Journal: Oxidative Medicine and Cellular Longevity

Article Title: Elucidating the Novel Mechanism of Ligustrazine in Preventing Postoperative Peritoneal Adhesion Formation

doi: 10.1155/2022/9226022

Figure Lengend Snippet: Ligustrazine inhibits hypoxia-induced PMC ECM deposition and EMT transition. (a) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α -SMA) in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. (b–g) qRT-PCR analysis of mRNA levels of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α -SMA) in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. (h–k) Immunofluorescence assay of mesothelial-related phenotypic biomarkers (E-cadherin, Cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail, α -SMA) in hypoxia-induced PMCs treated with 20 μ M ligustrazine for 24 h (200x).

Article Snippet: VEGF (sc-57496, Santa Cruz, USA), CTGF (sc-373936, Santa Cruz, USA), vascular cell adhesion molecule-1 (VCAM-1; ab134047, Abcam, USA), intercellular cell adhesion molecular-1 (ICAM-1; sc-8439, Santa Cruz, USA), cofilin (bs-2759R, Bioss, China), phospho-cofilin (p-cofilin, bs-20261R, Bioss, China), fibroblast-specific protein 1 (FSP1; bs-3759R, Bioss, China), matrix metalloproteinases 2 (MMP2; bs-4605R, Bioss, China), tissue inhibitor of metalloproteinases-1 (TIMP-1; bs-0415R, Bioss, China), E-cadherin (bs-1519R, Bioss, China), cytokeratin 18 (bs-2043R, Bioss, China), Snail (bs-1371R, Bioss, China), α -smooth muscle actin ( α -SMA; bs-10196R, Bioss, China), F-actin (FITC-conjugated phalloidin, C1033, Beyotime, China), vinculin (bs-6640R, Bioss, China), peroxisome proliferator-activated receptor γ (PPAR γ ; bs-4590R, Bioss, China), lamin B1 (sc-377000, Santa Cruz, USA), β -actin (CW0096M, CWBIO, China), and hypoxia-inducible factor 1 α (HIF-1 α ; sc-71247, Santa Cruz, USA) were used.

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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Elucidating the Novel Mechanism of Ligustrazine in Preventing Postoperative Peritoneal Adhesion Formation

doi: 10.1155/2022/9226022

Figure Lengend Snippet: Ligustrazine suppresses hypoxia-induced PMC functions by activating PPAR γ . (a, b) Western blot and qRT-PCR analysis of PPAR γ levels in hypoxia-induced PMCs treated with different concentrations of ligustrazine for 24 h. Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. (c) Immunofluorescence analysis of PPAR γ nuclear distribution in hypoxia-induced PMCs treated with 20 μ M ligustrazine for 24 h (200x). (d) Flow cytometry analysis of PPAR γ nuclear translocation in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h. (e) Western blot analysis of protein expression of profibrotic cytokines (VEGF and CTGF) in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h. (f, g) FDA staining assay of cell viability in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h (200x). Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. Compared with the ligustrazine group, # P < 0.05 and ## P < 0.01. (h) Western blot analysis of protein expression of migration and adhesion-associated molecules (VCAM-1 and ICAM-1) in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h. (i) Western blot analysis of cytoskeleton protein (vinculin) expression in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h. (j) Western blot analysis of protein expression of FSP1 in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h. (k) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α -SMA) in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h.

Article Snippet: VEGF (sc-57496, Santa Cruz, USA), CTGF (sc-373936, Santa Cruz, USA), vascular cell adhesion molecule-1 (VCAM-1; ab134047, Abcam, USA), intercellular cell adhesion molecular-1 (ICAM-1; sc-8439, Santa Cruz, USA), cofilin (bs-2759R, Bioss, China), phospho-cofilin (p-cofilin, bs-20261R, Bioss, China), fibroblast-specific protein 1 (FSP1; bs-3759R, Bioss, China), matrix metalloproteinases 2 (MMP2; bs-4605R, Bioss, China), tissue inhibitor of metalloproteinases-1 (TIMP-1; bs-0415R, Bioss, China), E-cadherin (bs-1519R, Bioss, China), cytokeratin 18 (bs-2043R, Bioss, China), Snail (bs-1371R, Bioss, China), α -smooth muscle actin ( α -SMA; bs-10196R, Bioss, China), F-actin (FITC-conjugated phalloidin, C1033, Beyotime, China), vinculin (bs-6640R, Bioss, China), peroxisome proliferator-activated receptor γ (PPAR γ ; bs-4590R, Bioss, China), lamin B1 (sc-377000, Santa Cruz, USA), β -actin (CW0096M, CWBIO, China), and hypoxia-inducible factor 1 α (HIF-1 α ; sc-71247, Santa Cruz, USA) were used.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Flow Cytometry, Translocation Assay, Expressing, Staining, Migration

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Elucidating the Novel Mechanism of Ligustrazine in Preventing Postoperative Peritoneal Adhesion Formation

doi: 10.1155/2022/9226022

Figure Lengend Snippet: The suppression effect of ligustrazine is achieved by the activated PPAR γ on the transrepression of SMRT-mediated HIF-1 α . (a) Western blot analysis of protein expression of profibrotic cytokines (VEGF and CTGF) in hypoxia-induced PMCs treated with KC7F2 for 24 h. (b, c) FDA staining assay of cell viability in hypoxia-induced PMCs treated with KC7F2 for 24 h (200x). Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. (d) Western blot analysis of protein expression of migration and adhesion-associated molecules (VCAM-1 and ICAM-1) in hypoxia-induced PMCs treated with KC7F2 for 24 h. (e) Western blot analysis of cytoskeleton protein (vinculin) expression in hypoxia-induced PMCs treated with KC7F2 for 24 h. (f) Western blot analysis of protein expression of FSP1 in hypoxia-induced PMCs treated with KC7F2 for 24 h. (g) Western blot analysis of protein expression of ECM-associated molecules (MMP2 and TIMP-1), mesothelial-related phenotypic biomarkers (E-cadherin and cytokeratin 18), and mesenchymal-related phenotypic biomarkers (Snail and α -SMA) in hypoxia-induced PMCs treated with KC7F2 for 24 h. (h, i) qRT-PCR and Western blot analysis of HIF-1 α levels in hypoxia-induced PMCs treated with 20 μ M ligustrazine and/or 1 μ M GW9662 for 24 h. Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. Compared with the ligustrazine group, # P < 0.05 and ## P < 0.01. (j, k) qRT-PCR analysis of mRNA levels of SMRT and NCoR in hypoxia-induced HPMCs after different transfection for 24 h. Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. (l) qRT-PCR analysis of mRNA levels of HIF-1 α in hypoxia-induced HPMCs treated with 20 μ M ligustrazine and/or with different transfection for 24 h. Compared with the control, ∗ P < 0.05 and ∗∗ P < 0.01. Compared with the ligustrazine group, # P < 0.05 and ## P < 0.01. (m) Western blot analysis of protein expression of HIF-1 α in hypoxia-induced HPMCs treated with 20 μ M ligustrazine and/or with different transfection for 24 h.

Article Snippet: VEGF (sc-57496, Santa Cruz, USA), CTGF (sc-373936, Santa Cruz, USA), vascular cell adhesion molecule-1 (VCAM-1; ab134047, Abcam, USA), intercellular cell adhesion molecular-1 (ICAM-1; sc-8439, Santa Cruz, USA), cofilin (bs-2759R, Bioss, China), phospho-cofilin (p-cofilin, bs-20261R, Bioss, China), fibroblast-specific protein 1 (FSP1; bs-3759R, Bioss, China), matrix metalloproteinases 2 (MMP2; bs-4605R, Bioss, China), tissue inhibitor of metalloproteinases-1 (TIMP-1; bs-0415R, Bioss, China), E-cadherin (bs-1519R, Bioss, China), cytokeratin 18 (bs-2043R, Bioss, China), Snail (bs-1371R, Bioss, China), α -smooth muscle actin ( α -SMA; bs-10196R, Bioss, China), F-actin (FITC-conjugated phalloidin, C1033, Beyotime, China), vinculin (bs-6640R, Bioss, China), peroxisome proliferator-activated receptor γ (PPAR γ ; bs-4590R, Bioss, China), lamin B1 (sc-377000, Santa Cruz, USA), β -actin (CW0096M, CWBIO, China), and hypoxia-inducible factor 1 α (HIF-1 α ; sc-71247, Santa Cruz, USA) were used.

Techniques: Western Blot, Expressing, Staining, Migration, Quantitative RT-PCR, Transfection

Journal: Scientific Reports

Article Title: Lipocalin 15 in the olfactory mucus is a biomarker for Bowman’s gland activity

doi: 10.1038/s41598-022-13464-y

Figure Lengend Snippet: Immunohistochemical staining of human olfactory cleft mucosa sections with rabbit anti-PGP9.5 antibody ( a and b ), rabbit anti-cytokeratin 18 antibody ( c ), and rabbit anti-LCN15 antibody ( d ). A, C, D: D, V, M, and L in A indicate dorsal, ventral, medial, and lateral zones of the nasal cavity, respectively. Left and right sides of the image correspond to superior and inferior ends of the olfactory cleft. Red rectangles (X and Y) indicate microscopic fields, shown as a magnified view in Supplementary Fig. . ( a ): PGP9.5 immunoreactivity is observed in sparse island areas of the olfactory mucosa area. ( b ): Magnified view of the field indicated by the blue rectangle in a . PGP9.5-positive ORNs are observed in the epithelium (black arrowheads). In the lamina propria, PGP9.5-positive olfactory nerve bundles are shown by the black arrows. ( c ): CK18 immunoreactivity in the epithelium and submucosal glands of the olfactory cleft mucosa. ( d ): LCN15 immunoreactivity in the submucosal glands, mainly in the superior end of the cleft. Scale bar in b = 100 μm and in d (also applies for a and c ) = 2 mm.

Article Snippet: The area distribution of PGP9.5 and LCN15-immunoreactivity in the olfactory cleft mucosa was analyzed quantitatively using serial sections stained with anti-CK18 antibody and double-stained with anti-LCN15 and anti-PGP9.5 antibodies at GeneticLab (Sapporo, Japan).

Techniques: Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Lipocalin 15 in the olfactory mucus is a biomarker for Bowman’s gland activity

doi: 10.1038/s41598-022-13464-y

Figure Lengend Snippet: Quantitative image analysis of the area for PGP9.5 and LCN15 immunoreactivity in the olfactory cleft mucosa. ( a ): Olfactory cleft mucosal lining with intact epithelium was morphologically identified using sections stained for cytokeratin 18. Using these sections, rectangle areas (1 × 1 mm) for analysis were serially set along the mucosal lining. ( b ): The same rectangular areas used in a were set on the serial sections stained for PGP9.5 and LCN15 for analysis. ( c ): A magnified view of the image in b . The immunoreactivity for PGP9.5 and LCN15 is visualized in gray and brown colors, respectively. ( d ): Areas of PGP9.5 and LCN15-immunoreactivity in each analysis area were automatically selected and marked in red (PGP9.5) and green (LCN15), respectively, by setting the color threshold. Scale bars in B and D are = 2 mm and 200 μm, respectively.

Article Snippet: The area distribution of PGP9.5 and LCN15-immunoreactivity in the olfactory cleft mucosa was analyzed quantitatively using serial sections stained with anti-CK18 antibody and double-stained with anti-LCN15 and anti-PGP9.5 antibodies at GeneticLab (Sapporo, Japan).

Techniques: Staining