Journal: Journal of Neuroinflammation

Article Title: A microglial LCN2-MC4R signaling axis drives silica-induced neuronal damage via C1q release

doi: 10.1186/s12974-026-03695-5

Figure Lengend Snippet: Transcriptome results showed that genes such as LCN2 and C1q were significantly upregulated in the hippocampus. A Volcano plot of differentially expressed genes (DEGs) in hippocampus from silica-exposed (Sil) versus vehicle (Veh) mice. Cut-off: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\left|{log}_{2}FC\right|\geqq\:O.O5$$\end{document} , \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:Pvalue\leqq\:0.05$$\end{document} , indicated by gray dashed lines in the plot. Red, significantly up-regulated; blue, significantly down-regulated; grey, non-significant. n = 3 biological replicates per group. B Gene set enrichment analysis of the 2,275 up-regulated genes using the Metascape database. C Inflammatory module network analysis of the DEGs enriched in GO:0006954 (GO database), circled by red in ( B ). D Neutrophil degranulation cascade module network analysis of the DEGs enriched in R-MMU-6,798,695 (Reactome database), circled by green in ( B ). E Microglial phagocytosis module network analysis of the DEGs enriched in WP3626 (WikiPathways database), circled by yellow in ( B ). F LCN2 and complement receptor C3ar1 were identified as having potential interactions. Interactions between genes among C-F are indicated by lines, whose colors represent interaction types from STRING database

Article Snippet: Cells were cultured in medium containing LCN2 (Sino Biological, 50409-M08H) at 100 ng/ml, the MC4R antagonist HS024 (MedChem Express, HY-P1215A) at 100 nM, or a combination of LCN2 and HS024 (LCN2-HS024) for 24 h. Cell supernatants were collected for subsequent ELISA analysis.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: A microglial LCN2-MC4R signaling axis drives silica-induced neuronal damage via C1q release

doi: 10.1186/s12974-026-03695-5

Figure Lengend Snippet: LCN2 further activates microglia, exacerbating neuroinflammation in the hippocampus. A Representative immunohistochemical staining of LCN2 in the CA1, CA3, and DG regions of paraffin-embedded tissue from Veh and Sil groups; scale bar: 50 μm. The black arrow indicates LCN2-positive cells, brown DAB precipitate indicates positive immunoreactivity. B Quantitative analysis of positive staining from panel A, ( n = 4). C Western blot analysis assessing LCN2 protein expression. D LCN2 expression levels in Veh and Sil group mice ( n = 9). E Representative images of LCN2 immunoreactivity (green) and Iba-1 (red) in the CA1, CA3, and DG regions of frozen tissue from Veh and Sil groups; scale bar: 50 μm. F Representative immunofluorescence images of frozen sections showing Iba-1-positive cells (red); scale bar: 50 μm. G Sholl analysis of microglia in frozen tissue from Veh and Sil groups, measuring the distance from the soma (in 2 μm increments) and the number of microglial processes intersecting concentric circles ( n = 4). Maxima for all measures were observed approximately 20 μm from the soma. Data are presented as mean ± SEM. For panels ( B ) and ( D ), data were analyzed by t-tests. For panel ( G ), data were analyzed by two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = not significant

Article Snippet: Cells were cultured in medium containing LCN2 (Sino Biological, 50409-M08H) at 100 ng/ml, the MC4R antagonist HS024 (MedChem Express, HY-P1215A) at 100 nM, or a combination of LCN2 and HS024 (LCN2-HS024) for 24 h. Cell supernatants were collected for subsequent ELISA analysis.

Techniques: Immunohistochemical staining, Staining, Western Blot, Expressing, Immunofluorescence

Journal: Journal of Neuroinflammation

Article Title: A microglial LCN2-MC4R signaling axis drives silica-induced neuronal damage via C1q release

doi: 10.1186/s12974-026-03695-5

Figure Lengend Snippet: LCN2 activates microglia through the MC4R receptor rather than the MC1R or MC3R receptors in the hippocampus. A Western blot analysis assessing MC4R, MC1R, and MC3R protein expression. B Protein expression (normalized to β-actin) of MC4R, MC1R, and MC3R in tissue from Veh and Sil group mice ( n = 9). C Representative images of MC4R immunoreactivity (green), LCN2 immunoreactivity (red), and Iba-1 (purple) in frozen tissue. Scale bar: 50 μm. D Quantitative analysis of the MC4R-positive cells of CA1, CA3, and DG regions ( n = 4). E Quantitative analysis of the LCN2-positive cells of CA1, CA3, and DG regions ( n = 4). F Quantitative analysis of the MC4R + LCN2 + Iba-1 + cells (cell/mm 2 ) in the CA1, CA3, and DG regions ( n = 4). Data are presented as mean ± SEM. For panels (B-C, E-F), data were analyzed by t-tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = not significant

Article Snippet: Cells were cultured in medium containing LCN2 (Sino Biological, 50409-M08H) at 100 ng/ml, the MC4R antagonist HS024 (MedChem Express, HY-P1215A) at 100 nM, or a combination of LCN2 and HS024 (LCN2-HS024) for 24 h. Cell supernatants were collected for subsequent ELISA analysis.

Techniques: Western Blot, Expressing

Journal: Journal of Neuroinflammation

Article Title: A microglial LCN2-MC4R signaling axis drives silica-induced neuronal damage via C1q release

doi: 10.1186/s12974-026-03695-5

Figure Lengend Snippet: LCN2-mediated cAMP/PKA/NF-κB signaling and C1q upregulation, with HS024 inhibiting C1q release in the BV2 cell culture. A ELISA measurement of cAMP in cell culture supernatant ( n = 4). The main effects of LCN2 (F = 92.206, p < 0.001) and HS024 (F = 18.470, p = 0.001) were both significant. B Western blot analysis assessing the expression levels of MC4R, PKA, NF-κB p65, and C1q in four groups. C Quantitative analysis of MC4R protein expression (normalized to β-actin) ( n = 3). The main effects of LCN2 (F = 6.863, p = 0.031) and HS024 (F = 17.455, p = 0.003) were significant. D Quantitative analysis of PKA protein expression (normalized to GAPDH) ( n = 3). The main effects of LCN2 (F = 15.842, p = 0.004) was significant; HS024 (F = 3.303, p = 0.107) was not. E Quantitative analysis of phosphorylated P65 (P-P65) protein expression (normalized to total P65) ( n = 3). The main effect of HS024 (F = 11.158, p = 0.010) was significant; LCN2 (F = 3.929, p = 0.083) was not. F Quantitative analysis of C1q protein expression (normalized to GAPDH) ( n = 3). The main effects of LCN2 (F = 14.909, p = 0.005) was significant; HS024 (F = 1.731, p = 0.225) was not. G Immunofluorescence analysis after conditional treatment. Representative images of P65 immunoreactivity (green) and DAPI nuclear staining (blue) in BV-2 cells. White arrows indicate P65 nuclear expression-positive cells. H Quantitative analysis of the results in G, ( n = 4). Scale bar: 50 μm. The main effects of LCN2 (F = 12.917, p = 0.004) was significant; HS024 (F = 2.696, p = 0.127) was not. I ELISA measurement of C1q in cell culture supernatant ( n = 4). The main effects of LCN2 (F = 37.783, p < 0.001) and HS024 (F = 9.739, p = 0.009) were both significant. J Western blot analysis to assess the expression levels of TNF-α and TGF-β in four groups. K Quantitative analysis of TNF-α protein expression (normalized to GAPDH) ( n = 3). The main effects of LCN2 (F = 22.812, p = 0.001) and HS024 (F = 96.627, p < 0.001) were both significant. L Quantitative analysis of TGF-β protein expression (normalized to GAPDH) ( n = 3). The main effect of HS024 (F = 65.778, p < 0.001) was significant; LCN2 (F = 0.994, p = 0.348) was not. Data are presented as mean ± SEM. For panels ( A , C - F , H - I , K - L ), data were analyzed by two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = not significant

Article Snippet: Cells were cultured in medium containing LCN2 (Sino Biological, 50409-M08H) at 100 ng/ml, the MC4R antagonist HS024 (MedChem Express, HY-P1215A) at 100 nM, or a combination of LCN2 and HS024 (LCN2-HS024) for 24 h. Cell supernatants were collected for subsequent ELISA analysis.

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

Journal: Journal of Neuroinflammation

Article Title: A microglial LCN2-MC4R signaling axis drives silica-induced neuronal damage via C1q release

doi: 10.1186/s12974-026-03695-5

Figure Lengend Snippet: PF ameliorates neuronal loss induced by silica exposure, which is accompanied by anxiety- and depression-like behaviors in the hippocampus. A Representative activity traces of mice in the OFT across four groups. B Total distance traveled by mice in the OFT ( n = 8). The main effects of Sil (F = 20.072, p < 0.001) and PF (F = 0.637, p = 0.432). C Immobility time in the OFT ( n = 8). The main effects of Sil (F = 20.513, p < 0.001) and PF (F = 5.440, p = 0.027). D Time spent in the central area of the OFT ( n = 8). The main effects of Sil (F = < 0.001, p = 0.983) and PF (F = 0.527, p = 0.474). E Activity trajectories of mice in the open arms and closed arms of the EPM. The red arrow indicates open arms; the green arrow indicates closed arms. F Total distance traveled by mice in the EPM ( n = 8). The main effects of Sil (F = 1.024, p = 0.320) and PF (F = 2.498, p = 0.125). G Time spent in the open arms ( n = 8). The main effects of Sil (F = 1.157, p = 0.291) and PF (F = 5.258, p = 0.030). H Number of entries into the open arms ( n = 8). The main effects of Sil (F = 8.321, p = 0.007) and PF (F = 0.771, p = 0.387). I Number of marbles buried by mice in the MBT ( n = 8). The main effects of Sil (F = 6.215, p = 0.019) and PF (F = 0.559, p = 0.461). J Western blot analysis assessing the expression levels of LCN2, MC4R, C1q, and BDNF in the four groups of mice. K Quantitative analysis of LCN2 protein expression ( n = 9). The main effects of Sil (F = 2.801, p = 0.104) and PF (F = 8.961, p = 0.005). L Quantitative analysis of MC4R protein expression ( n = 9). The main effects of Sil (F = 22.590, p < 0.001) and PF (F = 9.298, p = 0.005). M Quantitative analysis of C1q protein expression ( n = 9). The main effects of Sil (F = 5.380, p = 0.027) and PF (F = 65.328, p < 0.001). N Quantitative analysis of BDNF protein expression ( n = 9). The main effects of Sil (F = 18.296, p < 0.001) and PF (F = 24.246, p < 0.001). Data are presented as mean ± SEM. For panels (B-D, F-I, K-N), data were analyzed by two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns = not significant

Article Snippet: Cells were cultured in medium containing LCN2 (Sino Biological, 50409-M08H) at 100 ng/ml, the MC4R antagonist HS024 (MedChem Express, HY-P1215A) at 100 nM, or a combination of LCN2 and HS024 (LCN2-HS024) for 24 h. Cell supernatants were collected for subsequent ELISA analysis.

Techniques: Activity Assay, Western Blot, Expressing

Journal: Journal of Neuroinflammation

Article Title: A microglial LCN2-MC4R signaling axis drives silica-induced neuronal damage via C1q release

doi: 10.1186/s12974-026-03695-5

Figure Lengend Snippet: Proposed mechanism by which the microglial LCN2-MC4R signaling axis drives silica-induced neuronal loss. Silica exposure in the lungs triggers the release of LCN2. Both LCN2 and TNF-α then travel to the hippocampus, cross the blood-brain barrier, and activate microglia. In microglia, LCN2 binds to the MC4R, activating the cAMP/PKA/NF-κB signaling pathway. This activation stimulates microglia to release C1q, which initiates the complement cascade, leading to C3 deposition on neurons. These C3-tagged neurons are subsequently targeted by microglia, resulting in neuronal loss

Article Snippet: Cells were cultured in medium containing LCN2 (Sino Biological, 50409-M08H) at 100 ng/ml, the MC4R antagonist HS024 (MedChem Express, HY-P1215A) at 100 nM, or a combination of LCN2 and HS024 (LCN2-HS024) for 24 h. Cell supernatants were collected for subsequent ELISA analysis.

Techniques: Activation Assay