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: Antibodies used for western blotting

Article Snippet: rabbit anti-p-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 3033 , AB_331284.

Techniques: Western Blot

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: rabbit anti-p-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 3033 , AB_331284.

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: Tryp shifts microglia from M1 to M2 phenotype after spinal cord injury by targeting the cGAS/STING/NF-κB pathway. (A) Western blot analysis of cGAS and p-STING in spinal cord treated with Tryp at 3 dpi. (B) Quantitative analysis of cGAS and p-STING protein levels shown in A (normalized to β-actin, n = 6 mice per group). (C, E) Confocal laser scanning microscopy double immunostaining images (60× magnification) of cGAS (purple) and Iba1 (green) (C), and p-STING (purple) and Iba1 (green) (E) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 3 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. cGAS + Iba1 + cells and p-STING + Iba1 + cells were marked by white arrowheads. Scale bars, 50 μm (left) and 20 μm (enlarge). (D, F) Quantitative analysis of the percentages of cGAS + Iba1 + cells in Iba1 + cells (D, n = 3 mice per group), and p-STING + Iba1 + cells in Iba1 + cells (F, n = 3 mice per group). (G) Western blot analysis of p-p65, p65, p-IκBα and IκBα in spinal cord treated with Tryp at 7 dpi. (H) Quantitative analysis of p-p65 (normalized to p65), p65 (normalized to β-actin), p-IκBα (normalized to IκBα) and IκBα (normalized to β-actin) protein levels shown in G ( n = 10 mice per group). (I, K) Confocal laser scanning microscopy double immunostaining images (60× magnification) of CD86 (red) and CD68 (green) (I), and CD206 (red) and CD68 (green) (K) in the spinal cord lesion area of Sham + DMSO (T8–T10), Sham + Tryp (T8–T10), SCI + DMSO and SCI + Tryp mice at 7 dpi. Higher magnification panels are single optical Z sections of the areas marked by boxes. CD86 + CD68 + cells and CD206 + CD68 + cells were marked by white arrowheads. Scale bars: 50 μm (left) and 20μm (enlarge). (J, L) Quantitative analysis of the percentages of CD68 + /DAPI (left) and CD86 + CD68 + /CD68 + (right) (J, n = 6 mice per group), and CD68 + /DAPI (left) and CD206 + CD68 + /CD68 + (right) (L, n = 6 mice per group). Data were analyzed by one-way analysis of variance with Tukey’s multiple comparisons test (left) and unpaired two-tailed Student’s t -test (right). (M) Western blot analysis of CD206 and CD86 expression in spinal cord treated with Tryp at 7 dpi. (N, O) Quantitative analysis of CD206 (N) and CD86 (O) protein levels shown in M (normalized to β-actin, n = 9 mice per group). (P–T) Quantitative reverse transcription-polymerase chain reaction analysis of IL-6 (P), IL-1 β (Q), TNF-α (R), CD206 (S) and Arg-1 (T) gene expression levels in spinal cords of Sham + DMSO, Sham + Tryp, SCI + DMSO and SCI + Tryp mice at 7 dpi (normalized to β-actin, n = 6 mice 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; DMSO: dimethyl sulfoxide; dpi: days post-injury; Iba1: ionized calcium-binding adapter molecule 1; IL-6: interleukin-6; ns: not significant; NF-κB: nuclear factor-κB; ns: not significant; p: phosphorylated; p-STING: phosphorylated stimulator of interferon genes; TNF-α: tumor necrosis factor-α; Tryp: tryptanthrin.

Article Snippet: rabbit anti-p-NF-κB p65 , 1: 1000 , Cell Signaling Technology , 3033 , AB_331284.

Techniques: Western Blot, Confocal Laser Scanning Microscopy, Double Immunostaining, Two Tailed Test, Expressing, Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Binding Assay

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 2 | DACA reverses microglial activation, neuronal damage, and dendritic spine loss, and decreases the expression of HMGB1, NF-κB, and NLRP3 in the hippocampus of CUMS-induced mice. (A and B) Expression of IBA1 and MAP2 in the hippocampal CA1 region of mice. Scale bar, 20 μm. n = 3. (C) Expression levels of IL-6 and TNF-α in mouse serum. n = 3. (D and E) Dendritic spine density in the hippocampus of mice. Scale bar, 20 or 10 μm. n = 3. (F and G) Protein expression of MAP2, PSD95, and BDNF in the hippocampus of mice. n = 4. (H–I) Expression levels of HMGB1, NF-κB, NLRP3, IL-1β, and NRF2 in the hippocampus of mice. n = 4. #p < 0.5, ##p < 0.1, ###p < 0.01, ####p < 0.001, vs. control group; *p < 0.5, **p < 0.1, ***p < 0.01, vs. CUMS group.

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Activation Assay, Expressing, Control

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 3 | DACA reverses cell injury, inhibits the release of inflammatory factors, prevents HMGB1 nuclear translocation, and downregulates the expression of HMGB1, NF-κB, NLRP3, and IBA1 induced by LPS+ATP in BV2 cells. (A) Schematic timeline of BV2 cell experimental protocol. (B and C) CCK8 assay to assess BV2 cell viability. n = 5. (D) NO levels in BV2 cell supernatants measured by NO assay kit. n = 5. (E) ELISA detection of IL-6 and TNF-α levels in BV2 cell supernatants. n = 3. (F and G) Western blot analysis of HMGB1, P-P65/NF-κB, NLRP3, IL-1β, BDNF, and Nrf2 expression in BV2 cells. n = 4. (H and I) Immunofluorescence staining to detect IBA1 expression and HMGB1 nuclear translocation in BV2 cells. Scale bar, 20 μm. n = 3. #p < 0.5, ##p < 0.1, ###p < 0.01, ####p < 0.001, vs. control group; *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001, vs. LPS+ATP group.

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Translocation Assay, Expressing, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Control

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 5 | The HMGB1/NF-κB/NLRP3 pathway is involved in the regulation of DACA on CUMS-induced depressive-like behavior, memory impairment, microglial activation, and neuronal damage. (A) Schematic timeline of the animal experiment procedure. (B) Tracking plots of exper- imental animals in the OFT and MWM. (C) Sucrose preference percentage in the SPT, total distance traveled (mm) in the OFT, immobility time (s) in the TST and FST, escape latency (s), and number of platform area crossings in the MWM. n = 9. (D and E) Expression of IBA1 and MAP2 in the hippocampal CA1 region of mice. Scale bar, 20 μm. n = 3. (F) Expression levels of IL-6 and TNF-α in mouse serum. n = 3. (G and H) Dendritic spine density in the hippocampus of mice. Scale bar, 20 or 10 μm. n = 3. (I and J) Expression levels of MAP2, PSD95, and BDNF proteins in the hippocam- pus of mice. n = 4. (K and L) Western blot analysis of the expression levels of HMGB1, NF-κB, NLRP3, and IL-1β in the hippocampus of mice. n = 4.

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Activation Assay, Expressing, Western Blot

Journal: CNS neuroscience & therapeutics

Article Title: 11,12-Diacetyl-Carnosol Ameliorates Depression-Like Behaviors and Memory Dysfunction in CUMS Mouse Model via Inhibiting HMGB1-Mediated Neuroinflammation.

doi: 10.1111/cns.70406

Figure Lengend Snippet: FIGURE 6 | The HMGB1/NF-κB/NLRP3 pathway is involved in the regulation of DACA on LPS+ATP-induced BV2 cell injury, inflammation, and HMGB1 nuclear translocation. (A) Schematic timeline of the BV2 cell experimental procedure. (B) CCK-8 assay to assess BV2 cell viability. n = 5. (C) NO kit to measure the NO levels in BV2 cell supernatant. n = 5. (D and E) ELISA to measure IL-6 and TNF-α levels in BV2 cell super- natant. n = 3. (F and G) Western blot analysis of the expression of HMGB1, P-P65/NF-κB, NLRP3, IL-1β, and BDNF in BV2 cells. n = 4. (H and I) Immunofluorescence staining for IBA1 expression and HMGB1 nuclear translocation in BV2 cells. Scale bar, 20 μm. n = 3. ##p < 0.1, ###p < 0.01,

Article Snippet: For western blot analysis and immunofluorescence, we used the following antibodies: anti- Nrf2 antibody (1: 3000, Proteintech, 66,009- 1- IG), anti- HMGB1 antibody(1: 1000, Abcam, ab18256), anti- P- NF- κB antibody(1: 1000, Cell Signaling Technology, 3033S), anti- NF- κB p65 (D14E12) antibody (1: 1000, Cell Signaling Technology, 8242), anti- NLRP3 antibody (1: 1000, Cell Signaling Technology, 15,101), anti- IL- 1β antibody (1: 1000, Affinity, AF5103), anti- BDNF antibody (1: 1000, Cell Signaling Technology, 47,808), anti- Nrf2 antibody (1: 3000, Proteintech, 16,396- 1- AP), anti- IBA- 1 antibody (1: 1000, Abcam, ab178846), anti- MAP2 antibody (1: 1000, Abcam, ab11267), anti- PSD95 antibody (1: 1000, Abcam, ab18258), goat anti- rabbit IgG H&L (HRP) (1: 5000, Abcam, ab6721), goat anti- mouse IgG H&L (HRP) (1: 5000, Abcam, ab6789), goat anti- mouse IgG (H+L) cross- adsorbed secondary antibody, Alexa Fluor 488 (1: 500, nloaded from https://onlinelibrary.w iley.com /doi/10.1111/cns.70406 by IN A SP - N E PA L , W iley O nline L ibrary on [25/05/2025].

Techniques: Translocation Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Immunofluorescence, Staining