Fig 2: Sevoflurane activates the cGAS-STING pathway in POCD mice. A-B Tbk1 and Irf3 mRNA expression in the hippocampus were detected by real-time PCR (n=6). C-G Protein levels of cGAS, TBK1, STING, p-TBK1Ser172, p-IRF3Ser396, and IRF3 in the hippocampus were detected by western blot (n=6). H-J The fluorescence intensity of cGAS (green) and IBA1 (red) in hippocampal tissue was detected by immunofluorescence (n=3, bar=100 µm). K-L Protein levels of cGAS, STING, p-TBK1Ser172, and p-IRF3Ser396 in the BV2 cells were detected by western blot (n=3). Data are expressed as the mean ± SD. Comparisons between the two groups were made with an unpaired t-test. Differences among multiple groups were performed using ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: Inhibition of cGAS decreases cognitive dysfunction in POCD mice. A C57BL/6J mice were trained in the passive avoidance test. The next day, the mouse was given intraperitoneal injection of RU.521 (5 mg/kg) before 2 h. Then, 4% sevoflurane was exposed to mice for 6 h to induce cognitive impairment. On the first day after sevoflurane exposure, the mice underwent the passive avoidance test and the Y-maze test. B-C The dark avoidance test was conducted to assess the number of entering the darkroom and the dark latency (n=8). D The shuttle experiment was conducted to assess active avoidance (n=8). E Representative trajectories of each group in the training session of the Y-maze test. F-G Total distances and total resting times were assessed in the Y-maze training (n=8). H Representative trajectories of each group in the Y-maze testing. I-K The percentage of spontaneous alternation, the number of entries into the novel arm, and the duration spent in the novel arm were assessed in the Y-maze testing (n=8). L HE staining of hippocampal tissue (bar=50 µm). M-N Protein levels of p-TauT231 and APP in the hippocampus were detected by western blot (n=6). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: Blockade of DRP1 reduces sevoflurane-induced cGAS-STING pathway-dependent NLRP3 inflammasome in microglia. A BV2 cells were treated with 1 mM sevoflurane for 12 h after 100 nM Mdivi-1 intervention 30 min. Mitochondrial morphology stained with TOM20 in BV2 cells (bar=5 µm). B-C The morphological skeleton and quantification of mitochondrial morphology were detected in BV2 cells (n=3). D Representative images of BV2 cells loaded with the mitochondrial membrane potential indicator JC-1 (bar=50 μm). E-G mtDNA levels of Nd1, Cytb, and D-loop in the cytoplasm of BV2 cells were determined by real-time PCR (n=3). H-L Protein levels of cGAS, STING, p-TBK1Ser172, and p-IRF3Ser396 in the BV2 cells were detected by western blot (n=3). M-P The protein levels of NLRP3, pro-IL-1β, and IL-1β p17 in the BV2 cells were detected by western blot (n=3). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Inhibiting mPTP-VDAC channel opening attenuates sevoflurane-induced mtDNA cytosolic escape and reduces cGAS-STING pathway activation in microglia. A BV2 cells were treated with 1 mM sevoflurane for 12 h after 1 µM CsA intervention for 30 min and 10 µM VBIT-4 intervention for 30 min. mtDNA levels of Nd1, Cytb, and D-loop in cytoplasm of BV2 cells were determined by real-time PCR (n=3). B-I Protein levels of NLRP3, pro-IL-1β, IL-1β p17, cGAS, STING, p-TBK1Ser172, and p-IRF3Ser396 in the BV2 cells were detected by western blot (n=3). J-Q Protein levels of NLRP3, pro-IL-1β, IL-1β p17, cGAS, STING, p-TBK1Ser172, and p-IRF3Ser396 in the BV2 cells were detected by western blot (n=3). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001.