Recombinant chitinase-3-like protein 1 alleviates learning and memory impairments via M2 microglia polarization in postoperative cognitive dysfunction mice

Abstract

JOURNAL/nrgr/04.03/01300535-202509000-00032/figure1/v/2024-11-05T132919Z/r/image-tiff Postoperative cognitive dysfunction is a severe complication of the central nervous system that occurs after anesthesia and surgery, and has received attention for its high incidence and effect on the quality of life of patients. To date, there are no viable treatment options for postoperative cognitive dysfunction. The identification of postoperative cognitive dysfunction hub genes could provide new research directions and therapeutic targets for future research. To identify the signaling mechanisms contributing to postoperative cognitive dysfunction, we first conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the Gene Expression Omnibus GSE95426 dataset, which consists of mRNAs and long non-coding RNAs differentially expressed in mouse hippocampus 3 days after tibial fracture. The dataset was enriched in genes associated with the biological process "regulation of immune cells," of which Chil1 was identified as a hub gene. Therefore, we investigated the contribution of chitinase-3-like protein 1 protein expression changes to postoperative cognitive dysfunction in the mouse model of tibial fracture surgery. Mice were intraperitoneally injected with vehicle or recombinant chitinase-3-like protein 1 24 hours post-surgery, and the injection groups were compared with untreated control mice for learning and memory capacities using the Y-maze and fear conditioning tests. In addition, protein expression levels of proinflammatory factors (interleukin-1β and inducible nitric oxide synthase), M2-type macrophage markers (CD206 and arginase-1), and cognition-related proteins (brain-derived neurotropic factor and phosphorylated NMDA receptor subunit NR2B) were measured in hippocampus by western blotting. Treatment with recombinant chitinase-3-like protein 1 prevented surgery-induced cognitive impairment, downregulated interleukin-1β and nducible nitric oxide synthase expression, and upregulated CD206, arginase-1, pNR2B, and brain-derived neurotropic factor expression compared with vehicle treatment. Intraperitoneal administration of the specific ERK inhibitor PD98059 diminished the effects of recombinant chitinase-3-like protein 1. Collectively, our findings suggest that recombinant chitinase-3-like protein 1 ameliorates surgery-induced cognitive decline by attenuating neuroinflammation via M2 microglial polarization in the hippocampus. Therefore, recombinant chitinase-3-like protein 1 may have therapeutic potential for postoperative cognitive dysfunction.

Figure 1

Timeline of the experimental procedures. FC: Fear conditioning; rCHI3L1: recombinant chitinase-3–like protein 1.

Figure 2

Differential gene expression profile. (A) A volcano plot of the differentially expressed genes (DEGs) in the chip data. Upregulated genes are shown in red, downregulated genes in blue. (B, C) Gene Ontology (GO) analysis in biological process (B) and moelucar function (C) of the DEGs. (D, E) Protein–protein interaction (PPI) networks (D) and the top 10 genes of the clustering coefficient algorithms (E). (F, G) Freezing time (%) in the control (C) and surgery (S) groups of contextual memory (F) and cued memory (G) were conducted by contextual and cued fear conditioning tests on day 3 after surgery. (H) Representative western blots and densitometric analysis showing the expression of chitinase-3–like protein 1 (CHI3L1) on day 3 after surgery in the hippocampus. **P < 0.01, ***P < 0.001 (Student’s t-test). The experiments were repeated three times.

Figure 3

Representative western blots and densitometric analysis showing the protein expression of iNOS, IL-1β, CD206, Arg-1 and pERK in the hippocampus on days 3 and 7 after surgery and CHI3L1 treatment. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). The experiments were repeated three times. Arg-1: Arginase-1; C: control; CHI3L1: chitinase-3–like protein 1; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase; pERK: phosphorylated extracellular signal-regulated kinase; rCLP: recombinant CHI3L1; S: surgery; Veh: vehicle.

Figure 4

Changes in hippocampal BDNF, pNR2B protein expression and behavioral tests in all groups after CHI3L1 treatment. (A) Spontaneous alternation (%) in Y-maze and (B) freezing time (%) in fear conditioning test of each group on days 2, 6, and 13 (A) and days 3, 7, and 14 (B) after surgery. (C, D) Representative western blots and densitometric analysis showing the expression of BDNF and pNR2B in the hippocampus on days 3 and 7 after surgery and CHI3L1 treatment. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). The experiments were repeated three times. BDNF: Brain-derived neurotrophic factor; CHI3L1: chitinase-3–like protein 1; pNR2B: phosphorylated NMDA receptor subunit NR2B.

Figure 5

Changes in hippocampal iNOS, IL-1β, and CD206 protein expression and behavioral tests after ERK inhibitor treatment. (A) Representative western blots and densitometric analysis showing the expression of iNOS, IL-1β, and CD206 in the hippocampus on day 3 after surgery and treatment. (B) Spontaneous alternation (%) and (C) freezing time (%) of each group on day 3 after surgery. (D) Representative western blots and densitometric analysis showing the expression of BDNF and pNR2B in the hippocampus on day 3 after surgery. The experiments were repeated three times. *P < 0.05 (one-way analysis of variance followed by Tukey’s post hoc test). CHI3L1: Chitinase-3–like protein 1; ERK: extracellular signal–regulated kinase; IL-1β: interleukin-1β; iNOS: inducible nitric oxide synthase; PD: ERK inhibitor PD98059; rCLP: recombinant CHI3L1; S: surgery; Veh: vehicle.