Results

Thirty rats were enrolled and equally allocated to each group. There were no dropouts.

Behavioral test

The Y-maze alternation rate on the day before general anaesthesia did not differ between groups (p = 0.677). Repeated-measures ANOVA showed a significant main effect of time [F (1,28) = 32.408, p < 0.001] and a significant time × group interaction [F (1,28) = 29.283, p < 0.001], indicating that the change over time differed between groups (Fig. 1). In planned within-group comparisons, the alternation rate significantly decreased in the Control group (64.60% ± 17.11% → 43.56% ± 8.60%, p < 0.001), whereas no significant change was observed in the Ulinastatin group (67.27% ± 17.01% → 66.73% ± 15.58%, p = 0.345). On postoperative day 2, the alternation rate was significantly higher in the Ulinastatin group than in the Control group (Welch's t-test, p = 0.001) (Fig. 1).

Fig. 1: Cognitive function, using Y-maze test, on the day before general anaesthesia (represented with gray bar) and 2 days after general anaesthesia (represented with black bar). *: p < 0.001, compared with the day before general anaesthesia (represented with gray bar) and two days after general anaesthesia (represented with black bar) in Control group.†: p < 0.001, compared with Control group.

Microbiome analysis using lactobacillus viable count

Baseline faecal Lactobacillus counts did not differ between groups (p = 0.324). Repeated-measures ANOVA demonstrated a significant main effect of time (F (1,28) = 45.201, p < 0.001) and a significant time × group interaction (F (1,28) = 31.476, p < 0.001). In planned within-group comparisons, Lactobacillus counts significantly decreased in the Control group (11.47 ± 5.24 → 2.60 ± 1.30 log10 CFU/g, p < 0.001), whereas no significant change was observed in the Ulinastatin group (13.60 ± 6.34 → 12.80 ± 7.03 log10 CFU/g, p = 0.276). On postoperative day 2, Lactobacillus counts were significantly higher in the Ulinastatin group than in the Control group (Welch's t-test, p < 0.001) (Fig. 2).

Fig. 2: Lactobacillus viable countThe expression of Lactobacillus on the day before general anaesthesia (represented with gray bar) and 2 days after general anaesthesia (represented with black bar).*: p < 0.001, compared with the day before general anaesthesia (represented with gray bar) and two days after general anaesthesia (represented with black bar) in Control group.†: p < 0.001, compared with Control group.Abbreviations: CFU, Colony-forming unit.

SCFAs

The Ulinastatin group showed significantly higher SCFA levels than the Control group in the gut, blood, and brain (gut: 7.42 ± 1.89 vs. 0.57 ± 0.39 pg/mL, blood: 17.05 ± 6.33 vs. 1.32 ± 1.20 pg/mL, brain: 9.75 ± 2.75 vs. 0.47 ± 0.37 pg/mL; all p < 0.001) (Fig. 3).

Fig. 3: The expression of short-chain fatty acids (SCFAs). (A) Gut, (B) Blood and (C) Brain. *: p < 0.001, compared with Control group. Abbreviations: SCFAs, short-chain fatty acids.

Immunofluorescence staining and western blot for Nrf2

Immunofluorescence staining and western blot for Nrf2 in gut

Nrf2 expression assessed by immunohistochemistry was significantly higher in the Ulinastatin group than in the Control group in gut and (7.65% ± 1.02% vs. 1.81% ± 0.34%, p = 0.0001) (Fig. 4-A). Nrf2 expression assessed by Western blot was also significantly higher in the Ulinastatin group (1.04 ± 0.05 vs. 0.78 ± 0.08, p = 0.008) (Fig. 5-A).

Fig. 4: Immunofluorescence staining to detect nuclear factor erythroid-2-related factor 2 (Nrf2) (represented with white arrow). (A) Gut and (B) Brain. *: p = 0.0001, compared with Control group. Abbreviations: Nrf2, nuclear factor erythroid-2-related factor 2; DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 5: Western blot for quantification of nuclear factor erythroid-2-related factor 2 (Nrf2). (A) Gut and (B) Brain. *: p < 0.05, compared with Control group.Abbreviations: Nrf2, nuclear factor erythroid-2-related factor 2.

Immunofluorescence staining and western blot for Nrf2 in brain

Nrf2 expression assessed by immunohistochemistry was significantly higher in the Ulinastatin group than in the Control group in brain (7.79% ± 1.35% vs. 1.92% ± 0.50%, p = 0.0001) (Fig. 4-B). Nrf2 expression assessed by Western blot was also significantly higher in the Ulinastatin group (1.19 ± 0.28 vs. 0.66 ± 0.15, p = 0.013) (Fig. 5-B).