Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3aR ameliorates rotenone-induced neurodegeneration, synaptic loss, α-synuclein phosphorylation and cognitive deficits in mice. C57BL/6 mice were treated with rotenone for 3 weeks to establish PD models, with 30 min of SB290157 (C3aR inhibitor, 1 mg/kg, i.p.) administration after each rotenone injection. (A) Concentrations of C3a in the hippocampus of Con and Rot mice, measured by enzyme-linked immunosorbent assay (ELISA). n = 6. (B) Representative Western blot images and densitometric quantification of C3aR protein levels in the hippocampus and rotenone mice. n = 4. (C) The mRNA levels of C3aR in vehicle and rotenone mice. (D) Representative images of double-immunofluorescence staining with C3aR and Iba-1 or Neu-N antibodies and (E, F) the quantification of C3aR + Iba-1 + and C3aR + Neu-N + cells in vehicle and rotenone mice. n = 3. (G) The quantification of Neu-N + cell number and (H) optical density of PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. (I) The representative images of Neu-N and PSD95 staining in rotenone mice with or without C3aR inhibitor treatment. n = 6. (J, K) Representative bands of Neu-N, PSD95 and ser129-phosphorylated α-synuclein and the quantification of blots in rotenone mice with or without C3aR inhibitor treatment. n = 4. (L) Representative images of ser129-phosphorylated α-synuclein and (M) the quantification of staining density in rotenone mice with or without C3aR inhibitor treatment. n = 3. (N–Q) Escape latency, traveled distance, first platform crossing latency and platform crossing number in rotenone mice with or without C3aR inhibitor treatment. n = 15; ∗ p < 0.05, ∗∗ p < 0.01 for comparison between Rot & Rot + C3aR inhibitor groups; Scale bar = 50 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Phospho-proteomics, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Double Immunofluorescence Staining, Staining, Comparison

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis suppresses microglia-mediated neuroinflammation in rotenone-treated mice. WT mice, C3 −/− mice, and C3aR inhibitor (SB290157)-treated mice were exposed to rotenone for 3 weeks to induce PD-related neuroinflammation. (A) Representative images of Iba-1 immunostaining and (B) the quantification of staining density in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 6. (C) The mRNA levels of iNOS, IL-1β and TNFα in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. (D) Representative bands of p-MAPK, MAPK, p-p65, p65, p-IκBα and IκBα and the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4. ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Immunostaining, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis mitigates microglia-mediated abnormal synaptic pruning in rotenone-treated mice. WT mice, C3 −/− mice, and SB290157-treated mice were subjected to rotenone for 3 weeks. (A) Representative images of double fluorescence staining with anti-Iba-1 and anti-CD68 antibodies and (B) the quantification of staining in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 6. (C) Representative bands of PSD95, p-CREB, CREB, mBDNF and TrkB and (D) the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4. ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Fluorescence, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Inhibition of C3–C3aR axis attenuates rotenone-induced activation of dark microglia and related secretion of toxic lipids in vivo and in vitro. (A) Representative bands of p-eIF2α, eIF2α, PKR, PERK and ATF4 and (B) the quantification of blots in mice. n = 4. (C) Representative images of double-immunofluorescence staining with p-eIF2α and Iba-1 antibodies in mice. n = 3. (D) BV2 microglial cells were treated with C3 protein (with or without SB290157 or NOX2 inhibitor GSK2795039) for 24 h. Representative bands of p-eIF2α, eIF2α, PKR, PERK and ATF4 and the quantification of blots in BV2 cells were shown. (E) The mRNA levels of atf4 and asns in BV2 cells. n = 3. (F) 3D principal component analysis (PCA) score plot showing separation of lipid profiles. (G) The heatmap shows the differential lipid metabolites among groups. (H) Venn diagram showing the overlap of differential lipid metabolites. (I) Classification of differential lipid metabolites at the major category level between C3_C3aR vs. C3 and C3 vs. Control groups. (J) Subclass distribution of differential lipid metabolites between C3_C3aR vs. C3 and C3 vs. Control groups. n = 5. SP: sphingolipids; GL: glycerolipids; FA: fatty acyls; DL: derivatized lipids (biotinylation, diazomethane). ST: sterol lipids. PR: prenol lipids. GP: glycerophospholipids. ∗∗ p < 0.01; Scale bar = 50 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Inhibition, Activation Assay, In Vivo, In Vitro, Double Immunofluorescence Staining, Control

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: Blocking C3/C3aR axis suppresses rotenone-induced PANoptosis in mice. (A) Representative images of TUNEL staining and (B) the quantification of TUNEL + cells in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. (C) Representative bands of Bcl-2, Bax, active-caspase-3, p-MLKL, MLKL, p-RIP1, RIP1, p-RIP3, RIP3, NLRP3, caspase-1 and GSDMD and (D) the quantification of blots in rotenone-treated WT and C3 −/− mice and rotenone mice with or without C3aR inhibitor treatment. n = 4; ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm.

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, TUNEL Assay, Staining

Journal: Redox Biology

Article Title: The C3–C3aR axis drives rotenone-induced cognitive damage via synaptic engulfment, dark microglia and PANoptosis

doi: 10.1016/j.redox.2026.104062

Figure Lengend Snippet: C3–C3aR axis promotes PANoptosome formation and PANoptosis through z-DNA-ZBP-1 interaction via mitochondrial ROS. SH-SY5Y neuronal cells were treated with different concentrations of C3 protein (with or without SB290157, apoptosis inhibitor Z-VAD, necroptosis inhibitor Nec-1, inflammasome inhibitor MCC950, ROS scavenger NAC, NADPH oxidase inhibitor apocynin, mtROS scavenger mito-TEMPO, z-DNA inhibitor CQ, or z-DNA promoter CeCl 3 ) for 24 h. (A) The survival rate of cells treated with different concentrations of C3 protein. (B) The survival rate of cells treated with C3 with or without different inhibitors. (C) Representative pictures of ZBP-1, AIM2, RIP3 and caspase-1 interaction using COIP. (D) The ratio of A260/A295 absorbance. (E) Representative images of DHE staining and quantification of DHE density in SH-SY5Y cell among groups. (F) Representative images of z-DNA fluorescence staining and quantification of density in SH-SY5Y cell among groups. (G) Representative blots of z-DNA and ZBP-1 and quantification of blot density in SH-SY5Y cell among groups. (H) Representative images of double-immunofluorescence staining using antibodies against z-DNA and ZBP-1 and quantification of ration of z-DNA + ZBP-1 + /ZBP-1 + cells in SH-SY5Y cell among groups. (I) Representative blots of z-DNA and ZBP-1 interaction using COIP. (K) The survival rate of cells treated with C3 with or without apocynin, NAC, mito-TEMPO, CQ and CeCL 3 . n = 3; ∗ p < 0.05, ∗∗ p < 0.01; Scale bar = 100 μm in (E) and 25 μm in (F) and (H).

Article Snippet: Antibodies were obtained from various sources as follows: C3 (ab200999), Neu-N (ab177487), Fibrinogen (ab92572), ser129-phosphorylated α-synuclein (ab51253), and phosphorylated-RIP3 (ab95117) from Abcam (Waltham, MA, UK); C3aR and Iba-1 antibodies from Santa Cruz Biotechnology (sc-133172, Dallas, TX, USA) and Wako Chemicals (019–19741, Tokyo, Japan), respectively; GFAP (16825), Bcl2 (68103), and Bax (50599) antibodies from Proteintech (Wuhan, Hubei, China); PSD95 (3450S), ERK (9101S), p-ERK (4695T), p65 (8242T), p-p65 (3033S), IκBα (2859T), p-IκBα (4814T), RIP1 (53286S), p-RIP1 (3493S), MLKL (37333S), p-MLKL (37705S), and RIP3 (95702S) antibodies from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Fluorescence, Double Immunofluorescence Staining