Itaconate alleviates anesthesia/surgery-induced cognitive impairment by activating a Nrf2-dependent anti-neuroinflammation and neurogenesis via gut-brain axis

Abstract

Background: Postoperative cognitive dysfunction (POCD) is a common neurological complication of anesthesia and surgery in aging individuals. Neuroinflammation has been identified as a hallmark of POCD. However, safe and effective treatments of POCD are still lacking. Itaconate is an immunoregulatory metabolite derived from the tricarboxylic acid cycle that exerts anti-inflammatory effects by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. In this study, we investigated the effects and underlying mechanism of 4-octyl itaconate (OI), a cell-permeable itaconate derivative, on POCD in aged mice.

Methods: A POCD animal model was established by performing aseptic laparotomy in 18-month-old male C57BL/6 mice under isoflurane anesthesia while maintaining spontaneous ventilation. OI was intraperitoneally injected into the mice after surgery. Primary microglia and neurons were isolated and treated to lipopolysaccharide (LPS), isoflurane, and OI. Cognitive function, neuroinflammatory responses, as well as levels of gut microbiota and their metabolites were evaluated. To determine the mechanisms underlying the therapeutic effects of OI in POCD, ML385, an antagonist of Nrf2, was administered intraperitoneally. Cognitive function, neuroinflammatory responses, endogenous neurogenesis, neuronal apoptosis, and Nrf2/extracellular signal-related kinases (ERK) signaling pathway were evaluated.

Results: Our findings revealed that OI treatment significantly alleviated anesthesia/surgery-induced cognitive impairment, concomitant with reduced levels of the neuroinflammatory cytokines IL-1β and IL-6, as well as suppressed activation of microglia and astrocytes in the hippocampus. Similarly, OI treatment inhibited the expression of IL-1β and IL-6 in LPS and isoflurane-induced primary microglia in vitro. Intraperitoneal administration of OI led to alterations in the gut microbiota and promoted the production of microbiota-derived metabolites associated with neurogenesis. We further confirmed that OI promoted endogenous neurogenesis and inhibited neuronal apoptosis in the hippocampal dentate gyrus of aged mice. Mechanistically, we observed a decrease in Nrf2 expression in hippocampal neurons both in vitro and in vivo, which was reversed by OI treatment. We found that Nrf2 was required for OI treatment to inhibit neuroinflammation in POCD. The enhanced POCD recovery and promotion of neurogenesis triggered by OI exposure were, at least partially, mediated by the activation of the Nrf2/ERK signaling pathway.

Conclusions: Our findings demonstrate that OI can attenuate anesthesia/surgery-induced cognitive impairment by stabilizing the gut microbiota and activating Nrf2 signaling to restrict neuroinflammation and promote neurogenesis. Boosting endogenous itaconate or supplementation with exogenous itaconate derivatives may represent novel strategies for the treatment of POCD.

Keywords: 4-octyl itaconate; Gut microbiota; Itaconate; Neurogenesis; Neuroinflammation; Nrf2; Postoperative cognitive dysfunction.

Fig. 1

Experimental protocols. Aged mice were administered intraperitoneally with 4-Octyl itaconate (OI) or/and ML385 (an antagonist of Nrf2) 1 h after exploratory laparotomy under isoflurane anesthesia. Intestinal contents, blood, and brain tissue were collected for 16S rRNA gene sequencing, metabolomics, ELISA, and immunofluorescence analysis 24 h after surgery. Another cohort of aged mice underwent the open-field test (OFT) and new object recognition (NOR) test on postoperative days 5 and 6. Subsequently, 5-ethynyl-2′-deoxyuridine (EDU) was intraperitoneally injected into aged mice for 3 days, and brain tissue was collected for immunofluorescence analysis 9 days after surgery

Fig. 2

OI treatment alleviates POCD and inhibits hippocampal neuroinflammation in aged mice. a-c Open field test (OFT) was used to detect anxiety and motor activity in aged mice; a Road map of the OFT, b Time spent in the center region, c average velocity in the OFT. d, e The NOR test was performed to measure the recognition memory; d The typical routes in the NOR tests, e Discrimination index. Determination of IL-1β (f) and IL-6 (g) levels by ELISA in primary microglia. Determination of IL-1β (h) and IL-6 (i) levels by ELISA in the hippocampus of aged mice. Representative immunofluorescence staining and statistics of GFAP (j–m) and Iba1 (n–q) in the hippocampal DG and CA1 region of aged mice. Data are expressed as mean ± SD, n = 12 for behavioral experiments, n = 4 for inflammatory cytokines and immunofluorescence staining analysis, *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA. ns, no significant difference

Fig. 3

OI improves intestinal dysbiosis in aged mice after anesthesia and surgery. The intestinal contents of aged mice were subjected to 16S rRNA gene sequencing. a, b Differences in the intestinal flora composition of aged mice among the three groups of aged mice; a Relative abundance in the Phylum levels, Negativicutes and Bacteroidia increased after POCD and decreased after OI treatment, while Bacilli decreased after POCD and increased after OI treatment. b Relative abundance in the Genus levels, Lactobacillaceae and Erysipelotrichaceae decreased after POCD and increased after OI treatment, while Lachnospiraceae increased after POCD and decreased after OI treatment. c β-diversity by PCoA. d Chao1 index to characterize richness, Simpson and Shannon indices to characterize diversity, and OI treatment after POCD decreased richness and diversity (*p < 0.05 vs. POCD, one-way ANOVA). e, f Gut microbial markers identified by LEfSe analysis. g, h PICRUSt analysis determined the potential function of the gut microbiota in the administration of OI therapy after POCD. n = 5 for 16S rRNA gene sequencing

Fig. 4

OI treatment improves POCD in aged mice by modulating neurogenesis. The intestinal contents of aged mice were subjected to an untargeted metabolomic analysis. a Correlation hierarchical clustering heatmap showing the direct correlation between gut flora and metabolites in aged mice among the three groups. b Partial least squares discriminant analysis score plot showing good separation between the sham, POCD, and OI groups. c–f Differentially expressed metabolites were identified by Venn diagram (c), volcano plots (d, e) and heatmap (f). g, h Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis evaluated the enriched pathways for the representative profiles of these differential metabolites. n = 5 for an untargeted metabolomic analysis

Fig. 5

OI treatment promotes neurogenesis and reduces neuronal apoptosis. a Representative immunostaining images of DCX (green) and EDU (red) in the hippocampal DG region. b–d Quantification of DCX and EDU positively stained cells. e Representative TUNEL immunostaining images in the hippocampal DG region. f Statistics of TUNEL-positive cells. Data are expressed as mean ± SD. n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA

Fig. 6

Nrf2 expression is increased in hippocampal neurons after OI treatment. a Representative immunofluorescence staining of DCX (green) and Nrf2 (red) in the hippocampal DG region. b Quantification of Nrf2 intensity in immature neurons in the DG region. c Immunofluorescence staining of Nrf2 (red) and MAP2 (green) in primary neurons. d Quantification of integrated density of Nrf2 in the primary neurons. Data are expressed as mean ± SD (n = 4), *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA

Fig. 7

The Nrf2/ERK signaling pathway is involved in mediating OI-driven neurogenesis. a Representative immunostaining images of DCX (green) and EDU (red) in the hippocampal DG region. b–d Quantification of EDU and DCX positively stained cells. e Representative TUNEL immunostaining images in the hippocampal DG region. f Statistics of TUNEL-positive cells. g Representative immunostaining images of DCX (green) and p-ERK (red) in the hippocampal DG region. h Quantification of DCX and p-ERK positively stained cells. i Immunofluorescence staining of p-ERK (red) and MAP2 (green) in primary neurons. j Quantification of integrated density of p-ERK in the primary neurons. Data are expressed as mean ± SD. n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA

Fig. 8

Inhibition of Nrf2 signaling by ML385 weakens the capacity of OI to reverse POCD and inhibit neuroinflammation in the hippocampus. a–c Open field test (OFT) was used to detect anxiety and motor activity in aged mice; a Road map of the OFT, b Time spent in the center region, c average velocity in the OFT. d, e The NOR test was performed to measure recognition memory; d The typical routes in the NOR tests, e Discrimination index. Determination of IL-1β (f) and IL-6 (g) levels by ELISA in primary microglia. Determination of IL-1β (h) and IL-6 (i) levels by ELISA in the hippocampus of aged mice. Representative immunofluorescence staining and Statistics of GFAP (j, k) and Iba1 (l, m) in the hippocampal CA1 region of aged mice. Data are expressed as mean ± SD, n = 12 for behavioral experiments, n = 4 for inflammatory cytokines analysis. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA. ns, no significant difference

Fig. 9

Working hypothesis for OI-mediated therapeutic effect on POCD in aged mice. OI can be hydrolyzed to itaconate after administration, which may reverse anesthesia/surgery-induced gut dysbiosis to produce neuroprotective metabolites. OI may enter the brain parenchyma due to its high cell permeability and activate Nrf2 signaling to inhibit neuroinflammatory responses and restore neurogenesis by hydrolyzing into itaconate. These combined mechanisms contributed to cognitive improvement in aged mice with POCD. OI, 4-octyl itaconate; POCD, postoperative cognitive dysfunction; Nrf2, nuclear factor erythroid 2-related factor 2; ERK, extracellular signal-related kinases