Muscone ameliorates postoperative cognitive dysfunction in aged mice by modulating PPARγ to suppress TLR4/NF-κB/NLRP3 pathway-mediated neuroinflammation

Postoperative cognitive dysfunction (POCD) is closely associated with perioperative neuroinflammation, proinflammatory activation of microglia, and hippocampal neuronal injury. Muscone (Mus) exerts anti-inflammatory and neuroprotective effects, yet whether it can ameliorate anesthesia-and surgery-induced POCD and the underlying mechanisms remain unclear. This study aims to investigate whether Mus ameliorates POCD by modulating PPARγ -related signaling and suppressing inflammatory responses mediated by the TLR4/NF-κB/NLRP3 pathway.

① Network pharmacology: Mus target prediction was performed using SwissTargetPrediction, PharmMapper, SuperPred, and TargetNet databases. POCD-related targets were collected from GeneCards, DisGeNET, and OMIM databases. Potential targets through which Mus may act against POCD were screened, a protein-protein interaction (PPI) network was constructed, and core targets were selected. GO functional annotation and KEGG pathway enrichment analyses were subsequently performed. Molecular docking was used to evaluate the potential binding affinity of Mus with the core target proteins. ② Animal experiments: 32 aged male C57BL/6 mice (12 months old) were randomly divided into an NC group, a POCD group, a Mus group, and a Mus+GW9662 group, with 8 mice in each group initially and 6 mice per group finally included in behavioral analysis. Except for the NC group, all other groups underwent isoflurane anesthesia combined with tibial fracture internal fixation to establish a POCD model. The Mus group received an intraperitoneal injection of 10 mg/kg Mus, whereas the Mus+GW9662 group received PPARγ inhibitor, GW9662 at 1 mg/kg before Mus administration. Novel object recognition test was carried out on postoperative day 3, and Morris water maze test was conducted from postoperative days 3 to 8 to assess recognition memory and spatial learning and memory. After behavioral tests, 3 mice from each group were subjected to HE and Nissl staining to evaluate neuronal injury in the hippocampal CA1 region. RT-qPCR and Western blot were used to detect the expression of inflammatory factors, inflammation phenotype-related molecules, and PPARγ/TLR4/NF- κB/NLRP3-related molecules in hippocampal tissues. ③ Cell experiments: An inflammatory model was established in BV2 microglial cells induced by lipopolysaccharide (LPS), and then the cells were divided into NC, LPS, LPS+Mus, and LPS+Mus+GW9662 groups. CCK-8 assay was used to evaluate the effect of Mus on cell viability and determine the in vitro intervention concentration. Griess assay, flow cytometry, RT-qPCR, and Western blot were used to detect NO release, CD86/CD206 phenotypes, inflammatory factors, and expression of PPARγ/TLR4/NF-κB/NLRP3-related molecules. Conditioned media from the 4 groups of BV2 cells were subsequently collected to treat HT22 cells, and CCK-8 assay and flow cytometry were carried out to detect cell viability and apoptosis of HT22 cells, respectively.

① Potential targets of Mus intervention in POCD were mainly enriched in biological processes related to inflammatory responses, immune regulation and neural injury, and PPARG was identified as a potential target. Molecular docking suggested a potential binding interaction between Mus and PPARγ. ② The results of behavioral tests showed that, compared with the NC group, the POCD group exhibited fewer crossing number in Morris water maze test (1.33±1.03 vs 5.83±1.72, P=0.0003), a shorter duration in the target quadrant (22.11±2.94 s vs 44.13±6.95 s, P<0.0001), a lower novelty preference index (0.37±0.11 vs 0.72±0.05, P<0.0001), and a decreased cognitive index (−0.26±0.23 vs 0.43±0.11, P<0.0001). Compared with the POCD group, Mus treatment increased crossing number (5.83±0.75 vs 1.33±1.03, P<0.0001), prolonged time spent in the target quadrant (42.98±7.06 s vs 22.11±2.94 s, P<0.0001), and increased both the novelty preference index and cognitive index (both P<0.01). GW9662 partially attenuated the above improvements induced by Mus. Histological examination showed disorganized neuronal arrangement and reduced Nissl staining in the hippocampal CA1 region of the POCD mice. Mus treatment alleviated neuronal injury, whereas GW9662 weakened this protective effect. Molecular analysis showed that Mus reduced the expression of proinflammatory cytokines and iNOS, increased the expression of IL-10, Arg1, and PPARγ, and decreased the expression of TLR4, NF-κB p65, and NLRP3 in the hippocampus of POCD mice. GW9662 partially reversed the above effects. ③ CCK-8 assay showed that treatment of 10 μmol/L Mus for 24 h had no significant inhibitory effect on viability of BV2 or HT22 cells. Molecular analysis demonstrated that Mus reduced LPS-induced NO release and proinflammatory cytokine expression in BV2 cells, attenuated the shift toward a CD86-positive proinflammatory phenotype, and downregulated the TLR4/NF- κB/NLRP3 pathway-related molecules. The BV2 cell-conditioned medium co-culture experiment showed that LPS-induced inflammatory conditioned medium reduced the viability and increased the apoptosis of HT22 cells, whereas the conditioned medium from Mus-treated BV2 cells attenuated HT22 cell injury, and GW9662 partially attenuated this effect.

Mus ameliorates POCD induced by isoflurane anesthesia combined with surgery in aged mice. Its effects may be associated with regulation of PPARγ-related signaling, alleviating TLR4/NF-κB/NLRP3 pathway-mediated neuroinflammation, improving inflammatory status of microglial cells, and alleviating inflammatory microenvironment-mediated secondary neuronal injury.