Fig 1.
The effect of 3-DSC concentration on microglia apoptosis and viability.
(A, B) Primary microglia were treated with 3-DSC at 0, 5, 10, and 20 μM for 12 h. The apoptosis rate of microglia in different group was assessed Annexin V/PI staining followed by flow cytometry analysis. Annexin V and PI double negative cells were regarded as normal cells. Representative flow cytometry results were shown in (A), the quantification of flow cytometry results was shown in (B). (C) Cell viability of primary microglia treated with 3-DSC at 0, 5, 10, and 20 μM for 12h was assay assessed by CCK8 assay. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 2.
3-DSC inhibits the production of inflammatory cytokines in microglia.
(A) qRT-PCR analysis of TNFA, IL-6, and IL-1b mRNA expression in microglia treated with 3-DSC followed by stimulation of LPS for 5h. (B) Elisa assay showing TNFα, IL-6 and IL-1β level in the supernatants from the cultures of microglia treated with 3-DSC followed by stimulation of LPS for 9h. (C, D) Representative images of immunofluorescence (C) staining for Iba1 (red) and quantification (D) of mean fluorescence intensity normalized to control. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 3.
Inflammatory related signaling pathways in microglia are inhibited by 3-DSC.
(A, B) Representative immunoblot (A) and the quantification (B) of phosphorylated (p-) proteins of microglia stimulated with LPS in the presence or absence of 3-DSC for indicated times. (C, D) Representative immunoblot (C) and the quantification (D) of nuclear p65 of microglia stimulated with LPS in the presence or absence of 3-DSC for indicated times. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 4.
3-DSC treatment leads to changes in the expression of genes related to inflammatory responses.
(A) Differential expression analysis reveals that 3-DSC induces transcriptional changes in microglia under LPS stimulation. Volcano plots show differentially expressed genes (DEGs; fold change≥2.0 or ≤−2.0, false-discovery-rate-adjusted P-value<0.05). (B) The number of DEGs is shown, a total of 213 upregulated genes and 256 downregulated genes were indicated. (C) The heatmap of the top 10 downregulated and upregulated genes significantly affected by 3-DSC treatment in microglia. (D, E) GO analysis showing the top four terms significantly affected by 3-DSC treatment, from the most upregulated (D) to the most downregulated (E). (F) PPI networks among the DEGs and hub genes as computed by Cytoscape. Connectivity degree levels were used to screen hub genes. (G) qRT–PCR analysis verified the expression of representative genes discovered by RNA-seq analysis after 3-DSC treatment. β-actin was used as an endogenous control. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 5.
3-DSC reduces activated microglia-induced damage to neurons.
(A) the schematic diagram of co-culture assays. Microglia were pretreated with 3-DSC for 1h and stimulated with 1 μg/mL LPS for 12h. Conditioned medium (CM) from microglia was collected and neurons were cultured in conditioned medium for 24 h. (B) the apoptosis rate of neurons in different group was assessed Annexin V/PI staining followed by flow cytometry analysis. Annexin V positive cells were regarded as apoptotic cells. (C, D) Representative immunoblot (C) and the quantification (D) of apoptosis-related proteins in neurons cocultured with different conditioned medium. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 6.
3-DSC ameliorate microglia activation, neurological dysfunction, and cognitive impairment induced by TBI.
(A) Schematic diagram of the in vivo experimental protocols. (B) Neurological function between different group was evaluated by mNSS score. (C) Typical swimming paths in the Morris water maze during the training trials and probe trial. (D-F), swimming speed (E), escape latencies to find a hidden platform(E), and the frequency of crossing the hidden platform (F) were shown. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 7.
3-DSC ameliorate microglia activation and inflammatory cytokines production in vivo.
(A, B) Schematic diagram of the in vivo experimental protocols (A) and gross observation of brain tissue(B) in CCI model. Scale bar = 3 mm. (C, D) Image panels of brain sections (C) immunolabeled for Iba1and quantification (D) of mean fluorescence intensity normalized to sham. (E, F) Representatives immunoblot (E) and the quantification (F) of TNF-α, IL-6 and IL-1β of brain tissues with different treatment. Data are shown as mean ± SEM of three independent experiments, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.