Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Tryp inhibits microglia-derived inflammation through shifting polarization to the M2 phenotype. (A) Double immunostaining of phallotoxin (purple) and Iba1 (green) of BV2 cells incubated with different levels of Tryp (0, 2.5, 5.0, and 10.0 μM) with or without LPS for 24 hours. BV2 cells with hypertrophic bodies and multiple retracted cytoplasmic processes were identified as amoeboid cells (marked by white arrows). The areas marked by boxes are shown in the ‘Enlarge’ panels. Scale bars: 50 μm. (B) Quantitative analysis of the percentages of activated M1-like BV2 cells as shown in A ( n = 12 individual samples of cells per group). (C–G) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (C), IL-1 β (D), TNF-α (E), CD206 (F) and Arg-1 (G) gene expression levels in BV2 cells incubated with different levels of Tryp (0, 2.5, 5.0, 10.0 μM) with or without LPS for 24 hours (normalized to β-actin, n = 5 individual samples of cells per group). (H) Immunostaining of CD86 (CD86 + cells were marked by white arrows) and CD206 (CD206 + cells were marked by white arrows) in BV2 cells incubated with different levels of Tryp (0, 2.5, 5.0, 10.0 μM) with or without LPS for 24 hours. Scale bar, 50 μm. (I, J) Quantitative analysis of the percentages of CD86 + (I) and CD206 + (J) BV2 cells as shown in H ( n = 6 individual samples of cells per group). (K) Western blot analysis of CD206 and CD86 in BV2 cells incubated with various concentrations of Tryp (0, 2.5, 5.0, 10.0 μM) with or without LPS for 24 hours. (L, M) Quantitative analysis of CD86 (L) and CD206 (M) protein levels shown in (K) (normalized to β-actin, n = 6 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; Iba1: ionized calcium-binding adapter molecule 1; IB: immunoblotting; IL-6: interleukin-6; LPS: lipopolysaccharide; ns: not significant; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: Briefly, BV2 cells (4 × 10 5 /mL) were pretreated with LPS (1 μg/mL; Solarbio, Beijing, China) for 24 hours to induce a proinflammatory condition.

Techniques: Derivative Assay, Double Immunostaining, Incubation, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Immunostaining, Western Blot, Binding Assay

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Tryp promotes the M2 polarization of microglia through the cGAS/STING/NF-κB pathway. (A) Western blot analysis of cGAS and p-STING in BV2 cells incubated with Ctrl, LPS + DMSO (LD group) and LPS + Tryp (10.0 μM; LT group) for 1 hour. (B, C) Quantitative analysis of cGAS (B) and p-STING (C) protein levels as shown in A (normalized to β-actin, n = 8 individual samples of cells per group). (D) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells incubated with Ctrl, LD and LT for 1 hour. (E–H) Quantitative analysis of p-p65 (E, normalized to p65), p65 (F, normalized to β-actin), p-IκBα (G, normalized to IκBα), and IκBα (H, normalized to β-actin) protein levels shown in (D) (p-p65 and p65, n = 11 individual samples of cells per group; p-IκBα and IκBα, n = 8 individual samples of cells per group). (I) Western blot analysis of cGAS and p-STING in BV2 cells treated with Ctrl, LD, LT and LPS + Tryp + SR-717 (20.0 μM; LT + SR-717) for 1 hour. (J, K) Quantitative analysis of cGAS (J) and p-STING (K) protein levels shown in I (normalized to β-actin, n = 6 individual samples of cells per group). (L) Western blot analysis of p-p65, p65, p-IκBα and IκBα in BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 1 hour. (M–P) Quantitative analysis of p-p65 (M, normalized to p65), p65 (N, normalized to β-actin), p-IκBα (O, normalized to IκBα) and IκBα (P, normalized to β-actin) protein levels shown in L ( n = 6 individual samples of cells per group). (Q–U) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (Q), IL-1 β (R), TNF-α (S), CD206 (T) and Arg-1 (U) mRNA expression levels within BV2 cells treated with Ctrl, LD, LT and LT + SR-717 for 24 hours (normalized to β-actin, n = 3 individual samples of cells per group). (V) Immunostaining of CD86 (CD86 + BV2 cells and CD86 + primary cultured microglia were both marked by white arrows) and CD206 (CD206 + BV2 cells and CD206 + primary cultured microglia were both marked by white arrows) in BV2 cells and primary cultured microglia treated with Ctrl, LD, LT and LT + SR-717 for 24 hours. Scale bar: 100 μm. (W, X) Quantitative analysis of the percentages of CD86 + and CD206 + BV2 cells (W; n = 6 individual samples of cells per group), and primary cultured microglia (X; n = 3 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. Arg-1: Arginase-1; CD206: cluster of differentiation 206; cGAS: cyclic GMP-AMP synthase; Ctrl: control; DMSO: dimethyl sulfoxide; IκBα: nuclear factor-κB inhibitory protein α; IL-6: interleukin-6; ns: not significant; LPS: lipopolysaccharide; p: phosphorylated; STING: stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: Briefly, BV2 cells (4 × 10 5 /mL) were pretreated with LPS (1 μg/mL; Solarbio, Beijing, China) for 24 hours to induce a proinflammatory condition.

Techniques: Western Blot, Incubation, Reverse Transcription, Polymerase Chain Reaction, Expressing, Immunostaining, Cell Culture, Control

Journal: Neural Regeneration Research

Article Title: Pharmacological targeting cGAS/STING/NF-κB axis by tryptanthrin induces microglia polarization toward M2 phenotype and promotes functional recovery in a mouse model of spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01256

Figure Lengend Snippet: Microglia treated with tryptanthrin suppresses neuronal apoptosis through ER stress–related signaling. (A) Schematic of BV2 and N2a cell treatments and the conditional co-culture system. (B) Double immunostaining of CC-3 (green) and NeuN (red) in N2a cells incubated with conditional medium (CM) of BV2 cells treated by Ctrl (Ctrl-CM), LPS + DMSO (LD-CM) and LPS + Tryp (LT-CM). CC-3 + NeuN + cells were marked by white arrows. Scale bar: 100 μm. (C) Percentages of CC-3 + NeuN + cells shown in B ( n = 6 individual samples of cells per group). (D) Western blot analysis of Bcl-2 and Bax expression in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. (E, F) Quantitative analysis of Bcl-2 (E) and Bax (F) protein levels shown in D (normalized to β-actin, n = 6 individual samples of cells per group). (G) Western blot analysis of p-eIF2α, eIF2α, ATF4 and CHOP in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. (H–K) Quantitative analysis of p-eIF2α (H, normalized to eIF2α), eIF2α (I, normalized to β-actin), ATF4 (J, normalized to β-actin) and CHOP (K, normalized to β-actin) protein levels shown in G ( n = 6 individual samples of cells per group). (L) Double immunostaining of CHOP (green) and NeuN (red) in N2a cells incubated with Ctrl-CM, LD-CM and LT-CM. The areas marked by boxes were shown in the ‘Enlarge’ panels. Scale bars: 50 μm and 20 μm (enlarge). (M) Percentages of CHOP + NeuN + cells according to L ( n = 6 individual samples of cells per group). Data are expressed as the mean ± SEM and were analyzed using one-way analysis of variance with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. The data were from at least three separate and independent studies. ATF4: Activating transcription factor 4; CC-3: cleaved caspase-3; CHOP: CCAAT-enhancer-binding protein homologous protein; DMSO: dimethyl sulfoxide; eIF2α: eukaryotic initiation factor 2α; ER: endoplasmic reticulum; LPS: lipopolysaccharide; NeuN: neuron-specific nuclear protein; ns: not significant; p: phosphorylated; Tryp: tryptanthrin.

Article Snippet: Briefly, BV2 cells (4 × 10 5 /mL) were pretreated with LPS (1 μg/mL; Solarbio, Beijing, China) for 24 hours to induce a proinflammatory condition.

Techniques: Co-Culture Assay, Double Immunostaining, Incubation, Western Blot, Expressing, Binding Assay

Journal: Neural Regeneration Research

Article Title: Inhibiting ceramide synthase 5 expression in microglia decreases neuroinflammation after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01933

Figure Lengend Snippet: CerS5 knockdown inhibits LPS-induced BV2 cell pyroptosis in vitro. (A) Representative examples of immunofluorescence staining images used to quantify Iba1 + CerS5 + BV2 cells after treatment with si.CerS5 ( n = 5). There were significantly fewer Iba1 + CerS5 + cells in the LPS + si.CerS5 group compared with the LPS and LPS + NC groups. Iba1, green, CoraLite 488; CerS5, red, CoraLite 594; DAPI, blue. (B) Western blot analysis of CerS5 expression levels in BV2 cells after treatment with si.CerS5 ( n = 3). (C) Representative examples of immunofluorescence staining images used to quantify Iba1 + GSDMD + cells after treatment with si.CerS5 ( n = 5). There were significantly fewer Iba1 + GSDMD + /Iba1 + cells in the LPS + si.CerS5 group compared with the LPS and LPS + Si.NC groups. Iba1, green, CoraLite 488; GSDMD, red, CoraLite 594; DAPI, blue. Scale bars: 100 μm. (D) Western blot analysis of pyroptosis-associated protein expression levels in BV2 cells after treatment with siCerS5 ( n = 3). Data are expressed as the mean ± SEM. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test). CerS: Ceramide synthase; DAPI: 4′,6-diamidino-2-phenylindole; GSDMD: gasdermin D; Iba1: ionized calcium binding adaptor molecule 1; LPS: lipopolysaccharides; NC: negative control; SCI: spinal cord injury; si.CerS5: CerS5 siRNA.

Article Snippet: In the BV2 cell interference assay, inflammation was induced by treating BV2 cells at 80% confluence with lipopolysaccharide (LPS; 1 μg/mL; Beyotime, Shanghai, China) for 24 hours (Xu et al., 2021).

Techniques: Knockdown, In Vitro, Immunofluorescence, Staining, Western Blot, Expressing, Comparison, Binding Assay, Negative Control

Journal: Neural Regeneration Research

Article Title: Inhibiting ceramide synthase 5 expression in microglia decreases neuroinflammation after spinal cord injury

doi: 10.4103/NRR.NRR-D-23-01933

Figure Lengend Snippet: CerS5 knockdown inhibits neuronal apoptosis in the spinal cord after SCI. (A) Representative examples of immunofluorescence staining images used to quantify TUNEL + PC12 cells cultured in conditioned medium (CM) from BV2 cells treated with siCerS5 ( n = 5). There were significantly fewer TUNEL + cells in the CM (si.CerS5-BV2 + LPS) group compared with the CM (BV2 + LPS) group. NeuN, green, CoraLite 488; TUNEL, red, CoraLite 594; DAPI, blue. Scale bar: 100 μm. (B) Representative examples of immunofluorescence staining images used to quantify TUNEL + neurons in the SCI and Sham groups after treatment with siCerS5 ( n = 5). There were significantly fewer TUNEL + NeuN + cells in the SCI + si.CerS5 group compared with the SCI and SCI + si.NC groups. NeuN, green, CoraLite 488; TUNEL, red, CoraLite 594; DAPI, blue. Scale bar: 20 μm. (C) Representative examples of the HE staining images used to analyze the size of the lesion area 14 days after SCI. The injury area in the SCI + si.CerS5 group was smaller compared with that in the SCI and SCI + si.NC groups. The dashed line shows the injury area. Scale bar: 50 μm. (D) BMS scores were used to assess mouse motor ability at 1, 3, 7 and 14 days after SCI ( n = 6). Data are expressed as the mean ± SEM. ** P < 0.01 (one-way analysis of variance followed by Tukey’s multiple comparison test (A, C) or two-way analysis of variance followed by Tukey’s multiple comparison test (D). BMS: Basso Mouse Scale; CerS: ceramide synthase; DAPI: 4′,6-diamidino-2-phenylindole; HE: hematoxylin-eosin staining; LPS: lipopolysaccharides; NeuN: neuronal nuclei antigen; SCI: spinal cord injury; si.CerS5: CerS5 siRNA; TUNEL: TdT-mediated dUTP nick-end labeling.

Article Snippet: In the BV2 cell interference assay, inflammation was induced by treating BV2 cells at 80% confluence with lipopolysaccharide (LPS; 1 μg/mL; Beyotime, Shanghai, China) for 24 hours (Xu et al., 2021).

Techniques: Knockdown, Immunofluorescence, Staining, TUNEL Assay, Cell Culture, Comparison, End Labeling

Journal: Neural Regeneration Research

Article Title: High-dose dexamethasone regulates microglial polarization via the GR/JAK1/STAT3 signaling pathway after traumatic brain injury

doi: 10.4103/NRR.NRR-D-23-01772

Figure Lengend Snippet: Microglial activation in response to LPS and DEX treatment. (A) Representative immunofluorescence images showing that DEX treatment decreases M1 and M2 microglial polarization in cultured BV2 cells and primary microglia after exposure to LPS. Scale bars: 20 μm. Red represents Iba1, and green represents Arg1 or iNOS. (B) Counts of Arg1- and iNOS-positive cells normalized to Iba1-positive cells. * P < 0.05, vs . Control; # P < 0.05, vs . LPS (one-way analysis of variance with the least significant difference test). Data are presented as mean ± SD, n = 3. DAPI: 4′,6-Diamidino-2-phenylindole; DEX: dexamethasone; LPS: lipopolysaccharide.

Article Snippet: In the lipopolysaccharide (LPS) group, 1 µg/mL LPS (Sigma–Aldrich, St. Louis, MO, USA, Cat# L4391) was used to stimulate BV2 and primary microglia for 24 hours (Yin et al., 2020).

Techniques: Activation Assay, Immunofluorescence, Cell Culture, Control