Journal: Acta Pharmaceutica Sinica. B

Article Title: Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

doi: 10.1016/j.apsb.2024.06.020

Figure Lengend Snippet: ChP epithelial cells contribute to CSF CCL2 and macrophages enhance the expression of p-NKCC1 in ChP epithelial cells in vitro . (A) Quantification of CCL2 expression in ChP epithelial cells following LPS stimulation, via qPCR analysis. (B) Western blot analysis illustrating CCL2 expression levels. (C) In situ hybridization of CCL2 mRNA exclusively expressed in ChP epithelial cells in PHH. Scale bar = 50 μm. (D, E) Immunohistochemical staining of CCL2 distribution in ChP of ctrl and PHH rats. Scale bar = 50 μm. (F) Immunofluorescence analysis of pNKCC1 (red) in ChP. Scale bar = 50 μm. (G) Western blot results of CCL2 in Bindarit-treated ChP epithelial cells. (H) Western blot analysis comparing protein profiles among different groups. (I) Schematic diagram of 2D-transwell experiment. (J) Observation of NR8383 vertical migration in the co-culture system at 24 h using transwell images. (K) A 2D-transwell system to study the effect of macrophages on ChP epithelial cells. (L) Immunofluorescence staining of pNKCC1(green) in ChP epithelial cells in different groups. All data were presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. control/model.

Article Snippet: To inhibit TNF- α receptors, the ChP epithelial cells were treated with R7050 (cat: HY-110203, MCE, New Jersey, USA).

Techniques: Expressing, In Vitro, Western Blot, In Situ Hybridization, Immunohistochemical staining, Staining, Immunofluorescence, Migration, Co-Culture Assay, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

doi: 10.1016/j.apsb.2024.06.020

Figure Lengend Snippet: Macrophages enhance CSF hypersecretion in ChP epithelial cells via the TNF- α /TNFR1/NF- κ B pathway. (A) Schematic diagram of mechanisms of crosstalk between ChP epithelial cells and macrophages. (B) Representative images showed that TNFR1 but not TNFR2 is expressed by ChP in PHH rats. Scale bar = 20 μm. (C) The expression of NF- κ B and pNF- κ B in ChP epithelial cells treated with NR8383 derived conditional medium (NCM), TNF- α , and R7050. (D) CCL2 and IL-1 β mRNA expression in various groups. (E) The expression of NF- κ B, pNF- κ B, and CCL2 in epithelial cells treated with NCM or/and TNF- α neutralizing antibody. (F) The mRNA expression of proinflammatory cytokines such as CCL2, GM-CSF, IL-1 β , IL-6, and IL-1 α in ChP epithelial cells treated with TNF- α or/and Bay117082. (G) The expression of NF- κ B, pNF- κ B, and CCL2 in epithelial cells treated with TNF- α or/and Bay117082. (H) QPCR of CCL2 and NF- κ B in epithelial cells treated with LPS or/and Bay117082. (I) The expression of NF- κ B pathway, and CCL2 expression in ChP epithelial cells treated with LPS or/and TNF- α . (J) Western blot results of M1 marker (iNOS) and NF- κ B signaling in rat macrophages NR8383 cells treated with LPS or/and Bindarit. (K) QPCR of M1 markers (iNOS and CD86) and inflammatory cytokines such as TNF- α , IL-1 β , and GM-CSF in NR8383 cells treated with LPS or/and Bindarit. All data were presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001 and ∗∗∗∗ P < 0.0001 vs. control/model.

Article Snippet: To inhibit TNF- α receptors, the ChP epithelial cells were treated with R7050 (cat: HY-110203, MCE, New Jersey, USA).

Techniques: Expressing, Derivative Assay, Western Blot, Marker, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

doi: 10.1016/j.apsb.2024.06.020

Figure Lengend Snippet: Knockdown of ChP CCL2 using Bindarit relieves hydrocephalus and inhibits macrophage activation. (A) Representative T2-weighted images of lateral ventricles in the PHH group and Bindarit-treated group ( n = 5 animals per condition). (B) 3D reconstruction showcasing the lateral ventricles. (C) Quantification volumes of the lateral ventricle based on the T2-weighted images ( n = 5 animals per condition) and evaluation of CSF secretion rates ( n = 3 animals per condition). (D) Bindarit significantly inhibited the CCL2 mRNA expression in ChP and CSF CCL2 after Bindarit treatment ( n = 3 animals per condition). (E, F) The number of IBA1 + macrophages (green) in PHH and Bindarit-treated groups ( n = 5 animals per condition). Scale bar = 20 μm. (G) Bindarit decreased the number of phagocytic ChP macrophages (CD68 + ; Iba1 + ; % of total Iba1 + ) ( n = 5 animals per condition), Scale bar = 50 μm. (H) Quantitative analysis of the percent of macrophages in ChP cells, % of CD68 + macrophages in total macrophages, % of p65 + macrophages in ChP cells, and pNKCC1 expression in ChP, as well as the expression of TNF- α in CSF. (I) Bindarit reduced the expression of p-NKCC1 (red) in the ChP. Scale bar = 50 μm ( n = 5 animals per condition). (J) NF- κ B p65 signals (red) are seen in the cytoplasm of ChP epithelial cells (green arrow, b, and d) and cytoplasm-nuclear area in macrophages (white arrow, a and c) ( n = 5 animals per condition). Scale bar = 20 μm. All data were presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01 and ∗∗∗ P < 0.001 vs. control/model.

Article Snippet: To inhibit TNF- α receptors, the ChP epithelial cells were treated with R7050 (cat: HY-110203, MCE, New Jersey, USA).

Techniques: Knockdown, Activation Assay, Expressing, Control