Discussion

Summary of main findings

The present study showed that ulinastatin administration before general anaesthesia had a preventive effect on cognitive decline after anaesthesia, consistent with previous reports (Cho et al., 2024a, 2024b; Kim et al., 2023c). Ulinastatin also prevented the decline of Lactobacillus in the gut following general anaesthesia. This effect was supported by corresponding changes in SCFAs in the gut, blood, and brain and by changes in Nrf2 expression in the gut and brain.

The role of lactobacillus in neurologic disorders

We focused on Lactobacillus to explore the association between POCD and gut dysbiosis. Lactobacillus is a genus of Gram-positive, rod-shaped bacteria and constitutes a significant proportion of the microbiome in humans and animals. It is commonly referred to as acidophilus and is widely used as a probiotic in foods such as yoghurt and kimchi (Michael et al., 2020; Sauer and Han, 2021). Numerous studies have shown that Lactobacillus reduces inflammation in the central nervous system (CNS) and peripheral nervous system (Chen et al., 2022; Qin et al., 2022). Supplementation with Lactobacillus has antioxidant and neuroprotective effects (Ahmad Alwi et al., 2023; Kim et al., 2023a), and has been reported to improve or prevent pathological conditions, including neurological disorders (Gao et al., 2022; Oroojzadeh et al., 2022). It has also been shown to improve memory and learning by influencing neurotrophic factors (Aghamohammad et al., 2023) and to exert neuroprotective effects in traumatic brain injury (Ma et al., 2019). Meng et al. reported that dietary Lactobacillus regulated T-cell expression; reduced IL-1, IL-6, and tumour necrosis factor-α; and improved neural condition via modulation of intestinal status in neuritis (Meng et al., 2023). Moreover, Lactobacillus plays a key role in maintaining microecological balance through various mechanisms (Fan et al., 2021; Qin et al., 2022). These findings may underlie the preventive effect of ulinastatin on Lactobacillus decline and subsequent cognitive impairment observed in this study.

Gut-brain axis & SCFAs as metabolites from microbiomes in the gut

The gut–brain axis is defined as a bidirectional communication network between the intestine and the nervous system (Liu et al., 2025). This implies that the intestine can regulate nervous system function through multiple mechanisms (Liu et al., 2022b). Two conditions are necessary to support the gut–brain axis concept: metabolites from the microbiome must be identified in the gut, and these metabolites must be detected in the blood and brain after crossing the blood–brain barrier (BBB). To examine this in relation to Lactobacillus, we focused on SCFAs, which are produced in the gut as metabolites of Lactobacillus (Wang et al., 2021a). SCFAs are absorbed mainly in the caecum and colon (Martin-Gallausiaux et al., 2021) and can cross the BBB via monocarboxylate transporters in endothelial cells (Fock and Parnova, 2023). They contribute to maintenance of tight junction integrity and modulate inflammatory processes through inhibition of NF-κB and activation of Nrf2. SCFAs therefore influence BBB integrity and regulate CNS cells both morphologically and functionally, contributing to homeostasis (Dong and Cui, 2022; Silva et al., 2020). They have also been associated with effects on emotion, cognition, and the pathophysiology of CNS disorders (Cheng et al., 2024; Dong and Cui, 2022; Silva et al., 2020). The significantly increased SCFA levels observed in the Ulinastatin group across gut, blood, and brain may reflect preservation of Lactobacillus and provide evidence supporting the gut–brain axis.

Nrf2 as a regulator of SCFAs production

Nrf2 expression was evaluated by Western blot and immunohistochemistry as a marker of inflammatory regulation. Nrf2 plays a critical role in anti-inflammatory and antioxidant processes, regulating pro-inflammatory and anti-inflammatory cytokines and controlling gene expression via antioxidant response elements. Nrf2 signaling is also important for maintaining BBB tight junctions, and SCFA-induced activation of Nrf2 protects the BBB (Fock and Parnova, 2023). The major SCFAs produced in the human gut—acetate, propionate, and butyrate—are also regulated by Nrf2 (González-Bosch et al., 2021). Decreased Nrf2 expression is associated with the development and progression of neurodegenerative diseases (George et al., 2022). Conversely, activation of Nrf2 has been shown to slow progression of disorders such as Alzheimer's disease and Parkinson's disease by enhancing antioxidant defences and suppressing neuroinflammation (Qu et al., 2020; Yang et al., 2022a). Liang et al. reported that downregulation of Nrf2 in the brain contributed to the occurrence of POCD through neuroinflammation and oxidative stress (Li et al., 2023). In the present study, higher Nrf2 expression in the gut and brain after ulinastatin administration may be associated with increased SCFA levels and may further support the involvement of the gut–brain axis.

Study limitations

Several limitations should be considered in the presents study. First, Long-term storage of plasma and tissue samples is generally recommended at −80°C, particularly for periods exceeding 3 months (Xu and Kasprzyk-Hordern, 2023). However, we stored plasma and tissue sample at −20°C and analysed within 1 month. Previous studies have reported that short-term storage at −20°C has minimal effects on the stability and integrity of plasma and tissue samples (Flores et al., 2020). Therefore, the influence of the temperature on the results in the present study might be limited. Second, comprehensive analysis, such as 16 S ribosomal ribonucleic acid sequencing and metagenomics rather than assessment of a single microbiome in the gut, is required to evaluate overall gut dysbiosis. Although the present study has the strength that Lactobacillus was quantified as CFU, which reflects viability and functional status rather than composition profiling, the analysis should be interpreted as a targeted assessment of Lactobacillus-related changes rather than a comprehensive evaluation of gut dysbiosis. Third, although SCFAs were evaluated as representative microbial metabolites potentially linked to Lactobacillus in the present study, they are not exclusively produced by Lactobacillus and may originate from multiple gut microbial taxa (Ibrahim et al., 2024; Tang et al., 2023). The aim of the present study was not to establish a direct causal relationship between Lactobacillus and SCFA production, but rather to evaluate SCFAs as a functional readout of gut microbial metabolic activity potentially involved in the gut–brain axis. Moreover, combined SCFA levels rather than individual metabolites (acetate, propionate and butyrate) were measured in the present study. Individual SCFAs exert distinct biological functions and may have differential pro-inflammatory or anti-inflammatory effects (Cuciniello et al., 2023; Mann et al., 2024; Shin et al., 2023). Therefore, the association between CFUs of Lactobacillus and SCFA levels in the present study should be interpreted cautiously. Future studies will be necessary to clarify which SCFA component is most relevant to POCD.

Conclusion

In conclusion, the preventive effect of ulinastatin on cognitive decline after general anaesthesia was associated with control of gut dysbiosis, specifically through prevention of Lactobacillus decline, as reflected by changes in SCFAs in the gut, blood, and brain, and by altered Nrf2 expression in the gut and brain. Collectively, these findings highlighted the gut–brain axis as a clinically relevant therapeutic target and supported microbiome-targeted interventions as a promising strategy for preventing POCD in clinical practice.