorescence, Expressing, Fluorescence, TUNEL Assay

Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: Effects of RosA on H1N1 virus-induced apoptosis. A The apoptosis of H1N1 virus-infected cells was harvested for flow cytometry analysis after RosA treatment for 24 h. B Apoptosis percentage in H1N1 virus-infected A549 cells treated with or without RosA. C Western blot analysis of cleaved PARP and cleaved caspase 3 in H1N1 virus-infected cells treated with RosA. D The relative expression of cleaved PARP and cleaved caspase 3 was quantified relative to GAPDH. E H1N1 virus-infected A549 cells were treated with RosA alone or in combination with h-PGDS inhibitor for 24 h. Flow cytometry was used to identify the apoptosis of these cells. F The percentage of apoptosis in A549 cells with H1N1 virus-infected A549 cells treated for 24 h with RosA alone or in conjunction with an h-PGDS inhibitor. G Western blot analysis of cleaved PARP and cleaved caspase 3 in H1N1 virus-infected cells treated with RA alone or in combination with h-PGDS inhibitor. H The relative expression of cleaved PARP and cleaved caspase 3 expression was quantified relative to GAPDH. I The apoptosis of cells in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus-infection were detected by flow cytometry. J The percentage of apoptosis in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection. K The levels of cleaved PARP and cleaved caspase 3 in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection were detected by Western blotting. L The relative expression of cleaved PARP and cleaved caspase 3 expression was quantified relative to GAPDH. M Analysis of CD3 + CD8 + T lymphocytes by flow cytometry from peripheral blood. N Quantification of the proportions of CD3 + CD8 + T lymphocytes in the peripheral blood of H1N1 virus-infected mice. O Immunofluorescence staining of Granzyme B (pink) and TNF-α (red) in CD8 + T lymphocytes (green) of the lung tissues. P The relative fluorescence intensities of Granzyme B and TNF-α in CD8 + T lymphocytes were calculated. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: One-step TUNEL in Situ apoptosis kit (Green, FITC) was obtained from Elabscience Biotechnology Co., Ltd (E-CK-A320; Wuhan, China).

Techniques: Virus, Infection, Flow Cytometry, Western Blot, Expressing, Over Expression, Plasmid Preparation, Transfection, Immunofluorescence, Staining, Fluorescence

Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: Effects of h-PGDS inhibition on RosA’s protective effects against H1N1 virus-mediated lung injury. Two days before infection with mouse-adapted H1N1 virus (5LD 50 ), mice in the group with the combination of RosA with h-PGDS inhibitor were treated intraperitoneally with h-PGDS inhibitor for 30 min, and then administrated intragastrically (i.g.) with RosA. A At day 7 p.i., gross pathology showed edema and hemorrhage (white arrows) in the lung tissues. B The severity of exudation and edema were elevated by the lung index (lung/body weight ratio). C At day 7 p.i., H&E staining was used to assess the lung histological alterations caused by H1N1 viruses. Black arrows show: (i) bronchi with epithelial sloughing; (ii) peribronchitis; (iii) perivasculitis; (vi) alveolar collapse and leukocyte infiltration. D Histological scoring of lung injury. E Representative immunofluorescence images of active caspase 3 (red) and TUNEL (green) assay in lung SpC + (pink) alveolar epithelial cells. F Quantitative analysis of fluorescence intensities for active caspase 3 and TUNEL in SpC + alveolar epithelial cells. G The expression of IL-6 and TNF-α in lung SpC + alveolar epithelial cells was detected by immunofluorescence. H Quantitative analysis of fluorescence intensities for IL-6 (pink) and TNF-α (red) in SpC + (green) alveolar epithelial cells. I Levels of pro-inflammatory mediators (IL-6, MCP-1, RANTES and TNF-α) in the lung homogenates were determined by Luminex assay. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: One-step TUNEL in Situ apoptosis kit (Green, FITC) was obtained from Elabscience Biotechnology Co., Ltd (E-CK-A320; Wuhan, China).

Techniques: Inhibition, Virus, Infection, Staining, Immunofluorescence, TUNEL Assay, Fluorescence, Expressing, Luminex

Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: Schematic diagram depicting the mechanism by which RosA prevents H1N1 virus-induced severe lung injury. RosA treatment effectively increases the expression of h-PGDS, which catalyzes the conversion of PGH 2 to PGD 2 . Subsequently, PGD 2 binds to its receptor DP1, and thus triggers the expression of HO-1. The increased expression of HO-1 leads to attenuation of H1N1 virus-activated NF-κB and P38 MAPK signaling pathways, resulting in suppression of H1N1 virus-elicited pro-inflammatory responses and apoptosis

Article Snippet: One-step TUNEL in Situ apoptosis kit (Green, FITC) was obtained from Elabscience Biotechnology Co., Ltd (E-CK-A320; Wuhan, China).

Techniques: Virus, Expressing

Journal: Cell Death & Disease

Article Title: Death receptor 5 is required for intestinal stem cell activity during intestinal epithelial renewal at homoeostasis

doi: 10.1038/s41419-023-06409-4

Figure Lengend Snippet: Data were expressed as mean ± SEM. Statistical analyses were performed by the unpaired t -tests (* P < 0.05, *** P < 0.001, **** P < 0.0001). A Representative images of H&E staining in the ileum of WT mice and DR5 -/- mice (scale bars: 100 μm). B Quantification of villus length within two groups ( n = 6). At least 15 correctly aligned villi were counted randomly for each slide and the mean value was calculated. C The total number of epithelial cells in ileal villus of WT mice and DR5 -/- mice ( n = 6). At least 15 correctly aligned villi were measured for every slide to get the mean value. D qRT-PCR ( n = 6) and western blot ( n = 4) were performed to determine the relative mRNA and protein levels of lysozyme (Lyz) in ileum of WT mice and DR5 -/- mice. Lyz is the specific marker for Paneth cells. E qRT-PCR was performed to determine the relative mRNA levels of Muc2 and Tff3 in ileum of WT mice and DR5 -/- mice ( n = 6). Both are the specific marker genes for goblet cells. F Western blot to quantify the protein expression of MUC2 in ileum of WT mice and DR5 -/- mice ( n = 4). G qRT-PCR ( n = 6) and western blot ( n = 4) were performed to determine the relative mRNA and protein expression of ChgA in ileum of WT mice and DR5 -/- mice. It is the specific marker for enteroendocrine cells. H qRT-PCR was performed to determine the relative mRNA levels of Alpi in ileum of WT mice and DR5 -/- mice ( n = 6). It is the specific marker for enterocytes. I Relative mRNA expression of inflammatory factors in the ileum of WT mice and DR5 -/- mice ( n = 6). J Quantification of TUNEL + cells in ileal villus of WT mice and DR5 -/- mice ( n = 6). At least 10 villi were counted randomly for each slide and the mean value was calculated. K Total number of epithelial cells in ileal crypts of WT mice and DR5 -/- mice ( n = 6). At least 15 correctly aligned crypts were measured for every slide to get the mean value. L The volcano plot visualized the DEGs in ileal crypts from WT mice and DR5 -/- mice. Names listed in the figure were DEGs associated with proliferation or differentiation of ISC.

Article Snippet: Detection of apoptosis in intestinal epithelial cells was performed using the one-step TUNEL Apoptosis Assay Kit (Keygen biotech, Jiangsu, China).

Techniques: Staining, Quantitative RT-PCR, Western Blot, Marker, Expressing, TUNEL Assay

Journal: Cell Death & Disease

Article Title: Death receptor 5 is required for intestinal stem cell activity during intestinal epithelial renewal at homoeostasis

doi: 10.1038/s41419-023-06409-4

Figure Lengend Snippet: Data were expressed as mean ± SEM. Statistical analyses were performed by the unpaired t -tests or two-way ANOVA followed by Sidak’s multiple comparisons test (* P < 0.05, *** P < 0.001, **** P < 0.0001). A The analysis of KEGG apoptosis signalling pathway gene set based on RNA-seq. The top 20 genes are listed in the heatmap, which shows that the expression of apoptosis related genes is upregulated in intestinal crypts of DR5 -/- mice compared with that of WT mice ( n = 3). In this scheme, deep red corresponds to heightened gene expression levels, while light blue denotes lower expression. B Western blot was performed to quantify the cleaved caspase-3 protein expression in ileal crypts from WT mice and DR5 -/- mice ( n = 4). C Representative pictures of staining of Lyz, TUNEL, and DAPI (nuclei) in ileum of WT mice and DR5 -/- mice (scale bars: 100 μm). The red arrows indicate TUENL + Paneth cells, and the white arrows indicate the TUNEL + TA cells. D Statistical analysis to show the difference of the TUENL + Paneth cells and TUNEL + TA cells between WT mice and DR5 -/- mice ( n = 6). At least 20 crypts were counted randomly for each slide and the mean value was calculated. E Representative images showing organoids derived from crypts of WT and DR5 -/- mice 72 h after culture (scale bars: 500 μm). F Dynamic changes of organoid survival in WT and DR5 -/- mice ( n = 6). For each sample, four 40-fold visual fields were randomly selected to count the number of organoids under each visual field, and the average value was obtained. The number of organoids at different time points was normalized with the number of organoids at 0 h to reflect the organoid survival rate. G Immunofluorescence co-labelled Lyz and DR5 in ileum. The red arrows indicate the DR5 + Paneth cells and the yellow arrow indicates DR5 + ISC in the basement of crypt (scale bar: 50 μm).

Article Snippet: Detection of apoptosis in intestinal epithelial cells was performed using the one-step TUNEL Apoptosis Assay Kit (Keygen biotech, Jiangsu, China).

Techniques: RNA Sequencing, Expressing, Gene Expression, Western Blot, Staining, TUNEL Assay, Derivative Assay, Immunofluorescence

Journal: iScience

Article Title: CircSOBP suppresses the progression of glioma by disrupting glycolysis and promoting the MDA5-mediated immune response

doi: 10.1016/j.isci.2023.107897

Figure Lengend Snippet: Increased expression of circSOBP inhibits the proliferation, invasion, and migration of glioma cells (A) The spliced sequence of circSOBP was cloned into the pEGFP-C1 vector, with the upstream and downstream containing the identified active ALU elements. RT-qPCR analysis of circSOBP overexpression in U87 and U251 cells. (B) CCK-8 analysis of the effect of circSOBP overexpression on the proliferation of U87 and U251 cells. (C) Cell viability was detected at 0 h, 24 h, 48 h, and 72 h after transfection with pEGFP-C1 or pEGFP-C1-circSOBP plasmids in U87 and U251 cells. (D) Edu assay to analyze the effect on cell replication after overexpression of circSOBP in U87 and U251 cells. Scale bars, 50 μm. (E) TUNEL analysis of the effect of circSOBP overexpression on apoptosis of U87 and U251 cells. Scale bars, 50 μm. (F) Transwell assay to assess the invasive ability of glioma cells after overexpression of circSOBP. (G) Wound healing assay to detect the migration level of glioma cells after overexpression of circSOBP. All statistics of error bars, S.E.M. from three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001 by two-tailed Student’s t test. Normalized by the value of the control group.

Article Snippet: Next, TUNEL assay was performed using the One-Step TUNEL Apoptosis Kit (RiboBio, China) according to the manufacturer’s instructions.

Techniques: Expressing, Migration, Sequencing, Clone Assay, Plasmid Preparation, Quantitative RT-PCR, Over Expression, CCK-8 Assay, Transfection, EdU Assay, TUNEL Assay, Transwell Assay, Wound Healing Assay, Two Tailed Test, Control

Journal: iScience

Article Title: CircSOBP suppresses the progression of glioma by disrupting glycolysis and promoting the MDA5-mediated immune response

doi: 10.1016/j.isci.2023.107897

Figure Lengend Snippet:

Article Snippet: Next, TUNEL assay was performed using the One-Step TUNEL Apoptosis Kit (RiboBio, China) according to the manufacturer’s instructions.

Techniques: Virus, Recombinant, Staining, Transfection, Software, Fractionation, Labeling, Northern Blot, Cell Viability Assay, TUNEL Assay, Silver Staining, Luciferase, Reporter Gene Assay, ATP Assay, Enzyme-linked Immunosorbent Assay, Purification, Titration, Real-time Polymerase Chain Reaction