Probiotics relieve perioperative postoperative cognitive dysfunction induced by cardiopulmonary bypass through the kynurenine metabolic pathway

Postoperative cognitive dysfunction (POCD) has become the popular critical post-operative consequences, especially cardiopulmonary bypass surgery, leading to an increased risk of mortality. However, no therapeutic effect about POCD. Probiotics are beneficial bacteria living in the gut and help to reduce the risk of POCD. However, the detailed mechanism is still not entirely known. Therefore, our research aims to uncover the effect and mechanism of probiotics in relieving POCD and to figure out the possible relationship between kynurenine metabolic pathway. 36 rats were grouped into three groups: sham operated group (S group, n = 12), Cardiopulmonary bypass group (CPB group, n = 12), and probiotics+CPB (P group, n = 12). After CPB model preparation, water maze test and Garcia score scale was performed to identify the neurological function. Immunofluorescence and Hematoxylin and eosin staining has been used for hippocampal neurons detection. Brain injury related proteins, oxidative stress factors, and inflammatory factors were detected using enzyme-linked immunosorbent assays (ELISA). Neuronal apoptosis was detected by TdT-mediated dUTP nick end-labeling (TUNEL) staining and western blot. High-performance liquid chromatography/mass spectrometry (HPLC/MS) was performed to detect the key factors of the kynurenine metabolic pathway. Our results demonstrated that probiotics improved neurological function of post-CPB rats. The administration of probiotics ameliorated memory and learning in spatial terms CPB rats (P < 0.05). Hematoxylin and eosin (H&E) staining data, S‐100β and neuron-specific enolase (NSE) data convinced that probiotics agonists reduced brain damage in CPB rats (P < 0.05). Moreover, probiotics regulated inflammatory factors, meanwhile attenuated hippocampal neuronal apoptosis. Probiotics alleviated POCD in rats with CPB through regulation of kynurenine metabolic signaling pathway.

Cardiopulmonary bypass (CPB) is widely used in cardiac surgery that diverts the patient's blood into an external circuit replacing the function of heart 1 .Despite development for decades, CPB still lead to neurologic complications, such as stroke 2 , seizures 3 , delirium 4 , phrenic nerve palsy 5 , and perioperative neurocognitive disorders (PND) 6 .PND involves in preoperatively or postoperatively cognitive impairment or cognitive changes.PND contains the preoperatively diagnosed cognitive impairment, postoperative delirium (POD), cognitive decline, postoperative cognitive dysfunction (POCD) and neurocognitive disorders (NCD) 7 .CPB has independently contributed to POCD with incidence up to 60% 8 .Moreover, CPB may cause the inflammation-associated hippocampal damage and lead to POCD 9 .Interestingly, POCD is effectively relieved after the treatment of systemic inflammatory response syndrome (SIRS) 10,11 .However, the detailed pathogenesis and complexity of POCD is still not fully discovered.
SIRS has been implicated in blood-brain barrier leakage, cerebral edema, and inflammation 12,13 , and has become a regulator in the progress of POCD 14,15 .Recent studies have attempted to confirm the involvement of SIRS in POCD by administering various doses and types of corticosteroids during the perioperative period to reduce inflammation and lower the incidence of POCD 14,15 .In the inflammatory therapy, probiotics regulate the host's gut microbiota, reduce the inflammatory response, and thus play a role in preventing POCD.In addition, probiotics could also enhance gut barrier function, prevent harmful substances from entering the bloodstream, and protect the brain from damage 16 .Several researchers reported that oral probiotics can reduce the incidence of POCD [16][17][18] .A study on cardiac surgery patients found that oral probiotics significantly reduced the incidence of POCD and improved inflammatory markers 19 .In addition, another study found that oral probiotics improved cognitive function and reduced the risk of POCD in elderly patients 20 .Although current research indicates that probiotics may have potential in preventing POCD, more researches are needed to determine their molecule mechanism, and to provide more conclusive evidence for probiotics' promotion and application.The kynurenine pathway (KP) was involved in regulating the cognitive function [21][22][23] .Under normal physiological conditions, tryptophan (TRP) is metabolized by tryptophan-2,3-dioxygenase (TDO), which helps to maintain the KP in a dynamic equilibrium.However, during inflammatory states, the increasing inflammatory factors stimulate the activity of indoleamine-2,3-dioxygenase (IDO), which promotes degradation of TRP.As a result, IDO becomes the primary rate-limiting enzyme in the KP for the breakdown of TRP.An imbalance of kynurenic acid (KYNA) and quinolinic acid (QUIN) can affect central nervous system and regulate cognitive function 24,25 .Those reports suggest that the abnormal metabolism of the KP pathway due to systemic inflammation following CPB may contribute to the development of POCD.
According to our preliminary research, pre-treatment with probiotics can improve intestinal barrier function to a certain extent in rats after CPB 26 .A possible explanation for this effect is that CPB inhibits CPB-induced inflammatory responses, improves local intestinal immune function, and promotes the levels of tight junction proteins 26 .Therefore, our research aims to investigate the function of probiotics on the KP related molecules, inflammatory factors, and cognitive function in rats after CPB by establishing CPB model.We explored whether probiotics improve cognitive function after CPB by regulating peripheral blood TRP and KYNA, and seeks to identify new interventions targeting the gut microbiota to improve cognitive function after CPB, providing experimental evidence for its clinical application.

Experimental animals and groupings
Age matched 36 Sprague-Dawley male (n = 18) and female (n = 18) rats (6-8 weeks old; 300-350 g for males and 250-300 g for females) were used in this experiment.All the rats were procured from the animal center of General Hospital of Northern Theater Command.The protocols of animals were approved by the Ethics Committee of Experimental Animal Welfare (No. 2020079).The study was carried out obeying the guidelines of ARRIVE.Rats were categorized into three distinct groups (n = 12 each group): Sham operation (Sham group), CPB surgery (CPB group), probiotics (P group).One week before surgery, rats of P group were orally administered 2 ml of Jin Shuang qi suspension (a probiotic preparation containing live bacteria of Bifidobacterium, Streptococcus thermophilus, and Lactobacillus) with a concentration of 1 × 10 8 /ml per day.Rats of the S and C groups were orally administered 2 ml of saline.Anesthesia is initiated via an intraperitoneal injection of pentobarbital sodium, administered at a dosage of 50 mg/kg.After anesthesia, all rats underwent mechanical ventilation and intravenous puncture.Rats of S group did not undergo CPB surgery, while rats of the C and P groups underwent CPB for 1 h.Finally, euthanasia of the rats was performed through anaesthesia with isoflurane (4-5%; oxygen at 2 L per minute) followed by decapitation by guillotine.The bilateral hippocampus was immediately collected for further studies.No animals were sacrificed during the current study.

CPB model preparation procedure
In accordance with previous study, CPB surgery was performed 27 .The preparation involves the following steps: anesthesia is initiated via an intraperitoneal injection of pentobarbital sodium, administered at a dosage of 50 mg/ kg.Endotracheal intubation is performed using a 16-gauge catheter.A midline incision is made in the chest and cannulation is done in the right jugular vein and carotid artery using a 22-gauge needle and a 24-gauge needle respectively.Heparinization is performed by administering heparin (300 IU/kg) through the jugular vein.The rats are then placed on a bypass circuit consisting of a roller pump, an oxygenator, and a heat exchanger, draining blood from the inferior vena cava and returning it to the ascending aorta.Throughout the CPB period, detection of mean arterial pressure (MAP), central venous pressure (CVP), and blood gases is carried out.After 60 min of CPB, the rats were weaned off bypass and the chest were closed.

Water maze test
The experimental procedure involves setting up a large circular pool with a hidden platform just below the water's surface.Rats are allowed to habituate to the water before the acquisition phase begins, where they are trained to locate the hidden platform using spatial cues.During the probe trial, the platform is removed, and rats' behavior was recorded to assess their memory.Data analysis includes recording and analyzing swim paths, escape latencies, and time spent in each quadrant of the pool during the acquisition and probe trials.

Neurological function scores
Following one, three, and seven days of preparing the CPB model, Garcia score scale was employed to assess the neurological functions of rats across various groups 28,29 .

Hematoxylin and eosin (H&E) staining
Following with collection, hippocampus tissues were immobilized in a 4% paraformaldehyde and embedded.5 µm sections were sliced.To prepare the sections for observation, they were dewaxed using xylene, and stained with hematoxylin (Solarbio, Beijing, China), followed by rinsing with tap water.Slices were then subjected to ethanol hydrochloride treatment for 30 s, and stained with eosin.After transparentization and sealing, hippocampal CA1 region were observed under Olympus BX51 optical microscope (Olympus Company, Japan) at a magnification of ×400.

High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)
The quantitative analysis of TRP and kynurenine (KYN) was performed using HPLC-MS/MS.Both analytes were quantified using the peak area measurement method with a standard curve and the data were acquired and processed using Agilent Mass Hunter workstation.To prepare the samples, 50 μL of rat plasma was mixed with 50 μL of ultra-pure water and then 200 μL of acetonitrile was added for protein precipitation.For sample analysis, 20 μL of the supernatant was injected into the chromatographic system.The chromatographic conditions were as follows: a Waters HSS T3 column (100 mm × 2.1 mm, 1.8 μm), a mobile phase consisting of acetonitrile and 10 mmol/L ammonium acetate solution (containing 0.1% formic acid) in 0.5 mL/min, and an injection volume of 10 μL.Prior to analysis, 200 μL of each sample was mixed with 20 μL of 10% HClO 4 precipitant, vortexed, centrifuged to obtain the supernatant for injection.

Statistical analysis
Data were processed using SPSS19.0.Normally distributed metric data were expressed as mean ± standard deviation (X ± SD).All experimental results of the two-subgroup design were analyzed through independent student t-test.The results of the multi-group design were determined by one-way ANOVA and post-hoc Tukey test of multiple comparisons.P < 0.05 was considered statistically significant.

Ethics approval and consent to participate
This study and included experimental procedures were approved by the institutional animal care and use committee of General Hospital of Northern Theater Command.All animal housing and experiments conducted in strict accordance with the institutional guidelines for care and use of laboratory animals.The experimental protocol/s was approved by General Hospital of Northern Theater Command ethical committee on October 20, 2020.

CPB rat model performs the neurological deficit
All rats regained consciousness within 90 min after surgery.As illustrated in Fig. 1A, rats in the Sham group exhibited no discernible neurological impairments, while those in CPB group displayed varying degrees of neurological abnormalities.The neurological deficit scores, as shown in Fig. 1A, significantly increased after CPB (P < 0.05) compared to sham group.The CPB model rats maintained stable data for rectal temperature, pH, and arterial blood partial pressure of carbon dioxide (PaCO 2 ), but there was reduction in partial pressure of oxygen (PaO 2 ) and hemoglobin levels, as depicted in Fig. 1B.Our findings imply that the CPB rat model was effective set up, and CPB surgery unequivocally resulted in injury to the rats' neurological system.

Probiotics alleviate the neurological dysfunction in CPB rats
The administration of probiotics resulted in an enhancement of the neurological score within the probiotics group as compared to the CPB group (Fig. 2A).To evaluate cognitive function, we conducted the water maze test (Fig. 2B, C).Our data indicated that rats of CPB group exhibited prolonged latency in locating the platform compared to that in Sham group (P < 0.05).Conversely, the H group demonstrated a significantly shorter latency in finding the platform compared to the CPB group (P < 0.05).In the spatial exploration experiment, the CPB group displayed a notable reduction in the duration of staying in the original station quadrant and the number of times the original station was crossed in the target quadrant compared to the Sham group (P < 0.05).In contrast, the H group showed a significant increase in both the duration and frequency of staying in the original station quadrant compared to the CPB group (P < 0.05).The observed conclusions convinced that probiotics have the potential to alleviate neurological dysfunction.

Probiotics reduced the brain tissue injury in CPB rats
To evaluate the benificial impact of probiotics on brain tissue injury resulting from CPB, we utilized H&E staining to analyze hippocampal neurons (Fig. 3A).In the Sham group, hippocampal neurons exhibited a well-organized and tightly packed arrangement, characterized by clear boundaries and intact cell bands.Cells displayed normal structures, with distinct cytoplasmic and nuclear staining, indicating the absence of apparent lesions.In contrast, the hippocampus of CPB group rats showed significant damage, with disorganized cells, widened intercellular spaces, and increased astrocyte and vascular proliferation.Nerve cells appeared disarrayed, with nucleus dissolution, noticeable neuronal cell and cone cell death, resulting in a considerable reduction in cell count in the hippocampus.However, in the P group, the arrangement of hippocampal cells was more regular than in the CPB group, and the number of degenerative/necrotic nerve cells was significantly lower.Brain tissue staining demonstrated the less damage after probiotics therapy as neatly arranged cells and intact cell bands.These observations imply that CPB may induce severe damage to rat hippocampus, while probiotics may exert a protective effect against such damage.To delve further into brain damage, we employed ELISA to investigate alterations in the expression of indicators for brain damage (Fig. 3B).Increased serum levels of NSE and S-100β were noted in the CPB groups compared to the Sham group (P < 0.05).However, concentrations of NSE and S-100β in the P group were notably lower than those in the CPB group (P < 0.05).These results suggest that the administration of probiotics has the capability to alleviate brain damage in CPB rats.

Probiotics decrease the inflammation and oxidative stress in CPB rats
ELISA was used to monitor the levels of inflammatory elements (Fig. 4A) and oxidative stress factors (Fig. 4B) in the rat serum.CPB surgery had increased serum concentrations of IL-1β, IL-6, TNF-α, and IFN-γ, and decreased serum concentration of IL-10 (P < 0.05), indicating severe inflammation.Furthermore, the CPB group exhibited decreased superoxide dismutase (SOD) and nitric oxide (NO) and elevated malondialdeyde (MDA) level (P < 0.05), indicating severe oxidative stress.In contrast, the P group had significantly decreased levels of IL-1β, IL-6, TNF-α, and IFN-γ, and significantly increased level of IL-10 (P < 0.05), as well as significantly promoted SOD and NO and remarkably decreased concentration of MDA (P < 0.05), indicating that probiotics reverse inflammation and oxidative stress triggered by CPB.

Probiotics improve the neuronal apoptosis in CPB rats
TUNEL staining was employed to detect neuronal apoptosis in brain tissue (Fig. 5A).The C group exhibited a notably higher count of positive cells compared to the S group (P < 0.05), while the P group demonstrated a significantly lower count of positive cells than the CPB group in the hippocampal brain tissue (P < 0.05).The expression of apoptosis-related factors Bcl-2, Bax, pro-caspase-3, and cleaved caspase-3 was assessed through Western blotting (Fig. 5B).The CPB group displayed a significant reduction in Bcl-2 expression and a substantial increase in Bax expression compared to the Sham group (P < 0.05).Conversely, the P group manifested a significant elevation in Bcl-2 expression and a notable reduction in Bax expression relative to the CPB group (P < 0.05).www.nature.com/scientificreports/Moreover, pro-caspase-3 expression markedly decreased, and cleaved caspase-3 expression significantly increased in the CPB group compared to the Sham group (P < 0.05).In the P group, pro-caspase-3 expression significantly increased, and cleaved caspase-3 expression significantly decreased compared to the CPB group (P < 0.05).These findings indicate that probiotics have the potential to forestall neuronal degeneration and diminish neuronal apoptosis in CPB rats.

Probiotics regulates the kynurenine metabolic proteins
Many research investigations have highlighted anti-inflammatory role of the kynurenine metabolic pathwaysignaling pathway, illustrating its capacity to inhibit apoptosis in neurons undergoing cerebral ischemia.In the context of treating POCD in rats subjected to CPB with probiotics, HPLC/MS analysis revealed a significant elevation in serum levels in the CPB group compared to those in the Sham group (Fig. 6, P < 0.05).Specifically, Kyn level was increased, while the Trp level was decreased significantly in the CPB group; intriguingly, there was a noteworthy decrease in Kyn and an increase in Trp levels ((Fig.6, P < 0.05).Thus, probiotics may mitigate POCD in CPB rats by modulating the kynurenine metabolic pathway-signaling pathway.

Discussion
Cognitive dysfunction is a common problem of CNS, characterized by symptoms such as confusion, anxiety, personality changes, and impaired memory 30 .POCD, which occurs in several days to weeks after CPB, was shown in decreased memory, attention, and information processing ability.POCD may cause by combined action of multiple inflammatory factors 31,32 .However, there are no reliable treatment can effectively prevent or manage POCD.Researches have shown that the inflammatory response plays a critical role in cognitive impairment in patients after surgery 33,34 .It has been reported that inhibiting the inflammatory response can effectively improve patients' cognitive impairment 35 .However, specific drugs or biological agents that regulate the cognitive behavior of patients by modulating the inflammatory response remain to be developed.Probiotics, when provided in sufficient quantities, are living microorganisms that contribute to the well-being of the host 36 .Probiotics performed the regulation of gut microbiota, improvement of intestinal barrier function, and modulation of immune response 37 .Specifically, probiotics have been shown to have a positive impact on inflammation, which is an important contributor to various neurological disorders 38 .Probiotics have the potential to diminish the generation of pro-inflammatory cytokines and hinder the initiation of inflammatory pathways, leading to a decrease in inflammation and an improvement in related symptoms 39 .Additionally, studies have shown that probiotics may have a beneficial effect on neurological disorders such as depression, anxiety, and cognitive impairment, potentially through their anti-inflammatory effects and regulation of the gut-brain axis [39][40][41] .Oral administration of specific strain of Bifidobacterium, Streptococcus thermophilus, and Lactobacillus has been proved that can effectively reduce postoperative infection rates, postoperative complication, and hospital stay duration.some experiments show that treatment with a specific strain of Bifidobacterium or Lactobacillus have a positive impact on cognitive processes and emotional behavior 42 .Bifidobacterium and Lactobacillus intake can reduce memory impairment by regulating central GABA receptor, increasing the expression of brain-derived neurotrophic factor 43,44 .Bacteroides and the closely related genus Prevotella have been shown to be associated with cognitive impairment.Probiotic capsule (BIFICO, Sine Pharmaceuticals, Shanghai, China) contains bifidobacterium longum (> 10 7 colony-forming units [CFU]/210 mg), Lactobacillus acidophil (> 10 7 CFU/210 mg), and Enterococcus faecalis (> 10 7 CFU/210 mg), which have been shown to reduce postoperative cognitive impairment in elderly patients with hip or knee replacement 45 .
Overall, the use of probiotics holds great promise for improving both the prevention and treatment of various neurological disorders.Our research demonstrated that the administration of probiotics led to a significant improvement in cognitive dysfunction in CPB rats.The results of S-100β and NSE examination revealed that probiotics reduced the brain damage in CPB rats.Additionally, we demonstrated that probiotics significantly decrease inflammatory response and oxidative stress based on ELISA detection.TUNEL staining and western blotting analysis showed that probiotics attenuated the hippocampal neuronal apoptosis.These findings suggested that probiotics could improve the POCD-related cognitive dysfunction in CPB rats.
KYN is a metabolite of the essential amino acid TRP in the human body.The KP is important in TRP metabolism of brain.This pathway is composed of different metabolites and various enzymes and is regulated by pro-inflammatory mediators and hormones, which activate or change the synthesis of metabolites in the pathway and regulate neurotransmitter release and functional execution.Moreover, many studies have found that the physicochemical changes in various CNS diseases are related to KP pathway metabolic disorders [46][47][48] .Therefore, the KP pathway plays a critical role in cognitive function.
SIRS is a significant pathological and physiological change after CPB heart surgery, and it is also an important factor in the damage to multiple target organs in the body 49 .Inflammatory elements encompassing TNF-α, IL-1β and IL-6 are related with SIRS response.These inflammatory factors can destroy the blood brain barrier (BBB), leading to brain tissue damage 50 .Numerous inflammatory factors infiltrated the brain tissue via diverse pathways, triggering the activation of glial cells and initiating a cascade of immune responses that lead to an overproduction of inflammatory mediators within CNS, and lead to neuronal damage, resulting in a decline in behavioral and memory abilities 51 .During CPB, because blood is exposed to the external environment, the body's immune system is activated, leading to SIRS during the perioperative period, and many pro-inflammatory factors such as TNF-α and IL-1β are released.In the blood after CPB surgery, endotoxins cause the release of cytokines.These cause an increase in the quinolinic acid (QUIN)/KYNA ratio.QUIN is an N-methyl-D-aspartate (NMDA) receptor activator, and injection of it can cause a decline in cognitive function accompanied by neuronal loss, similar with cognitive impairment.KYNA is the only endogenous inhibitor of NMDA receptors and plays an important role in the regulation of neural function.KYNA can block the toxicity of excitatory amino acids by inhibiting NMDA receptors, thereby preventing damage to brain tissues and protecting the nervous system 52 .Zwilling et al. 53 found that an increase in KYNA concentration and a decrease in QUIN concentration in the brain can significantly reduce the occurrence of Alzheimer disease (AD), Huntington's disease, and neurodegeneration.Solvang et al. 54 found that the KYN/TRP ratio (KTR) and folic acid levels were positively correlated with overall cognitive impairment in the elderly.Forrest et al. 55 found that the KTR in peripheral blood undergoing CPB increased significantly at 18 h and 6 days after CPB, indicating an increase in IDO activity; KYNA and 3-HAA increased significantly on the 6th day, and 3-HAA increased more significantly; Folate and TNF-α also increased on the 6th day post-CPB.Their regression analysis showed that the substances in the KP pathway were important predictors of cognitive function changes after surgery.Forrest proved that the KP pathway and folate are involved in cognitive impairment after heart surgery, and more importantly, inhibition of the KP pathway can prevent brain damage caused by ischemia and reduce central inflammation-related mortality, suggesting that the KP pathway can be used as an important potential target for the promotion of drugs for the prevention and treatment of brain damage or POCD.In our study, we found that probiotics could regulate the levels of KYN and TRP in Rats serum, balance the ratio of KYN/TRP, leading to reduce the inflammatory factors and promote the cognitive impairment.
To summarize, our study suggests that probiotics have neuroprotective effects against CPB-induced POCD brain damage, partially through modulation of the kynurenine metabolic pathway.Our research performed novel finding for molecular mechanisms and signaling pathways underlying the beneficial effects of probiotics in POCD brain damage and may offer a novel therapeutic target.Future studies should investigate other potential relationships between the gut microbiota and the kynurenine metabolic pathway in improving the specific mechanisms of POCD.

Figure 1 .
Figure 1.CPB rats were prepared successfully.The CPB model was used to study rats, and their neurological function scores and hemodynamic parameters were evaluated after the procedure.(A) The neurological function scores of rats in the sham and CPB groups.(B) Hemodynamic parameters after CPB surgery.*P < 0.05.

Figure 2 .
Figure 2. The administration of probiotics has been shown to alleviate neurological dysfunctions in rats undergoing CPB.To evaluate postoperative cognitive dysfunction, we conducted water maze tests and measured neurological function scores.The results were presented as follows: (A) neurological function score, (B) hidden platform training test, and (C) space exploration experiment.*P < 0.05.CPB cardiopulmonary bypass.

Figure 6 .
Figure 6.Probiotics regulates kynurenine metabolic proteins in CPB rats.HPLC/MS results of Kyn and Trp levels.